Protein Kinase A is a Dependent Factor and Therapeutic Target in Fibrous Dysplasia

Protein Kinase A is a Dependent Factor and Therapeutic Target in Fibrous Dysplasia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Protein Kinase A is a Dependent Factor and Therapeutic Target in Fibrous Dysplasia Xuefeng Zhao, Zhongyu Liu, Lu Xing, Wenlong Huang, Ning Ji, Hang ZHAO, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5522083/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Fibrous dysplasia (FD) is a skeletal disorder caused by activating mutations in Gα s , leading to bone fractures, deformities, and pain. Protein kinase A (PKA), the principal effector of Gα s /cAMP signaling, plays critical roles in various biological processes. However, its role in FD is unknown. Here, we demonstrate that PKA activation replicates FD-like lesions in a transgenic mouse model expressing an activating mutation of the PKA catalytic subunit α (PKAcα W197R ) in the skeletal stem cell (SSC) lineage. Mechanistically, PKA promotes osteoclastogenesis and aberrant osteogenic differentiation and proliferation of SSCs, while impairing mineralization. Interestingly, downregulating PKA activity, using either a genetically engineered PKA inhibitor peptide or small-molecule inhibitors, effectively alleviates FD lesions in an FD mouse model and safeguards bone health by increasing trabecular bone volume in an osteoporosis mouse model. These findings demonstrate that PKA is a dependent factor in FD initiation and progression, underscoring its potential as a therapeutic target. Biological sciences/Stem cells/Mesenchymal stem cells Health sciences/Medical research/Experimental models of disease Health sciences/Diseases/Cancer/Bone cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Fibrous dysplasia (FD) is a skeletal disease characterized by the replacement of normal bone with fibrous tissue and irregularly mineralized woven bone, leading to bone fractures, deformities, and pain 1 . FD is a disease of skeletal stem cells (SSCs) harboring activating mutations in GNAS , which encodes the α-subunit of the G s stimulatory protein (Gα s ) 2,3 . Currently, there is no cure for FD. Antiresorptive agents such as bisphosphonates (BPs) and Denosumab are used off-label to alleviate FD-related bone pain and reduce fracture risk 1,4 . However, BPs cannot prevent disease progression and may not alleviate pain effectively for some patients 5,6 . Although Denosumab has been shown to effectively reduce the progression of FD lesions and control bone pain 7–9 , the possibility of life-threatening bone-turnover rebound with hypercalcemia after discontinuation raises concerns about its safety when used to treat FD 10 . This issue highlights the urgency of understanding the pathophysiological mechanisms of FD and identifying new therapeutic targets. We previously proposed that a cure for FD could be achieved by reducing the expression of the disease-causing mutation and/or its associated downstream molecular events 3 . Protein kinase A (PKA), the principal intracellular effector of cyclic AMP (cAMP), is ubiquitously expressed in eukaryotic cells and is involved in numerous biological processes and disease conditions 11 . Under normal physiological conditions, PKA is tightly regulated by the stability of the holoenzyme 12 . The inactive tetrameric PKA dissociates after the cooperative binding of cAMP to the regulatory subunits (R), releasing two catalytic subunits (C) to phosphorylate PKA substrates. PRKACA encodes the catalytic α subunit of PKA (PKAcα). The W197R mutant of PRKACA remains active after the binding of R subunits 13,14 , which has been associated with cortisol-secreting adrenal adenomas and Cushing syndrome 15 . The R subunit and protein kinase inhibitor peptide (PKI) are two known physiological inhibitors of PKA that competitively bind to the C subunits via their inhibitory consensus sequence 16 . Our previous study revealed markedly elevated phosphorylation levels of PKA in FD lesions, corresponding to hyperactivated Gα s /cAMP signaling 3 . We hypothesized that PKA plays an important role in the initiation and progression of FD and thus could be a potential therapeutic target. The PKA signaling pathway plays a crucial yet ambiguous role in controlling skeletal homeostasis. Studies have reported opposite effects of PKA signaling on osteogenic differentiation and bone formation. Some studies indicate that PKA activation promotes the osteogenic differentiation of osteoprogenitor cells and bone formation 17–20 , whereas others show that it inhibits these processes 21–23 . Moreover, several studies have revealed differential roles of cAMP/PKA signaling in the regulation of bone remodeling, with varying effects at different stages of differentiation 24,25 . In addition to osteogenic differentiation and bone formation, Gα s /cAMP/PKA signaling is also involved in regulating osteoclastogenesis and bone resorption through receptor activator of nuclear factor kappa-B ligand (RANKL) 26,27 or nuclear factor of activated T cells c1 (NFATc1) 28,29 , although the results are conflicting. Global or macrophage/osteoclast-specific deletion of Gα s led to reduced trabecular bone quality and increased trabecular osteoclasts 30 . However, activation of PKA in osteocytes and late osteoblasts has been reported to cause no significant bone resorption 19 . Inconsistencies in existing studies largely stem from variations in the experimental materials and methods used across studies and data interpretation. These inconsistencies emphasize the need for further research to better understand PKA’s function and therapeutic potential in bone development, remodeling, and related diseases, including FD. This study demonstrated that PKA activation in SSCs is sufficient to induce FD-like lesions in a conditional, tetracycline-inducible transgenic mouse model expressing PKAcα W197R . PKA inhibition in our previously established FD mouse model 3 , either by PKI or by small molecular inhibitors of PKA (H89 and Rp-8-Br-cAMPs) 31,32 , largely rescued FD lesions. Together, our findings demonstrated that FD initiation and progression are strictly PKA-dependent. PKA promotes osteoclastogenesis, induces aberrant osteogenic differentiation and proliferation of SSCs, but suppresses bone formation, leading to imbalanced osteolytic lesions in FD. Downregulating the PKA signaling pathway effectively alleviates FD lesions in mice and safeguards bone health, suggesting that PKA is a potential therapeutic target in FD. RESULTS PKA activation in skeletal stem cells results in FD-like lesions PKAcα W197R , the constitutively active mutant of PKAcα, was generated via a site-directed mutagenesis approach to introduce the W197R amino acid substitution in recombinant human PKAcα 13 . Compared with wild-type PKAcα, PKAcα W197R resulted in more robust transcriptional activation of the cAMP-responsive element (CRE) luciferase reporter in 293T cells (Fig. 1 a). Immunoblotting for the HA tag, which was added to the PKAcα W197R construct, confirmed the successful expression of the PKAcα W197R transgene in 293T cells following transfection (Fig. 1 b). Furthermore, increased phosphorylation levels of both PKA and CREB, downstream effectors of the Gα s /PKA pathway, were observed, as shown by immunoblotting and ELISA analyses (Fig. 1 b, c). These data support the efficient activation of the PKA signaling pathway by PKAcα W197R . Next, we developed conditional transgenic mice expressing PKAcα W197R and reverse tetracycline-regulated transactivator (rtTA) under the control of Tet-responsive element (TRE) and Loxp-STOP-Loxp (LSL) cassettes, respectively (Tet-PKAcα W197R -rtTA). rtTA is transcribed and translated after the LSL cassette is eliminated by Cre recombinase and binds to the TRE. However, it only activates transcription of the target gene PKAcα W197R in the presence of doxycycline (Dox). By crossing the Tet-PKAcα W197R -rtTA mouse with the Prrx1 - Cre mouse, we created an SSC-specific, tetracycline-inducible transgenic mouse model, Tet-PKAcα W197R -rtTA/ Prrx1 - Cre (referred to as PKA mice). In PKA mice, the expression of PKAcα W197R is restricted to Prrx1 -expressing SSCs in limb and craniofacial bones (Fig. 1 d). All pups were born healthy following a Mendelian distribution with no obvious differences in either size or behavior. The expression of the PKAcα W197R transgene in limb bone was confirmed via quantitative PCR (qPCR) analysis and HA tag immunoblotting and was associated with increased PKA phosphorylation levels (Fig. 1 e, f) in PKA mice treated with Dox. These data support the efficient and tissue-specific activation of the PKA signaling pathway in PKA mice. To investigate whether the expression of PKAcα W197R in SSCs is sufficient to initiate FD, PKA mice were treated with Dox (0.1 g/L in drinking water) at the age of 6 weeks and evaluated visually for signs of bone lesions daily. While littermates appeared normal, all PKA mutant mice developed typical FD-like lesions, as judged by limb swelling (Fig. 2 a, red arrows) and limping behaviors (Supplementary Fig. 1a) rapidly following Dox induction (Fig. 2 b). Radiological analyses revealed FD with a “ground-glass” appearance and severe osteolytic changes in limb bones and skulls (Fig. 2 c, d, red arrows). Notably, PKA mice presented significant but fewer expansile bone lesions than did those with the disease-causing mutant Gα s R201C (Fig. 2 d) 3 . The osteolytic changes in PKA mice were further confirmed by a reduced bone volume fraction (BV/TV) and decreased bone mineral density (BMD), as shown by µCT analysis (Fig. 2 e). Histopathological examinations revealed classical histological features of human FD lesions in PKA mice (Fig. 2 f) 1 . H&E staining revealed that normal lamellar bone was replaced by a fibrous matrix (white stars) and irregularly shaped, immature woven bone (black stars), which lacked osteoblasts rimming its surface. Abundant osteoclasts (black arrows) were observed within the lesional area, suggesting osteoclastic bone resorption and increased bone remodeling. Von Kossa staining of undecalcified limb bone revealed severe mineralization defects and prominent osteoid. The cortical bone was compromised by widespread undermineralized tissue (white stars). Sirius Red staining revealed widespread green birefringent type III collagen under polarized light, indicating bone tissue damage. In conclusion, the expression of the activating mutant PKAcα W197R in SSCs induces FD-like lesions. PKA inhibition halts FD-like bone lesions induced by Gα The endogenous protein kinase inhibitor peptide (PKI) is a potent PKA-specific inhibitor that has been widely used in PKA-related research 16 . The synthetic peptide of the PKA-inhibiting domain of PKI (amino acids 1–24) and its nonsense mutant, PKI4A, were employed in our study to modulate PKA activity 33,34 . In vitro analyses demonstrated the robust inhibitory effect of PKI on the PKA signaling pathway. The phosphorylation of both PKA and CREB induced by a mixture of forskolin and 3-isobutyl-1-methylxanthine (FI) was suppressed after PKI plasmid transfection but not after PKI4A transfection (Fig. 3 a, b). A similar strategy as that used for the PKA mice was employed to develop conditional transgenic mice expressing PKI or PKI4A under the control of TRE, along with LSL -rtTA and EGFP elements (Tet-PKI-rtTA and Tet-PKI4A-rtTA, respectively). PKA activity modulation in our previously established FD mouse model (Tet-Gα s R201C / LSL -rtTA/ Prrx1 - Cre ) 3 was achieved by replacing LSL -rtTA with Tet-PKI-rtTA or Tet-PKI4A-rtTA mice, creating Tet-Gα s R201C /Tet-PKI-rtTA/ Prrx1-Cre (referred to as FD-PKI mice) and Tet-Gα s R201C /Tet-PKI4A-rtTA/ Prrx1-Cre (referred to as FD-PKI4A mice), respectively (Fig. 3 c). Only heterozygous FD-PKI and FD-PKI4A mice were utilized in the study. Homozygous FD-PKI mice were excluded due to their ineffectiveness in lowering the PKA signaling pathway for unknown reasons (Supplementary Fig. 2a). Additionally, homozygous FD-PKI4A mice presented a low survival rate (Supplementary Fig. 2b), possibly because of excessive rtTA expression in Tet-PKI4A-rtTA mice. The specific expression of the PKI transgene in limb bones of FD-PKI mice was confirmed by qPCR analysis (Fig. 3 d). The Gα s R201C mutation upregulated the Gα s /cAMP signaling pathway in both FD-PKI and FD-PKI4A mice, as evidenced by increased GNAS expression in their limb bones (Fig. 3 d) and elevated cAMP levels in the serum (Fig. 3 e). However, the phosphorylation of PKA was significantly suppressed in FD-PKI mice but not in FD-PKI4A mice (Fig. 3 f). These data demonstrated that PKA activity was effectively suppressed in FD-PKI mice without affecting the activation of the upstream Gα s /cAMP signaling pathway. To test whether the progression of FD is PKA dependent, both FD-PKI4A and FD-PKI mice were treated with various doses of Dox (0.005–0.1 g/L in drinking water) at the age of 6 weeks. The disease burden worsened and caused death in both FD-PKI4A and FD-PKI mice as the dose of Dox increased, although FD-PKI mice presented a milder disease burden than FD-PKI4A mice did (Supplementary Fig. 4a). All FD-PKI4A mice developed severe FD-like bone lesions within 1 week of Dox induction at a dose as low as 0.006 g/L (Fig. 2 a–e, and Supplementary Fig. 1a). The histopathological changes in FD-PKI4A mice (Fig. 2 f) mirrored those observed in our previously established FD mice (Supplementary Fig. 3a–c). Interestingly, most FD-PKI mice failed to develop an FD-like phenotype after 2 weeks of Dox induction at the same dose despite the mild swelling observed in the limbs and paws (Fig. 2 a, b). The cortical bone and skull of FD-PKI mice appeared continuous and intact, with no evidence of deformities or osteolytic changes (Fig. 2 c, d). The anatomical structure and quality of limb bones in FD-PKI mice did not differ from those in control mice (Fig. 2 e). Histopathologically, typical FD-like changes were absent in FD-PKI mice (Fig. 2 f). The cortical bone was well mineralized without the presence of woven bone. Locomotor activity analysis, which is used to assess movement ability and quality of life, revealed no significant difference between FD-PKI and control mice, unlike in PKA or FD-PKI4A mice (Supplementary Fig. 1a). Upon close examination, only 3 out of 32 FD-PKI mice developed minor FD-like lesions under this dose of Dox, which were confined to the metaphysis with no invasion into the bone marrow cavity (Fig. 2 b and Supplementary Fig. 4a). Collectively, these findings suggest that PKA inhibition halts FD-like bone lesions induced by Gα s R201C . PKA inhibition in FD-SSCs suppresses osteoclastogenesis and promotes bone formation Next, we investigated the bone remodeling events regulated by PKA. The increase in osteoclastogenesis caused by Gα s R201C was suppressed to normal levels in FD-PKI mice, as evidenced by the absence of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the cortical bone (Fig. 4 a). In contrast, the differentiation of osteoclasts was significantly increased in PKA and FD-PKI4A mice, which led to severe bone resorption (Fig. 4 a). Consistently, the serum level of tartrate-resistant acid phosphatase 5b (TRACP-5b) (Fig. 4 b) and the transcriptional level of osteoclast-specific markers ( Csf1r , Rank , Nfatc1 , and Acp5 ) were increased in the bone lesions of PKA and FD-PKI4A mice, similar to those of FD mice (Supplementary Fig. 5a). However, these osteoclast markers demonstrated downregulated expression in FD-PKI mice. Rankl was suppressed to normal levels in FD-PKI mice in both gene and circulating protein forms (Supplementary Fig. 5b and Fig. 4 c). Besides the unchanged expression levels of Osteoprotegerin ( Opg ), the decoy receptor for Rankl , upon PKA signaling modulation, the Rankl / Opg ratio was significantly reduced in FD-PKI mice (Supplementary Fig. 5b, c). However, Rankl level and Rankl / Opg ratio were significantly elevated upon PKA activation in PKA and FD-PKI4A mice, stimulating osteoclast differentiation (Fig. 4 c and Supplementary Fig. 5b, c). These data suggest that osteoclastic bone resorption in FD lesions is driven by Rankl-dependent osteoclastogenesis, which is promoted by PKA activation. Conversely, PKA activation enhanced the osteogenic lineage commitment of SSCs but inhibited osteoblast maturation. Significantly elevated expression of osteoblast transcription factors, runt-related transcription factor 2 ( Runx2 ) and Osterix ( Osx ), and other osteogenic genes, such as alkaline phosphatase ( Alp ) and collagen type 1 ( Col1a1 ), and marginally decreased expression of the mature osteoblast marker osteocalcin ( Ocn ) were detected in the bone lesions of PKA mice (Fig. 4 d). The aberrant osteogenic differentiation of SSCs disrupted by Gα s signaling activation was restored in FD-PKI mice following PKA inhibition. Ocn-expressing matured osteoblasts (lining cells) were observed on the surface of the trabecular bone in FD-PKI mice (Fig. 4 e). Notably, early osteogenic differentiation was not completely blocked in FD-PKI mice, as demonstrated by the expression of Osx on the surface of trabecular bone (Fig. 4 e). Furthermore, the highly proliferative activities of the fibroblast-like cells were revealed by a 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay in PKA and FD-PKI4A mice (Fig. 4 f, g) 3,35 . Collectively, our data demonstrated that the PKA signaling pathway promotes Rankl-dependent osteoclastogenesis and induces early osteogenic differentiation and proliferation of SSCs but impairs their maturation and mineralization. Downregulating ectopically activated PKA signaling could reduce osteoclastogenesis and restore the balance between osteogenic differentiation and osteoclastogenesis, resulting in bone formation. Downregulated PKA signaling safeguards bone health To further study the effects of PKA on bone remodeling without hyperactivation of Gα s /cAMP signaling, we created a Tet-PKI-rtTA/ Prrx1 - Cre mouse model (referred to as PKI mice) (Fig. 5 a). We hypothesized that the bone volume and density would increase in PKI mice upon PKA inhibition on the basis of accumulated data from PKA and FD-PKI mice. To downregulate PKA signaling in the SSCs of limb bone and observe phenotypic changes, various doses of Dox were administered to PKI mice for up to 6 months (Fig. 5 b). The expression of the PKI transgene in limb bone was confirmed by qPCR analysis and EGFP tag immunofluorescence staining in mutant mice (Fig. 5 c, d). The phosphorylation of PKA was decreased in limb bones of PKI mice following Dox administration (Fig. 5 e). Surprisingly, there were no significant phenotypic (Fig. 5 f) or behavioral (data not shown) differences between PKI and control mice after 6 months of high-dose Dox induction (6 g/kg in the diet). The cortical bone of the limbs was continuous and well mineralized without significant pathological changes in the PKI mice (Fig. 5 g, h). Osteoclastogenesis remained at a normal level compared with that in control mice (Fig. 5 i). Interestingly, abundant adipocytes were observed in the bone marrow of PKI mice (Fig. 5 h, j) 25,36 . Further analyses via µCT imaging revealed no significant thickening of cortical bone or skull in PKI mice (Fig. 5 k). The cortical bone mass in PKI mice was unaffected by PKA inhibition, as shown by the BV/TV and tissue mineral density (TMD) (Fig. 6 l). However, significant increases in both the number and thickness of trabecular bone were observed in PKI mice, resulting in a subtle increase in trabecular bone mass (Fig. 5 m, n). Mirroring the findings in PKI and PKA mice, MC3T3-E1-14 osteoblastic cells exhibited severely impaired mineralization capacity upon PKA activation and significantly increased mineralization upon PKA inhibition, modulated by FI and H89, respectively (Supplementary Fig. 6a–c). To further assess the effect of PKA inhibition on bone remodeling, we utilized a mouse model of osteoporosis induced by ovariectomy (OVX) in both female PKI mice and their littermate controls. All OVX mice, along with a group of blank control mice, then underwent 11 weeks of Dox induction at a dose of 6 g/kg (Fig. 6 a). As expected, the ovariectomized control group (Control + OVX) exhibited significant bone loss, as evidenced by reductions in BMD, BV/TV, and trabecular number, along with an increase in trabecular separation, compared with those of the non-ovariectomized control group (Control) (Fig. 6 b–d) 37 . However, downregulation of PKA signaling in SSCs mitigated bone loss in ovariectomized PKI mice (PKI/ Prrx1-cre + OVX). There was no statistically significant difference in the BV/TV, BMD, or trabecular variables between the blank control and ovariectomized PKI mice (Fig. 6 b–d). These data suggest that downregulated PKA signaling safeguards both the quantity and quality of bone under normal and disease conditions. PKA is a potential therapeutic target for FD Inspired by the above data, we explored therapeutic options for FD by targeting PKA. Two small-molecule inhibitors of PKA, H89 and Rp-8-Br-cAMPS, both of which are potent and selective with distinct mechanisms, were utilized to decrease PKA activity in our FD mouse model. The therapeutic regimen is shown in Fig. 7 a. First, the FD phenotype was established in all FD mice after 3 days of Dox induction (0.2 g/L in the drinking water) (T1). FD mice were then randomly divided into the FD, FD + H89, and FD + Rp-8-Br-cAMPs groups. While all the mice were subjected to the same dose of Dox throughout the study, the FD mice in the two PKA inhibitor groups received either H89 or Rp-8-Br-cAMPs via intraperitoneal injection every other day for 11 days (T2). At T2, all FD mice presented significantly upregulated GNAS expression in limb bones (Fig. 7 b). PKA signaling was effectively suppressed in FD mice from both the FD + H89 and FD + Rp-8-Br-cAMPs groups, as evidenced by the decreased phosphorylation level of PKA in limb bones (Fig. 7 c). Limb swelling and reduced mobility observed in FD mice at T1 were significantly improved in both PKA inhibitor groups within 1 week of treatment (Fig. 7 d). By T2, FD mice in the PKA inhibitor groups exhibited less limb swelling and significantly improved mobility compared with those in the FD group, although their mobility remained below normal levels (Fig. 7 e, f). Similarly, FD mice treated with PKA inhibitors presented reduced ground-glass opacities (Fig. 7 g) and osteolytic changes (Fig. 7 h). H&E staining demonstrated that FD lesions were restricted to the metaphysis area in the mice treated with the PKA inhibitors and did not affect the diaphysis. In contrast, mice in the FD group exhibited fully developed FD lesions expanding throughout the entire limb bones, accompanied by severe osteolytic bone damage (Fig. 7 i). In conclusion, our findings demonstrate that PKA inhibition effectively alleviates FD lesions in mice, confirming that PKA is a potential therapeutic target in FD. DISCUSSION In this study, we investigated the role of PKA in the pathophysiological mechanisms of FD via a combination of molecular and genetic approaches. Our findings demonstrate that FD initiation and progression are strictly PKA-dependent. PKA promotes osteoclastogenesis, induces aberrant osteogenic differentiation and proliferation of SSCs, but suppresses bone formation, leading to imbalanced osteolytic lesions in FD. Downregulating the PKA signaling pathway effectively alleviates FD lesions in mice and safeguards bone health, suggesting that PKA is a potential therapeutic target in FD. FD is caused by activating mutations of Gα s in SSCs, which lead to elevated cAMP concentrations and phosphorylation levels of PKA. Dysregulation of PKA signaling is associated with a wide range of diseases. PKAcα W197R , the mutant used in our study, is a constitutively active variant of the catalytic subunit of PKA 13,14 . Unlike some other PKAcα mutants, in silico analysis revealed no changes in the PKA substrate specificity of PKAcα W197R 38 , making it an ideal candidate for studying the mechanisms of PKA in FD pathogenesis. To examine whether PKA activation is sufficient to initiate FD, we developed a transgenic mouse model expressing PKAcα W197R in SSCs under the control of Dox (Tet-PKAcα W197R -rtTA/ Prrx1 - Cre ). Typical FD-like bone lesions rapidly develop in the limb and skull areas following the upregulation of PKA signaling through the expression of PKAcα W197R . This finding parallels others’ study in which the upregulation of PKA activity through R type I α subunit (R1α) haploinsufficiency led to FD-like bone lesions 39 . These findings demonstrate that the activation of PKA is sufficient to initiate FD and reinforces the pivotal role of PKA in FD. Notably, the FD-like lesions in the abovementioned mouse model exhibit more severe osteoclastic resorption and less expansion than those initiated by disease-causing Gα s R201C mutants (Tet-Gα s R201C / LSL -rtTA/ Prrx1 - Cre and Tet-Gα s R201C /Tet-PKI4A-rtTA/ Prrx1-Cre ), suggesting that PKA plays a dominant role in osteoclastogenesis. PKA inhibition in our previously established FD mouse model was achieved by introducing PKI into a transgenic system (Tet-Gα s R201C /Tet-PKI-rtTA/ Prrx1-Cre ). Both Gα s R201C and PKI were expressed in SSCs in mice upon induction with Dox. PKA activity was effectively suppressed in these mice without affecting the activation of the upstream Gα s /cAMP signaling pathway. As a result, the progression of FD was effectively halted by PKI but not its nonsense mutant PKI4A. Notably, different doses of Dox were used in our transgenic mouse models, including PKA, FD-PKI (4A), and FD mice. This variation may be attributed to the newer third generations of TRE and rtTA and the high-efficiency promoters used in the PKA and FD-PKI (4A) mice. In PKA and PKI (4A) mice, rtTA transgene expression driven by the CAG promoter might be greater than that driven by the PKG promoter in previous LSL -rtTA mice 3,40,41 , leading to increased sensitivity to Dox. This could explain the low survival rate of homozygous FD-PKI4A mice treated with Dox. As a result, only heterozygous FD-PKI (4A) mice were utilized in the study. For better comparison, the Dox dose for FD-PKI mice was adjusted to match that of FD-PKI4A mice, resulting in comparable lesions to those observed in FD mice. Together, our data demonstrate that FD initiation and progression are strictly PKA-dependent. Next, we assessed PKA as a therapeutic target for FD. Our data demonstrate that FD lesions in mice were effectively alleviated through the use of the pharmacological PKA inhibitors H89 and Rp-8-Br-cAMPs, highlighting the potential of targeting PKA as a novel therapeutic approach for FD. Notably, many studies have identified the non-PKA-specific actions of H89 42 . Although Rp-8-Br-cAMPs is considered selective inhibitor of type I PKA due to its cAMP analog structure and mechanisms of action 32 , their inhibitory efficacy may be compromised when the cAMP level is extremely high, as in our FD mouse model. Additionally, Rp-8-Br-cAMPs may bind to exchange protein activated by cAMP (Epac), another direct target of cAMP that shares similar cyclic nucleotide–binding sites with PKA 43 . However, our preliminary data revealed that FD lesions could not be rescued by ESI-09 44 , a small-molecule inhibitor of Epac (data not shown). Epac is a guanine nucleotide exchange factor for the small GTPases Rap1/2, and it has been implicated in various cellular processes, such as cell adhesion, cell–cell junction formation, insulin secretion, and neurotransmitter release 45 . There is a complex interconnection between the Epac- and PKA-mediated signaling pathways. For example, a recent study showed that inhibiting PKA via PKI diverts GPCR/Gα s /cAMP signaling toward EPAC and ERK activation and is involved in tumor growth 46 . More studies are needed to elucidate the role of Epac in FD pathogenesis. Undeniably, Gα s is a negative phenotypic regulator of bone mass, as evidenced by the opposing bone manifestations observed in Albright's hereditary osteodystrophy (AHO), progressive osseous heteroplasia (POH), and FD 47,48 . AHO and POH are associated with inactivating Gα s mutations, which are clinically characterized by heterotopic ossification 49 , whereas FD is linked to activating Gα s mutations, leading to bone destruction 2 . However, the role of Gα s /cAMP/PKA signaling in regulating bone remodeling is controversial, largely due to variations in the experimental materials and methods used across studies and data interpretation. For example, Gα s or PKA dysregulation in different cell populations often leads to complicated or opposing results 19,20,30,50–52 . PKA signaling appears to regulate stem cells and progenitor cells differently from bone cells, such as osteoblasts and osteocytes, although the underlying mechanisms remain unclear. Importantly, the expression levels of osteogenic transcription factors, such as Runx2 and Osterix, do not necessarily correlate with the quantity or quality of bone formation in vivo , as observed phenotypically. Conclusions concerning how PKA regulates osteogenic differentiation should not rely solely on changes in the expression levels of osteogenic markers, which may help explain discrepancies between in vitro and in vivo studies 18,20,22,50 . Accumulating evidence supports a stage-dependent regulatory model in which Gα s /cAMP/PKA promotes early osteogenic differentiation but impairs maturation 3,25,53,54 . Additionally, Gα s has been shown to exert differential effects on trabecular and cortical bone 29,30,50 and age-dependent 55 and sex-dependent 19,20,56 influences on osteogenic differentiation or bone formation, suggesting the involvement of complex regulatory mechanisms in bone remodeling. Our findings suggest a dual regulatory model of bone remodeling in which PKA induces early osteogenic differentiation and proliferation of SSCs but impairs their maturation and mineralization while significantly enhancing RANKL-dependent osteoclastogenesis. However, unlike Gα s , the downstream effector PKA does not appear to function as a straightforward negative regulator of bone mass. Compared with the dramatic bone loss caused by hyperactivation in this study, PKA inhibition had only a subtle effect on trabecular bone anabolism, suggesting the presence of compensatory feedback mechanisms that counterbalance the downregulation of PKA signaling. This bone safeguard effect makes PKA inhibition a promising anabolic therapy for bone destruction diseases, including FD and osteoporosis 57 . This hypothesis is preliminarily supported by observations in an osteoporosis disease model, where the quantity and quality of bone were preserved in ovariectomized mice following PKA inhibition. FD typically manifests in adolescence and progresses into adulthood 58 , necessitating early intervention and ongoing treatment. Our study highlights, for the first time, the dependency of PKA on the initiation and progression of FD and suggests the promising therapeutic potential of PKA inhibition in treating FD and other similar bone destruction diseases. The safety of the antiresorptive medications currently used in FD management is an increasing concern. We demonstrated that long-term PKA inhibition in mice, restricted to SSCs in the limb and craniofacial areas, caused no significant side effects on bone or soft tissue. Instead, it slightly increased the trabecular bone volume. A cure for FD could be achieved by specifically targeting PKA, rather than the upstream disease-causing Gα s mutations, to avoid broader impacts and minimize side effects. Encouragingly, the pathophysiological role of the Gα s /PKA pathway has recently gained increased attention from researchers 11 . Various promising approaches and delivery systems targeting different components of the Gα s /PKA pathway have been developed and continue to evolve 59–62 , paving the way for molecular therapies for FD and other Gα s /PKA-related diseases. This study has several limitations. The regulatory mechanisms of PKA in bone remodeling remain elusive. More in-depth studies are needed to reveal the underlying mechanisms, particularly the possible compensatory crosstalk mechanisms that counterbalance the downregulation of PKA signaling in bone remodeling, as observed in this study. This approach could lead to the identification of additional therapeutic targets for bone metabolism diseases. Moreover, given the broad involvement of PKA in physiological processes, the safety of the general use of PKA inhibition requires further evaluation. MATERIALS AND METHODS Study design This study aimed to evaluate the influence of PKA on FD development, its regulatory impact on differentiation, and its potential as a therapeutic target. These objectives were addressed by (i) creating a series of tetracycline-inducible, tissue-specific transgenic models (PKA mice, PKI mice, FD-PKI mice, and FD-PKI4A mice) to achieve in vivo regulation of the Gα s /cAMP/PKA signaling axis, (ii) examining the bone tissue phenotype in these mice, (iii) investigating the cellular and molecular changes induced by PKA regulation both in vivo and in vitro , and (iv) studying the effects of the intraperitoneal injection of two small-molecule PKA inhibitors on attenuating FD progression. Sample sizes were determined by the investigators on the basis of previous experimental experience, with exact numbers provided in the respective figure captions. In vivo experiments included all genetically screened animals in which the dosing strategy was adhered to. For experiments involving exogenous drug administration, animals and samples were randomly assigned to experimental and control groups. Endpoints were set at 2 weeks for all models except for PKI mice, with an endpoint of 6 months. The investigators were not blinded during the data analysis. All in vivo and in vitro experiments were replicated three or more times. DNA constructs Recombinant human PKAcα was amplified via PCR from pRSET PKAcα, a gift from Dr. Susan Taylor, with a C-terminal HA tag inserted into pCEFL (pCEFL PKAcα-HA). The sequence was verified by Sanger sequencing. The W197R amino acid substitution was carried out using the QuikChange II site-directed mutagenesis kit (Agilent, Santa Clara, CA, 200523) 13 . The primers used for mutagenesis are as follows: PKAcα W197R forward: 5'-cgtgtgaaaggccgtact A ggaccttgtgtg-3', PKAcα W197R reverse: 5'-cacacaaggtcc T agtacggcctttcacacg-3'. The synthetic peptide of the PKA-inhibiting domain of PKI (amino acids 1–24) and its nonsense mutant, PKI4A, were employed in our study to modulate PKA activity 34 . Briefly, EGFP-PKI was cloned by inserting the 24 coding amino acids of human PKI-alpha (PKIA) into the C-terminus of EGFP. For use as a control, the phenylalanine and arginine residues of the PKI peptide were replaced with alanine to disrupt binding to protein kinase A (PKA), and the resulting protein was named EGFP-PKI4A. Mice To generate Tet-PKAcα W197R -rtTA, Tet-PKI-rtTA, and Tet-PKI4A-rtTA transgenic mice, the coding sequences were cloned downstream of the tet-responsive elements (TREs) within an improved Tet-On (3G) vector (Clontech® Laboratories, Inc., Mountain View, CA, USA, PT5148-1) 63 . Mice carrying the mutated human Gα s R201C gene were previously described 34 , and they are referred to as “Tet-Gα s R201C ” mice. Mice expressing Cre recombinase in mouse limb buds driven by a Prrx1 -derived enhancer (B6. Cg-Tg(Prrx1-cre)1Cjt/J), referred to as “Prrx1-Cre mice”, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) 64 . We confirmed the presence of transgenes through PCR analysis of tail DNA. The presence of Gα s R201C transgenes was analyzed as previously described 34 . Specific primers were used to verify the presence of the Tet-PKAcα W197R , Tet-PKI, and Tet-PKI4A transgenes, each producing bands of the expected sizes (601, 349, and 356 bp, respectively). The generation and characterization of the Tet-Gα s R201C /LSL-rtTA/ Prrx1-Cre mouse model (referred to as “FD mice”) have been previously described 3 . To create other models, Tet-PKAcα W197R -rtTA mice were crossed with Prrx1-Cre mice to generate Tet-PKAcα W197R -rtTA/ Prrx1-Cre mice (referred to as “PKA mice”). Similarly, Tet-PKI-rtTA mice were crossed with Prrx1-Cre mice to produce PKI-rtTA/ Prrx1-Cre mice (referred to as “PKI mice”). By crossing Tet-PKI-rtTA mice with Tet-Gα s R201C and Prrx1-Cre mice, we generated Tet-Gα s R201C /Tet-PKI-rtTA/ Prrx1-Cre (referred to as “FD-PKI mice”). Finally, by replacing Tet-PKI-rtTA with Tet-PKI4A-rtTA, we generated Tet-Gα s R201C /Tet-PKI4A-rtTA/ Prrx1-Cre (referred to as “FD-PKI mice”). All the experiments used littermates, including male and female mice, as controls. Dox (MedChemExpress, Monmouth Junction, NJ, USA, HY-N0565B) was administered at concentrations ranging from 0.006 to 0.1 g/L in the drinking water or 6 g/kg in the diet. Dox induction commenced at 6 weeks of age and continued for up to 2 weeks, 11 weeks, or 6 months, as specified for each experiment. Bilateral ovariectomies were performed at the age of 9 weeks to mimic postmenopausal estrogen deficiency 65,66 . Two small-molecule PKA inhibitors were used in the FD mice rescue study and were administered via intraperitoneal injection every 2 days: H89 (MedChemExpress, Monmouth Junction, NJ, HY-15979A) at a dose of 50 mg/kg and Rp-8-Br-cAMPs (MedChemExpress, Monmouth Junction, NJ, HY-100530D) at a dose of 100 mg/kg. The animal experiments were conducted in accordance with protocols approved by the Sichuan University Institutional Animal Care and Use Committee (IACUC) and complied with the Guide for the Care and Use of Laboratory Animals. The mice were housed in specific pathogen-free (SPF) barrier facilities at the Laboratory Animal Center of West China Second University Hospital, Sichuan University. The use of live animals was approved by the Ethics Committee of West China Hospital of Stomatology (WCHSIRB-D-2019-197). Cell lines, osteogenic induction and transfection The MC3T3-E1 subclone 14 cell line was obtained from SAIOS Biotechnology Co., Ltd. (Wuhan, China, CL-077 m). The cells were cultured in complete alpha-MEM supplemented with 90% alpha-MEM (Gibco, Waltham, MA, USA, #12571063) and 10% FBS (Gibco, Waltham, MA, USA, #10099141C) at 5% CO2 and 37°C. For osteogenic differentiation, the cells were seeded into 24-well plates at a density of 3×10 4 cells/well and cultured in osteogenic (OS) induction medium for 2 weeks. The OS medium comprised complete alpha-MEM supplemented with 150 µM ascorbic acid (MilliporeSigma, Burlington, MA, USA, A4403), 10 mM β-glycerophosphate (MilliporeSigma, Burlington, MA, USA, G9422), and 10 nM dexamethasone (MilliporeSigma, Burlington, MA, USA, D4902). Forskolin (MilliporeSigma, Burlington, MA, USA, F6886) was used at a concentration of 10 µM in combination with IBMX (MilliporeSigma, Burlington, MA, USA, I7018) at 100 µM in OS medium (FI medium). H89 (MedChemExpress, Monmouth Junction, NJ, USA, HY-15979A) was used at a concentration of 10 µM in OS medium. The 293T cell line was obtained from ATCC and cultured in complete DMEM supplemented with 90% DMEM (Gibco, Waltham, MA, USA, #C11995500BT) and 10% FBS. The cells were seeded into 24-well plates at a density of 2.5×10 4 cells per well and transfected with Lipofectamine 3000 (Invitrogen, Waltham, MA, USA, L3000001) following the manufacturer's instructions. At 48 h posttransfection, the cells were harvested. Intracellular total-CREB and phospho-CREB levels were measured via the Total CREB ELISA Kit (Cell Signaling Technology, Danvers, MA, USA, #36001C) and Phospho-CREB (Ser133) Sandwich ELISA Kit (Cell Signaling Technology, Danvers, MA, USA, #7385), respectively, according to the manufacturers’ instructions. Luciferase assay A cAMP-responsive-element–driven reporter luciferase (CRE-luc) assay was employed to measure PKA activity 67 . 293T cells in 24-well plates were co-transfected with CRE (0.05 µg cm − 2 ) plus the DNA constructs indicated in the Fig. 1 A: Gα S (0.1 µg cm − 2 ), PKAcα (0.1 µg cm − 2 ), and PKAcα W197R (0.1 µg cm − 2 ). The cells were harvested 24 hours after transfection, and the luciferase activity was subsequently measured via a Dual-Glo Luciferase Assay Kit (Promega, Madison, WI, USA, E1910) and a microtiter plate luminometer (Dynex Tech, Chantilly, VA, USA). Luciferase normalization was performed in every case by co-transfecting a Renilla luciferase vector (0.005 µg cm − 2 ; Promega). Alizarin red staining (ARS) and quantitative analysis ARS was conducted after 14 days of osteogenic induction. The cells were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. After being washed with PBS three times, the cells were stained with freshly prepared ARS solution (Beyotime, Shanghai, China, C0148S) for 15 min and then washed at least five times with distilled water until the rinsing fluid was clear. Images were captured via a stereomicroscope (Leica, Wetzlar, Germany, EZ4 HD), and quantitative analysis was performed with ImageJ software 68 , with a consistent HSB threshold (hue: 0–30, saturation: 160–222, brightness: 174–255). Tissue collection and processing Fresh bone tissues were ground into shatters via a tissue homogenizer (Servicebio, Wuhan, China, #KZ-5F-3D) in TRIzol reagent (Invitrogen, Carlsbad, CA, USA, #15596–026) immediately after collection. The resulting mixture was frozen at − 80°C for subsequent RNA isolation. Bone tissue samples were collected and fixed in zinc 10% formalin fixative (MilliporeSigma, Burlington, MA, USA, #Z2902) at room temperature overnight and then stored in 70% ethanol for further processing. Fixed skeletal samples were either decalcified in 4% buffered EDTA for subsequent paraffin and optimal cutting temperature (OCT) frozen embedding or directly embedded in polymethyl methacrylate (PMMA) without decalcification. RNA isolation and quantitative real-time polymerase chain reaction (qPCR) RNA was isolated from bone tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, #15596–026) and from cell lines using the Cell Total RNA Isolation Kit (Foregene, Chengdu, China, RE-03111) following the manufacturer's instructions. Reverse transcription was conducted with the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan, #RR037A) on an S1000 Thermal Cycler Platform (BIO–RAD, Irvine, CA, USA). Subsequent quantitative PCR (qPCR) was performed using SYBR Select Master Mix (Applied Biosystems, Waltham, MA, USA, #44729080) on a QuanStudio 3 Real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The target gene expression levels were normalized to Gapdh . The oligonucleotides used for amplification were as follows: (gene, forward sequence 5' → 3', reverse sequence 5' → 3'): Gapdh 5'-TCATTGACCTCAACTACATG-3', 5'-TCGCTCCTGGAAGATGGTGAT-3'; Gnas 5'-GCAGAAGGACAAGCAGGTCT-3', 5'-CCCTCTCCGTTAAACCCATT-3'; PRKACA 5'-GCGTGTGAAAGGCCGTACT-3', 5'-GGATAGGCTGGTCAGCGAAG-3', PKI 5'-AACGAGAAGCGCGATCACATG-3', 5'-TGCATTTCTTCTACCTGTTCTTCCTG-3', Csflr 5'-TGGATGCCTGTGAATGGCTCTG-3', 5'-GTGGGTGTCATTCCAAACCTGC-3'; Rank 5'-GGACAACGGAATCAGATGTGGTC-3', 5'-CCACAGAGATGAAGAGGAGCAG-3'; Nfatc1 5'-GGTGCCTTTTGCGAGCAGTATC-3', 5'-CGTATGGACCAGAATGTGACGG-3'; Acp5 5'-GCGACCATTGTTAGCCACATACG-3', 5'-CGTTGATGTCGCACAGAGGGAT-3'; Rankl 5'-CACAGCGCTTCTCAGGAGCTC-3', 5'-GAGATCTTGGCCCAGCCTCGA-3'; Opg 5'-AGTCCGTGAAGCAGGAGTGCA-3', 5'-AAGTCTCACCTGAGAAGAACC-3'; Runx2 5'-GACTGTGGTTACCGTCATGGC-3', 5'-ACTTGGTTTTTCATAACAGCGGA-3'; Osx 5'-ATGGCGTCCTCTCTGCTTG-3', 5'-TGAAAGGTCAGCGTATGGCTT-3'; Alpl 5'-CACGGCCATCCTATATGGTAA-3', 5'-GGGCCTGGTAGTTGTTGTGA-3'; Col1a1 5'-CACCCTCAAGAGCCTGAGTC-3', 5'-GTT CGGGCTGATGTACCAGT-3'; Ocn 5'-ACCCTGGCTGCGCTCTGTCTCT-3', 5'-GATGCGTTTGTAGGCGGTCTTCA-3'. Immunoblot analysis Western blot assays were performed and repeated at least three times, as previously described 34 . The antibodies used were as follows: GAPDH (Cell Signaling Technology, Danvers, MA, USA, #2118), CREB (Cell Signaling Technology, Danvers, MA, USA, #9197), phospho-CREB Ser133 (Cell Signaling Technology, Danvers, MA, USA, #9198), HA-tag (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-805), and phospho-PKA substrate (RRXS*/T*) (Cell Signaling Technology, Danvers, MA, USA, #9624). Serum collection and measurements Mouse blood (> 1 ml) was collected via retro-orbital eye collection prior to euthanasia. The blood was stored at room temperature for 30 min. Subsequently, the serum was separated via centrifugation (30 min, 5000 rcf, 4°C) and frozen at − 80°C for future analysis. Serum levels of cAMP, TRACP-5b, and Rankl were quantified via enzyme-linked immunosorbent assay (ELISA). The ELISA kits used were the Mouse Cyclic Adenosine Monophosphate (cAMP) ELISA Kit (Jianglai Industry Co., Shanghai, China, JL13362), the Mouse Tartrate-Resistant Acid Phosphatase 5b (TRACP-5b) ELISA Kit (US Biological, Salem, MA, USA, 359452), and the Mouse TRANCE (TNFSF11) ELISA Kit (RayBiotech, Norcross, GA, USA, O35235). Animal behavior analyses An apparatus (XinRuan Technology, Shanghai, China) consisting of an acrylic test box (internal measurements: 50 cm × 50 cm × 45 cm) equipped with a video camera was used for the animal behavior analyses. The 300-second test was started immediately after the animals were transported to the test box, given that the novelty aspect of the environment is a crucial component of the test. The parameters analyzed were the total distance traveled, average speed, walking periods, and number of standing times. Microcomputed tomography (µCT) The specimens were scanned using a SCANCO Medical AG VivaCT 80 scanner with an isotropic voxel size of 10 µm or a SCANCO Medical µCT 100 scanner (SCANCO Medical, Brüttisellen, Switzerland) with an isotropic voxel size of 7.5 µm, following standard guidelines 69 . The region of interests (ROIs) is defined as the area 2 mm and 3 mm proximal to the tibial growth plate or middle of diaphysis. The threshold set at 220 for 3D image reconstruction. Sirius red staining Following deparaffinization and rehydration, the paraffin sections were stained with Picro-Sirius Red solution for 1 h. The Picrosirius Red solution consisted of 0.1% (wt/vol) Direct Red 80 (MilliporeSigma, Burlington, MA, USA, #2610-10-8) in a saturated aqueous picric acid solution. After two washes in 0.5% acetic acid water, the sections were dehydrated in graded alcohol and mounted with Permount mounting medium (Biosharp, Anhui, China, BL704A). The evaluation was conducted under transmitted and polarized light microscopy (Nikon, Tokyo, Japan, Eclipse Ci-Pol). Tartrate-resistant acid phosphatase (TRAP) staining The paraffin sections were incubated at 37°C in freshly prepared TRAP staining solution for 15 min, with periodic monitoring. Following staining, the sections were washed in distilled water, counterstained with methyl green (Wako, Richmond, VA, USA, #184-67001), dehydrated in graded alcohol, and mounted with Permount mounting medium (Biosharp, Anhui, China, BL704A). The TRAP staining solution was composed of 0.02% naphthol AS-TR phosphate (MilliporeSigma, Burlington, MA, USA, #N6125-1G) and 0.03% Fast Red violet LB salt (MilliporeSigma, Burlington, MA, USA, #F3381-5G) dissolved in 0.1 M sodium acetate buffer (pH 5), containing 50 mM sodium tartrate (MilliporeSigma, Burlington, MA, USA, #S4797-100G) and 0.1 M sodium acetate (MilliporeSigma, Burlington, MA, USA, #S1111500GM). Immunohistochemistry Paraffin sections were deparaffinized and rehydrated through xylene and a graded alcohol series, followed by microwave antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The sections were then blocked with 10% goat serum (Boster Biological Technology, Wuhan, China, AR1009) for 1 h and incubated with primary antibodies overnight at 4°C. Detection was performed with the avidin-biotin complex (ABC) and DAB systems using the VECTASTAIN ABC-HRP Kit (Vector Laboratories, Newark, CA, USA, PK-6101) and the DAB Detection Kit (GeneTech, Shanghai, China, GK600510). The sections were subsequently counterstained with hematoxylin. The slides were scanned using a brightfield slide scanner (Leica, Wetzlar, Germany; Aperio VERSA). Each immunostaining was performed in at least three mice, with multiple fields reviewed. The antibodies used in this study included anti-Sp7/osterix (Osx) (Abcam, Cambridge, MA, USA, ab22552) and anti-osteocalcin (Ocn) (Abcam, Cambridge, MA, USA, ab93876). Immunofluorescence OCT-embedded frozen sections were blocked and incubated with primary EGFP-tag antibody (Proteintech, Rosemont, IL, USA, 50430-2-AP) overnight at 4°C. The sections were subsequently incubated with anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA, Alexa Fluor 546), followed by nuclear counterstaining and mounting with mounting medium containing DAPI (Vector Laboratories, Newark, CA, USA, H-1200-10). Each immunostaining was performed in at least three mice, with multiple fields reviewed. Images were captured using an A1 HD25 confocal microscope (Nikon, Tokyo, Japan). EdU proliferation assay The proliferation assay was conducted as previously described. Briefly, 100 µL of 10 mM EdU (Thermo Fisher Scientific, Waltham, MA, USA, A10044) per 10 g of mouse body weight was administered 3 h prior to euthanasia, followed by decalcification and sectioning. Click staining and subsequent immunofluorescence staining were performed according to the manufacturer's instructions for the Click-it EdU Alexa Fluor 555 Imaging Kit (Thermo Fisher Scientific, Waltham, MA, USA, C10338). Images were captured using a confocal microscope (Nikon, Tokyo, Japan, A1 HD25), and quantitative analysis was performed using ImageJ software 68 , with a consistent threshold (65–225). Statistical analysis All the data were analyzed using GraphPad Prism software (San Diego, CA, USA). Significance was determined by the P value. One-way ANOVA, two-way ANOVA, and t tests were performed as appropriate to analyze significant differences among groups. A P value less than 0.05 was considered statistically significant. All the statistical tests were two-tailed. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”. Declarations Acknowledgments: We thank Dr. J. Silvio Gutkind and Dr. Susan S. Taylor for generously sharing pRSET PKAcα plasmid with us. We thank Dr. Ramiro Iglesias-Bartolome and Dr. Xiaodong Feng for meaningful discussion. We thank Dr. Li Chen from Analytical & Testing Center Sichuan University for her help with micro-CT scanning and analysis. Funding: This project was supported by National Natural Science Foundation of China Grants (82170995, 82470940, 82071146, 82371002, 81991502); National Key R&D Program of China (2022YFC2402900); Sichuan Science and Technology Program, China (2024NSFSC0540); charitable funds from Mr. Mingxu Zhou family for fibrous dysplasia related research (Sichuan University internal grants 18H1134, 19H1134 and 23H0924). 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Gs G protein-coupled receptor signaling in osteoblasts elicits age-dependent effects on bone formation. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 25 , 584-593 (2010). McMullan, P., Maye, P., Yang, Q., Rowe, D. W. & Germain-Lee, E. L. Parental Origin of Gsalpha Inactivation Differentially Affects Bone Remodeling in a Mouse Model of Albright Hereditary Osteodystrophy. JBMR Plus 6 , e10570 (2022). Basha, G. et al. Lipid nanoparticle-mediated silencing of osteogenic suppressor GNAS leads to osteogenic differentiation of mesenchymal stem cells in vivo. Mol Ther 30 , 3034-3051 (2022). Florenzano, P. et al. Age-Related Changes and Effects of Bisphosphonates on Bone Turnover and Disease Progression in Fibrous Dysplasia of Bone. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 34 , 653-660 (2019). Manschwetus, J. T. et al. A Stapled Peptide Mimic of the Pseudosubstrate Inhibitor PKI Inhibits Protein Kinase A. Molecules 24 (2019). Rinaldi, L. et al. Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system. Nature communications 10 , 2572 (2019). Coles, G. L. et al. Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells. Cancer cell 38 , 129-143 e127 (2020). Visscher, M., Arkin, M. R. & Dansen, T. B. Covalent targeting of acquired cysteines in cancer. Curr Opin Chem Biol 30 , 61-67 (2016). Das, A. T., Tenenbaum, L. & Berkhout, B. Tet-On Systems For Doxycycline-inducible Gene Expression. Curr Gene Ther 16 , 156-167 (2016). Logan, M. et al. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 33 , 77-80 (2002). Jilka, R. L. et al. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 257 , 88-91 (1992). Finkelman, R. D., Bell, N. H., Strong, D. D., Demers, L. M. & Baylink, D. J. Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss. Proceedings of the National Academy of Sciences of the United States of America 89 , 12190-12193 (1992). Castellone, M. D., Teramoto, H., Williams, B. O., Druey, K. M. & Gutkind, J. S. Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science 310 , 1504-1510 (2005). Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9 , 671-675 (2012). Bouxsein, M. L. et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 25 , 1468-1486 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files 20241124V41NCSI.pdf Supplementary Figures Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5522083","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":385843358,"identity":"010b7980-73a9-41e2-9f07-d619527208bc","order_by":0,"name":"Xuefeng Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYDACCQjFzw8iEwpI0CI5swGkxYAULRsOgChitMjPbn74gOHPYQnj86sTPzwwYJDnFzuAXwvjnGPGBgw8aRJmN95ulgA6zHDm7AT8WpglEswkGCRs6sxunN0A0pJgcJuAFjaJ9G8SDAYSEsYzzm7+QZQWHokcoC0JNhIG/L3biLNFQiKn2CDhQJqExA3ebRYJBhKE/SI/I33jgw/AEOPvP7v55o8KG3l+aQJawACsRgJCEqEcDvgPkKJ6FIyCUTAKRhIAADDTPWscj90aAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6860-7739","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Xuefeng","middleName":"","lastName":"Zhao","suffix":""},{"id":385843359,"identity":"9e6b95c1-a7da-4af1-8862-7fc382a59b00","order_by":1,"name":"Zhongyu Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhongyu","middleName":"","lastName":"Liu","suffix":""},{"id":385843360,"identity":"45f1216c-b4c1-446c-972d-b6ff701600a6","order_by":2,"name":"Lu Xing","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Xing","suffix":""},{"id":385843361,"identity":"71bab98d-a970-4296-af66-cd4ddf12eeda","order_by":3,"name":"Wenlong Huang","email":"","orcid":"https://orcid.org/0000-0002-8641-7872","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wenlong","middleName":"","lastName":"Huang","suffix":""},{"id":385843362,"identity":"af91d4c4-34dc-4fc7-9723-a5fb8ba31632","order_by":4,"name":"Ning Ji","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Ji","suffix":""},{"id":385843363,"identity":"b05fabad-808c-4546-84e5-0ead1459f0e4","order_by":5,"name":"Hang ZHAO","email":"","orcid":"https://orcid.org/0000-0003-1268-0616","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"ZHAO","suffix":""},{"id":385843364,"identity":"8965466d-a0ba-4021-89dd-58a5b9b64908","order_by":6,"name":"Qianming Chen","email":"","orcid":"https://orcid.org/0000-0002-5371-4432","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Qianming","middleName":"","lastName":"Chen","suffix":""},{"id":385843365,"identity":"f519caec-2a0f-40c0-a5b9-6fc3edbff00a","order_by":7,"name":"Xianglong Han","email":"","orcid":"https://orcid.org/0000-0001-7650-8204","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xianglong","middleName":"","lastName":"Han","suffix":""},{"id":385843366,"identity":"64795122-bc6e-4af4-af6f-f50b312c2a88","order_by":8,"name":"Ding Bai","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Ding","middleName":"","lastName":"Bai","suffix":""}],"badges":[],"createdAt":"2024-11-25 16:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5522083/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5522083/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-61402-z","type":"published","date":"2025-07-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70644359,"identity":"fe1ec679-c745-4531-9b64-61f0207de318","added_by":"auto","created_at":"2024-12-05 07:55:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":345815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKAcα\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eW197R\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutation enhances PKA signaling.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Luciferase assay showing cAMP-responsive element-binding protein (CREB) expression in PKAcα\u003csup\u003eW197R\u003c/sup\u003e-transfected cells. (\u003cstrong\u003eb\u003c/strong\u003e) Immunoblot showing the phosphorylation of PKA and CREB in PKAcα\u003csup\u003eW197R\u003c/sup\u003e-transfected cells, with HA-tag detection confirming transfection. (\u003cstrong\u003ec\u003c/strong\u003e) ELISA results showing the level of phosphorylated CREB in PKAcα\u003csup\u003eW197R\u003c/sup\u003e-transfected cells (n = 6, two-way ANOVA). (\u003cstrong\u003ed\u003c/strong\u003e) Schematic of the Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e/\u003cem\u003ePrrx1-Cre\u003c/em\u003e transgenic mouse model (PKA mice) for tissue-specific expression of PKAcα\u003csup\u003eW197R\u003c/sup\u003e upon Dox administration. (\u003cstrong\u003ee\u003c/strong\u003e) qPCR analysis of \u003cem\u003ePRKACA\u003c/em\u003e expression in limb bones of PKA mice (n = 8, unpaired t test). (\u003cstrong\u003ef\u003c/strong\u003e) Immunoblotting confirming phosphorylated PKA levels in limb bones. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/3f92a6f06cb31e036a800649.png"},{"id":70644364,"identity":"8703251d-6bef-4203-9151-179e9353ae52","added_by":"auto","created_at":"2024-12-05 07:55:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1655081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA activation induces FD-like lesions, while its inhibition halts Gα\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/sub\u003e\u003csup\u003e\u003cstrong\u003eR201C\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-induced FD lesions. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative limb images of Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (PKA mice), Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI4a/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (FD-PKI4a mice), Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (FD-PKI mice), and littermate controls. (\u003cstrong\u003eb\u003c/strong\u003e) Kaplan‒Meier curve of mice free from visible disease symptoms (limb swelling and limping). (\u003cstrong\u003ec\u003c/strong\u003e) μCT images of long bones showing ground-glass deformities (red arrows) in PKA and FD-PKI4a mice, which were not observed in FD-PKI mice. (\u003cstrong\u003ed\u003c/strong\u003e) Representative images of three-dimensional reconstructions of limb bone showing expansile lesions in PKA and FD-PKI4a mice. Skull images revealing bone defects in PKA and FD-PKI4a mice. (\u003cstrong\u003ee\u003c/strong\u003e) Quantitative μCT analysis of the bone volume fraction (BV/TV) and bone mineral density (BMD) in PKA, FD-PKI4a, FD-PKI, and littermate controls (n ≥ 5, one-way ANOVA). (\u003cstrong\u003ef\u003c/strong\u003e) Histopathological examination (H\u0026amp;E, von Kossa, and Sirius red staining) of bone showing FD-like lesions in PKA and FD-PKI4a mice. FD-PKI and control mice exhibit dense cortical bone with defined boundaries (dashed lines). Multinucleated osteoclasts (black arrows) are observed on woven bone surfaces in PKA and FD-PKI4a mice. Sirius red staining revealed disorganized woven bone and type III collagen fibrosis in PKA and FD-PKI4a mice. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/c98c0b7bd6e1873aaf667be0.png"},{"id":70644537,"identity":"14f63ced-938f-4ee3-97df-5bf9fc03a013","added_by":"auto","created_at":"2024-12-05 08:03:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":496914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA inhibitor peptide (PKI) suppresses PKA signaling. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Immunoblotting and (\u003cstrong\u003eb\u003c/strong\u003e) ELISA showing the phosphorylation levels of PKA and CREB in 293T cells stimulated with forskolin and 3-isobutyl-1-methylxanthine (FI) with or without PKI plasmid transfection (n = 6, one-way ANOVA). (\u003cstrong\u003ec\u003c/strong\u003e) Schematic representation of the Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (FD-PKI mice) transgenic mouse model, which is engineered to express the Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e mutation, thereby upregulating the Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling pathway while concurrently expressing PKI to inhibit PKA signaling in a tissue-specific manner upon Dox administration. The Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI4a/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (FD-PKI4a mice) model was generated via the same strategy. (\u003cstrong\u003ed\u003c/strong\u003e) qPCR analysis confirming PKI mRNA transcription in FD-PKI mice and showing increased \u003cem\u003eGNAS\u003c/em\u003e mRNA expression in limb bones of both FD-PKI and FD-PKI4a mice (n ≥ 6, one-way ANOVA). (\u003cstrong\u003ee\u003c/strong\u003e) ELISA results showing cAMP levels in the serum of FD-PKI and FD-PKI4a mice (n ≥ 6, one-way ANOVA). (\u003cstrong\u003ef\u003c/strong\u003e) Immunoblotting showing the phosphorylation of PKA in PKA, FD-PKI4a, and FD-PKI mice. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/bc87f2be87d960eca31044e0.png"},{"id":70644539,"identity":"79b52350-994b-46a3-94a9-ee427c15785a","added_by":"auto","created_at":"2024-12-05 08:03:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1252368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA inhibition in FD-SSCs suppresses osteoclastogenesis and promotes osteogenic differentiation. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) TRAP staining of limb bone showing TRAP-positive cells in PKA, FD-PKI4A and FD-PKI mice. (\u003cstrong\u003eb\u003c/strong\u003e) ELISA results showing the serum TRACP-5b levels in PKA, FD-PKI4A and FD-PKI mice (n ≥ 5, one-way ANOVA). (\u003cstrong\u003ec\u003c/strong\u003e) ELISA results of serum Rankl levels (n ≥ 5, one-way ANOVA). (\u003cstrong\u003ed\u003c/strong\u003e) qPCR analyses of early osteogenic markers in limb bones (n ≥ 6, two-way ANOVA). (\u003cstrong\u003ee\u003c/strong\u003e) Immunohistochemistry showing elevated early osteogenic markers in PKA and FD-PKI4A mice, which returned to baseline levels in FD-PKI mice. In contrast, late osteogenic marker levels decreased in PKA and FD-PKI4A mice and increased in the FD-PKI mice. (\u003cstrong\u003ef\u003c/strong\u003e) EdU staining and (\u003cstrong\u003eg\u003c/strong\u003e) quantitative analysis demonstrating increased proliferation in PKA and FD-PKI4A mice, with no significant difference in proliferation between PKA mice and FD-PKI4A mice (n ≥ 4, unpaired t test). The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/5fdc0943f5dc757c31d8aa27.png"},{"id":70644546,"identity":"7d755ebb-1743-4b22-8c6f-0ceedca5de56","added_by":"auto","created_at":"2024-12-05 08:03:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":590294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA inhibition in SSCs only modestly increases trabecular bone mass. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic representation of the Tet-PKI/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (PKI mice) transgenic mouse model, which is designed to express PKA inhibitor peptide (PKI) in a tissue-specific manner upon Dox administration. (\u003cstrong\u003eb\u003c/strong\u003e) Study regimen outlining the Dox treatment schedule for PKI and control mice, along with the timing of sample collection (V = video, H = histology, TC = tissue collection, SC = serum collection). (\u003cstrong\u003ec\u003c/strong\u003e) qPCR analysis confirming the transcription of PKI mRNA in PKI mice. (\u003cstrong\u003ed\u003c/strong\u003e) Immunofluorescence staining showing EGFP-tag expression, verifying successful PKI expression in PKI mice. (\u003cstrong\u003ee\u003c/strong\u003e) Immunoblotting demonstrating the suppression of PKA phosphorylation in limb bone. (\u003cstrong\u003ef\u003c/strong\u003e) Representative images of limb morphology and (\u003cstrong\u003eg\u003c/strong\u003e) μCT images showing no significant differences in limb swelling or bone deformity between PKI mice and littermate controls. (\u003cstrong\u003eh\u003c/strong\u003e) H\u0026amp;E staining and (\u003cstrong\u003ei\u003c/strong\u003e) TRAP staining of limb bone revealed no significant differences in bone histomorphology or osteoclast activity between PKI mice and littermate controls. (\u003cstrong\u003ej\u003c/strong\u003e) Quantitative analysis of the number of adipocytes in PKI mice (n ≥ 4, unpaired t test). (\u003cstrong\u003ek\u003c/strong\u003eand \u003cstrong\u003el\u003c/strong\u003e) Representative images of three-dimensional reconstruction and quantitative analyses of cortical bone and skull (n ≥ 6, unpaired t test). (\u003cstrong\u003em\u003c/strong\u003e and \u003cstrong\u003en\u003c/strong\u003e) Representative images of three-dimensional reconstruction and quantitative analyses of trabecular bone (n ≥ 6, unpaired t test). The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/17aea8092a59c0aa8dfefda8.png"},{"id":70645641,"identity":"960ceba5-3b57-48e9-943d-a7c985ec1ee0","added_by":"auto","created_at":"2024-12-05 08:11:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":604272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA inhibition in SSCs mitigates bone loss in ovariectomized mice. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Study regimen outlining the Dox treatment and ovariectomy (OVX) schedule for PKI/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (PKI mice) and control mice, along with the timing of sample collection (H = histology, TC = tissue collection, SC = serum collection). (\u003cstrong\u003eb\u003c/strong\u003e) Representative images of limbs at T1 from OVX-operated PKI mice, OVX-operated control mice, and untreated control mice. (\u003cstrong\u003ec\u003c/strong\u003eand \u003cstrong\u003ed\u003c/strong\u003e) μCT and quantitative analyses of trabecular bone at T1 in OVX-operated control mice and PKI mice. The parameters assessed included the bone volume fraction (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), and trabecular space (Tb.Sp) (n ≥ 4, unpaired t test). The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/63fa92d7f69c40106d6e4755.png"},{"id":70644547,"identity":"0d9201c9-2fde-459a-a2a0-ef833fbf32dc","added_by":"auto","created_at":"2024-12-05 08:03:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1054985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKA is a potential therapeutic target in fibrous dysplasia. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Therapeutic regimen outlining Dox and PKA inhibitor administration in Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/\u003cem\u003eLSL\u003c/em\u003e-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (FD mice), along with the timing of sample collection (V = video, H = histology, TC = tissue collection, SC = serum collection). (\u003cstrong\u003eb\u003c/strong\u003e) qPCR analysis at T2 showing \u003cem\u003eGNAS\u003c/em\u003e mRNA expression in limb bones (n ≥ 4, one-way ANOVA). (\u003cstrong\u003ec\u003c/strong\u003e) Immunoblotting at T2 demonstrating PKA phosphorylation in limb bones. (\u003cstrong\u003ed\u003c/strong\u003e) Kaplan‒Meier curve showing the persistence of FD-like symptoms (limb swelling and limping behavior) in the FD group (n = 11), whereas the FD + H89 (n = 11) and FD + Rp-8-Br-cAMPs (n = 7) groups presented a reversal of symptoms within 1 week of PKA inhibitor treatment. (\u003cstrong\u003ee\u003c/strong\u003e) Locomotor activity analyses assessing movement ability before and after PKA inhibitor treatment (n ≥ 4, one-way ANOVA; data not shown). (\u003cstrong\u003ef\u003c/strong\u003e) Representative limb images at T2. (\u003cstrong\u003eg\u003c/strong\u003e) μCT images at T2. (\u003cstrong\u003eh\u003c/strong\u003e) Representative images of three-dimensional reconstructions of limb bone at T2. (\u003cstrong\u003ei\u003c/strong\u003e) H\u0026amp;E staining at T2 showing the restricted bone lesions in the inhibitor-treated groups. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th–75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as “+”.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/c491ad5ef0601af8c8d3e9c1.png"},{"id":85826763,"identity":"d431d4e6-8758-4740-aecb-5a7afb31bc39","added_by":"auto","created_at":"2025-07-02 07:18:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7931418,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/c347e7d3-264d-4a91-93f8-ee12bff171ff.pdf"},{"id":70645640,"identity":"5c481562-8391-4cd4-9f19-b2525c87e649","added_by":"auto","created_at":"2024-12-05 08:11:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":909200,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"20241124V41NCSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5522083/v1/b7320711104832391667e040.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Protein Kinase A is a Dependent Factor and Therapeutic Target in Fibrous Dysplasia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFibrous dysplasia (FD) is a skeletal disease characterized by the replacement of normal bone with fibrous tissue and irregularly mineralized woven bone, leading to bone fractures, deformities, and pain\u003csup\u003e1\u003c/sup\u003e. FD is a disease of skeletal stem cells (SSCs) harboring activating mutations in \u003cem\u003eGNAS\u003c/em\u003e, which encodes the α-subunit of the G\u003csub\u003es\u003c/sub\u003e stimulatory protein (Gα\u003csub\u003es\u003c/sub\u003e)\u003csup\u003e2,3\u003c/sup\u003e. Currently, there is no cure for FD. Antiresorptive agents such as bisphosphonates (BPs) and Denosumab are used off-label to alleviate FD-related bone pain and reduce fracture risk\u003csup\u003e1,4\u003c/sup\u003e. However, BPs cannot prevent disease progression and may not alleviate pain effectively for some patients\u003csup\u003e5,6\u003c/sup\u003e. Although Denosumab has been shown to effectively reduce the progression of FD lesions and control bone pain\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e, the possibility of life-threatening bone-turnover rebound with hypercalcemia after discontinuation raises concerns about its safety when used to treat FD\u003csup\u003e10\u003c/sup\u003e. This issue highlights the urgency of understanding the pathophysiological mechanisms of FD and identifying new therapeutic targets.\u003c/p\u003e \u003cp\u003eWe previously proposed that a cure for FD could be achieved by reducing the expression of the disease-causing mutation and/or its associated downstream molecular events\u003csup\u003e3\u003c/sup\u003e. Protein kinase A (PKA), the principal intracellular effector of cyclic AMP (cAMP), is ubiquitously expressed in eukaryotic cells and is involved in numerous biological processes and disease conditions\u003csup\u003e11\u003c/sup\u003e. Under normal physiological conditions, PKA is tightly regulated by the stability of the holoenzyme\u003csup\u003e12\u003c/sup\u003e. The inactive tetrameric PKA dissociates after the cooperative binding of cAMP to the regulatory subunits (R), releasing two catalytic subunits (C) to phosphorylate PKA substrates. \u003cem\u003ePRKACA\u003c/em\u003e encodes the catalytic α subunit of PKA (PKAcα). The W197R mutant of \u003cem\u003ePRKACA\u003c/em\u003e remains active after the binding of R subunits\u003csup\u003e13,14\u003c/sup\u003e, which has been associated with cortisol-secreting adrenal adenomas and Cushing syndrome\u003csup\u003e15\u003c/sup\u003e. The R subunit and protein kinase inhibitor peptide (PKI) are two known physiological inhibitors of PKA that competitively bind to the C subunits via their inhibitory consensus sequence\u003csup\u003e16\u003c/sup\u003e. Our previous study revealed markedly elevated phosphorylation levels of PKA in FD lesions, corresponding to hyperactivated Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling\u003csup\u003e3\u003c/sup\u003e. We hypothesized that PKA plays an important role in the initiation and progression of FD and thus could be a potential therapeutic target.\u003c/p\u003e \u003cp\u003eThe PKA signaling pathway plays a crucial yet ambiguous role in controlling skeletal homeostasis. Studies have reported opposite effects of PKA signaling on osteogenic differentiation and bone formation. Some studies indicate that PKA activation promotes the osteogenic differentiation of osteoprogenitor cells and bone formation\u003csup\u003e17\u0026ndash;20\u003c/sup\u003e, whereas others show that it inhibits these processes\u003csup\u003e21\u0026ndash;23\u003c/sup\u003e. Moreover, several studies have revealed differential roles of cAMP/PKA signaling in the regulation of bone remodeling, with varying effects at different stages of differentiation\u003csup\u003e24,25\u003c/sup\u003e. In addition to osteogenic differentiation and bone formation, Gα\u003csub\u003es\u003c/sub\u003e/cAMP/PKA signaling is also involved in regulating osteoclastogenesis and bone resorption through receptor activator of nuclear factor kappa-B ligand (RANKL)\u003csup\u003e26,27\u003c/sup\u003e or nuclear factor of activated T cells c1 (NFATc1)\u003csup\u003e28,29\u003c/sup\u003e, although the results are conflicting. Global or macrophage/osteoclast-specific deletion of Gα\u003csub\u003es\u003c/sub\u003e led to reduced trabecular bone quality and increased trabecular osteoclasts\u003csup\u003e30\u003c/sup\u003e. However, activation of PKA in osteocytes and late osteoblasts has been reported to cause no significant bone resorption\u003csup\u003e19\u003c/sup\u003e. Inconsistencies in existing studies largely stem from variations in the experimental materials and methods used across studies and data interpretation. These inconsistencies emphasize the need for further research to better understand PKA\u0026rsquo;s function and therapeutic potential in bone development, remodeling, and related diseases, including FD.\u003c/p\u003e \u003cp\u003eThis study demonstrated that PKA activation in SSCs is sufficient to induce FD-like lesions in a conditional, tetracycline-inducible transgenic mouse model expressing PKAcα\u003csup\u003eW197R\u003c/sup\u003e. PKA inhibition in our previously established FD mouse model\u003csup\u003e3\u003c/sup\u003e, either by PKI or by small molecular inhibitors of PKA (H89 and Rp-8-Br-cAMPs)\u003csup\u003e31,32\u003c/sup\u003e, largely rescued FD lesions. Together, our findings demonstrated that FD initiation and progression are strictly PKA-dependent. PKA promotes osteoclastogenesis, induces aberrant osteogenic differentiation and proliferation of SSCs, but suppresses bone formation, leading to imbalanced osteolytic lesions in FD. Downregulating the PKA signaling pathway effectively alleviates FD lesions in mice and safeguards bone health, suggesting that PKA is a potential therapeutic target in FD.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePKA activation in skeletal stem cells results in FD-like lesions\u003c/h2\u003e \u003cp\u003ePKAcα\u003csup\u003eW197R\u003c/sup\u003e, the constitutively active mutant of PKAcα, was generated via a site-directed mutagenesis approach to introduce the W197R amino acid substitution in recombinant human PKAcα\u003csup\u003e13\u003c/sup\u003e. Compared with wild-type PKAcα, PKAcα\u003csup\u003eW197R\u003c/sup\u003e resulted in more robust transcriptional activation of the cAMP-responsive element (CRE) luciferase reporter in 293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Immunoblotting for the HA tag, which was added to the PKAcα\u003csup\u003eW197R\u003c/sup\u003e construct, confirmed the successful expression of the PKAcα\u003csup\u003eW197R\u003c/sup\u003e transgene in 293T cells following transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Furthermore, increased phosphorylation levels of both PKA and CREB, downstream effectors of the Gα\u003csub\u003es\u003c/sub\u003e/PKA pathway, were observed, as shown by immunoblotting and ELISA analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). These data support the efficient activation of the PKA signaling pathway by PKAcα\u003csup\u003eW197R\u003c/sup\u003e. Next, we developed conditional transgenic mice expressing PKAcα\u003csup\u003eW197R\u003c/sup\u003e and reverse tetracycline-regulated transactivator (rtTA) under the control of Tet-responsive element (TRE) and \u003cem\u003eLoxp-STOP-Loxp (LSL)\u003c/em\u003e cassettes, respectively (Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA). rtTA is transcribed and translated after the \u003cem\u003eLSL\u003c/em\u003e cassette is eliminated by \u003cem\u003eCre\u003c/em\u003e recombinase and binds to the TRE. However, it only activates transcription of the target gene PKAcα\u003csup\u003eW197R\u003c/sup\u003e in the presence of doxycycline (Dox). By crossing the Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA mouse with the \u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e mouse, we created an SSC-specific, tetracycline-inducible transgenic mouse model, Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA/\u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e (referred to as PKA mice). In PKA mice, the expression of PKAcα\u003csup\u003eW197R\u003c/sup\u003e is restricted to \u003cem\u003ePrrx1\u003c/em\u003e-expressing SSCs in limb and craniofacial bones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). All pups were born healthy following a Mendelian distribution with no obvious differences in either size or behavior. The expression of the PKAcα\u003csup\u003eW197R\u003c/sup\u003e transgene in limb bone was confirmed via quantitative PCR (qPCR) analysis and HA tag immunoblotting and was associated with increased PKA phosphorylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f) in PKA mice treated with Dox. These data support the efficient and tissue-specific activation of the PKA signaling pathway in PKA mice.\u003c/p\u003e \u003cp\u003eTo investigate whether the expression of PKAcα\u003csup\u003eW197R\u003c/sup\u003e in SSCs is sufficient to initiate FD, PKA mice were treated with Dox (0.1 g/L in drinking water) at the age of 6 weeks and evaluated visually for signs of bone lesions daily. While littermates appeared normal, all PKA mutant mice developed typical FD-like lesions, as judged by limb swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, red arrows) and limping behaviors (Supplementary Fig.\u0026nbsp;1a) rapidly following Dox induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Radiological analyses revealed FD with a \u0026ldquo;ground-glass\u0026rdquo; appearance and severe osteolytic changes in limb bones and skulls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d, red arrows). Notably, PKA mice presented significant but fewer expansile bone lesions than did those with the disease-causing mutant Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed)\u003csup\u003e3\u003c/sup\u003e. The osteolytic changes in PKA mice were further confirmed by a reduced bone volume fraction (BV/TV) and decreased bone mineral density (BMD), as shown by \u0026micro;CT analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Histopathological examinations revealed classical histological features of human FD lesions in PKA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef)\u003csup\u003e1\u003c/sup\u003e. H\u0026amp;E staining revealed that normal lamellar bone was replaced by a fibrous matrix (white stars) and irregularly shaped, immature woven bone (black stars), which lacked osteoblasts rimming its surface. Abundant osteoclasts (black arrows) were observed within the lesional area, suggesting osteoclastic bone resorption and increased bone remodeling. Von Kossa staining of undecalcified limb bone revealed severe mineralization defects and prominent osteoid. The cortical bone was compromised by widespread undermineralized tissue (white stars). Sirius Red staining revealed widespread green birefringent type III collagen under polarized light, indicating bone tissue damage. In conclusion, the expression of the activating mutant PKAcα\u003csup\u003eW197R\u003c/sup\u003e in SSCs induces FD-like lesions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePKA inhibition halts FD-like bone lesions induced by Gα\u003c/h3\u003e\n\u003cp\u003eThe endogenous protein kinase inhibitor peptide (PKI) is a potent PKA-specific inhibitor that has been widely used in PKA-related research\u003csup\u003e16\u003c/sup\u003e. The synthetic peptide of the PKA-inhibiting domain of PKI (amino acids 1\u0026ndash;24) and its nonsense mutant, PKI4A, were employed in our study to modulate PKA activity\u003csup\u003e33,34\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e analyses demonstrated the robust inhibitory effect of PKI on the PKA signaling pathway. The phosphorylation of both PKA and CREB induced by a mixture of forskolin and 3-isobutyl-1-methylxanthine (FI) was suppressed after PKI plasmid transfection but not after PKI4A transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). A similar strategy as that used for the PKA mice was employed to develop conditional transgenic mice expressing PKI or PKI4A under the control of TRE, along with \u003cem\u003eLSL\u003c/em\u003e-rtTA and EGFP elements (Tet-PKI-rtTA and Tet-PKI4A-rtTA, respectively). PKA activity modulation in our previously established FD mouse model (Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/\u003cem\u003eLSL\u003c/em\u003e-rtTA/\u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e)\u003csup\u003e3\u003c/sup\u003e was achieved by replacing \u003cem\u003eLSL\u003c/em\u003e-rtTA with Tet-PKI-rtTA or Tet-PKI4A-rtTA mice, creating Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (referred to as FD-PKI mice) and Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI4A-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (referred to as FD-PKI4A mice), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Only heterozygous FD-PKI and FD-PKI4A mice were utilized in the study. Homozygous FD-PKI mice were excluded due to their ineffectiveness in lowering the PKA signaling pathway for unknown reasons (Supplementary Fig.\u0026nbsp;2a). Additionally, homozygous FD-PKI4A mice presented a low survival rate (Supplementary Fig.\u0026nbsp;2b), possibly because of excessive rtTA expression in Tet-PKI4A-rtTA mice. The specific expression of the PKI transgene in limb bones of FD-PKI mice was confirmed by qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e mutation upregulated the Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling pathway in both FD-PKI and FD-PKI4A mice, as evidenced by increased \u003cem\u003eGNAS\u003c/em\u003e expression in their limb bones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and elevated cAMP levels in the serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). However, the phosphorylation of PKA was significantly suppressed in FD-PKI mice but not in FD-PKI4A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). These data demonstrated that PKA activity was effectively suppressed in FD-PKI mice without affecting the activation of the upstream Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling pathway.\u003c/p\u003e \u003cp\u003eTo test whether the progression of FD is PKA dependent, both FD-PKI4A and FD-PKI mice were treated with various doses of Dox (0.005\u0026ndash;0.1 g/L in drinking water) at the age of 6 weeks. The disease burden worsened and caused death in both FD-PKI4A and FD-PKI mice as the dose of Dox increased, although FD-PKI mice presented a milder disease burden than FD-PKI4A mice did (Supplementary Fig.\u0026nbsp;4a). All FD-PKI4A mice developed severe FD-like bone lesions within 1 week of Dox induction at a dose as low as 0.006 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;e, and Supplementary Fig.\u0026nbsp;1a). The histopathological changes in FD-PKI4A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) mirrored those observed in our previously established FD mice (Supplementary Fig.\u0026nbsp;3a\u0026ndash;c). Interestingly, most FD-PKI mice failed to develop an FD-like phenotype after 2 weeks of Dox induction at the same dose despite the mild swelling observed in the limbs and paws (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The cortical bone and skull of FD-PKI mice appeared continuous and intact, with no evidence of deformities or osteolytic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). The anatomical structure and quality of limb bones in FD-PKI mice did not differ from those in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Histopathologically, typical FD-like changes were absent in FD-PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The cortical bone was well mineralized without the presence of woven bone. Locomotor activity analysis, which is used to assess movement ability and quality of life, revealed no significant difference between FD-PKI and control mice, unlike in PKA or FD-PKI4A mice (Supplementary Fig.\u0026nbsp;1a). Upon close examination, only 3 out of 32 FD-PKI mice developed minor FD-like lesions under this dose of Dox, which were confined to the metaphysis with no invasion into the bone marrow cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;4a). Collectively, these findings suggest that PKA inhibition halts FD-like bone lesions induced by Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePKA inhibition in FD-SSCs suppresses osteoclastogenesis and promotes bone formation\u003c/h3\u003e\n\u003cp\u003eNext, we investigated the bone remodeling events regulated by PKA. The increase in osteoclastogenesis caused by Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e was suppressed to normal levels in FD-PKI mice, as evidenced by the absence of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the cortical bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, the differentiation of osteoclasts was significantly increased in PKA and FD-PKI4A mice, which led to severe bone resorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Consistently, the serum level of tartrate-resistant acid phosphatase 5b (TRACP-5b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and the transcriptional level of osteoclast-specific markers (\u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eRank\u003c/em\u003e, \u003cem\u003eNfatc1\u003c/em\u003e, and \u003cem\u003eAcp5\u003c/em\u003e) were increased in the bone lesions of PKA and FD-PKI4A mice, similar to those of FD mice (Supplementary Fig.\u0026nbsp;5a). However, these osteoclast markers demonstrated downregulated expression in FD-PKI mice. Rankl was suppressed to normal levels in FD-PKI mice in both gene and circulating protein forms (Supplementary Fig.\u0026nbsp;5b and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Besides the unchanged expression levels of \u003cem\u003eOsteoprotegerin\u003c/em\u003e (\u003cem\u003eOpg\u003c/em\u003e), the decoy receptor for \u003cem\u003eRankl\u003c/em\u003e, upon PKA signaling modulation, the \u003cem\u003eRankl\u003c/em\u003e/\u003cem\u003eOpg\u003c/em\u003e ratio was significantly reduced in FD-PKI mice (Supplementary Fig.\u0026nbsp;5b, c). However, Rankl level and \u003cem\u003eRankl\u003c/em\u003e/\u003cem\u003eOpg\u003c/em\u003e ratio were significantly elevated upon PKA activation in PKA and FD-PKI4A mice, stimulating osteoclast differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Supplementary Fig.\u0026nbsp;5b, c). These data suggest that osteoclastic bone resorption in FD lesions is driven by Rankl-dependent osteoclastogenesis, which is promoted by PKA activation.\u003c/p\u003e \u003cp\u003eConversely, PKA activation enhanced the osteogenic lineage commitment of SSCs but inhibited osteoblast maturation. Significantly elevated expression of osteoblast transcription factors, \u003cem\u003erunt-related transcription factor 2\u003c/em\u003e (\u003cem\u003eRunx2\u003c/em\u003e) and \u003cem\u003eOsterix\u003c/em\u003e (\u003cem\u003eOsx\u003c/em\u003e), and other osteogenic genes, such as \u003cem\u003ealkaline phosphatase\u003c/em\u003e (\u003cem\u003eAlp\u003c/em\u003e) and \u003cem\u003ecollagen type 1\u003c/em\u003e (\u003cem\u003eCol1a1\u003c/em\u003e), and marginally decreased expression of the mature osteoblast marker \u003cem\u003eosteocalcin\u003c/em\u003e (\u003cem\u003eOcn\u003c/em\u003e) were detected in the bone lesions of PKA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The aberrant osteogenic differentiation of SSCs disrupted by Gα\u003csub\u003es\u003c/sub\u003e signaling activation was restored in FD-PKI mice following PKA inhibition. Ocn-expressing matured osteoblasts (lining cells) were observed on the surface of the trabecular bone in FD-PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Notably, early osteogenic differentiation was not completely blocked in FD-PKI mice, as demonstrated by the expression of Osx on the surface of trabecular bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Furthermore, the highly proliferative activities of the fibroblast-like cells were revealed by a 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) proliferation assay in PKA and FD-PKI4A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g)\u003csup\u003e3,35\u003c/sup\u003e. Collectively, our data demonstrated that the PKA signaling pathway promotes Rankl-dependent osteoclastogenesis and induces early osteogenic differentiation and proliferation of SSCs but impairs their maturation and mineralization. Downregulating ectopically activated PKA signaling could reduce osteoclastogenesis and restore the balance between osteogenic differentiation and osteoclastogenesis, resulting in bone formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDownregulated PKA signaling safeguards bone health\u003c/h3\u003e\n\u003cp\u003eTo further study the effects of PKA on bone remodeling without hyperactivation of Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling, we created a Tet-PKI-rtTA/\u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e mouse model (referred to as PKI mice) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We hypothesized that the bone volume and density would increase in PKI mice upon PKA inhibition on the basis of accumulated data from PKA and FD-PKI mice. To downregulate PKA signaling in the SSCs of limb bone and observe phenotypic changes, various doses of Dox were administered to PKI mice for up to 6 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The expression of the PKI transgene in limb bone was confirmed by qPCR analysis and EGFP tag immunofluorescence staining in mutant mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). The phosphorylation of PKA was decreased in limb bones of PKI mice following Dox administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Surprisingly, there were no significant phenotypic (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) or behavioral (data not shown) differences between PKI and control mice after 6 months of high-dose Dox induction (6 g/kg in the diet). The cortical bone of the limbs was continuous and well mineralized without significant pathological changes in the PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h). Osteoclastogenesis remained at a normal level compared with that in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Interestingly, abundant adipocytes were observed in the bone marrow of PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh, j)\u003csup\u003e25,36\u003c/sup\u003e. Further analyses via \u0026micro;CT imaging revealed no significant thickening of cortical bone or skull in PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). The cortical bone mass in PKI mice was unaffected by PKA inhibition, as shown by the BV/TV and tissue mineral density (TMD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el). However, significant increases in both the number and thickness of trabecular bone were observed in PKI mice, resulting in a subtle increase in trabecular bone mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em, n). Mirroring the findings in PKI and PKA mice, MC3T3-E1-14 osteoblastic cells exhibited severely impaired mineralization capacity upon PKA activation and significantly increased mineralization upon PKA inhibition, modulated by FI and H89, respectively (Supplementary Fig.\u0026nbsp;6a\u0026ndash;c).\u003c/p\u003e \u003cp\u003eTo further assess the effect of PKA inhibition on bone remodeling, we utilized a mouse model of osteoporosis induced by ovariectomy (OVX) in both female PKI mice and their littermate controls. All OVX mice, along with a group of blank control mice, then underwent 11 weeks of Dox induction at a dose of 6 g/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). As expected, the ovariectomized control group (Control\u0026thinsp;+\u0026thinsp;OVX) exhibited significant bone loss, as evidenced by reductions in BMD, BV/TV, and trabecular number, along with an increase in trabecular separation, compared with those of the non-ovariectomized control group (Control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u0026ndash;d)\u003csup\u003e37\u003c/sup\u003e. However, downregulation of PKA signaling in SSCs mitigated bone loss in ovariectomized PKI mice (PKI/\u003cem\u003ePrrx1-cre\u003c/em\u003e\u0026thinsp;+\u0026thinsp;OVX). There was no statistically significant difference in the BV/TV, BMD, or trabecular variables between the blank control and ovariectomized PKI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u0026ndash;d). These data suggest that downregulated PKA signaling safeguards both the quantity and quality of bone under normal and disease conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePKA is a potential therapeutic target for FD\u003c/h3\u003e\n\u003cp\u003eInspired by the above data, we explored therapeutic options for FD by targeting PKA. Two small-molecule inhibitors of PKA, H89 and Rp-8-Br-cAMPS, both of which are potent and selective with distinct mechanisms, were utilized to decrease PKA activity in our FD mouse model. The therapeutic regimen is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea. First, the FD phenotype was established in all FD mice after 3 days of Dox induction (0.2 g/L in the drinking water) (T1). FD mice were then randomly divided into the FD, FD\u0026thinsp;+\u0026thinsp;H89, and FD\u0026thinsp;+\u0026thinsp;Rp-8-Br-cAMPs groups. While all the mice were subjected to the same dose of Dox throughout the study, the FD mice in the two PKA inhibitor groups received either H89 or Rp-8-Br-cAMPs via intraperitoneal injection every other day for 11 days (T2). At T2, all FD mice presented significantly upregulated \u003cem\u003eGNAS\u003c/em\u003e expression in limb bones (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). PKA signaling was effectively suppressed in FD mice from both the FD\u0026thinsp;+\u0026thinsp;H89 and FD\u0026thinsp;+\u0026thinsp;Rp-8-Br-cAMPs groups, as evidenced by the decreased phosphorylation level of PKA in limb bones (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Limb swelling and reduced mobility observed in FD mice at T1 were significantly improved in both PKA inhibitor groups within 1 week of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). By T2, FD mice in the PKA inhibitor groups exhibited less limb swelling and significantly improved mobility compared with those in the FD group, although their mobility remained below normal levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). Similarly, FD mice treated with PKA inhibitors presented reduced ground-glass opacities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg) and osteolytic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). H\u0026amp;E staining demonstrated that FD lesions were restricted to the metaphysis area in the mice treated with the PKA inhibitors and did not affect the diaphysis. In contrast, mice in the FD group exhibited fully developed FD lesions expanding throughout the entire limb bones, accompanied by severe osteolytic bone damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). In conclusion, our findings demonstrate that PKA inhibition effectively alleviates FD lesions in mice, confirming that PKA is a potential therapeutic target in FD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we investigated the role of PKA in the pathophysiological mechanisms of FD via a combination of molecular and genetic approaches. Our findings demonstrate that FD initiation and progression are strictly PKA-dependent. PKA promotes osteoclastogenesis, induces aberrant osteogenic differentiation and proliferation of SSCs, but suppresses bone formation, leading to imbalanced osteolytic lesions in FD. Downregulating the PKA signaling pathway effectively alleviates FD lesions in mice and safeguards bone health, suggesting that PKA is a potential therapeutic target in FD.\u003c/p\u003e \u003cp\u003eFD is caused by activating mutations of Gα\u003csub\u003es\u003c/sub\u003e in SSCs, which lead to elevated cAMP concentrations and phosphorylation levels of PKA. Dysregulation of PKA signaling is associated with a wide range of diseases. PKAcα\u003csup\u003eW197R\u003c/sup\u003e, the mutant used in our study, is a constitutively active variant of the catalytic subunit of PKA\u003csup\u003e13,14\u003c/sup\u003e. Unlike some other PKAcα mutants, \u003cem\u003ein silico\u003c/em\u003e analysis revealed no changes in the PKA substrate specificity of PKAcα\u003csup\u003eW197R 38\u003c/sup\u003e, making it an ideal candidate for studying the mechanisms of PKA in FD pathogenesis.\u003c/p\u003e \u003cp\u003eTo examine whether PKA activation is sufficient to initiate FD, we developed a transgenic mouse model expressing PKAcα\u003csup\u003eW197R\u003c/sup\u003e in SSCs under the control of Dox (Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA/\u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e). Typical FD-like bone lesions rapidly develop in the limb and skull areas following the upregulation of PKA signaling through the expression of PKAcα\u003csup\u003eW197R\u003c/sup\u003e. This finding parallels others\u0026rsquo; study in which the upregulation of PKA activity through R type I α subunit (R1α) haploinsufficiency led to FD-like bone lesions\u003csup\u003e39\u003c/sup\u003e. These findings demonstrate that the activation of PKA is sufficient to initiate FD and reinforces the pivotal role of PKA in FD. Notably, the FD-like lesions in the abovementioned mouse model exhibit more severe osteoclastic resorption and less expansion than those initiated by disease-causing Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e mutants (Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/\u003cem\u003eLSL\u003c/em\u003e-rtTA/\u003cem\u003ePrrx1\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e and Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI4A-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e), suggesting that PKA plays a dominant role in osteoclastogenesis.\u003c/p\u003e \u003cp\u003ePKA inhibition in our previously established FD mouse model was achieved by introducing PKI into a transgenic system (Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e). Both Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e and PKI were expressed in SSCs in mice upon induction with Dox. PKA activity was effectively suppressed in these mice without affecting the activation of the upstream Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling pathway. As a result, the progression of FD was effectively halted by PKI but not its nonsense mutant PKI4A. Notably, different doses of Dox were used in our transgenic mouse models, including PKA, FD-PKI (4A), and FD mice. This variation may be attributed to the newer third generations of TRE and rtTA and the high-efficiency promoters used in the PKA and FD-PKI (4A) mice. In PKA and PKI (4A) mice, rtTA transgene expression driven by the CAG promoter might be greater than that driven by the PKG promoter in previous \u003cem\u003eLSL\u003c/em\u003e-rtTA mice\u003csup\u003e3,40,41\u003c/sup\u003e, leading to increased sensitivity to Dox. This could explain the low survival rate of homozygous FD-PKI4A mice treated with Dox. As a result, only heterozygous FD-PKI (4A) mice were utilized in the study. For better comparison, the Dox dose for FD-PKI mice was adjusted to match that of FD-PKI4A mice, resulting in comparable lesions to those observed in FD mice. Together, our data demonstrate that FD initiation and progression are strictly PKA-dependent.\u003c/p\u003e \u003cp\u003eNext, we assessed PKA as a therapeutic target for FD. Our data demonstrate that FD lesions in mice were effectively alleviated through the use of the pharmacological PKA inhibitors H89 and Rp-8-Br-cAMPs, highlighting the potential of targeting PKA as a novel therapeutic approach for FD. Notably, many studies have identified the non-PKA-specific actions of H89\u003csup\u003e42\u003c/sup\u003e. Although Rp-8-Br-cAMPs is considered selective inhibitor of type I PKA due to its cAMP analog structure and mechanisms of action\u003csup\u003e32\u003c/sup\u003e, their inhibitory efficacy may be compromised when the cAMP level is extremely high, as in our FD mouse model. Additionally, Rp-8-Br-cAMPs may bind to exchange protein activated by cAMP (Epac), another direct target of cAMP that shares similar cyclic nucleotide\u0026ndash;binding sites with PKA\u003csup\u003e43\u003c/sup\u003e. However, our preliminary data revealed that FD lesions could not be rescued by ESI-09\u003csup\u003e44\u003c/sup\u003e, a small-molecule inhibitor of Epac (data not shown). Epac is a guanine nucleotide exchange factor for the small GTPases Rap1/2, and it has been implicated in various cellular processes, such as cell adhesion, cell\u0026ndash;cell junction formation, insulin secretion, and neurotransmitter release\u003csup\u003e45\u003c/sup\u003e. There is a complex interconnection between the Epac- and PKA-mediated signaling pathways. For example, a recent study showed that inhibiting PKA via PKI diverts GPCR/Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling toward EPAC and ERK activation and is involved in tumor growth\u003csup\u003e46\u003c/sup\u003e. More studies are needed to elucidate the role of Epac in FD pathogenesis.\u003c/p\u003e \u003cp\u003eUndeniably, Gα\u003csub\u003es\u003c/sub\u003e is a negative phenotypic regulator of bone mass, as evidenced by the opposing bone manifestations observed in Albright's hereditary osteodystrophy (AHO), progressive osseous heteroplasia (POH), and FD\u003csup\u003e47,48\u003c/sup\u003e. AHO and POH are associated with inactivating Gα\u003csub\u003es\u003c/sub\u003e mutations, which are clinically characterized by heterotopic ossification\u003csup\u003e49\u003c/sup\u003e, whereas FD is linked to activating Gα\u003csub\u003es\u003c/sub\u003e mutations, leading to bone destruction\u003csup\u003e2\u003c/sup\u003e. However, the role of Gα\u003csub\u003es\u003c/sub\u003e/cAMP/PKA signaling in regulating bone remodeling is controversial, largely due to variations in the experimental materials and methods used across studies and data interpretation. For example, Gα\u003csub\u003es\u003c/sub\u003e or PKA dysregulation in different cell populations often leads to complicated or opposing results\u003csup\u003e19,20,30,50\u0026ndash;52\u003c/sup\u003e. PKA signaling appears to regulate stem cells and progenitor cells differently from bone cells, such as osteoblasts and osteocytes, although the underlying mechanisms remain unclear. Importantly, the expression levels of osteogenic transcription factors, such as Runx2 and Osterix, do not necessarily correlate with the quantity or quality of bone formation \u003cem\u003ein vivo\u003c/em\u003e, as observed phenotypically. Conclusions concerning how PKA regulates osteogenic differentiation should not rely solely on changes in the expression levels of osteogenic markers, which may help explain discrepancies between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies\u003csup\u003e18,20,22,50\u003c/sup\u003e. Accumulating evidence supports a stage-dependent regulatory model in which Gα\u003csub\u003es\u003c/sub\u003e/cAMP/PKA promotes early osteogenic differentiation but impairs maturation\u003csup\u003e3,25,53,54\u003c/sup\u003e. Additionally, Gα\u003csub\u003es\u003c/sub\u003e has been shown to exert differential effects on trabecular and cortical bone\u003csup\u003e29,30,50\u003c/sup\u003e and age-dependent\u003csup\u003e55\u003c/sup\u003e and sex-dependent\u003csup\u003e19,20,56\u003c/sup\u003e influences on osteogenic differentiation or bone formation, suggesting the involvement of complex regulatory mechanisms in bone remodeling.\u003c/p\u003e \u003cp\u003eOur findings suggest a dual regulatory model of bone remodeling in which PKA induces early osteogenic differentiation and proliferation of SSCs but impairs their maturation and mineralization while significantly enhancing RANKL-dependent osteoclastogenesis. However, unlike Gα\u003csub\u003es\u003c/sub\u003e, the downstream effector PKA does not appear to function as a straightforward negative regulator of bone mass. Compared with the dramatic bone loss caused by hyperactivation in this study, PKA inhibition had only a subtle effect on trabecular bone anabolism, suggesting the presence of compensatory feedback mechanisms that counterbalance the downregulation of PKA signaling. This bone safeguard effect makes PKA inhibition a promising anabolic therapy for bone destruction diseases, including FD and osteoporosis\u003csup\u003e57\u003c/sup\u003e. This hypothesis is preliminarily supported by observations in an osteoporosis disease model, where the quantity and quality of bone were preserved in ovariectomized mice following PKA inhibition.\u003c/p\u003e \u003cp\u003eFD typically manifests in adolescence and progresses into adulthood\u003csup\u003e58\u003c/sup\u003e, necessitating early intervention and ongoing treatment. Our study highlights, for the first time, the dependency of PKA on the initiation and progression of FD and suggests the promising therapeutic potential of PKA inhibition in treating FD and other similar bone destruction diseases. The safety of the antiresorptive medications currently used in FD management is an increasing concern. We demonstrated that long-term PKA inhibition in mice, restricted to SSCs in the limb and craniofacial areas, caused no significant side effects on bone or soft tissue. Instead, it slightly increased the trabecular bone volume. A cure for FD could be achieved by specifically targeting PKA, rather than the upstream disease-causing Gα\u003csub\u003es\u003c/sub\u003e mutations, to avoid broader impacts and minimize side effects. Encouragingly, the pathophysiological role of the Gα\u003csub\u003es\u003c/sub\u003e/PKA pathway has recently gained increased attention from researchers\u003csup\u003e11\u003c/sup\u003e. Various promising approaches and delivery systems targeting different components of the Gα\u003csub\u003es\u003c/sub\u003e/PKA pathway have been developed and continue to evolve\u003csup\u003e59\u0026ndash;62\u003c/sup\u003e, paving the way for molecular therapies for FD and other Gα\u003csub\u003es\u003c/sub\u003e/PKA-related diseases.\u003c/p\u003e \u003cp\u003eThis study has several limitations. The regulatory mechanisms of PKA in bone remodeling remain elusive. More in-depth studies are needed to reveal the underlying mechanisms, particularly the possible compensatory crosstalk mechanisms that counterbalance the downregulation of PKA signaling in bone remodeling, as observed in this study. This approach could lead to the identification of additional therapeutic targets for bone metabolism diseases. Moreover, given the broad involvement of PKA in physiological processes, the safety of the general use of PKA inhibition requires further evaluation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate the influence of PKA on FD development, its regulatory impact on differentiation, and its potential as a therapeutic target. These objectives were addressed by (i) creating a series of tetracycline-inducible, tissue-specific transgenic models (PKA mice, PKI mice, FD-PKI mice, and FD-PKI4A mice) to achieve \u003cem\u003ein vivo\u003c/em\u003e regulation of the Gα\u003csub\u003es\u003c/sub\u003e/cAMP/PKA signaling axis, (ii) examining the bone tissue phenotype in these mice, (iii) investigating the cellular and molecular changes induced by PKA regulation both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, and (iv) studying the effects of the intraperitoneal injection of two small-molecule PKA inhibitors on attenuating FD progression. Sample sizes were determined by the investigators on the basis of previous experimental experience, with exact numbers provided in the respective figure captions. \u003cem\u003eIn vivo\u003c/em\u003e experiments included all genetically screened animals in which the dosing strategy was adhered to. For experiments involving exogenous drug administration, animals and samples were randomly assigned to experimental and control groups. Endpoints were set at 2 weeks for all models except for PKI mice, with an endpoint of 6 months. The investigators were not blinded during the data analysis. All \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments were replicated three or more times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA constructs\u003c/h2\u003e \u003cp\u003eRecombinant human PKAcα was amplified via PCR from pRSET PKAcα, a gift from Dr. Susan Taylor, with a C-terminal HA tag inserted into pCEFL (pCEFL PKAcα-HA). The sequence was verified by Sanger sequencing. The W197R amino acid substitution was carried out using the QuikChange II site-directed mutagenesis kit (Agilent, Santa Clara, CA, 200523)\u003csup\u003e13\u003c/sup\u003e. The primers used for mutagenesis are as follows: PKAcα\u003csup\u003eW197R\u003c/sup\u003e forward: 5'-cgtgtgaaaggccgtact\u003cb\u003eA\u003c/b\u003eggaccttgtgtg-3', PKAcα\u003csup\u003eW197R\u003c/sup\u003e reverse: 5'-cacacaaggtcc\u003cb\u003eT\u003c/b\u003eagtacggcctttcacacg-3'. The synthetic peptide of the PKA-inhibiting domain of PKI (amino acids 1\u0026ndash;24) and its nonsense mutant, PKI4A, were employed in our study to modulate PKA activity\u003csup\u003e34\u003c/sup\u003e. Briefly, EGFP-PKI was cloned by inserting the 24 coding amino acids of human PKI-alpha (PKIA) into the C-terminus of EGFP. For use as a control, the phenylalanine and arginine residues of the PKI peptide were replaced with alanine to disrupt binding to protein kinase A (PKA), and the resulting protein was named EGFP-PKI4A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eTo generate Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA, Tet-PKI-rtTA, and Tet-PKI4A-rtTA transgenic mice, the coding sequences were cloned downstream of the tet-responsive elements (TREs) within an improved Tet-On (3G) vector (Clontech\u0026reg; Laboratories, Inc., Mountain View, CA, USA, PT5148-1)\u003csup\u003e63\u003c/sup\u003e. Mice carrying the mutated human Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e gene were previously described\u003csup\u003e34\u003c/sup\u003e, and they are referred to as \u0026ldquo;Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e\u0026rdquo; mice. Mice expressing \u003cem\u003eCre\u003c/em\u003e recombinase in mouse limb buds driven by a \u003cem\u003ePrrx1\u003c/em\u003e-derived enhancer (B6. Cg-Tg(Prrx1-cre)1Cjt/J), referred to as \u0026ldquo;Prrx1-Cre mice\u0026rdquo;, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA)\u003csup\u003e64\u003c/sup\u003e. We confirmed the presence of transgenes through PCR analysis of tail DNA. The presence of Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e transgenes was analyzed as previously described\u003csup\u003e34\u003c/sup\u003e. Specific primers were used to verify the presence of the Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e, Tet-PKI, and Tet-PKI4A transgenes, each producing bands of the expected sizes (601, 349, and 356 bp, respectively). The generation and characterization of the Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/LSL-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e mouse model (referred to as \u0026ldquo;FD mice\u0026rdquo;) have been previously described\u003csup\u003e3\u003c/sup\u003e. To create other models, Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA mice were crossed with \u003cem\u003ePrrx1-Cre\u003c/em\u003e mice to generate Tet-PKAcα\u003csup\u003eW197R\u003c/sup\u003e-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e mice (referred to as \u0026ldquo;PKA mice\u0026rdquo;). Similarly, Tet-PKI-rtTA mice were crossed with \u003cem\u003ePrrx1-Cre\u003c/em\u003e mice to produce PKI-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e mice (referred to as \u0026ldquo;PKI mice\u0026rdquo;). By crossing Tet-PKI-rtTA mice with Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e and \u003cem\u003ePrrx1-Cre\u003c/em\u003e mice, we generated Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (referred to as \u0026ldquo;FD-PKI mice\u0026rdquo;). Finally, by replacing Tet-PKI-rtTA with Tet-PKI4A-rtTA, we generated Tet-Gα\u003csub\u003es\u003c/sub\u003e\u003csup\u003eR201C\u003c/sup\u003e/Tet-PKI4A-rtTA/\u003cem\u003ePrrx1-Cre\u003c/em\u003e (referred to as \u0026ldquo;FD-PKI mice\u0026rdquo;).\u003c/p\u003e \u003cp\u003eAll the experiments used littermates, including male and female mice, as controls. Dox (MedChemExpress, Monmouth Junction, NJ, USA, HY-N0565B) was administered at concentrations ranging from 0.006 to 0.1 g/L in the drinking water or 6 g/kg in the diet. Dox induction commenced at 6 weeks of age and continued for up to 2 weeks, 11 weeks, or 6 months, as specified for each experiment. Bilateral ovariectomies were performed at the age of 9 weeks to mimic postmenopausal estrogen deficiency\u003csup\u003e65,66\u003c/sup\u003e. Two small-molecule PKA inhibitors were used in the FD mice rescue study and were administered via intraperitoneal injection every 2 days: H89 (MedChemExpress, Monmouth Junction, NJ, HY-15979A) at a dose of 50 mg/kg and Rp-8-Br-cAMPs (MedChemExpress, Monmouth Junction, NJ, HY-100530D) at a dose of 100 mg/kg. The animal experiments were conducted in accordance with protocols approved by the Sichuan University Institutional Animal Care and Use Committee (IACUC) and complied with the Guide for the Care and Use of Laboratory Animals. The mice were housed in specific pathogen-free (SPF) barrier facilities at the Laboratory Animal Center of West China Second University Hospital, Sichuan University. The use of live animals was approved by the Ethics Committee of West China Hospital of Stomatology (WCHSIRB-D-2019-197).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, osteogenic induction and transfection\u003c/h2\u003e \u003cp\u003eThe MC3T3-E1 subclone 14 cell line was obtained from SAIOS Biotechnology Co., Ltd. (Wuhan, China, CL-077 m). The cells were cultured in complete alpha-MEM supplemented with 90% alpha-MEM (Gibco, Waltham, MA, USA, #12571063) and 10% FBS (Gibco, Waltham, MA, USA, #10099141C) at 5% CO2 and 37\u0026deg;C. For osteogenic differentiation, the cells were seeded into 24-well plates at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured in osteogenic (OS) induction medium for 2 weeks. The OS medium comprised complete alpha-MEM supplemented with 150 \u0026micro;M ascorbic acid (MilliporeSigma, Burlington, MA, USA, A4403), 10 mM β-glycerophosphate (MilliporeSigma, Burlington, MA, USA, G9422), and 10 nM dexamethasone (MilliporeSigma, Burlington, MA, USA, D4902). Forskolin (MilliporeSigma, Burlington, MA, USA, F6886) was used at a concentration of 10 \u0026micro;M in combination with IBMX (MilliporeSigma, Burlington, MA, USA, I7018) at 100 \u0026micro;M in OS medium (FI medium). H89 (MedChemExpress, Monmouth Junction, NJ, USA, HY-15979A) was used at a concentration of 10 \u0026micro;M in OS medium.\u003c/p\u003e \u003cp\u003eThe 293T cell line was obtained from ATCC and cultured in complete DMEM supplemented with 90% DMEM (Gibco, Waltham, MA, USA, #C11995500BT) and 10% FBS. The cells were seeded into 24-well plates at a density of 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and transfected with Lipofectamine 3000 (Invitrogen, Waltham, MA, USA, L3000001) following the manufacturer's instructions. At 48 h posttransfection, the cells were harvested. Intracellular total-CREB and phospho-CREB levels were measured via the Total CREB ELISA Kit (Cell Signaling Technology, Danvers, MA, USA, #36001C) and Phospho-CREB (Ser133) Sandwich ELISA Kit (Cell Signaling Technology, Danvers, MA, USA, #7385), respectively, according to the manufacturers\u0026rsquo; instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase assay\u003c/h2\u003e \u003cp\u003eA cAMP-responsive-element\u0026ndash;driven reporter luciferase (CRE-luc) assay was employed to measure PKA activity\u003csup\u003e67\u003c/sup\u003e. 293T cells in 24-well plates were co-transfected with CRE (0.05\u0026ensp;\u0026micro;g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) plus the DNA constructs indicated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA: Gα\u003csub\u003eS\u003c/sub\u003e (0.1 \u0026micro;g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), PKAcα (0.1 \u0026micro;g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), and PKAcα\u003csup\u003eW197R\u003c/sup\u003e (0.1 \u0026micro;g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). The cells were harvested 24 hours after transfection, and the luciferase activity was subsequently measured via a Dual-Glo Luciferase Assay Kit (Promega, Madison, WI, USA, E1910) and a microtiter plate luminometer (Dynex Tech, Chantilly, VA, USA). Luciferase normalization was performed in every case by co-transfecting a \u003cem\u003eRenilla\u003c/em\u003e luciferase vector (0.005 \u0026micro;g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e; Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlizarin red staining (ARS) and quantitative analysis\u003c/h2\u003e \u003cp\u003eARS was conducted after 14 days of osteogenic induction. The cells were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. After being washed with PBS three times, the cells were stained with freshly prepared ARS solution (Beyotime, Shanghai, China, C0148S) for 15 min and then washed at least five times with distilled water until the rinsing fluid was clear. Images were captured via a stereomicroscope (Leica, Wetzlar, Germany, EZ4 HD), and quantitative analysis was performed with ImageJ software\u003csup\u003e68\u003c/sup\u003e, with a consistent HSB threshold (hue: 0\u0026ndash;30, saturation: 160\u0026ndash;222, brightness: 174\u0026ndash;255).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection and processing\u003c/h2\u003e \u003cp\u003eFresh bone tissues were ground into shatters via a tissue homogenizer (Servicebio, Wuhan, China, #KZ-5F-3D) in TRIzol reagent (Invitrogen, Carlsbad, CA, USA, #15596\u0026ndash;026) immediately after collection. The resulting mixture was frozen at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent RNA isolation. Bone tissue samples were collected and fixed in zinc 10% formalin fixative (MilliporeSigma, Burlington, MA, USA, #Z2902) at room temperature overnight and then stored in 70% ethanol for further processing. Fixed skeletal samples were either decalcified in 4% buffered EDTA for subsequent paraffin and optimal cutting temperature (OCT) frozen embedding or directly embedded in polymethyl methacrylate (PMMA) without decalcification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative real-time polymerase chain reaction (qPCR)\u003c/h2\u003e \u003cp\u003eRNA was isolated from bone tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, #15596\u0026ndash;026) and from cell lines using the Cell Total RNA Isolation Kit (Foregene, Chengdu, China, RE-03111) following the manufacturer's instructions. Reverse transcription was conducted with the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan, #RR037A) on an S1000 Thermal Cycler Platform (BIO\u0026ndash;RAD, Irvine, CA, USA). Subsequent quantitative PCR (qPCR) was performed using SYBR Select Master Mix (Applied Biosystems, Waltham, MA, USA, #44729080) on a QuanStudio 3 Real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The target gene expression levels were normalized to \u003cem\u003eGapdh\u003c/em\u003e. The oligonucleotides used for amplification were as follows: (gene, forward sequence 5' \u0026rarr; 3', reverse sequence 5' \u0026rarr; 3'): \u003cem\u003eGapdh\u003c/em\u003e 5'-TCATTGACCTCAACTACATG-3', 5'-TCGCTCCTGGAAGATGGTGAT-3'; \u003cem\u003eGnas\u003c/em\u003e 5'-GCAGAAGGACAAGCAGGTCT-3', 5'-CCCTCTCCGTTAAACCCATT-3'; \u003cem\u003ePRKACA\u003c/em\u003e 5'-GCGTGTGAAAGGCCGTACT-3', 5'-GGATAGGCTGGTCAGCGAAG-3', PKI 5'-AACGAGAAGCGCGATCACATG-3', 5'-TGCATTTCTTCTACCTGTTCTTCCTG-3', \u003cem\u003eCsflr\u003c/em\u003e 5'-TGGATGCCTGTGAATGGCTCTG-3', 5'-GTGGGTGTCATTCCAAACCTGC-3'; \u003cem\u003eRank\u003c/em\u003e 5'-GGACAACGGAATCAGATGTGGTC-3', 5'-CCACAGAGATGAAGAGGAGCAG-3'; \u003cem\u003eNfatc1\u003c/em\u003e 5'-GGTGCCTTTTGCGAGCAGTATC-3', 5'-CGTATGGACCAGAATGTGACGG-3'; \u003cem\u003eAcp5\u003c/em\u003e 5'-GCGACCATTGTTAGCCACATACG-3', 5'-CGTTGATGTCGCACAGAGGGAT-3'; \u003cem\u003eRankl\u003c/em\u003e 5'-CACAGCGCTTCTCAGGAGCTC-3', 5'-GAGATCTTGGCCCAGCCTCGA-3'; \u003cem\u003eOpg\u003c/em\u003e 5'-AGTCCGTGAAGCAGGAGTGCA-3', 5'-AAGTCTCACCTGAGAAGAACC-3'; \u003cem\u003eRunx2\u003c/em\u003e 5'-GACTGTGGTTACCGTCATGGC-3', 5'-ACTTGGTTTTTCATAACAGCGGA-3'; \u003cem\u003eOsx\u003c/em\u003e 5'-ATGGCGTCCTCTCTGCTTG-3', 5'-TGAAAGGTCAGCGTATGGCTT-3'; \u003cem\u003eAlpl\u003c/em\u003e 5'-CACGGCCATCCTATATGGTAA-3', 5'-GGGCCTGGTAGTTGTTGTGA-3'; \u003cem\u003eCol1a1\u003c/em\u003e 5'-CACCCTCAAGAGCCTGAGTC-3', 5'-GTT CGGGCTGATGTACCAGT-3'; \u003cem\u003eOcn\u003c/em\u003e 5'-ACCCTGGCTGCGCTCTGTCTCT-3', 5'-GATGCGTTTGTAGGCGGTCTTCA-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eWestern blot assays were performed and repeated at least three times, as previously described\u003csup\u003e34\u003c/sup\u003e. The antibodies used were as follows: GAPDH (Cell Signaling Technology, Danvers, MA, USA, #2118), CREB (Cell Signaling Technology, Danvers, MA, USA, #9197), phospho-CREB Ser133 (Cell Signaling Technology, Danvers, MA, USA, #9198), HA-tag (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-805), and phospho-PKA substrate (RRXS*/T*) (Cell Signaling Technology, Danvers, MA, USA, #9624).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSerum collection and measurements\u003c/h2\u003e \u003cp\u003eMouse blood (\u0026gt;\u0026thinsp;1 ml) was collected via retro-orbital eye collection prior to euthanasia. The blood was stored at room temperature for 30 min. Subsequently, the serum was separated via centrifugation (30 min, 5000 rcf, 4\u0026deg;C) and frozen at \u0026minus;\u0026thinsp;80\u0026deg;C for future analysis. Serum levels of cAMP, TRACP-5b, and Rankl were quantified via enzyme-linked immunosorbent assay (ELISA). The ELISA kits used were the Mouse Cyclic Adenosine Monophosphate (cAMP) ELISA Kit (Jianglai Industry Co., Shanghai, China, JL13362), the Mouse Tartrate-Resistant Acid Phosphatase 5b (TRACP-5b) ELISA Kit (US Biological, Salem, MA, USA, 359452), and the Mouse TRANCE (TNFSF11) ELISA Kit (RayBiotech, Norcross, GA, USA, O35235).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnimal behavior analyses\u003c/h2\u003e \u003cp\u003eAn apparatus (XinRuan Technology, Shanghai, China) consisting of an acrylic test box (internal measurements: 50 cm \u0026times; 50 cm \u0026times; 45 cm) equipped with a video camera was used for the animal behavior analyses. The 300-second test was started immediately after the animals were transported to the test box, given that the novelty aspect of the environment is a crucial component of the test. The parameters analyzed were the total distance traveled, average speed, walking periods, and number of standing times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMicrocomputed tomography (\u0026micro;CT)\u003c/h2\u003e \u003cp\u003eThe specimens were scanned using a SCANCO Medical AG VivaCT 80 scanner with an isotropic voxel size of 10 \u0026micro;m or a SCANCO Medical \u0026micro;CT 100 scanner (SCANCO Medical, Br\u0026uuml;ttisellen, Switzerland) with an isotropic voxel size of 7.5 \u0026micro;m, following standard guidelines\u003csup\u003e69\u003c/sup\u003e. The region of interests (ROIs) is defined as the area 2 mm and 3 mm proximal to the tibial growth plate or middle of diaphysis. The threshold set at 220 for 3D image reconstruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSirius red staining\u003c/h2\u003e \u003cp\u003eFollowing deparaffinization and rehydration, the paraffin sections were stained with Picro-Sirius Red solution for 1 h. The Picrosirius Red solution consisted of 0.1% (wt/vol) Direct Red 80 (MilliporeSigma, Burlington, MA, USA, #2610-10-8) in a saturated aqueous picric acid solution. After two washes in 0.5% acetic acid water, the sections were dehydrated in graded alcohol and mounted with Permount mounting medium (Biosharp, Anhui, China, BL704A). The evaluation was conducted under transmitted and polarized light microscopy (Nikon, Tokyo, Japan, Eclipse Ci-Pol).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTartrate-resistant acid phosphatase (TRAP) staining\u003c/h2\u003e \u003cp\u003eThe paraffin sections were incubated at 37\u0026deg;C in freshly prepared TRAP staining solution for 15 min, with periodic monitoring. Following staining, the sections were washed in distilled water, counterstained with methyl green (Wako, Richmond, VA, USA, #184-67001), dehydrated in graded alcohol, and mounted with Permount mounting medium (Biosharp, Anhui, China, BL704A). The TRAP staining solution was composed of 0.02% naphthol AS-TR phosphate (MilliporeSigma, Burlington, MA, USA, #N6125-1G) and 0.03% Fast Red violet LB salt (MilliporeSigma, Burlington, MA, USA, #F3381-5G) dissolved in 0.1 M sodium acetate buffer (pH 5), containing 50 mM sodium tartrate (MilliporeSigma, Burlington, MA, USA, #S4797-100G) and 0.1 M sodium acetate (MilliporeSigma, Burlington, MA, USA, #S1111500GM).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin sections were deparaffinized and rehydrated through xylene and a graded alcohol series, followed by microwave antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The sections were then blocked with 10% goat serum (Boster Biological Technology, Wuhan, China, AR1009) for 1 h and incubated with primary antibodies overnight at 4\u0026deg;C. Detection was performed with the avidin-biotin complex (ABC) and DAB systems using the VECTASTAIN ABC-HRP Kit (Vector Laboratories, Newark, CA, USA, PK-6101) and the DAB Detection Kit (GeneTech, Shanghai, China, GK600510). The sections were subsequently counterstained with hematoxylin. The slides were scanned using a brightfield slide scanner (Leica, Wetzlar, Germany; Aperio VERSA). Each immunostaining was performed in at least three mice, with multiple fields reviewed. The antibodies used in this study included anti-Sp7/osterix (Osx) (Abcam, Cambridge, MA, USA, ab22552) and anti-osteocalcin (Ocn) (Abcam, Cambridge, MA, USA, ab93876).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eOCT-embedded frozen sections were blocked and incubated with primary EGFP-tag antibody (Proteintech, Rosemont, IL, USA, 50430-2-AP) overnight at 4\u0026deg;C. The sections were subsequently incubated with anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) cross-adsorbed secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA, Alexa Fluor 546), followed by nuclear counterstaining and mounting with mounting medium containing DAPI (Vector Laboratories, Newark, CA, USA, H-1200-10). Each immunostaining was performed in at least three mice, with multiple fields reviewed. Images were captured using an A1 HD25 confocal microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEdU proliferation assay\u003c/h2\u003e \u003cp\u003eThe proliferation assay was conducted as previously described. Briefly, 100 \u0026micro;L of 10 mM EdU (Thermo Fisher Scientific, Waltham, MA, USA, A10044) per 10 g of mouse body weight was administered 3 h prior to euthanasia, followed by decalcification and sectioning. Click staining and subsequent immunofluorescence staining were performed according to the manufacturer's instructions for the Click-it EdU Alexa Fluor 555 Imaging Kit (Thermo Fisher Scientific, Waltham, MA, USA, C10338). Images were captured using a confocal microscope (Nikon, Tokyo, Japan, A1 HD25), and quantitative analysis was performed using ImageJ software\u003csup\u003e68\u003c/sup\u003e, with a consistent threshold (65\u0026ndash;225).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were analyzed using GraphPad Prism software (San Diego, CA, USA). Significance was determined by the P value. One-way ANOVA, two-way ANOVA, and t tests were performed as appropriate to analyze significant differences among groups. A P value less than 0.05 was considered statistically significant. All the statistical tests were two-tailed. The data are presented as box-and-whisker plots, with boxes representing the interquartile range (25th\u0026ndash;75th percentiles), the minimum and maximum values reached by bars, the median plotted as a line in the middle, and the mean marked as \u0026ldquo;+\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Dr. J. Silvio Gutkind and Dr. Susan S. Taylor for generously sharing pRSET PKAc\u0026alpha; plasmid with us. We thank Dr. Ramiro Iglesias-Bartolome and Dr. Xiaodong Feng for meaningful discussion. We thank Dr. Li Chen from Analytical \u0026amp; Testing Center Sichuan University for her help with micro-CT scanning and analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This project was supported by National Natural Science Foundation of China Grants (82170995, 82470940, 82071146, 82371002, 81991502); National Key R\u0026amp;D Program of China (2022YFC2402900); Sichuan Science and Technology Program, China (2024NSFSC0540); charitable funds from Mr. Mingxu Zhou family for fibrous dysplasia related research (Sichuan University internal grants 18H1134, 19H1134 and 23H0924).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e X.Z. conceptualized and designed research; X.Z. and Z.L. performed research; L.X., W.H. and N.J. provided technical support; X.Z., Z.L, Q.C., X.H. and D.B. analyzed data; X.Z. and Z.L. wrote the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoyce, A. 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L.\u003cem\u003e et al.\u003c/em\u003e Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. \u003cem\u003eJournal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1468-1486 (2010).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5522083/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5522083/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibrous dysplasia (FD) is a skeletal disorder caused by activating mutations in Gα\u003csub\u003es\u003c/sub\u003e, leading to bone fractures, deformities, and pain. Protein kinase A (PKA), the principal effector of Gα\u003csub\u003es\u003c/sub\u003e/cAMP signaling, plays critical roles in various biological processes. However, its role in FD is unknown. Here, we demonstrate that PKA activation replicates FD-like lesions in a transgenic mouse model expressing an activating mutation of the PKA catalytic subunit α (PKAcα\u003csup\u003eW197R\u003c/sup\u003e) in the skeletal stem cell (SSC) lineage. Mechanistically, PKA promotes osteoclastogenesis and aberrant osteogenic differentiation and proliferation of SSCs, while impairing mineralization. Interestingly, downregulating PKA activity, using either a genetically engineered PKA inhibitor peptide or small-molecule inhibitors, effectively alleviates FD lesions in an FD mouse model and safeguards bone health by increasing trabecular bone volume in an osteoporosis mouse model. These findings demonstrate that PKA is a dependent factor in FD initiation and progression, underscoring its potential as a therapeutic target.\u003c/p\u003e","manuscriptTitle":"Protein Kinase A is a Dependent Factor and Therapeutic Target in Fibrous Dysplasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-05 07:55:32","doi":"10.21203/rs.3.rs-5522083/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a1064a3b-36f6-4b1f-9e27-195e689772bb","owner":[],"postedDate":"December 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41109135,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"},{"id":41109136,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":41109137,"name":"Health sciences/Diseases/Cancer/Bone cancer"}],"tags":[],"updatedAt":"2025-07-02T07:18:44+00:00","versionOfRecord":{"articleIdentity":"rs-5522083","link":"https://doi.org/10.1038/s41467-025-61402-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-12-05 07:55:32","video":"","vorDoi":"10.1038/s41467-025-61402-z","vorDoiUrl":"https://doi.org/10.1038/s41467-025-61402-z","workflowStages":[]},"version":"v1","identity":"rs-5522083","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5522083","identity":"rs-5522083","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}