Non-pyroptotic caspase-11 activity regulates osteoclastogenesis and pathological bone loss

Non-pyroptotic caspase-11 activity regulates osteoclastogenesis and pathological bone loss | 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 Non-pyroptotic caspase-11 activity regulates osteoclastogenesis and pathological bone loss Jeong-Tae Koh, Xianyu Piao, Ju Han Song, Jung-Woo Kim, Seung-Hee Kwon, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6181572/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted 5 You are reading this latest preprint version Abstract Osteoclasts are essential for bone remodeling; however, their hyperactivity leads to pathological bone loss. While inflammasome-activated caspases are known to influence osteoclastogenesis, the role of caspase-11, beyond its conventional function in pyroptosis, remains unclear. Here, we identified caspase-11 as a pivotal regulator of RANKL-induced osteoclast differentiation. Caspase-11 expression and activity were elevated in bone tissues exhibiting excessive resorption and in RANKL-stimulated bone marrow-derived macrophages. Unlike inflammasome activation, RANKL-induced caspase-11 did not trigger typical inflammasome-associated inflammatory responses. Caspase-11 knockout mice displayed increased bone mass and resistance to RANKL-induced bone resorption; in parallel, genetic or pharmacological inhibition of caspase-11 impaired osteoclast differentiation in vitro. Notably, mechanistic studies revealed that RANKL-activated caspase-11 translocates to the nucleus, where it cleaves and inactivates poly(ADP-ribose) polymerase 1 (PARP1), a transcriptional repressor of osteoclastogenesis. In addition, using the caspase-11 inhibitor, VX-765, substantially reduced ovariectomy-induced bone loss. These findings collectively reveal a novel, non-inflammatory function of caspase-11 in osteoclastogenesis, positioning it as a promising therapeutic target for osteolytic diseases. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Pathogenesis/Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bone remodeling is a complex, continuous process involving the coordinated actions of osteoclasts and osteoblasts to maintain skeletal integrity throughout life [ 1 ]. Dysregulation of this process leads to osteoporosis, a debilitating condition characterized by increased bone resorption, decreased bone mass, and a significantly increased fracture risk. Osteoporosis is a global health problem affecting millions of individuals and is projected to increase with aging populations and changing lifestyles [ 2 ]. Given the significant burden of osteoporosis, elucidating the underlying mechanisms is critical for developing novel therapeutic strategies for treating and preventing this disease. Osteoclasts, the cells responsible for bone resorption, arise from the differentiation of mononuclear pre-osteoclasts. This process is critically regulated by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling [ 3 ]. The binding of RANKL to its receptor, RANK, on pre-osteoclasts initiates signaling pathways involving nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), culminating in the activation of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master regulator of osteoclast differentiation. NFATc1 activation promotes the expression of key osteoclast-related genes such as tartrate-resistant acid phosphatase ( Trap ) and cathepsin K ( Ctsk ) [ 4 ]. This process is intricately modulated by chromatin remodeling mechanisms, including methylation, acetylation, and poly(ADP)-ribosylation (PARylation) [5 − 7]. Inflammatory caspases (caspase-1, -4, -5, and − 11), a subset of cysteine-dependent aspartate-specific proteases, are primarily responsible for inflammatory responses. These enzymes promote the maturation of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, and cleave gasdermin D (GSDMD), triggering pyroptotic cell death [8 − 10]. Caspase-1 is predominantly activated by canonical inflammasomes, such as the NLRP3 inflammasome, in response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [ 11 ]. Conversely, caspase-11 (or caspase-4/-5 in humans) orchestrates the non-canonical inflammasome pathway [ 12 ]. Activation of caspase-11 requires a priming phase, driven by factors such as bacterial lipopolysaccharide (LPS), interferons (IFNs), IL-1, and high mobility group box 1 [ 13 , 14 ]. Unlike the canonical pathway, caspase-11 directly binds to cytosolic LPS, leading to oligomerization and autoproteolysis, thus bypassing traditional inflammasome components [ 12 , 15 , 16 ]. Excessive activation of caspase-11 has been strongly associated with immune-related diseases, particularly sepsis [ 12 , 17 ]. While caspase-11 is primarily known for its role in pyroptosis, recent studies suggest its involvement in non-inflammatory cellular processes [ 14 , 18 ]. Our previous study demonstrated that NLRP3 inflammasome-related caspase-1 contributes to age-related alveolar bone loss through inflammation-dependent and independent regulation of osteoclast differentiation [ 19 ]. Thus, this present study aimed to investigate the role of caspase-11 in osteoclastogenesis. Our results indicate that caspase-11 plays a unique role in the initiation of osteoclast differentiation in vitro, distinct from traditional inflammasome activation. Furthermore, we demonstrate that genetic ablation and pharmacological inhibition of caspase-11 preserve bone integrity in osteoporotic conditions. This research uncovers a novel non-pyroptotic function of caspase-11 in osteoclastogenesis and suggests new therapeutic avenues to mitigate osteoclast-associated bone loss. Results Caspase-11 is upregulated in experimental models of bone loss To investigate the involvement of caspase-11 in bone loss, we examined its expression in three experimental models: aging, ovariectomy (OVX), and periodontitis. In the aging model, micro-computed tomography (µ-CT) analysis confirmed significant bone mass reduction, accompanied by an increased number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (Fig. 1 a). Western blot analysis revealed a statistically significant increase in caspase-11 protein (p43/p38 forms) in femoral bone tissue, correlating with increased expression of the osteoclast marker CTSK (Fig. 1 b). In the OVX model, µ-CT analysis showed substantial trabecular bone loss in OVX-treated mice, consistent with an increase in TRAP-positive osteoclasts compared to sham-operated controls (Fig. 1 c). Western blot analysis confirmed a significant upregulation of caspase-11 (p38 form) in the femurs of OVX-treated mice, whereas its expression was barely detectable in sham-operated controls. While CTSK levels also increased modestly, caspase-11 upregulation exhibited a stronger correlation with osteoclastic bone resorption (Fig. 1 d). In the periodontitis model, ligature-induced alveolar bone loss was observed via µ-CT analysis, along with increased TRAP staining of osteoclasts in affected regions (Fig. 1 e). Consistently, Western blot analysis demonstrated elevated expression of caspase-11 and CTSK in alveolar bone samples (Fig. 1 f). Collectively, these findings suggest that caspase-11 upregulation is strongly associated with osteoclast-mediated bone loss across multiple pathological conditions. Caspase-11 expression and activity increase during RANKL-induced osteoclast differentiation Given the observed increase in caspase-11 levels in osteoporotic bone (Fig. 1 ), we next investigated its expression dynamics during RANKL-induced osteoclast differentiation. In bone marrow-derived macrophages (BMMs), RANKL stimulation significantly increased caspase-11 mRNA and protein expression, coinciding with upregulation of key osteoclastogenic markers, including c-Fos, Nfatc1, Trap, and Ctsk (Fig. 2 a, b). Interestingly, caspase-11 expression peaked at early differentiation stages, preceding the maximal expression of most osteoclast markers. Moreover, its expression increased dose-dependently with RANKL stimulation, in parallel with c-Fos upregulation and NF-κB p65 phosphorylation (S Fig. 1 ). Enzymatic activity assays further confirmed that caspase-11 activity significantly increased during early osteoclastogenesis, consistent with its expression pattern (Fig. 2 c). To ascertain the upstream signaling pathway involved, we treated with BMMs with specific inhibitors targeting NF-κB (Bay11-7082), ERK (U0126), JNK (SP600125), and p38 (SB202190). Notably, only NF-κB inhibition effectively suppressed RANKL-induced caspase-11 expression (Fig. 2 d). Western blot analysis confirmed that NF-κB blockage led to reduced caspase-11 protein levels, underscoring the critical role of the RANKL/RANK/NF-κB axis in caspase-11 induction (Fig. 2 e). RANKL-induced caspase-11 upregulation is independent of inflammasome activation To determine whether RANKL-induced caspase-11 upregulation involves inflammasome activation, we assessed key pyroptosis markers, including lactate dehydrogenase (LDH) release and IL-1β secretion. Unlike LPS transfection (non-canonical inflammasome activation) or LPS plus ATP treatment (canonical inflammasome activation), which resulted in significant LDH release, RANKL treatment exhibited minimal cytotoxicity by day 3 (Fig. 2 f). Similarly, IL-1β secretion was negligible in RANKL-treated cells, in contrast to inflammasome-activating conditions (Fig. 2 g). Western blot analysis of culture supernatants further confirmed that inflammasome-associated markers (IL-1β, caspase-1, or cleaved-GSDMD) were undetectable in RANKL-treated cells, despite caspase-11 upregulation (Fig. 2 h). These results indicate that RANKL-induced caspase-11 expression occurs independently of classical inflammasome activation, suggesting a distinct, non-inflammatory role of caspase-11 in osteoclast differentiation. Caspase-11 positively regulates osteoclast differentiation To assess the functional role of caspase-11 in osteoclastogenesis, we performed siRNA-mediated knockdown in BMMs. The knockdown efficiency was confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analyses (S Fig. 2 a, b). Caspase-11 knockdown led to a marked reduction in the expression of key osteoclast markers, including Trap and Ctsk, and the master regulator NFATc1 (Fig. 3 a, b). TRAP staining further confirmed a significant decrease in osteoclast formation (Fig. 3 c). Similarly, pharmacological inhibition of caspase-11 using Ac-LEVD-CHO suppressed RANKL-induced osteoclastogenesis. Inhibitor treatment significantly reduced caspase-11 activity and osteoclast marker expression (S Fig. 2 c, d and Fig. 3 d, e), as well as osteoclast formation and hydroxyapatite resorption (Fig. 3 f, g). Given the dynamic expression of caspase-11 during osteoclast differentiation (Fig. 2 ), we next investigated the critical time points for its function. Time-course experiments revealed that caspase-11 inhibition during the early differentiation phase (E) significantly impaired osteoclastogenesis, with effects comparable to continuous inhibition (EL). In contrast, inhibition during the later phase (L) had minimal impact (Fig. 3 h − k). These results indicate that caspase-11 is crucial for the initiation of RANKL-induced osteoclastogenesis, expanding its functional repertoire beyond non-canonical inflammasome activation. Genetic ablation of caspase-11 attenuates RANKL-induced bone loss in vivo To further evaluate the in vivo role of caspase-11 in osteoclastogenesis, we injected RANKL into caspase-11 wild-type and knockout mice and assessed bone mass alterations. µ-CT analysis revealed that caspase-11 knockout mice exhibited significantly higher baseline trabecular and cortical bone mass compared to wild-type controls (Fig. 4 a, b and S Fig. 3 ). Following RANKL administration, both groups experienced bone loss; however, caspase-11 knockout mice showed significantly attenuated reductions. In the femur, trabecular bone volume fraction (BV/TV) decreased by 63% in wild-type mice but only by 16% in knockout mice. Trabecular thickness (Tb.Th) exhibited an 8% decrease in wild-type mice and a 3% increase in knockout mice. Trabecular number (Tb.N) decreased by 60% in wild-type mice but only by 20% in knockout mice. Similarly, bone mineral density (BMD) decreased by 35% in wild-type mice and 12% in knockout mice, whereas trabecular separation (Tb.Sp) increased by 33% in wild-type mice compared to 12% in knockout mice (Fig. 4 a, b). Comparable protective effects were observed in vertebral trabecular bone (S Fig. 3 a, b). Meanwhile, femoral cortical bone remained largely unaffected by RANKL administration (S Fig. 3 c, d). Histological analysis corroborated these findings. Hematoxylin and eosin (H&E) staining confirmed increased trabecular bone mass in caspase-11 knockout mice, while TRAP staining revealed significantly fewer TRAP-positive osteoclasts in the primary spongiosa compared to wild-type controls (Fig. 4 c, d). Although osteoclast numbers increased following RANKL injection, the magnitude of the increase was significantly lower in knockout mice (Fig. 4 c, d). qRT-PCR analysis of femoral bones from wild-type mice further confirmed RANKL-induced upregulation of caspase-11 , accompanied by elevated expression of Nfatc1 , Trap , and Ctsk (Fig. 4 e). To determine whether increased bone mass in caspase-11 knockout mice resulted from cumulative effects during growth, we examined femoral bones from 4-week-old mice. Despite no difference in body weight or growth plate thickness (S Fig. 4 a, b), H&E staining and µ-CT analysis revealed significantly higher trabecular bone mass in the knockout mice at this early age (S Fig. 4 c − e). These findings suggest that caspase-11 plays a crucial role in RANKL-induced osteoclastogenesis and continuous bone remodeling throughout life. Caspase-11 deficiency impairs RANKL-induced osteoclastogenesis in vitro To further explore the mechanism underlying the reduced bone loss in caspase-11 knockout mice, we examined RANKL-induced osteoclastogenesis in vitro using BMMs from wild-type and caspase-11 knockout mice. RANKL stimulation significantly upregulated osteoclast markers (c-Fos, Nfatc1, Trap, Ctsk, and Mmp-9) in wild-type cells; however, this response was markedly blunted in caspase-11-deficient BMMs (Fig. 5 a, b). Consistently, TRAP staining and pit formation assays confirmed significant defects in osteoclast formation and bone resorptive activity in caspase-11-deficient cells (Fig. 5 c, d). To determine whether impaired osteoclastogenesis resulted from defects in RANKL/RANK signaling, we examined RANK expression and downstream signaling in wild-type and knockout BMMs. Flow cytometry and qRT-PCR analyses revealed no significant differences in RANK surface expression or mRNA levels between the two groups (Fig. 5 e, f). Furthermore, RANKL-induced phosphorylation of NF-κB p65, a key downstream effector, was comparable in both wild-type and knockout cells (Fig. 5 g), indicating that the RANKL/RANK pathway remains intact. To confirm that the observed defects were directly attributable to caspase-11 loss, we reintroduced caspase-11 into knockout BMMs via retroviral transduction. Western blot analysis confirmed successful restoration of caspase-11 expression (Fig. 5 h). Notably, caspase-11-reconstituted cells exhibited a significant recovery in osteoclast differentiation upon RANKL treatment (Fig. 5 h − j). These findings establish caspase-11 as a critical regulator of RANKL-induced osteoclastogenesis, reinforcing its essential role in bone resorption. Caspase-11 mediates PARP1 cleavage to promote osteoclast differentiation Previous studies have highlighted the pivotal role of poly(ADP-ribose) polymerase-1 (PARP1) in osteoclast differentiation, acting through transcriptional regulation via PARylation and its proteolytic degradation by inflammatory caspases [ 6 , 20 ]. To examine the involvement of caspase-11 in PARP1 regulation during osteoclast differentiation, we first confirmed the inhibitory role of PARP1. Knockdown of PARP1 via siRNA significantly increased the formation of TRAP-positive cells and upregulated the expression of osteoclast markers (TRAP, CTSK, and NFATc1) following RANKL treatment (S Fig. 5 a − c). Similarly, pharmacological inhibition of PARP1 with rucaparib, a specific PARP1 inhibitor, led to increased osteoclast marker expression and osteoclast formation, along with reduced PAR levels (S Fig. 5 d − f). These results confirm that PARP1 functions as a negative regulator of osteoclast differentiation, consistent with previous findings. We next investigated whether caspase-11 mediates PARP1 cleavage during osteoclast differentiation. Western blot analysis showed that RANKL stimulation induced PARP1 cleavage, as evidenced by the appearance of the p89 PARP1 fragment. However, both caspase-11 knockdown and pharmacological inhibition markedly reduced RANKL-induced PARP1 cleavage (Fig. 6 a, b). Similarly, BMMs derived from caspase-11 knockout mice exhibited significantly diminished PARP1 cleavage upon RANKL stimulation (Fig. 6 c). Restoration of caspase-11 expression in knockout BMMs via viral transduction successfully rescued PARP1 cleavage, confirming the essential role of caspase-11 in this process (Fig. 6 d). To determine whether inhibition of PARP1 could bypass the requirement for caspase-11 in osteoclast differentiation, we treated BMMs from wild-type and caspase-11 knockout mice with rucaparib in combination with RANKL. As expected, rucaparib treatment significantly enhanced RANKL-induced osteoclast differentiation in wild-type cells (Fig. 6 e − g). Notably, blocking PARP1 activity in caspase-11-deficient BMMs partially rescued osteoclast differentiation (Fig. 6 e − g). These findings suggest that caspase-11 functions as an upstream regulator of PARP1 cleavage, promoting osteoclast differentiation. Caspase-11 translocates to the nucleus and directly cleaves PARP1 upon RANKL stimulation To elucidate the mechanism by which caspase-11 regulates the nuclear protein PARP1 during osteoclast differentiation, we examined its subcellular localization in cytoplasmic and nuclear fractions. Western blot analysis revealed that caspase-11 predominantly resides in the cytoplasm under basal conditions. However, upon RANKL stimulation, caspase-11 translocated to the nucleus, coinciding with increased PARP1 cleavage (Fig. 6 h). In contrast, although caspase-1 level was also elevated following RANKL treatment, it did not undergo nuclear translocation (Fig. 6 h). These results indicate that caspase-11, but not caspase-1, plays a critical role in nuclear PARP1 processing during osteoclastogenesis. Further supporting these findings, experiments using RAW 264.7 cells, which differentiate into osteoclasts upon RANKL stimulation, confirmed the nuclear translocation of caspase-11 and the cleavage of PARP1. Western blot analysis demonstrated caspase-11 localization in the nucleus and PARP1 cleavage following RANKL stimulation, consistent with observations in BMMs (S Fig. 6 ). Immunofluorescence staining further demonstrated that caspase-11, initially confined to the cytoplasm, translocated to the nucleus upon RANKL treatment (Fig. 6 i, j), validating its regulatory role in PARP1 processing during osteoclast differentiation. To determine whether caspase-11 directly cleaves PARP1, we performed in vitro enzyme assays. Whole-cell lysates from naive BMMs were incubated with recombinant caspase-11, and Western blot analysis using an antibody specific for the PARP1 cleavage site (D214) confirmed the generation of the p89 PARP1 fragment production. This cleavage was effectively blocked by the caspase-11 inhibitor Ac-LEVD-CHO (Fig. 6 k). To exclude the possibility of indirect effects mediated by other proteases, recombinant full-length PARP1 was incubated with recombinant caspase-11. Direct cleavage of PARP1 at D214 by caspase-11 was confirmed, and this reaction was completely inhibited by Ac-LEVD-CHO (Fig. 6 l). Collectively, these results demonstrate that upon RANKL stimulation, caspase-11 translocates to the nucleus and directly cleaves PARP1, thereby regulating osteoclast differentiation. Targeting caspase-11 attenuates ovariectomy-induced bone loss To assess the therapeutic potential of caspase-11 inhibition, we selected VX-765 (Belnacasan), a selective inhibitor of interleukin-converting enzymes (ICEs), as a candidate caspase-11 inhibitor [ 21 ]. In vitro experiments were conducted to evaluate its effects on caspase-11 status and osteoclastogenesis. Western blot analysis confirmed that VX-765 effectively inhibited RANKL-induced caspase-11 activation and PARP1 cleavage (Fig. 7 a). Additionally, VX-765 suppressed the expression of osteoclast differentiation markers in a dose-dependent manner (Fig. 7 a, b), accompanied by a reduction in osteoclast formation and activity (Fig. 7 c, d). These findings indicate that VX-765 impairs RANKL-induced osteoclastogenesis by inhibiting caspase-11 activity. Next, we evaluated the in vivo efficacy of VX-765 using an OVX-induced osteoporosis model. Mice received intraperitoneal injection of VX-765 starting one week after OVX surgery and continuing for four weeks. No significant changes in body weight were observed, suggesting minimal drug toxicity and a favorable safety profile (Fig. 7 e). µ-CT analysis of femurs revealed that OVX significantly reduced bone mass parameters, including BV/TV, Tb.N and BMD, in vehicle-treated mice compared to sham-operated controls. However, VX-765 treatment significantly attenuated these reductions (Fig. 7 f, g), although no significant differences were observed in Tb.Th and Tb.Sp between the vehicle and VX-765 groups. TRAP staining of femur sections further demonstrated the efficacy of VX-765 in reducing osteoclast activity. OVX surgery markedly increased the number of osteoclasts per bone surface in the primary spongiosa but not in the endosteal surface. VX-765 treatment significantly reduced osteoclast numbers, confirming its role in suppressing osteoclast-mediated bone resorption (Fig. 7 h, i). Furthermore, qRT-PCR analysis of bone tissue showed that VX-765 partially suppressed OVX-induced upregulation of osteoclast differentiation markers (S Fig. 7 a). Western blot analysis of bone samples detected a modest increase in the caspase-11 p30 fragment following OVX surgery. However, contrary to our initial hypothesis, this increase persisted despite VX-765 administration (S Fig. 7 b). Overall, these findings demonstrate that VX-765 attenuates OVX-induced bone loss by suppressing osteoclast differentiation and activity, supporting its potential as a therapeutic strategy for osteoporosis and other osteoclast-mediated bone diseases. Discussion Caspase-11 was preferentially recognized for its role in non-canonical inflammasome activation through direct sensing of intracellular LPS. While its function in pyroptosis-mediated immune diseases is well established, its role in bone biology remains largely unexplored. Here, we identify caspase-11 as a novel regulator of osteoclastogenesis, independent of its inflammatory functions. Specifically, we demonstrate that caspase-11 directly cleaves PARP1, a suppressor of osteoclast differentiation, thereby promoting osteoclastogenesis (Fig. 8 ). These findings reveal an unrecognized function of caspase-11 in bone homeostasis and highlight its potential as a therapeutic target for osteoclast-mediated bone diseases such as osteoporosis. We identified RANKL as an endogenous regulator of caspase-11 expression and activity. Caspase-11 levels were consistently elevated in bone tissues from animal models of osteoporosis associated with increased RANKL expression, including aging, menopause, and periodontitis [22 − 24]. Additionally, RANKL administration directly induced caspase-11 expression in bone tissue, resembling LPS-induced upregulation via NF-κB signaling in bacterial infection models [ 25 ]. However, unlike LPS, which triggers secondary amplification via type 1 IFNs, RANKL-induced caspase-11 expression occurs independently of IFN signaling (data not shown). This may explain the more moderate increase in caspase-11 expression compared to LPS stimulation. In addition to upregulating caspase-11 expression, RANKL enhanced its enzymatic activity, as evidenced by the presence of its active forms (p38/p30) in the Western blot analysis and increased activity in the enzymatic assay. Notably, unlike classical non-canonical inflammasome activation, RANKL-induced caspase-11 activity did not lead to cytokine release or GSDMD-mediated pyroptosis. Instead, caspase-11 exhibited sublytic activity, promoting osteoclast differentiation without triggering cell death. This aligns with reports suggesting non-pyroptotic roles for caspase-11, such as facilitating cell migration [ 14 , 18 ]. However, the precise mechanism of caspase-11 activation in osteoclastogenesis‒whether through homodimerization and autoproteolysis, or cleavage by an upstream protease‒remains unclear and warrants further investigation. PARP1 is a nuclear enzyme involved in DNA repair, genome integrity, and transcriptional regulation via ADP-ribosylation [ 26 ]. While extensively studied as a therapeutic target in oncology [ 27 ], its role in osteoclastogenesis is increasingly recognized. PARP1 functions as a transcriptional repressor of osteoclast-related genes such as Trap and brain-type cytoplasmic creatine kinase [ 28 , 29 ]. Upon RANKL stimulation, PARP1 dissociates from these promoters, facilitating osteoclast differentiation. Previous studies have implicated caspase-1 in NLRP3 inflammasome-driven PARP1 degradation, which modulates NFATc1 activity during osteoclastogenesis [ 6 , 20 ]. Here, we confirm the inhibitory role of PARP1 in osteoclast differentiation and provide novel evidence that caspase-11 directly mediates its cleavage. In vitro enzyme assays using recombinant caspase-11 demonstrated direct PARP1 cleavage. Furthermore, Western blot analysis using an antibody recognizing cleaved PARP1 (Asp214) revealed a cleavage mechanism analogous to apoptosis-related caspases such as caspase-3 and caspase-7 [ 30 ]. The role of caspase-11 in osteoclastogenesis was further validated using caspase-11 knockout BMMs. RANKL-induced PARP1 cleavage was significantly reduced in knockout cells compared to wild-type cells. Additionally, treatment with the PARP1 inhibitor rucaparib restored osteoclast differentiation in caspase-11-deficient BMMs, confirming that caspase-11 modulates osteoclastogenesis via PARP1. However, rucaparib treatment did not fully rescue osteoclast differentiation to wild-type levels, suggesting that caspase-11 may influence osteoclastogenesis through additional substrates or pathways beyond PARP1. Further studies are needed to identify these potential targets and fully elucidate the broader role of caspase-11 in osteoclast biology. Although caspase-11, like most caspases, lacks a nuclear localization signal motif, our study provides novel evidence that it translocates to the nucleus upon RANKL stimulation. Western blot analysis confirmed the presence of both pro- and mature forms of caspase-11 in the nucleus following RANKL treatment. However, whether caspase-11 undergoes maturation in the cytoplasm before nuclear import or is directly processed within the nucleus remains unclear. Identifying RANKL-responsive substrate-like proteins that facilitate as cytoplasmic-to-nuclear shuttling carriers for caspase-11 is an important area for further investigation [ 31 ]. In vivo studies revealed that adult caspase-11 knockout mice exhibit greater bone volume than wild-type mice, likely due to reduced osteoclast number and activity. In vitro studies using BMMs further confirmed that caspase-11 deficiency impairs osteoclast differentiation and activity, consistent with increase bone mass in knockout mice. Interestingly, even young caspase-11 knockout mice (4 weeks old) displayed increased bone mass, a developmental stage where bone formation dominates over resorption [ 32 ]. This finding suggests that caspase-11 may regulate both bone resorption and formation, a hypothesis warranting further exploration. A key limitation of this study is the use of conventional knockout mice, which do not allow for cell type-specific analysis of caspase-11 function. While in vitro experiments with caspase-11-deficient BMMs clarified its role in osteoclastogenesis, bone remodeling involves multiple cell types, including osteoblasts, osteoclasts, and chondrocytes. Future studies employing conditional knockout models will be essential to delineate the cell-specific roles of caspase-11 in bone remodeling and provide a more physiologically relevant understanding of its function. Concerns regarding the long-term use of current osteoporosis treatments, such as bisphosphonates and RANKL inhibitors, stem from potential adverse effects, including atypical fractures and osteonecrosis of the jaw [ 33 , 34 ]. This underscores the need for novel therapeutic strategies targeting osteoclast-specific pathways. Our previous study demonstrated that the NLRP3 inflammasome contributes to age-related alveolar bone loss through both inflammation-dependent and -independent mechanisms, and that the NLRP3 inhibitor MCC950 effectively prevents this condition [ 19 ]. Similarly, inflammasome-targeted approaches are being explored for treating alveolar bone loss [ 35 ]. Given their role in inflammasome activation, inflammatory caspases are emerging as promising drug targets, with several inhibitors currently under development [ 36 ]. While pyroptosis inhibitors have been proposed for caspase-11-mediated diseases, they predominantly target GSDMD rather than caspase-11 itself [ 37 ]. The absence of selective caspase-11 inhibitors may be due to its limited substrate specificity compared to caspase-1 [ 38 ]. Nevertheless, elucidating the role of caspase-11 in osteoporosis may facilitate the development of selective inhibitors with potential therapeutics. To explore the feasibility of targeting caspase-11 in osteoclast-mediated bone loss, we evaluated the dual caspase-1/-11 inhibitor VX-765. VX-765, an orally bioavailable prodrug metabolized to VRT-043198, exhibits potent inhibition of both caspase-1 (Ki: 0.8 nM) and caspase-11 (Ki: <0.6 nM) in vitro [ 21 ]. Initially developed for caspase-1-mediated diseases involving IL-1β [ 39 , 40 ], VX-765 demonstrated efficacy in inhibiting osteoclastogenesis in vitro and significantly reduced OVX-induced bone loss by approximately 65% in vivo. This reduction correlated with decreased osteoclast numbers and expression of osteoclast-specific markers. Notably, while OVX-induced bone loss was associated with increased caspase-11 activation, VX-765 treatment did not reduce the levels of mature caspase-11 subunits (p38/p30), consistent with previous findings that VX-765, as a reversible inhibitor, does not prevent the proteolytic conversion of pro-caspase-1 to its active subunits [ 39 ]. The relative contributions of caspase-1 and caspase-11 to the anti-resorptive effects of VX-765 remain to be clarified. Estrogen deficiency induced by OVX has been shown to activate the NLRP3–caspase-1–IL-1β axis [ 41 ], suggesting that part of VX-765’s effect may be mediated through caspase-1 inhibition. However, this effect is likely secondary to reduced systemic inflammation, whereas caspase-11 inhibition may directly suppress osteoclast differentiation and activity. To delineate the specific role of caspase-11 in osteoclast-mediated bone resorption, future studies utilizing selective inhibitors or conditional knockout models will be essential. In summary, this study identifies caspase-11 as a novel regulator of osteoclast differentiation through a unique mechanism involving the proteolytic degradation of PARP1, a transcriptional repressor of osteoclastogenesis. These findings provide new insights into the role of caspase-11 in bone metabolism and highlight its potential as a therapeutic target for osteolytic diseases. Materials and Methods Mice C57BL/6J mice were obtained from Damool Science (Daejeon, Korea). The Jackson Laboratory (Bar Harbor, ME) supplied the caspase-11 knockout mice ( Casp4 tm1Yuan , #024698) [ 42 ]. The genotypes of the mice were confirmed by semi-quantitative polymerase chain reaction (PCR) using primers from the indicated supplier (S Table 1). This study included both male and female animals to ensure the generalizability of the findings across sexes. Male mice were primarily used to minimize the influence of hormonal fluctuations, particularly estrogen, on bone remodeling and RANKL-induced osteoclastogenesis. Conversely, female mice were exclusively utilized for the OVX-induced bone loss model to study the impact of estrogen deficiency on bone resorption. Unless otherwise stated, key in vivo and in vitro experiments yielded consistent findings across each sex. All mice were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YB-2024-84). This study also followed the ARRIVE guidelines for preclinical studies. Cell cultures Bone marrow cells were isolated from the long bones of 6- to 8-week-old C57BL/6 wild-type or caspase-11 knockout mice. To generate BMMs, the isolated bone marrow cells were cultured for 4 days in α-MEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin − streptomycin (Gibco), and recombinant mouse M-CSF (30 ng/mL; Biolegend, San Diego, CA). Mouse monocyte/macrophage Raw264.7 cells (Korean Cell Line Bank, Seoul, Korea) were maintained in α-MEM containing 10% FBS and antibiotics. To induce osteoclast differentiation, BMMs were seeded at a density of 3 × 10 5 cells/mL and cultured for 3 to 4 days in a medium containing M-CSF (30 ng/mL) and recombinant mouse RANKL (100 ng/mL; Peprotech, Rocky Hill, NJ). In a parallel experiment, Raw 264.7 cells were seeded at a 1.5 × 10 5 cells/mL density and cultured with 100 ng/mL RANKL. During this period, the cells were treated with Ac-LEVD-CHO (caspase-11 inhibitor; Sigma, St. Louis, MO), BAY 11-7082 (NF-κB inhibitor; Sigma), or VX-765 (caspase-1/-11 inhibitor; AdooQ, Irvine, CA), as indicated. The medium was replaced daily. Osteoclast formation was assessed by tartrate-resistant acid phosphatase (TRAP) staining. Inflammasome activation For canonical inflammasome activation, BMMs (5 × 10 5 cells/mL) were primed with 100 ng/mL E. coli LPS (Sigma) for 6 hours in a complete medium, followed by replacement with Opti-MEM (Gibco). The cells were then stimulated with 3 mM ATP (Sigma) for 30 minutes. The culture supernatant and cell lysate were subsequently collected for further analyses. For non-canonical inflammasome activation, BMMs (5 × 10 5 cells/mL) were primed with 100 ng/mL LPS for 16 hours. The medium was then replaced with Opti-MEM and cells were transfected with LPS (final concentration of 25 µg/mL) for 8 hours using FuGENE HD transfection reagent (final concentration of 0.6% v/v; Promega, Madison, WI). Culture supernatants were concentrated by methanol/chloroform protein precipitation. Inflammasome activation was assessed using Western blot analysis of supernatants and cell lysates. Micro-computed tomography (µ-CT) analysis Formalin-fixed bone specimens were scanned using a Skyscan 1172 X-ray microtomography system (Bruker, Kontich, Belgium) with an isotropic voxel size of 15 µm and an X-ray voltage of 50 kV and current of 200 µA. The acquisition of three-dimensional (3D) images was facilitated using Skyscan NRecon software, followed by CT-analyzer (CTan) software analysis. The 3D rendering of bone structures was accomplished using Mimics software (version 14.0, Materialise). For the femur, trabecular bone volume was quantified within a volume of interest (VOI) located 0.54 mm proximal to the distal epiphyseal growth plate, extending a height of 1 mm. Cortical bone volume was measured at the mid-diaphysis over a length of 0.5 mm. Vertebral bone volume was assessed at the middle (50%) and central (45%) regions of the fifth lumbar vertebra (L5). The integrity of the alveolar bone was assessed by measuring the cementoenamel junction to the alveolar bone crest (CEJ-ABC) distance on the buccal side of the mandible. Specifically, the measurement was taken at the distal root of the first molar, both roots of the second molar, and the root of the third molar. Additionally, bone volume was measured at the interproximal regions between the first and second molars and the second and third molars. Declarations Conflict-of-Interest : The authors have declared that no conflict of interest exists. Author contributions XP, JHS, and JTK conceived and designed the study. XP, JHS, JWK, SHK, SHO, SS, SGP, ZW, and ZF conducted experiments. XP, JHS, JWK, SHO and JTK analyzed the data. JHR, NK, and JTK contributed to the discussion and data interpretation. JHS, JWK, and JTK acquired funding. XP and JHS wrote the initial draft. JWK, JHR, NK, and JTK reviewed and edited the manuscript. JTK supervised the study. XP and JHS share first authorship, and the order in which they are listed was determined by workload. All authors approved the final manuscript. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A5A2027521 to JTK), the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A1A01061824 to JHS), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2009626 to JWK). References Bolamperti S, Villa I, Rubinacci A. Bone remodeling: an operational process ensuring survival and bone mechanical competence. Bone Res. 2022;10:48. Sfeir JG, Drake MT, Khosla S, Farr JN. Skeletal aging. Mayo Clin Proc. 2022;97:1194-1208. Tsai J, Kaneko K, Suh AJ, Bockman R, Park-Min KH. Origin of osteoclasts: osteoclast precursor cells. J Bone Metab. 2023;30:127-140. 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Receptor activator of nuclear factor-κB ligand and sclerostin expression in osteocytes of alveolar bone in rats with ligature-induced periodontitis. J Periodontol. 2014;85:e370-378. Schauvliege R, Vanrobaeys J, Schotte P, Beyaert R. Caspase-11 gene expression in response to lipopolysaccharide and interferon-gamma requires nuclear factor-kappa B and signal transducer and activator of transcription (STAT) 1. J Biol Chem. 2002;277:41624-41630. Alemasova EE, Lavrik OI. Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins. Nucleic Acids Res. 2019;47:3811-3827. Curtin NJ, Szabo C. Poly(ADP-ribose) polymerase inhibition: past, present and future. Nat Rev Drug Discov. 2020;19:711-736. Beranger GE, Momier D, Rochet N, Carle GF, Scimeca JC. Poly(adp-ribose) polymerase-1 regulates Tracp gene promoter activity during RANKL-induced osteoclastogenesis. J Bone Miner Res. 2008;23:564-571. Chen J, Sun Y, Mao X, Liu Q, Wu H, Chen Y. 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McKenzie BA, Mamik MK, Saito LB, Boghozian R, Monaco MC, Major EO, et al. Caspase-1 inhibition prevents glial inflammasome activation and pyroptosis in models of multiple sclerosis. Proc Natl Acad Sci U S A. 2018;115:E6065-E6074. Ruan H, Zhang H, Feng J, Luo H, Fu F, Yao S, et al. Inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, offering a therapeutic target for osteoporosis. Int Immunopharmacol. 2023;124:110901. Wang S, Miura M, Jung YK, Zhu H, Li E, Yuan J. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell. 1998;92:501-509. Additional Declarations There is no duality of interest Supplementary Files CDDOriginalWB.pdf SupplCDDCasp111.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted Reviewer # 1 agreed at journal 11 Mar, 2025 Reviewers invited by journal 11 Mar, 2025 Submission checks completed at journal 10 Mar, 2025 Editor assigned by journal 07 Mar, 2025 First submitted to journal 07 Mar, 2025 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. 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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-6181572","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427129789,"identity":"cca48597-c1ca-4e44-9ee4-08dd7bed877b","order_by":0,"name":"Jeong-Tae 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1","display":"","copyAsset":false,"role":"figure","size":1939680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase-11 is upregulated in bone tissues from experimental bone loss models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative 3D m-CT images and TRAP-stained sections of distal femoral trabecular bone from young (6 months; n = 5) and aged (29 months; n = 5) mice. Morphometric parameters, including BMD (bone mineral density), BV/TV (bone volume/tissue volume), Tb.N (trabecular number), and Tb.Sp (trabecular separation), are presented as the mean ± SD. Abbreviations: BMD, bone mineral density; BV/TV, bone volume/tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular separation. Scale bar, 0.1 mm for TRAP-stained sections. (\u003cstrong\u003eb\u003c/strong\u003e) Western blot analysis of caspase-11 and cathepsin K (CTSK) expression in tibiae of young and aged mice. (\u003cstrong\u003ec\u003c/strong\u003e) Representative 3D m-CT images and TRAP-stained sections of distal femoral trabecular bone from sham-operated (n = 4) and ovariectomized (OVX; n = 5) mice. Scale bar, 0.1 mm for TRAP-stained sections. (\u003cstrong\u003ed\u003c/strong\u003e) Western blot analysis of caspase-11 and CTSK expression in femurs of sham-operated or OVX mice. (\u003cstrong\u003ee\u003c/strong\u003e) Representative 3D m-CT images and TRAP-stained sections of maxillary alveolar bone from control mice (n = 4) and mice with silk ligature-induced periodontitis (PD; n = 4). The cementoenamel junction to alveolar bone crest (CEJ-ABC) distance was measured at three buccal sites: the distal root of the first molar (M1), both roots of the second molar (M2), and the third molar (M3). BV/TV measurements were performed on the alveolar bone between M1 and M2 (M1−M2) and between M2 and M3 (M2−M3). Scale bar, 0.2 mm for TRAP-stained sections. (\u003cstrong\u003ef\u003c/strong\u003e) Western blot analysis of caspase-11 and CTSK expression in alveolar bone from control and PD mice. Protein levels were quantified using ImageJ software and normalized to b-actin. Data are expressed as fold change relative to control and presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/c8718014cffd091554dae38c.png"},{"id":78450888,"identity":"76eb7905-e59e-45c5-8482-198d69ba6e92","added_by":"auto","created_at":"2025-03-13 11:13:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":672726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRANKL induces caspase-11 activation independent of pyroptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e−\u003cstrong\u003ec\u003c/strong\u003e) Bone marrow-derived macrophages (BMMs) were treated with RANKL for the indicated time points. (\u003cstrong\u003ea\u003c/strong\u003e) qRT-PCR analysis of caspase-11 and osteoclast marker mRNA levels. Results represent fold change relative to untreated cells and are expressed as the mean ± SD from three independent experiments. (\u003cstrong\u003eb\u003c/strong\u003e) Western blot analysis of caspase-11 and osteoclast marker protein levels, with b-actin as the internal control. (\u003cstrong\u003ec\u003c/strong\u003e) Caspase-11 enzymatic activity was measured in whole-cell extracts. Data are expressed as fold changes relative to untreated samples and presented as the mean ± SD from at least three independent experiments. (\u003cstrong\u003ed\u003c/strong\u003e) BMMs were pretreated with specific inhibitors for 15 minutes, followed by RANKL stimulation for 6 hours. \u003cem\u003eCaspase-11\u003c/em\u003e mRNA levels were analyzed by qRT-PCR and normalized to untreated controls. Data are presented as the mean ± SD of three independent experiments. BA, Bay11-7082 (NF-kB); U, U0126 (ERK); SP, SP600125 (JNK); and SB, SB202190 (p38). (\u003cstrong\u003ee\u003c/strong\u003e) BMMs were pretreated with BA for 30 minutes and then stimulated with RANKL for the indicated periods. Western blot analysis assessed caspase-11 protein levels, with p-p65 and p-IkB levels used as positive controls. (\u003cstrong\u003ef\u003c/strong\u003e−\u003cstrong\u003eh\u003c/strong\u003e) BMMs were treated with RANKL for the indicated time points. For positive controls of non-canonical and canonical inflammasome activation, cells were transfected with LPS (LPS\u003csup\u003eFuHD\u003c/sup\u003e) or treated with LPS and ATP (LPS+ATP). (\u003cstrong\u003ef\u003c/strong\u003e) Cytotoxicity was measured by LDH release assay. (\u003cstrong\u003eg\u003c/strong\u003e) IL-1b levels in culture supernatants were measured by ELISA. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e. untreated control. n.s, not significant. (\u003cstrong\u003eh\u003c/strong\u003e) Levels of inflammasome-related proteins in conditioned supernatants (Sup) and whole cell lysates (Lys) were analyzed by Western blotting.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/263339ff0c69e30ed7c50b80.png"},{"id":78451763,"identity":"d41360a7-f2b9-4b14-ad2f-0e666f8be3cc","added_by":"auto","created_at":"2025-03-13 11:29:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1377047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase-11 positively regulates osteoclast differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ec\u003c/strong\u003e) Bone marrow-derived macrophages (BMMs) were transfected with control siRNA (si-Ctrl) or caspase-11 siRNA (si-\u003cem\u003eC11\u003c/em\u003e) and stimulated with RANKL for 3 days. (\u003cstrong\u003ea\u003c/strong\u003e) qRT-PCR analysis of osteoclast marker mRNA levels. Data are expressed as the fold change relative to si-Ctrl and shown as the mean ± SD from three independent experiments. (\u003cstrong\u003eb\u003c/strong\u003e) Western blot analysis of osteoclast marker protein levels, with b-actin as the internal control. (\u003cstrong\u003ec\u003c/strong\u003e) TRAP staining was performed to assess osteoclast formation and number. Scale bar, 500 mm. (\u003cstrong\u003ed\u003c/strong\u003e–\u003cstrong\u003eg\u003c/strong\u003e) BMMs were cultured with RANKL in the presence or absence of the caspase-11 inhibitor Ac-LEVD-CHO (C11\u003cem\u003einhi\u003c/em\u003e, 50 mM) for 3 days. (\u003cstrong\u003ed\u003c/strong\u003e) qRT-PCR analysis of osteoclast marker mRNA levels. Results are expressed as the fold change relative to untreated control and presented as the mean ± SD from three independent experiments. (\u003cstrong\u003ee\u003c/strong\u003e) Western blot analysis of osteoclast marker protein levels, with b-actin as the internal control. (\u003cstrong\u003ef\u003c/strong\u003e) Osteoclast formation was assessed by TRAP staining. Scale bar, 500 mm. (\u003cstrong\u003eg\u003c/strong\u003e) Bone resorption activity was measured using a pit formation assay. (\u003cstrong\u003eh\u003c/strong\u003e–k) BMMs were treated with Ac-LEVD-CHO (C11\u003cem\u003einhi\u003c/em\u003e) in the presence of RANKL, as indicated. (\u003cstrong\u003eh\u003c/strong\u003e) Schematic diagram of the experimental design. (\u003cstrong\u003ei\u003c/strong\u003e) qRT-PCR analysis of osteoclast marker mRNA levels. Results are expressed as the fold change relative to untreated controls and presented as the mean ± SD from three independent experiments. (\u003cstrong\u003ej\u003c/strong\u003e) Western blot analysis of osteoclast marker proteins, with b-actin as the loading control. (\u003cstrong\u003ek\u003c/strong\u003e) Osteoclast formation and number were evaluated by TRAP staining. Scale bar, 500 mm.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/e2e6e2f1f324680bbe7bf1d8.png"},{"id":78451328,"identity":"e88e2126-41e4-4549-865e-4386dca91232","added_by":"auto","created_at":"2025-03-13 11:21:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2177085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase-11 deficiency attenuates RANKL-induced bone loss in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative 3D m-CT images of the distal femur of wild-type (WT) and \u003cem\u003ecaspase-11\u003c/em\u003e knockout (\u003cem\u003eCasp-11\u003c/em\u003eKO) mice after vehicle or RANKL injection. Longitudinal (left), transverse (upper right), and coronal (lower right) views of trabecular bone at the distal metaphysis are shown. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of trabecular bone morphometric parameters, including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and trabecular separation (Tb.Sp). Data are presented as box-plots with median (horizontal line) and minimum and maximum values (whiskers). Vehicle-treated groups: n = 5; RANKL-treated groups: n = 6. n.s, not significant. (\u003cstrong\u003ec\u003c/strong\u003e) Representative images of hematoxylin and eosin (H\u0026amp;E, upper left) and TRAP staining of the distal femur. Magnified TRAP-stained images (lower) show osteoclasts (arrowheads) on the primary spongiosa. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of TRAP-positive osteoclasts is expressed as the number of osteoclasts per bone surface (Oc.N/BS). Box-plots as in (\u003cstrong\u003eb\u003c/strong\u003e). (\u003cstrong\u003ee\u003c/strong\u003e) qRT-PCR analysis of caspase-11 and osteoclast-related gene expression in whole bone tissues from WT mice. Data are expressed as the mean ± SD.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/69351502d425c5237112f0fd.png"},{"id":78451329,"identity":"a0626bdd-afe0-4399-bbd7-2a8847622ed7","added_by":"auto","created_at":"2025-03-13 11:21:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1467084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic ablation of caspase-11 inhibits osteoclastogenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e) Bone marrow-derived macrophages (BMMs) from wild-type (WT) and caspase-11 knockout (KO) mice were stimulated using RANKL for the indicated times. (\u003cstrong\u003ea\u003c/strong\u003e) qRT-PCR analysis of osteoclast-related gene expression. Data are expressed as the mean ± SD of three independent experiments. (\u003cstrong\u003eb\u003c/strong\u003e) Western blot analysis of osteoclast marker proteins, with b-actin as the internal control. (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) Representative images of TRAP staining (\u003cstrong\u003ec\u003c/strong\u003e) and pit formation assay (\u003cstrong\u003ed\u003c/strong\u003e) in RANKL-treated BMMs from WT and KO mice. Quantification of TRAP-positive cells and pit area is presented as the mean ± SD. Scale bar, 500 mm. (\u003cstrong\u003ee\u003c/strong\u003e) Flow cytometric analysis of RANK expression on cell surface of WT and caspase-11 KO BMMs. Representative histograms are shown, with mean fluorescence intensity (MFI) obtained from three independent experiments. (\u003cstrong\u003ef\u003c/strong\u003e) qRT-PCR analysis of \u003cem\u003eTnfrsf11a\u003c/em\u003e (RANK) mRNA levels in WT and KO BMMs. Data are expressed as the mean ± SD. n.s, not significant. (\u003cstrong\u003eg\u003c/strong\u003e) Western blot analysis of NF-kB activation in WT and KO BMMs, with b-actin as internal control. (\u003cstrong\u003eh\u003c/strong\u003e–\u003cstrong\u003ej\u003c/strong\u003e) BMMs from WT and \u003cem\u003ecaspase-11\u003c/em\u003e KO mice were infected with retrovirus expressing either a control vector (pMX–Ctrl) or caspase-11 (pMX−C11) and stimulated with or without RANKL for 72 hours. (\u003cstrong\u003eh\u003c/strong\u003e) Western blot analysis of caspase-11 and osteoclast markers, with b-actin as the internal control. (\u003cstrong\u003ei\u003c/strong\u003e) qRT-PCR analysis of osteoclast-related gene expression. (\u003cstrong\u003ej\u003c/strong\u003e) Representative TRAP-stained images and quantification of TRAP-positive cells. Data are expressed as the mean ± SD. Scale bar, 500 mm.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/dec3f809fa6ebf9018c1b8de.png"},{"id":78451326,"identity":"cf4ae25f-d72d-4153-8f25-25ac828071a8","added_by":"auto","created_at":"2025-03-13 11:21:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1655226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase-11 regulates PARP1 proteolytic processing, a suppressor of osteoclast differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Western blot analysis of cleaved-PARP1 (cl-PARP1) levels in bone marrow-derived macrophages (BMMs) transfected with control siRNA or \u003cem\u003ecaspase-11\u003c/em\u003e siRNA (si-\u003cem\u003eC11\u003c/em\u003e) and treated with RANKL for 24 hours. b-actin was used as the loading control. (\u003cstrong\u003eb\u003c/strong\u003e) Western blot analysis of cl-PARP1 levels in BMMs treated with RANKL in the presence or absence of the caspase-11 inhibitor Ac-LEVD-CHO (C11\u003cem\u003einhi\u003c/em\u003e) for 24 hours. (\u003cstrong\u003ec\u003c/strong\u003e) Western blot analysis of caspase-11 and cl-PARP1 in BMMs isolated from wild-type (WT) and \u003cem\u003ecaspase-11\u003c/em\u003eknockout (KO) mice, treated with RANKL for 24 hours. (\u003cstrong\u003ed\u003c/strong\u003e) BMMs from WT and KO mice were infected with retrovirus expressing either a control vector (pMX–Ctrl) or caspase-11 (pMX−C11) and treated with RANKL for an additional 24 hours. cl-PARP1 levels were analyzed by Western blotting. (\u003cstrong\u003ee\u003c/strong\u003e–\u003cstrong\u003eg\u003c/strong\u003e) BMMs from WT and \u003cem\u003ecaspase-11\u003c/em\u003e KO mice were treated with RANKL for 3 days in the presence or absence of rucaparib (Ruca). (\u003cstrong\u003ee\u003c/strong\u003e) Western blot analysis of PAR, NFATc1, and CTSK. (\u003cstrong\u003ef\u003c/strong\u003e) Representative TRAP-stained images of osteoclasts with quantification of TRAP-positive cells (mean ± SD). Scale bar, 200 mm. (\u003cstrong\u003eg\u003c/strong\u003e) qRT-PCR analysis of osteoclast-related marker expression, expressed as fold change relative to untreated WT cells (mean ±SD). (\u003cstrong\u003eh\u003c/strong\u003e) Western blot analysis of cytosolic and nuclear fractions from BMMs treated with RANKL for 24 hours. MEK2 and lamin B were used as loading controls for the cytosolic and nuclear fractions, respectively. (\u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) Immunofluorescence analysis of caspase-11 localization in RAW 264.7 cells treated with RANKL for 24 hours. (\u003cstrong\u003ei\u003c/strong\u003e) Cells were stained with an anti-caspase-11 antibody, followed by an Alexa 488-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. Scale bars, 10 mm. (\u003cstrong\u003ej\u003c/strong\u003e) Fluorescence intensity profiles along the white line were generated using ImageJ. PRV (Pearson’s R value) indicates colocalization efficiency. (\u003cstrong\u003ek\u003c/strong\u003e) In vitro cleavage assay of PARP1 by recombinant caspase-11 (10 U/reaction) in whole-cell lysates from BMMs, with or without the caspase-11 inhibitor Ac-LEVD-CHO (C11\u003cem\u003einhi\u003c/em\u003e, 500 mM). Western blotting was employed to detect cl-PARP1 and GSDMD (positive control). NT, N-terminal fragment. (\u003cstrong\u003el\u003c/strong\u003e) Recombinant caspase-11 was incubated with recombinant full-length PARP1 in the presence or absence of Ac-LEVD-CHO. Western blotting was employed to detect cl-PARP1.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/ba44e9b598063294acc1dce9.png"},{"id":78450893,"identity":"484f0f83-a870-4862-8f9f-0fd4c2d2e929","added_by":"auto","created_at":"2025-03-13 11:13:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2206706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe caspase-11 inhibitor VX-765 attenuates osteoclastogenesis and prevents OVX-induced bone loss.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ed\u003c/strong\u003e) Bone marrow-derived macrophages (BMMs) were treated with RANKL in increasing concentrations of VX-765 (50, 100, and 200 mM; + indicates 200 mM). (\u003cstrong\u003ea\u003c/strong\u003e) Western blot analysis of the specified proteins at the indicated time points. (\u003cstrong\u003eb\u003c/strong\u003e) qRT-PCR analysis of osteoclast-related gene expression after 3 days of treatment. Data are expressed as mean ± SD of three independent experiments. (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) Representative images of TRAP staining (\u003cstrong\u003ec\u003c/strong\u003e) and pit formation assay (\u003cstrong\u003ed\u003c/strong\u003e) with quantification of TRAP-positive osteoclasts and resorbed pit area, expressed as the mean ±SD. Scale bar, 500 mm. (\u003cstrong\u003ee\u003c/strong\u003e–\u003cstrong\u003ei\u003c/strong\u003e) Ovariectomized (OVX) or sham-operated mice were treated with VX-765 or vehicle (n = 8 per group). (\u003cstrong\u003ee\u003c/strong\u003e) Changes in body weight during the treatment period. (\u003cstrong\u003ef\u003c/strong\u003e) Representative 3D m-CT images of the distal femur, showing coronal (top) and transverse (bottom) views of trabecular bone at the distal metaphysis. (\u003cstrong\u003eg\u003c/strong\u003e) m-CT-based quantification of trabecular bone morphometric parameters, including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and trabecular separation (Tb.Sp). Box−plots represent the median (horizontal line within each box) and minimum/maximum values (whiskers). n.s, not significant. (\u003cstrong\u003eh\u003c/strong\u003e) Representative TRAP-stained images of distal femur sections. Scale bar, 0.2 mm. (\u003cstrong\u003ei\u003c/strong\u003e) Quantification of osteoclast numbers at the primary spongiosa and endosteal surfaces of the femurs, expressed as number of osteoclasts per bone surface (Oc.N/BS). n.s, not significant.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/877bb355863d868b17f46808.png"},{"id":78450891,"identity":"614491f1-cd85-4a97-818c-818c0068da57","added_by":"auto","created_at":"2025-03-13 11:13:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":721132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed models for caspase-11-mediated regulation of osteoclastogenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Indirect regulation via caspase-11 non-canonical inflammasome activation. Caspase-11 indirectly promotes osteoclastogenesis by activating the non-canonical inflammasome. This activation facilitates pyroptosis and/or IL-1bsecretion in cooperation with canonical inflammasome components such as NLRP3. The resulting inflammatory milieu enhances osteoclast differentiation. (\u003cstrong\u003eb\u003c/strong\u003e) Direct regulation independent of inflammasome activation. In this pathway, RANKL stimulation upregulates the expression and activity of caspase-11 in pre-osteoclasts, leading to its nuclear translocation. In the nucleus, caspase-11 cleaves PARP1, a suppressor of osteoclastogenesis, thereby directly promoting osteoclast differentiation. This pathway operates independently of the non-canonical inflammasome. Abbreviations: ASC, apoptosis-associated speck-like protein containing a CARD; Ctsk, cathepsin K; DAMPs, damage-associated molecular patterns; GSDMD, gasdermin D; Mmp-9, matrix metalloproteinase-9; NF-kB, nuclear factor kB; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; NLRP3, NLR family pyrin domain containing 3; Oscar, osteoclast-associated Ig-like receptor; PAMPs, pathogen-associated molecular patterns; PARP1, poly (ADP-ribose) polymerase 1; Pre-OCs, pre-osteoclasts; TLRs, toll-like receptors; Trap, tartrate-resistant acid phosphatase.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/8a73595096d3e8cd20c3eb45.png"},{"id":94169283,"identity":"a7970851-f20c-4a25-909f-0279ad912b82","added_by":"auto","created_at":"2025-10-23 07:06:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15136658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/8a880cea-48ad-4a1f-a4e1-21a598695f08.pdf"},{"id":78450892,"identity":"9cebe598-648a-437f-b97e-e09d42df1b2c","added_by":"auto","created_at":"2025-03-13 11:13:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1191652,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CDDOriginalWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/8d7cbf20cbb28b6116494404.pdf"},{"id":78450895,"identity":"4c1f3be9-2446-4895-91b9-d4c51e3eba2f","added_by":"auto","created_at":"2025-03-13 11:13:29","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1022871,"visible":true,"origin":"","legend":"","description":"","filename":"SupplCDDCasp111.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6181572/v1/8d152a29bd56f64696efaef8.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Non-pyroptotic caspase-11 activity regulates osteoclastogenesis and pathological bone loss","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBone remodeling is a complex, continuous process involving the coordinated actions of osteoclasts and osteoblasts to maintain skeletal integrity throughout life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Dysregulation of this process leads to osteoporosis, a debilitating condition characterized by increased bone resorption, decreased bone mass, and a significantly increased fracture risk. Osteoporosis is a global health problem affecting millions of individuals and is projected to increase with aging populations and changing lifestyles [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Given the significant burden of osteoporosis, elucidating the underlying mechanisms is critical for developing novel therapeutic strategies for treating and preventing this disease.\u003c/p\u003e \u003cp\u003eOsteoclasts, the cells responsible for bone resorption, arise from the differentiation of mononuclear pre-osteoclasts. This process is critically regulated by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The binding of RANKL to its receptor, RANK, on pre-osteoclasts initiates signaling pathways involving nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), culminating in the activation of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master regulator of osteoclast differentiation. NFATc1 activation promotes the expression of key osteoclast-related genes such as \u003cem\u003etartrate-resistant acid phosphatase\u003c/em\u003e (\u003cem\u003eTrap\u003c/em\u003e) and \u003cem\u003ecathepsin K\u003c/em\u003e (\u003cem\u003eCtsk\u003c/em\u003e) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This process is intricately modulated by chromatin remodeling mechanisms, including methylation, acetylation, and poly(ADP)-ribosylation (PARylation) [5\u0026thinsp;\u0026minus;\u0026thinsp;7].\u003c/p\u003e \u003cp\u003eInflammatory caspases (caspase-1, -4, -5, and \u0026minus;\u0026thinsp;11), a subset of cysteine-dependent aspartate-specific proteases, are primarily responsible for inflammatory responses. These enzymes promote the maturation of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, and cleave gasdermin D (GSDMD), triggering pyroptotic cell death [8\u0026thinsp;\u0026minus;\u0026thinsp;10]. Caspase-1 is predominantly activated by canonical inflammasomes, such as the NLRP3 inflammasome, in response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Conversely, caspase-11 (or caspase-4/-5 in humans) orchestrates the non-canonical inflammasome pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Activation of caspase-11 requires a priming phase, driven by factors such as bacterial lipopolysaccharide (LPS), interferons (IFNs), IL-1, and high mobility group box 1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Unlike the canonical pathway, caspase-11 directly binds to cytosolic LPS, leading to oligomerization and autoproteolysis, thus bypassing traditional inflammasome components [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Excessive activation of caspase-11 has been strongly associated with immune-related diseases, particularly sepsis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile caspase-11 is primarily known for its role in pyroptosis, recent studies suggest its involvement in non-inflammatory cellular processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our previous study demonstrated that NLRP3 inflammasome-related caspase-1 contributes to age-related alveolar bone loss through inflammation-dependent and independent regulation of osteoclast differentiation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, this present study aimed to investigate the role of caspase-11 in osteoclastogenesis. Our results indicate that caspase-11 plays a unique role in the initiation of osteoclast differentiation in vitro, distinct from traditional inflammasome activation. Furthermore, we demonstrate that genetic ablation and pharmacological inhibition of caspase-11 preserve bone integrity in osteoporotic conditions. This research uncovers a novel non-pyroptotic function of caspase-11 in osteoclastogenesis and suggests new therapeutic avenues to mitigate osteoclast-associated bone loss.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-11 is upregulated in experimental models of bone loss\u003c/h2\u003e \u003cp\u003eTo investigate the involvement of caspase-11 in bone loss, we examined its expression in three experimental models: aging, ovariectomy (OVX), and periodontitis. In the aging model, micro-computed tomography (\u0026micro;-CT) analysis confirmed significant bone mass reduction, accompanied by an increased number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Western blot analysis revealed a statistically significant increase in caspase-11 protein (p43/p38 forms) in femoral bone tissue, correlating with increased expression of the osteoclast marker CTSK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the OVX model, \u0026micro;-CT analysis showed substantial trabecular bone loss in OVX-treated mice, consistent with an increase in TRAP-positive osteoclasts compared to sham-operated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Western blot analysis confirmed a significant upregulation of caspase-11 (p38 form) in the femurs of OVX-treated mice, whereas its expression was barely detectable in sham-operated controls. While CTSK levels also increased modestly, caspase-11 upregulation exhibited a stronger correlation with osteoclastic bone resorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the periodontitis model, ligature-induced alveolar bone loss was observed via \u0026micro;-CT analysis, along with increased TRAP staining of osteoclasts in affected regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Consistently, Western blot analysis demonstrated elevated expression of caspase-11 and CTSK in alveolar bone samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Collectively, these findings suggest that caspase-11 upregulation is strongly associated with osteoclast-mediated bone loss across multiple pathological conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCaspase-11 expression and activity increase during RANKL-induced osteoclast differentiation\u003c/h3\u003e\n\u003cp\u003eGiven the observed increase in caspase-11 levels in osteoporotic bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we next investigated its expression dynamics during RANKL-induced osteoclast differentiation. In bone marrow-derived macrophages (BMMs), RANKL stimulation significantly increased caspase-11 mRNA and protein expression, coinciding with upregulation of key osteoclastogenic markers, including c-Fos, Nfatc1, Trap, and Ctsk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Interestingly, caspase-11 expression peaked at early differentiation stages, preceding the maximal expression of most osteoclast markers. Moreover, its expression increased dose-dependently with RANKL stimulation, in parallel with \u003cem\u003ec-Fos\u003c/em\u003e upregulation and NF-κB p65 phosphorylation (S Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Enzymatic activity assays further confirmed that caspase-11 activity significantly increased during early osteoclastogenesis, consistent with its expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo ascertain the upstream signaling pathway involved, we treated with BMMs with specific inhibitors targeting NF-κB (Bay11-7082), ERK (U0126), JNK (SP600125), and p38 (SB202190). Notably, only NF-κB inhibition effectively suppressed RANKL-induced caspase-11 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Western blot analysis confirmed that NF-κB blockage led to reduced caspase-11 protein levels, underscoring the critical role of the RANKL/RANK/NF-κB axis in caspase-11 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\n\u003ch3\u003eRANKL-induced caspase-11 upregulation is independent of inflammasome activation\u003c/h3\u003e\n\u003cp\u003eTo determine whether RANKL-induced caspase-11 upregulation involves inflammasome activation, we assessed key pyroptosis markers, including lactate dehydrogenase (LDH) release and IL-1β secretion. Unlike LPS transfection (non-canonical inflammasome activation) or LPS plus ATP treatment (canonical inflammasome activation), which resulted in significant LDH release, RANKL treatment exhibited minimal cytotoxicity by day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Similarly, IL-1β secretion was negligible in RANKL-treated cells, in contrast to inflammasome-activating conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Western blot analysis of culture supernatants further confirmed that inflammasome-associated markers (IL-1β, caspase-1, or cleaved-GSDMD) were undetectable in RANKL-treated cells, despite caspase-11 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). These results indicate that RANKL-induced caspase-11 expression occurs independently of classical inflammasome activation, suggesting a distinct, non-inflammatory role of caspase-11 in osteoclast differentiation.\u003c/p\u003e\n\u003ch3\u003eCaspase-11 positively regulates osteoclast differentiation\u003c/h3\u003e\n\u003cp\u003eTo assess the functional role of caspase-11 in osteoclastogenesis, we performed siRNA-mediated knockdown in BMMs. The knockdown efficiency was confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analyses (S Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Caspase-11 knockdown led to a marked reduction in the expression of key osteoclast markers, including Trap and Ctsk, and the master regulator NFATc1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). TRAP staining further confirmed a significant decrease in osteoclast formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Similarly, pharmacological inhibition of caspase-11 using Ac-LEVD-CHO suppressed RANKL-induced osteoclastogenesis. Inhibitor treatment significantly reduced caspase-11 activity and osteoclast marker expression (S Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e), as well as osteoclast formation and hydroxyapatite resorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the dynamic expression of caspase-11 during osteoclast differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we next investigated the critical time points for its function. Time-course experiments revealed that caspase-11 inhibition during the early differentiation phase (E) significantly impaired osteoclastogenesis, with effects comparable to continuous inhibition (EL). In contrast, inhibition during the later phase (L) had minimal impact (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u0026thinsp;\u0026minus;\u0026thinsp;k). These results indicate that caspase-11 is crucial for the initiation of RANKL-induced osteoclastogenesis, expanding its functional repertoire beyond non-canonical inflammasome activation.\u003c/p\u003e\n\u003ch3\u003eGenetic ablation of caspase-11 attenuates RANKL-induced bone loss in vivo\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the in vivo role of caspase-11 in osteoclastogenesis, we injected RANKL into caspase-11 wild-type and knockout mice and assessed bone mass alterations. \u0026micro;-CT analysis revealed that caspase-11 knockout mice exhibited significantly higher baseline trabecular and cortical bone mass compared to wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b and S Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Following RANKL administration, both groups experienced bone loss; however, caspase-11 knockout mice showed significantly attenuated reductions. In the femur, trabecular bone volume fraction (BV/TV) decreased by 63% in wild-type mice but only by 16% in knockout mice. Trabecular thickness (Tb.Th) exhibited an 8% decrease in wild-type mice and a 3% increase in knockout mice. Trabecular number (Tb.N) decreased by 60% in wild-type mice but only by 20% in knockout mice. Similarly, bone mineral density (BMD) decreased by 35% in wild-type mice and 12% in knockout mice, whereas trabecular separation (Tb.Sp) increased by 33% in wild-type mice compared to 12% in knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Comparable protective effects were observed in vertebral trabecular bone (S Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Meanwhile, femoral cortical bone remained largely unaffected by RANKL administration (S Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological analysis corroborated these findings. Hematoxylin and eosin (H\u0026amp;E) staining confirmed increased trabecular bone mass in caspase-11 knockout mice, while TRAP staining revealed significantly fewer TRAP-positive osteoclasts in the primary spongiosa compared to wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). Although osteoclast numbers increased following RANKL injection, the magnitude of the increase was significantly lower in knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). qRT-PCR analysis of femoral bones from wild-type mice further confirmed RANKL-induced upregulation of \u003cem\u003ecaspase-11\u003c/em\u003e, accompanied by elevated expression of \u003cem\u003eNfatc1\u003c/em\u003e, \u003cem\u003eTrap\u003c/em\u003e, and \u003cem\u003eCtsk\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo determine whether increased bone mass in caspase-11 knockout mice resulted from cumulative effects during growth, we examined femoral bones from 4-week-old mice. Despite no difference in body weight or growth plate thickness (S Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b), H\u0026amp;E staining and \u0026micro;-CT analysis revealed significantly higher trabecular bone mass in the knockout mice at this early age (S Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026thinsp;\u0026minus;\u0026thinsp;e). These findings suggest that caspase-11 plays a crucial role in RANKL-induced osteoclastogenesis and continuous bone remodeling throughout life.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-11 deficiency impairs RANKL-induced osteoclastogenesis in vitro\u003c/h2\u003e \u003cp\u003eTo further explore the mechanism underlying the reduced bone loss in caspase-11 knockout mice, we examined RANKL-induced osteoclastogenesis in vitro using BMMs from wild-type and caspase-11 knockout mice. RANKL stimulation significantly upregulated osteoclast markers (c-Fos, Nfatc1, Trap, Ctsk, and Mmp-9) in wild-type cells; however, this response was markedly blunted in caspase-11-deficient BMMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Consistently, TRAP staining and pit formation assays confirmed significant defects in osteoclast formation and bone resorptive activity in caspase-11-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether impaired osteoclastogenesis resulted from defects in RANKL/RANK signaling, we examined RANK expression and downstream signaling in wild-type and knockout BMMs. Flow cytometry and qRT-PCR analyses revealed no significant differences in RANK surface expression or mRNA levels between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f). Furthermore, RANKL-induced phosphorylation of NF-κB p65, a key downstream effector, was comparable in both wild-type and knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), indicating that the RANKL/RANK pathway remains intact.\u003c/p\u003e \u003cp\u003eTo confirm that the observed defects were directly attributable to caspase-11 loss, we reintroduced caspase-11 into knockout BMMs via retroviral transduction. Western blot analysis confirmed successful restoration of caspase-11 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Notably, caspase-11-reconstituted cells exhibited a significant recovery in osteoclast differentiation upon RANKL treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh\u0026thinsp;\u0026minus;\u0026thinsp;j). These findings establish caspase-11 as a critical regulator of RANKL-induced osteoclastogenesis, reinforcing its essential role in bone resorption.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCaspase-11 mediates PARP1 cleavage to promote osteoclast differentiation\u003c/h3\u003e\n\u003cp\u003ePrevious studies have highlighted the pivotal role of poly(ADP-ribose) polymerase-1 (PARP1) in osteoclast differentiation, acting through transcriptional regulation via PARylation and its proteolytic degradation by inflammatory caspases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To examine the involvement of caspase-11 in PARP1 regulation during osteoclast differentiation, we first confirmed the inhibitory role of PARP1. Knockdown of PARP1 via siRNA significantly increased the formation of TRAP-positive cells and upregulated the expression of osteoclast markers (TRAP, CTSK, and NFATc1) following RANKL treatment (S Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c). Similarly, pharmacological inhibition of PARP1 with rucaparib, a specific PARP1 inhibitor, led to increased osteoclast marker expression and osteoclast formation, along with reduced PAR levels (S Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026thinsp;\u0026minus;\u0026thinsp;f). These results confirm that PARP1 functions as a negative regulator of osteoclast differentiation, consistent with previous findings.\u003c/p\u003e \u003cp\u003eWe next investigated whether caspase-11 mediates PARP1 cleavage during osteoclast differentiation. Western blot analysis showed that RANKL stimulation induced PARP1 cleavage, as evidenced by the appearance of the p89 PARP1 fragment. However, both caspase-11 knockdown and pharmacological inhibition markedly reduced RANKL-induced PARP1 cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Similarly, BMMs derived from caspase-11 knockout mice exhibited significantly diminished PARP1 cleavage upon RANKL stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Restoration of caspase-11 expression in knockout BMMs via viral transduction successfully rescued PARP1 cleavage, confirming the essential role of caspase-11 in this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether inhibition of PARP1 could bypass the requirement for caspase-11 in osteoclast differentiation, we treated BMMs from wild-type and caspase-11 knockout mice with rucaparib in combination with RANKL. As expected, rucaparib treatment significantly enhanced RANKL-induced osteoclast differentiation in wild-type cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026thinsp;\u0026minus;\u0026thinsp;g). Notably, blocking PARP1 activity in caspase-11-deficient BMMs partially rescued osteoclast differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026thinsp;\u0026minus;\u0026thinsp;g). These findings suggest that caspase-11 functions as an upstream regulator of PARP1 cleavage, promoting osteoclast differentiation.\u003c/p\u003e\n\u003ch3\u003eCaspase-11 translocates to the nucleus and directly cleaves PARP1 upon RANKL stimulation\u003c/h3\u003e\n\u003cp\u003e To elucidate the mechanism by which caspase-11 regulates the nuclear protein PARP1 during osteoclast differentiation, we examined its subcellular localization in cytoplasmic and nuclear fractions. Western blot analysis revealed that caspase-11 predominantly resides in the cytoplasm under basal conditions. However, upon RANKL stimulation, caspase-11 translocated to the nucleus, coinciding with increased PARP1 cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). In contrast, although caspase-1 level was also elevated following RANKL treatment, it did not undergo nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). These results indicate that caspase-11, but not caspase-1, plays a critical role in nuclear PARP1 processing during osteoclastogenesis.\u003c/p\u003e \u003cp\u003eFurther supporting these findings, experiments using RAW 264.7 cells, which differentiate into osteoclasts upon RANKL stimulation, confirmed the nuclear translocation of caspase-11 and the cleavage of PARP1. Western blot analysis demonstrated caspase-11 localization in the nucleus and PARP1 cleavage following RANKL stimulation, consistent with observations in BMMs (S Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Immunofluorescence staining further demonstrated that caspase-11, initially confined to the cytoplasm, translocated to the nucleus upon RANKL treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j), validating its regulatory role in PARP1 processing during osteoclast differentiation.\u003c/p\u003e \u003cp\u003eTo determine whether caspase-11 directly cleaves PARP1, we performed in vitro enzyme assays. Whole-cell lysates from naive BMMs were incubated with recombinant caspase-11, and Western blot analysis using an antibody specific for the PARP1 cleavage site (D214) confirmed the generation of the p89 PARP1 fragment production. This cleavage was effectively blocked by the caspase-11 inhibitor Ac-LEVD-CHO (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). To exclude the possibility of indirect effects mediated by other proteases, recombinant full-length PARP1 was incubated with recombinant caspase-11. Direct cleavage of PARP1 at D214 by caspase-11 was confirmed, and this reaction was completely inhibited by Ac-LEVD-CHO (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el). Collectively, these results demonstrate that upon RANKL stimulation, caspase-11 translocates to the nucleus and directly cleaves PARP1, thereby regulating osteoclast differentiation.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTargeting caspase-11 attenuates ovariectomy-induced bone loss\u003c/h2\u003e \u003cp\u003eTo assess the therapeutic potential of caspase-11 inhibition, we selected VX-765 (Belnacasan), a selective inhibitor of interleukin-converting enzymes (ICEs), as a candidate caspase-11 inhibitor [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In vitro experiments were conducted to evaluate its effects on caspase-11 status and osteoclastogenesis. Western blot analysis confirmed that VX-765 effectively inhibited RANKL-induced caspase-11 activation and PARP1 cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Additionally, VX-765 suppressed the expression of osteoclast differentiation markers in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b), accompanied by a reduction in osteoclast formation and activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, d). These findings indicate that VX-765 impairs RANKL-induced osteoclastogenesis by inhibiting caspase-11 activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we evaluated the in vivo efficacy of VX-765 using an OVX-induced osteoporosis model. Mice received intraperitoneal injection of VX-765 starting one week after OVX surgery and continuing for four weeks. No significant changes in body weight were observed, suggesting minimal drug toxicity and a favorable safety profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). \u0026micro;-CT analysis of femurs revealed that OVX significantly reduced bone mass parameters, including BV/TV, Tb.N and BMD, in vehicle-treated mice compared to sham-operated controls. However, VX-765 treatment significantly attenuated these reductions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, g), although no significant differences were observed in Tb.Th and Tb.Sp between the vehicle and VX-765 groups. TRAP staining of femur sections further demonstrated the efficacy of VX-765 in reducing osteoclast activity. OVX surgery markedly increased the number of osteoclasts per bone surface in the primary spongiosa but not in the endosteal surface. VX-765 treatment significantly reduced osteoclast numbers, confirming its role in suppressing osteoclast-mediated bone resorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, i). Furthermore, qRT-PCR analysis of bone tissue showed that VX-765 partially suppressed OVX-induced upregulation of osteoclast differentiation markers (S Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Western blot analysis of bone samples detected a modest increase in the caspase-11 p30 fragment following OVX surgery. However, contrary to our initial hypothesis, this increase persisted despite VX-765 administration (S Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Overall, these findings demonstrate that VX-765 attenuates OVX-induced bone loss by suppressing osteoclast differentiation and activity, supporting its potential as a therapeutic strategy for osteoporosis and other osteoclast-mediated bone diseases.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCaspase-11 was preferentially recognized for its role in non-canonical inflammasome activation through direct sensing of intracellular LPS. While its function in pyroptosis-mediated immune diseases is well established, its role in bone biology remains largely unexplored. Here, we identify caspase-11 as a novel regulator of osteoclastogenesis, independent of its inflammatory functions. Specifically, we demonstrate that caspase-11 directly cleaves PARP1, a suppressor of osteoclast differentiation, thereby promoting osteoclastogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings reveal an unrecognized function of caspase-11 in bone homeostasis and highlight its potential as a therapeutic target for osteoclast-mediated bone diseases such as osteoporosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified RANKL as an endogenous regulator of caspase-11 expression and activity. Caspase-11 levels were consistently elevated in bone tissues from animal models of osteoporosis associated with increased RANKL expression, including aging, menopause, and periodontitis [22\u0026thinsp;\u0026minus;\u0026thinsp;24]. Additionally, RANKL administration directly induced caspase-11 expression in bone tissue, resembling LPS-induced upregulation via NF-κB signaling in bacterial infection models [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, unlike LPS, which triggers secondary amplification via type 1 IFNs, RANKL-induced caspase-11 expression occurs independently of IFN signaling (data not shown). This may explain the more moderate increase in caspase-11 expression compared to LPS stimulation.\u003c/p\u003e \u003cp\u003eIn addition to upregulating caspase-11 expression, RANKL enhanced its enzymatic activity, as evidenced by the presence of its active forms (p38/p30) in the Western blot analysis and increased activity in the enzymatic assay. Notably, unlike classical non-canonical inflammasome activation, RANKL-induced caspase-11 activity did not lead to cytokine release or GSDMD-mediated pyroptosis. Instead, caspase-11 exhibited sublytic activity, promoting osteoclast differentiation without triggering cell death. This aligns with reports suggesting non-pyroptotic roles for caspase-11, such as facilitating cell migration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the precise mechanism of caspase-11 activation in osteoclastogenesis‒whether through homodimerization and autoproteolysis, or cleavage by an upstream protease‒remains unclear and warrants further investigation.\u003c/p\u003e \u003cp\u003ePARP1 is a nuclear enzyme involved in DNA repair, genome integrity, and transcriptional regulation via ADP-ribosylation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While extensively studied as a therapeutic target in oncology [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], its role in osteoclastogenesis is increasingly recognized. PARP1 functions as a transcriptional repressor of osteoclast-related genes such as \u003cem\u003eTrap\u003c/em\u003e and \u003cem\u003ebrain-type cytoplasmic creatine kinase\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Upon RANKL stimulation, PARP1 dissociates from these promoters, facilitating osteoclast differentiation. Previous studies have implicated caspase-1 in NLRP3 inflammasome-driven PARP1 degradation, which modulates NFATc1 activity during osteoclastogenesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Here, we confirm the inhibitory role of PARP1 in osteoclast differentiation and provide novel evidence that caspase-11 directly mediates its cleavage. In vitro enzyme assays using recombinant caspase-11 demonstrated direct PARP1 cleavage. Furthermore, Western blot analysis using an antibody recognizing cleaved PARP1 (Asp214) revealed a cleavage mechanism analogous to apoptosis-related caspases such as caspase-3 and caspase-7 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of caspase-11 in osteoclastogenesis was further validated using caspase-11 knockout BMMs. RANKL-induced PARP1 cleavage was significantly reduced in knockout cells compared to wild-type cells. Additionally, treatment with the PARP1 inhibitor rucaparib restored osteoclast differentiation in caspase-11-deficient BMMs, confirming that caspase-11 modulates osteoclastogenesis via PARP1. However, rucaparib treatment did not fully rescue osteoclast differentiation to wild-type levels, suggesting that caspase-11 may influence osteoclastogenesis through additional substrates or pathways beyond PARP1. Further studies are needed to identify these potential targets and fully elucidate the broader role of caspase-11 in osteoclast biology.\u003c/p\u003e \u003cp\u003e Although caspase-11, like most caspases, lacks a nuclear localization signal motif, our study provides novel evidence that it translocates to the nucleus upon RANKL stimulation. Western blot analysis confirmed the presence of both pro- and mature forms of caspase-11 in the nucleus following RANKL treatment. However, whether caspase-11 undergoes maturation in the cytoplasm before nuclear import or is directly processed within the nucleus remains unclear. Identifying RANKL-responsive substrate-like proteins that facilitate as cytoplasmic-to-nuclear shuttling carriers for caspase-11 is an important area for further investigation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn vivo studies revealed that adult caspase-11 knockout mice exhibit greater bone volume than wild-type mice, likely due to reduced osteoclast number and activity. In vitro studies using BMMs further confirmed that caspase-11 deficiency impairs osteoclast differentiation and activity, consistent with increase bone mass in knockout mice. Interestingly, even young caspase-11 knockout mice (4 weeks old) displayed increased bone mass, a developmental stage where bone formation dominates over resorption [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This finding suggests that caspase-11 may regulate both bone resorption and formation, a hypothesis warranting further exploration. A key limitation of this study is the use of conventional knockout mice, which do not allow for cell type-specific analysis of caspase-11 function. While in vitro experiments with caspase-11-deficient BMMs clarified its role in osteoclastogenesis, bone remodeling involves multiple cell types, including osteoblasts, osteoclasts, and chondrocytes. Future studies employing conditional knockout models will be essential to delineate the cell-specific roles of caspase-11 in bone remodeling and provide a more physiologically relevant understanding of its function.\u003c/p\u003e \u003cp\u003eConcerns regarding the long-term use of current osteoporosis treatments, such as bisphosphonates and RANKL inhibitors, stem from potential adverse effects, including atypical fractures and osteonecrosis of the jaw [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This underscores the need for novel therapeutic strategies targeting osteoclast-specific pathways. Our previous study demonstrated that the NLRP3 inflammasome contributes to age-related alveolar bone loss through both inflammation-dependent and -independent mechanisms, and that the NLRP3 inhibitor MCC950 effectively prevents this condition [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, inflammasome-targeted approaches are being explored for treating alveolar bone loss [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Given their role in inflammasome activation, inflammatory caspases are emerging as promising drug targets, with several inhibitors currently under development [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. While pyroptosis inhibitors have been proposed for caspase-11-mediated diseases, they predominantly target GSDMD rather than caspase-11 itself [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The absence of selective caspase-11 inhibitors may be due to its limited substrate specificity compared to caspase-1 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Nevertheless, elucidating the role of caspase-11 in osteoporosis may facilitate the development of selective inhibitors with potential therapeutics.\u003c/p\u003e \u003cp\u003eTo explore the feasibility of targeting caspase-11 in osteoclast-mediated bone loss, we evaluated the dual caspase-1/-11 inhibitor VX-765. VX-765, an orally bioavailable prodrug metabolized to VRT-043198, exhibits potent inhibition of both caspase-1 (Ki: 0.8 nM) and caspase-11 (Ki: \u0026lt;0.6 nM) in vitro [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Initially developed for caspase-1-mediated diseases involving IL-1β [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], VX-765 demonstrated efficacy in inhibiting osteoclastogenesis in vitro and significantly reduced OVX-induced bone loss by approximately 65% in vivo. This reduction correlated with decreased osteoclast numbers and expression of osteoclast-specific markers. Notably, while OVX-induced bone loss was associated with increased caspase-11 activation, VX-765 treatment did not reduce the levels of mature caspase-11 subunits (p38/p30), consistent with previous findings that VX-765, as a reversible inhibitor, does not prevent the proteolytic conversion of pro-caspase-1 to its active subunits [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe relative contributions of caspase-1 and caspase-11 to the anti-resorptive effects of VX-765 remain to be clarified. Estrogen deficiency induced by OVX has been shown to activate the NLRP3\u0026ndash;caspase-1\u0026ndash;IL-1β axis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], suggesting that part of VX-765\u0026rsquo;s effect may be mediated through caspase-1 inhibition. However, this effect is likely secondary to reduced systemic inflammation, whereas caspase-11 inhibition may directly suppress osteoclast differentiation and activity. To delineate the specific role of caspase-11 in osteoclast-mediated bone resorption, future studies utilizing selective inhibitors or conditional knockout models will be essential.\u003c/p\u003e \u003cp\u003eIn summary, this study identifies caspase-11 as a novel regulator of osteoclast differentiation through a unique mechanism involving the proteolytic degradation of PARP1, a transcriptional repressor of osteoclastogenesis. These findings provide new insights into the role of caspase-11 in bone metabolism and highlight its potential as a therapeutic target for osteolytic diseases.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eC57BL/6J mice were obtained from Damool Science (Daejeon, Korea). The Jackson Laboratory (Bar Harbor, ME) supplied the caspase-11 knockout mice (\u003cem\u003eCasp4\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Yuan\u003c/em\u003e\u003c/sup\u003e, #024698) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The genotypes of the mice were confirmed by semi-quantitative polymerase chain reaction (PCR) using primers from the indicated supplier (S Table\u0026nbsp;1). This study included both male and female animals to ensure the generalizability of the findings across sexes. Male mice were primarily used to minimize the influence of hormonal fluctuations, particularly estrogen, on bone remodeling and RANKL-induced osteoclastogenesis. Conversely, female mice were exclusively utilized for the OVX-induced bone loss model to study the impact of estrogen deficiency on bone resorption. Unless otherwise stated, key in vivo and in vitro experiments yielded consistent findings across each sex. All mice were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YB-2024-84). This study also followed the ARRIVE guidelines for preclinical studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures\u003c/h2\u003e \u003cp\u003eBone marrow cells were isolated from the long bones of 6- to 8-week-old C57BL/6 wild-type or caspase-11 knockout mice. To generate BMMs, the isolated bone marrow cells were cultured for 4 days in α-MEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin\u0026thinsp;\u0026minus;\u0026thinsp;streptomycin (Gibco), and recombinant mouse M-CSF (30 ng/mL; Biolegend, San Diego, CA). Mouse monocyte/macrophage Raw264.7 cells (Korean Cell Line Bank, Seoul, Korea) were maintained in α-MEM containing 10% FBS and antibiotics.\u003c/p\u003e \u003cp\u003eTo induce osteoclast differentiation, BMMs were seeded at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL and cultured for 3 to 4 days in a medium containing M-CSF (30 ng/mL) and recombinant mouse RANKL (100 ng/mL; Peprotech, Rocky Hill, NJ). In a parallel experiment, Raw 264.7 cells were seeded at a 1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL density and cultured with 100 ng/mL RANKL. During this period, the cells were treated with Ac-LEVD-CHO (caspase-11 inhibitor; Sigma, St. Louis, MO), BAY 11-7082 (NF-κB inhibitor; Sigma), or VX-765 (caspase-1/-11 inhibitor; AdooQ, Irvine, CA), as indicated. The medium was replaced daily. Osteoclast formation was assessed by tartrate-resistant acid phosphatase (TRAP) staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInflammasome activation\u003c/h2\u003e \u003cp\u003eFor canonical inflammasome activation, BMMs (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were primed with 100 ng/mL \u003cem\u003eE. coli\u003c/em\u003e LPS (Sigma) for 6 hours in a complete medium, followed by replacement with Opti-MEM (Gibco). The cells were then stimulated with 3 mM ATP (Sigma) for 30 minutes. The culture supernatant and cell lysate were subsequently collected for further analyses. For non-canonical inflammasome activation, BMMs (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were primed with 100 ng/mL LPS for 16 hours. The medium was then replaced with Opti-MEM and cells were transfected with LPS (final concentration of 25 \u0026micro;g/mL) for 8 hours using FuGENE HD transfection reagent (final concentration of 0.6% v/v; Promega, Madison, WI). Culture supernatants were concentrated by methanol/chloroform protein precipitation. Inflammasome activation was assessed using Western blot analysis of supernatants and cell lysates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMicro-computed tomography (\u0026micro;-CT) analysis\u003c/h2\u003e \u003cp\u003eFormalin-fixed bone specimens were scanned using a Skyscan 1172 X-ray microtomography system (Bruker, Kontich, Belgium) with an isotropic voxel size of 15 \u0026micro;m and an X-ray voltage of 50 kV and current of 200 \u0026micro;A. The acquisition of three-dimensional (3D) images was facilitated using Skyscan NRecon software, followed by CT-analyzer (CTan) software analysis. The 3D rendering of bone structures was accomplished using Mimics software (version 14.0, Materialise). For the femur, trabecular bone volume was quantified within a volume of interest (VOI) located 0.54 mm proximal to the distal epiphyseal growth plate, extending a height of 1 mm. Cortical bone volume was measured at the mid-diaphysis over a length of 0.5 mm. Vertebral bone volume was assessed at the middle (50%) and central (45%) regions of the fifth lumbar vertebra (L5). The integrity of the alveolar bone was assessed by measuring the cementoenamel junction to the alveolar bone crest (CEJ-ABC) distance on the buccal side of the mandible. Specifically, the measurement was taken at the distal root of the first molar, both roots of the second molar, and the root of the third molar. Additionally, bone volume was measured at the interproximal regions between the first and second molars and the second and third molars.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict-of-Interest\u003c/strong\u003e: The authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXP, JHS, and JTK conceived and designed the study. XP, JHS, JWK, SHK, SHO, SS, SGP, ZW, and ZF conducted experiments. XP, JHS, JWK, SHO and JTK analyzed the data. JHR, NK, and JTK contributed to the discussion and data interpretation. JHS, JWK, and JTK acquired funding. XP and JHS wrote the initial draft. JWK, JHR, NK, and JTK reviewed and edited the manuscript. JTK supervised the study. XP and JHS share first authorship, and the order in which they are listed was determined by workload. All authors approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A5A2027521 to JTK), the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A1A01061824 to JHS), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2009626 to JWK).\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBolamperti S, Villa I, Rubinacci A. Bone remodeling: an operational process ensuring survival and bone mechanical competence. Bone Res. 2022;10:48.\u003c/li\u003e\n\u003cli\u003eSfeir JG, Drake MT, Khosla S, Farr JN. Skeletal aging. 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Inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, offering a therapeutic target for osteoporosis. Int Immunopharmacol. 2023;124:110901.\u003c/li\u003e\n\u003cli\u003eWang S, Miura M, Jung YK, Zhu H, Li E, Yuan J. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell. 1998;92:501-509.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6181572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6181572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoclasts are essential for bone remodeling; however, their hyperactivity leads to pathological bone loss. While inflammasome-activated caspases are known to influence osteoclastogenesis, the role of caspase-11, beyond its conventional function in pyroptosis, remains unclear. Here, we identified caspase-11 as a pivotal regulator of RANKL-induced osteoclast differentiation. Caspase-11 expression and activity were elevated in bone tissues exhibiting excessive resorption and in RANKL-stimulated bone marrow-derived macrophages. Unlike inflammasome activation, RANKL-induced caspase-11 did not trigger typical inflammasome-associated inflammatory responses. Caspase-11 knockout mice displayed increased bone mass and resistance to RANKL-induced bone resorption; in parallel, genetic or pharmacological inhibition of caspase-11 impaired osteoclast differentiation in vitro. Notably, mechanistic studies revealed that RANKL-activated caspase-11 translocates to the nucleus, where it cleaves and inactivates poly(ADP-ribose) polymerase 1 (PARP1), a transcriptional repressor of osteoclastogenesis. In addition, using the caspase-11 inhibitor, VX-765, substantially reduced ovariectomy-induced bone loss. These findings collectively reveal a novel, non-inflammatory function of caspase-11 in osteoclastogenesis, positioning it as a promising therapeutic target for osteolytic diseases.\u003c/p\u003e","manuscriptTitle":"Non-pyroptotic caspase-11 activity regulates osteoclastogenesis and pathological bone loss","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 11:13:25","doi":"10.21203/rs.3.rs-6181572/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-11T12:03:42+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-11T09:24:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-10T13:08:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-08T02:36:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2025-03-08T02:36:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a880f1bf-492f-4a39-90dc-fe0bfa7a2bfb","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45512819,"name":"Biological sciences/Cell biology"},{"id":45512820,"name":"Health sciences/Diseases"},{"id":45512821,"name":"Health sciences/Pathogenesis/Inflammation"}],"tags":[],"updatedAt":"2025-10-23T07:05:51+00:00","versionOfRecord":{"articleIdentity":"rs-6181572","link":"https://doi.org/10.1038/s41418-025-01596-3","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2025-10-22 04:00:00","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-03-13 11:13:25","video":"","vorDoi":"10.1038/s41418-025-01596-3","vorDoiUrl":"https://doi.org/10.1038/s41418-025-01596-3","workflowStages":[]},"version":"v1","identity":"rs-6181572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6181572","identity":"rs-6181572","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}