The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: Insights into therapeutic targets for lung fibrosis

The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: Insights into therapeutic targets for lung fibrosis | 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 Research Article The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: Insights into therapeutic targets for lung fibrosis Sarah Orfanos, Brian T. Deeney, Gaoyuan Cao, Nikhil Karmacharya, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7321140/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Respiratory Research → Version 1 posted 11 You are reading this latest preprint version Abstract Background: Injury-repair responses typically induce tissue or organ scarring. Generally, cAMP-mobilizing agents inhibit the activation of fibroblasts and deposition of extracellular matrix. Some but not all cAMP-mobilizing agents inhibit fibrosis. Evidence suggests that inhaled treprostinil, a prostacyclin (IP) analog, increases intracellular cAMP levels [cAMP] I , and slows the decline in pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the molecular mechanisms by which cAMP-mobilizing agents, including treprostinil, alter the expression of matrix proteins in human lung fibroblasts (HLF) remain unclear. Unlike other G αs -coupled receptors, we posit that the antifibrotic properties of treprostinil are driven by cAMP-mobilizing-dependent and -independent responses mediated by the IP receptor activation. Methods: As a model of lung fibrosis, primary HLF derived from non-IPF and IPF donors were stimulated with TGF-β; collagen 1A1 and plasminogen activator inhibitor-1 (PAI) expression were then measured in the presence and absence of cAMP mobilizing agents. The necessity of receptor activation for inhibiting TGF-β-induced markers of fibrosis was determined by using soluble receptor inhibitors and decreasing receptor expression with siRNA. Results: Treprostinil decreased TGF-β-induced extracellular matrix production by HLF, and the magnitude of the inhibition was greater than that of other cAMP-mobilizing GPCR agonists despite these agents comparably increasing cAMP levels. There was no difference in the sensitivity and magnitude of the treprostinil inhibition in HLF derived from non-fibrosis and lung fibrosis donors. Treprostinil inhibition of TGF-β-induced collagen 1A1and PAI-1 was mediated through the activation of the IP receptor. The activation of the EP2 receptor, in part, inhibited TGF-β-induced collagen 1A1 expression by treprostinil or prostaglandin E2. β2 agonists had little effect on TGF-β-induced expression of collagen 1A1 and PAI-1. The inhibitory effects of treprostinil on TGF-β-induced collagen 1A1 expression required G αs activation, while G αs only partially mediated treprostinil inhibition of PAI-1. Conclusion: The anti-fibrotic properties of treprostinil are primarily mediated by the IP receptor, acting through both G αs -dependent and -independent pathways. Understanding the differential effects of cAMP-mobilizing pathways on HLF fibrotic signatures can provide insight into developing novel targets to manage IPF. treprostinil idiopathic pulmonary fibrosis human lung fibroblasts cAMP signaling prostacyclin receptor GPCR Gαs subunit Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Intracellular cyclic adenosine monophosphate (cAMP) can play a significant role in modulating fibrosis, specifically by influencing fibroblast activity and the production of extracellular matrix (ECM) (1, 2). While cAMP can be beneficial in some contexts by inhibiting fibrosis, its effects can vary depending on the specific tissue and signaling pathways involved (3). Concerning lung fibrosis, idiopathic pulmonary fibrosis (IPF) represents a devastating, progressive disease with limited therapeutic options. Despite recent efforts to advance the field, the prognosis remains bleak, with a median survival of 3.8 years in a cohort of patients over 65 years old (4, 5). Fibrotic diseases in other organs can be partly due to aberrant injury-repair response driven by TGF-β effects on mesenchymal-derived cells such as fibroblasts and myofibroblasts (6). Current IPF therapies, including nintedanib and pirfenidone, are encouraging; however, despite slowing the decline in the forced vital capacity (FVC), no benefit in survival was reported (7, 8). Only lung transplantation in IPF improves survival, yet many IPF patients are ineligible for lung transplants due to advanced age and contraindications for immunosuppressive therapies. Recently, treprostinil, a therapeutic used to treat pulmonary arterial hypertension, improved lung function in a small cohort of patients with pulmonary hypertension secondary to IPF (9, 10). Motivated by these findings, clinical trials are now examining the efficacy of treprostinil in IPF patients; the precise antifibrotic mechanisms of action of treprostinil and other cAMP-mobilizing agents remain unclear. Treprostinil activates several G protein-coupled receptors (GPCR), including the IP (Prostacyclin), EP2 (Prostaglandin E2), DP1 (Prostaglandin D2 receptor 1), and EP4 (Prostaglandin E2 receptor 4) receptors (11). As an agonist for GPCRs coupled to G αs , treprostinil increases intracellular cAMP ([cAMP] I ) by activating adenylyl cyclase. Increases in [cAMP] I stimulate protein kinase A (PKA), whose substrates modulate gene expression. Some G αs- coupled GPCRs co-localize with specific adenylyl cyclase isoforms (12-14). Adenylyl cyclase then forms a complex with surrounding effector molecules, including the tetrameric PKA unit, the A-kinase anchoring proteins (AKAPs), and the exchange protein activated by cAMP (EPAC). These mechanisms may contribute to the precise spatial and temporal regulation of cAMP signaling within cells (15-20). Whether the anti-fibrotic effects of treprostinil are cAMP-dependent and -independent, and whether specific receptors mediate treprostinil effects, remains unclear (21, 22). In this study, we posit that the anti-fibrotic effects observed with treprostinil and other cAMP-mobilizing agents are mediated through the activation of receptors coupled to the G αs subunit and can be attributed, in part, to selective downstream cAMP signaling rather than solely relying on the magnitude of agonist-induced intracellular cAMP levels. Understanding how treprostinil compares with other cAMP-mobilizing agents in mediating their anti-fibrotic effects can provide insight into the development of novel targeted therapies for fibrosis. Materials and Methods Materials: SDS PAGE/immunoblotting supplies were purchased from Life Technologies (Grand Island, NY). Odyssey blocking buffer and secondary antibodies were purchased from Li-Cor (Lincoln, NE). cAMP ELISA kit was purchased from Applied Biosystems, Thermo Fisher Scientific (Bedford, MA). Green nucleus-targeted cADDis cAMP was obtained from Montana Molecular (Bozeman, MT). Trichostatin A was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for detection of collagen 1A1, vimentin and S100A4 were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies for detection of recombinant anti-Smad3 (phospho S423 + S425), phospho-HSP20, PTGER2 and PAI-1 were obtained from Abcam (Waltham, MA). Antibodies for detection of G αs were purchased from Santacruz Biotechnologies (Santa Cruz, CA). Antibodies for detection of SMA (actin α) were purchased from Sigma (Burlington, MA). PTGER2 silencer siRNA was obtained from Invitrogen, Thermo Fisher Scientific (Waltham, MA). GNAS silencer siRNA was purchased from Dharmacon (Lafayette, CO). Treprostinil and prostaglandin E2 were procured from Cayman chemical (Ann Arbor, MI), isoproterenol from Sigma Aldrich (St. Louis, MO) and TGF-β from R&D Systems (Minneapolis, MN). The IP antagonist CAY10441 was purchased from Cayman Chemical (Ann Arbor, MI). Human lung fibroblasts (HLF) were isolated and characterized from lung parenchyma tissue: De-identified human lungs were obtained from the National Disease Research Interchange (Philadelphia, PA), the International Institute for the Advancement of Medicine (New York, NY), or BioIVT (Gladstone, NJ). Since the tissues are de-identified, Rutgers deemed the tissue procurement as non-human subject research and exempted it from IRB approval. All fibroblast cells were isolated from these tissues as previously described by our laboratory (23). In some experiments, the antifibrotic effects of cAMP-mobilizing agents were compared between HLF derived from donors with non-lung fibrosis and those with lung fibrosis. The clinical characteristics of these donors are shown in Supplement Figure 2. Briefly, HLF isolation was obtained after all vessels and bronchioles were removed from the tissue, the tissue was then cut into 1x1mm or smaller pieces. The tissue was transferred to a 30 ml digestion solution containing collagenase D (1.5mg/ml, Roche ) in F12 medium (Invitrogen, Waltham, MA), supplemented with 2mM calcium chloride, and incubated for 60 minutes. At the end of digestion, 20 ml 1X PBS was added and the solution was vortexed for 1min, then filtered through a 70 μm cell strainer. The flow-through was centrifuged at 1500 rpm for 5min, and the supernatant was discarded. Cells were washed with 50 ml of 1X PBS and centrifuged. Cells were seeded into T75 flasks and cultured in F-12 medium with 10% FBS for 7-10 days before passage (23). Our laboratory has extensive experience in characterizing HLF cell lines as previously described (23). Briefly, HLF were characterized using vimentin and S100A4 immunocytochemistry. HLF were S100A4 (fibroblast-specific protein 1) (+) and vimentin (+) whereas smooth muscle cells were vimentin (-) and S100A4 (-). At confluence, cultured HLF monolayers revealed that over 98% of the cells were S100A4 (+), vimentin (+), SMA (smooth muscle actin) (-) or weak (+), and EpCAM (epithelial cell adhesion molecule) (-) (Supplemental Figure 1). HLF cells at passage numbers 2-4 were used for all experiments (23). Treatment of HLF: Cells were pretreated with varying doses of isoproterenol (1 nM,10 nM, 100 nM, 1µM), treprostinil (1nM, 10 nM, 100 nM, 1µM), or prostaglandin E2 (10nM, 100 nM, 1µM, 10 µM) for 30 minutes, then TGF-β (5 ng/ml) was added. Levels of collagen 1A1 and PAI-1 were measured at baseline and after 24 hr of TGF-β stimulation. Collagen 1A1 and PAI-1 are proteins associated with lung fibrosis, and accordingly, we used these proteins as markers of a fibrotic signature (23-29). Measurements were performed in the presence and absence of the IP receptor antagonist CAY10441, as well as with the G αs ( GNAS ) or PTGER2 (EP2 receptor) siRNA. Immunoblotting: After treatment, the cell monolayers were collected by adding 0.1% perchloric acid. Cells were pelleted, lysed, and incubated with NuPage® reducing agent and sample buffer. Proteins were separated using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with the primary antibodies described in the reagents section. Protein bands were detected using near-infrared conjugated secondary antibodies with Li-Cor Odyssey CLX, and the intensity of the protein bands was calculated using Image Studio v. 5.2 (30). The collagen 1A1, PAI-1, SMAD3, pHSP20, and G αs were normalized to tubulin. cAMP ELISA assay: Sub-confluent HLF (grown to 50-70% confluence) were treated for 5 min with varying concentrations of treprostinil, isoproterenol, and prostaglandin E2. Cells were lysed, and total cAMP was measured using the Applied Biosystems cAMP Screen Immunoassay System® (Thermo Fisher Scientific). Measurements were performed using a luminometer (BMG Labtech CLARIOstar microplate reader, Cary, NC) (31). Live cell cAMP cADDis assay: HLF were seeded in a 4-well chamber slide (50,000 cells/chamber). HLF were then incubated with the BacMam® viral vector with a promoter expressing the green nucleus-targeted cAMP sensor and trichostatin A. Under live microscopy (Image Pro Plus 6.0), cells were stimulated with treprostinil (10nM) or isoproterenol (100nM), and sequential images every 2 sec were captured. Nuclei were selected prospectively, and a decrease in the fluorescence of the cADDis® vectors, which correlates with increasing [cAMP] I in vivo , was measured using Image J (32). Statistical analysis: All experiments were performed in four or more unique cell lines (Supplemental Fig. 2). GraphPad Prism software (version 10, Boston, MA) was used for data analysis. Statistical significance was determined using the Wilcoxon’s paired test or Friedman’s test for multiple comparisons (30). Nonlinear regression was used to quantify the nuclear cAMP response. Statistical significance was as follows: NS: not significant, * p<0.05, ** p<0.01, *** p<0.001; the data presentation represents means and SEM from biological and technical replicates. Results Treprostinil decreases TGF-β-induced extracellular matrix markers in HLF. To assess the effects of treprostinil on TGF-β–induced extracellular matrix protein expression in HLF derived from non-fibrosis (Fig. 1A) and fibrosis donors (Fig. 1B), we performed immunoblot analysis for collagen 1A1 (Col1A1) and plasminogen activator inhibitor-1 (PAI-1). Stimulation with TGF-β (5 ng/mL, 24 hours) increased Col1A1 and PAI-1 expression in HLF. Although a slight variation in baseline Col1A1 and PAI-1 expression was observed in HLF from non-fibrosis donors, substantial heterogeneity in response to TGF-β stimulation was seen. Despite this variation, treprostinil inhibited TGF-β–induced expression in a dose-dependent manner, with 10 nM identified as the lowest effective concentration (Fig. 1). In HLF from donors with fibrosis, there was an increase in baseline Col1A1 but not PAI-1 compared with HLF from non-fibrosis donors. However, the magnitude of TGF-β-induced Col 1A1 and PAI-1 expression was comparable, as was the inhibitory response to treprostinil. In separate experiments, we investigated whether treprostinil inhibited TGF-β-induced SMAD phosphorylation (pSMAD), a necessary signaling event in regulating TGF-β-induced cellular function. Treprostinil had little effect on TGF-β-induced pSMAD (Supplemental Fig. 3). The dose and time interval of TGF-β exposure of HLF are those experimentally determined to be physiologically and pharmacologically relevant, as previously described (23-29) Levels of [cAMP] I induced by treprostinil, PGE 2 , or isoproterenol failed to predict their ability to inhibit TGF-β-induced extracellular matrix. We reasoned that agonists that increase [cAMP] i by activating GPCRs coupled to G αs would have comparable inhibitory effects on TGF-β-induced Col1A1 and PAI-1 expression. We compared the lowest effective inhibitory dose of treprostinil (10 nM) on TGF-β-induced Col1A1 and PAI-1 expression with increasing doses of isoproterenol (1 nM to 1 µM) and PGE 2 (10 nM to 10 µM). Isoproterenol had little inhibitory effect on TGF-β-induced Col1A1 and PAI-1 expression. Although PGE₂ reduced TGF-β-induced Col1A1 and PAI-1 expression, this inhibition was realized only at the highest dose tested (10 µM) (Fig. 2A). The lack of an inhibitory effect by isoproterenol, a well-known inducer of intracellular cAMP, suggests that the inhibition of TGF-β–induced extracellular matrix protein expression is unlikely to be mediated solely by increased [cAMP] I . Comparing [cAMP] I induced by treprostinil, isoproterenol, and PGE 2 , all agents increased [cAMP] I (Fig. 2B). Treprostinil and PGE 2 , arithmetically induced higher levels of [cAMP] I compared to isoproterenol; however, this difference was not statistically significant. Since nuclear cAMP levels can impact gene expression, we also assessed whether treprostinil and isoproterenol manifested differential effects on nuclear cAMP levels. Interestingly, isoproterenol, which has little impact on TGF-β-induced extracellular matrix expression, induced comparable levels of nuclear cAMP to that induced by 10 nM treprostinil (Fig. 2C). Collectively, these data suggest that the activation of GPCR differentially modulates TGF-β-induced Col1A1 and PAI-1 expression, and that levels of [cAMP] I and nuclear cAMP failed to predict the inhibitory effects of the agonists on TGF-β-induced extracellular matrix responses. Collectively, our data suggest that the robust inhibitory impact of treprostinil on TGF-β-induced Col1A1 and PAI-1 expression can be mediated by activating a G αs -independent signaling pathway and a G αs -selective response through treprostinil’s activation of its multiple cognate GPCRs. G αs partially inhibits TGF-β-induced collagen 1A1 by treprostinil and PGE 2 . However, the inhibition of TGF-β-induced PAI-1 by treprostinil and PGE 2 in part is G αs -independent. To determine whether G αs is required to inhibit TGF-β-induced collagen 1A1 and PAI-1 by GPCR agonists, G αs expression was decreased using siRNA, and the effects of agonists on inhibition of TGF-β-induced protein expression were assessed (Fig. 3). In these experiments, the G αs knockdown efficiency was greater than 98%. The physiological effects of the knockdown were confirmed by the inability of treprostinil and PGE 2 to activate phosphorylation of HSP20, a known signaling event that is G αs -, cAMP-, and protein kinase A-dependent (Fig. 3A). Inhibition of G αs reversed treprostinil inhibition of TGF-β-induced collagen 1A1; the reversal of PGE 2 inhibition was modest. G αs knockdown had far less effect on treprostinil- or PGE 2 -induced inhibition of TGF-β-induced PAI-1 expression (Fig. 3B and C). These data suggest that treprostinil and PGE₂ differentially activate G αs -coupled GPCRs and their downstream signaling pathways to inhibit TGF-β-induced collagen 1A1 and PAI-1 expression. Treprostinil inhibits TGF-β-induced protein expression by activating the prostacyclin (IP) receptor. Treprostinil activates several GPCRs, including the prostacyclin (IP) receptor, prostaglandin D2 receptor 1 (DP1), prostaglandin E2 receptor 2 (EP2), and the receptor 4 (EP4). To address the receptor specificity mediating treprostinil’s inhibitory effects on TGF-β-induced protein expression, HLF were stimulated with TGF-β in the presence and absence of treprostinil, soluble IP, DP1, and EP4 receptor antagonists, as well as siRNA to the gene encoding the prostaglandin E receptor 2 (PTGER2). The IP receptor antagonist (CAY1044) profoundly reversed treprostinil-mediated inhibition of TGF-β-induced collagen 1A1 and PAI-1 expression (supplemental Fig. 4, Fig. 4A). As expected, the IP receptor antagonist had little effect on the inhibition of TGF-β-induced collagen 1A1 and PAI-1 expression induced by prostaglandin E2 (Fig. 4A). After the knockdown of the EP2 receptor, the inhibition of TGF-β- induced collagen 1A1 by treprostinil or prostaglandin E2 was partially reversed. However, EP2 knockdown had little impact on treprostinil, and prostaglandin E2-mediated inhibition of PAI-1 expression induced by TGF-β was also unaffected (Fig. 4B). Similarly, EP4 and DP1 receptor antagonists had little effect on treprostinil-mediated inhibition of PAI-1 and collagen 1A1 expression (Fig. 4C and 4D). IP antagonism and EP2 knockdown were confirmed by measuring treprostinil- and PGE 2 -induced phosphorylation of HSP20, which was profoundly decreased, suggesting inhibition of the cognate receptor activation of G αs (Fig. 4E and 4F). These data suggest that the activation of the IP receptor plays a key role in inhibiting TGF-β-induced collagen 1A1 and PAI1 expression. In contrast, the EP2 receptor can partially block TGF-β-induced collagen 1A1 expression. Discussion Despite agonists inducing comparable levels of [cAMP] I or nuclear cAMP, treprostinil and PGE 2 , but not isoproterenol, decreased TGF-β-induced extracellular matrix protein. These findings support our hypothesis that G as -coupled GPCRs selectively modulate TGF-β-induced collagen and PAI-1 in HLF. Treprostinil activation of the IP receptor-linked G αs subunit robustly inhibits TGF-β-induced extracellular matrix production. This was specific to the IP receptor, while a modest inhibitory effect was observed with treprostinil activation of the EP2 receptor. The concept that GPCRs can induce selective cAMP responses through a spatial colocalization of their linked G αs subunit with specific adenylyl cyclase complexes has been suggested (33-35). Evidence suggests that overexpression of adenylyl cyclase 6 decreased collagen 1A1 synthesis and bleomycin-induced fibrosis through specific colocalization of adenylyl cyclase 6 with β1, β2 and IP receptors but not with EP2 or EP4 receptors (33-35). These data support our findings that G αs subunits can evoke differential downstream signaling pathway that modulate cellular function. GPCR- G αs interactions vary among different GPCRs and can modulate differential cellular functions. Notably, the EP2 receptor-coupled G αs complex differs from the β 2 AR-coupled G αs complex regarding transmembrane domain distance and alignment. Such signaling events can be explained by differential coupling to the same G αs subunit, potentially modulating specific TGF-β responses, as observed in our study (36). The dynamic spatial localization and the conformational state of the activated IP receptor coupled G αs may also serve as an initial step in generating selective cAMP responses, activating receptor-associated independent cAMP nanodomains and ultimately contributing to the anti-fibrotic effects of treprostinil (37, 38). The dissociation among the antifibrotic function of GPCRs and the agonist-induced levels of intracellular cAMP was described by Roberts et al , who suggested the possibility of nuclear cAMP compartmentalization. However, our data did not support differential increases in nuclear cAMP levels by G αs -coupled receptor agonists (39, 40). Activation of IPR coupled-G αs was the primary mechanism by which treprostinil inhibited TGF-β-induced collagen 1A1. However, our findings suggest this is not the sole pathway involved, particularly in inhibiting TGF-β-induced PAI1 expression. Therefore, exploring other G αs -independent hypotheses that may also mediate the anti-fibrotic function of treprostinil is warranted, particularly concerning the involvement of additional G α protein subunits. This includes G αq activation of phospholipase C, which generates inositol triphosphate and diacylglycerol, and G α12/13 activation of Rho guanine exchange factors. Additionally, the potential role of the G β/γ subunit is particularly intriguing and warrants further investigation. G β/γ regulates adenylyl cyclase, phospholipase C, and ion channels. G β/γ complex is involved in cardiac fibroblasts activation and fibrosis (41). Therefore, an interesting next step would be to determine if treprostinil is a biased agonist decreasing the recruitment of the G β/γ complex. The existence of “functional selectivity” in certain GPCR/ligand pairs, characterized by an imbalance between G-protein signaling and β-arrestin pathways, poses an alternative hypothesis. Treprostinil and prostaglandin E2, despite both targeting the same EP2 receptor, may act as biased ligands. This bias could promote distinct conformational changes in β-arrestin, creating an imbalance between the β-arrestin and G αs pathway (42, 43). Highlighting the ability of treprostinil to influence a SMAD-independent TGF-β-induced fibrosis pathway could offer novel therapeutic options for treating IPF (Supplemental Fig. 4). Further investigation is needed into how treprostinil can serve as a co-agonist via non-canonical GPCR pathways and through GPCR cross-talk (44, 45). Based on our data, we propose that treprostinil inhibits TGF-β-induced extracellular matrix production through a selective cAMP response mediated by the recruitment of IP receptor–linked Gαs. This effect is not replicated by activation of EP2-linked G αs . Additionally, treprostinil exerts anti-fibrotic effects through a G αs -independent mechanism, particularly by inhibiting TGF-β-induced PAI-1 expression. Potentially, the anti-fibrotic action of treprostinil is mediated by activation of the IP receptor, coupled to Gαs, and by modulating canonical and non-canonical pathways of GPCR signaling. A better understanding of these pathways may lead to the identification of novel therapeutic targets for treating IPF and fibrosis. Abbreviations cAMP Cyclic adenosine monophosphate EP2 Prostaglandin E2 receptor 2 GPCR G protein coupled receptor HLF Human lung fibroblasts HSP20 Heat shock protein 20 IP Prostacyclin receptor IPF Idiopathic pulmonary fibrosis PAI1 Plasminogen activator inhibitor 1 PGE 2 Prostaglandin E2 PKA Protein kinase A TGF-β Transforming growth factor beta Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: Not applicable. Authors' contributions: SO, BTD, GC, NK, AR, CKW, QI, RO and RAP designed the experiments. SO, BTD, GC, NK and AR performed the experiments and analyzed the data. SO, BTD, GC drafted the manuscript. CKW, QI, RO, RAP reviewed and edited manuscript. 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A disease module in the interactome explains disease heterogeneity, drug response and captures novel pathways and genes in asthma. Hum Mol Genet. 2015;24(11):3005-20. Curtis MJ, Alexander SPH, Cirino G, George CH, Kendall DA, Insel PA, et al. Planning experiments: Updated guidance on experimental design and analysis and their reporting III. Br J Pharmacol. 2022;179(15):3907-13. Cao G, Lam H, Jude JA, Karmacharya N, Kan M, Jester W, et al. Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation. Am J Respir Cell Mol Biol. 2022;66(1):96-106. Nuñez FJ, Johnstone TB, Corpuz ML, Kazarian AG, Mohajer NN, Tliba O, et al. Glucocorticoids rapidly activate cAMP production via G(αs) to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects. Faseb j. 2020;34(2):2882-95. Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, Insel PA. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem. 2001;276(45):42063-9. Liu X, Li F, Sun SQ, Thangavel M, Kaminsky J, Balazs L, et al. Fibroblast-specific expression of AC6 enhances beta-adrenergic and prostacyclin signaling and blunts bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2010;298(6):L819-29. Liu X, Ostrom RS, Insel PA. cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts. Am J Physiol Cell Physiol. 2004;286(5):C1089-99. Qu C, Mao C, Xiao P, Shen Q, Zhong YN, Yang F, et al. Ligand recognition, unconventional activation, and G protein coupling of the prostaglandin E(2) receptor EP2 subtype. Sci Adv. 2021;7(14). Buyanov I, Popov P. Characterizing conformational states in GPCR structures using machine learning. Sci Rep. 2024;14(1):1098. Anton SE, Kayser C, Maiellaro I, Nemec K, Möller J, Koschinski A, et al. Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling. Cell. 2022;185(7):1130-42.e11. Roberts MJ, Broome RE, Kent TC, Charlton SJ, Rosethorne EM. The inhibition of human lung fibroblast proliferation and differentiation by Gs-coupled receptors is not predicted by the magnitude of cAMP response. Respir Res. 2018;19(1):56. Roberts MJ, May LT, Keen AC, Liu B, Lam T, Charlton SJ, et al. Inhibition of the Proliferation of Human Lung Fibroblasts by Prostacyclin Receptor Agonists is Linked to a Sustained cAMP Signal in the Nucleus. Front Pharmacol. 2021;12:669227. Khan SM, Martin RD, Bayne A, Pétrin D, Bourque K, Jones-Tabah J, et al. Gβγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts. J Biol Chem. 2023;299(4):103064. Shukla AK, Violin JD, Whalen EJ, Gesty-Palmer D, Shenoy SK, Lefkowitz RJ. Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors. Proc Natl Acad Sci U S A. 2008;105(29):9988-93. Masuho I, Kise R, Gainza P, Von Moo E, Li X, Tany R, et al. Rules and mechanisms governing G protein coupling selectivity of GPCRs. Cell Rep. 2023;42(10):113173. Crudden C, Shibano T, Song D, Suleymanova N, Girnita A, Girnita L. Blurring Boundaries: Receptor Tyrosine Kinases as functional G Protein-Coupled Receptors. Int Rev Cell Mol Biol. 2018;339:1-40. Nürnberg B, Beer-Hammer S, Reisinger E, Leiss V. Non-canonical G protein signaling. Pharmacology & Therapeutics. 2024;255:108589. Additional Declarations No competing interests reported. Supplementary Files Supplementalfigurelegeds.docx Orfanosrespresearch.pptx Supplementary Figures uncroppedblotsRR.pptx Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Respiratory Research → Version 1 posted Editorial decision: Revision requested 06 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 27 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers invited by journal 15 Aug, 2025 Editor assigned by journal 14 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 07 Aug, 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. 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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-7321140","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503253678,"identity":"4ebe5eaf-fca1-471a-81fc-083800d5c9c9","order_by":0,"name":"Sarah Orfanos","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Orfanos","suffix":""},{"id":503253679,"identity":"d56f6016-294c-4fd2-b58e-2168ac9a8bc8","order_by":1,"name":"Brian T. Deeney","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"T.","lastName":"Deeney","suffix":""},{"id":503253680,"identity":"b8f7c0e4-261f-4b9b-bf10-85c5984ad231","order_by":2,"name":"Gaoyuan Cao","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gaoyuan","middleName":"","lastName":"Cao","suffix":""},{"id":503253681,"identity":"b3dbd703-eb75-4548-85ac-07231d3467b7","order_by":3,"name":"Nikhil Karmacharya","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nikhil","middleName":"","lastName":"Karmacharya","suffix":""},{"id":503253682,"identity":"d63bba81-a975-409f-a1f4-8e753acf36c7","order_by":4,"name":"Anjani Ravi","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Anjani","middleName":"","lastName":"Ravi","suffix":""},{"id":503253683,"identity":"a7c8e7a6-f019-40f5-a2df-da06732cdfa9","order_by":5,"name":"Cynthia J. Koziol-White","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cynthia","middleName":"J.","lastName":"Koziol-White","suffix":""},{"id":503253684,"identity":"e28c75d5-3136-4413-97b2-a3732fadef6c","order_by":6,"name":"Qi Yang","email":"","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Yang","suffix":""},{"id":503253685,"identity":"6a82b7f6-675d-47ef-9d80-0fdaf7335191","order_by":7,"name":"Rennolds S. Ostrom","email":"","orcid":"","institution":"Chapman University School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Rennolds","middleName":"S.","lastName":"Ostrom","suffix":""},{"id":503253686,"identity":"d9ae64cf-17e8-47b6-9348-882cdec4a7bb","order_by":8,"name":"Reynold A. Panettieri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFAC5uMfPlQwJBigCPLg1cKWxjjjDExLAlFaeMyYOdtI0WI+uy3tMeO8e3nm/AfYPvP+qJUz7z/A+OBtG24tMncOHzcu3FZcbDkjgXk2T8JxY5kbCcyGc/FokZBIS5CeuS0hccMNBmZmnoRjiTMkGNikefFqyTGQ5p0D1HL+AFhL/Qz+A+y/CWgxk+ZtAGo5kADSUpMgwZDAxoxXi8yxZMMZxxKKDW4kNjPOSTtgOEMisVlyzjk8WqSbDz74UJOQZ3D+8GGGNzZ18hL8hw9+eFOGWwuDBJzF2AAkDsMYRGkBgzr8qkfBKBgFo2BEAgBvHlBaf8M+ggAAAABJRU5ErkJggg==","orcid":"","institution":"Robert Wood Johnson School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Reynold","middleName":"A.","lastName":"Panettieri","suffix":""}],"badges":[],"createdAt":"2025-08-07 17:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7321140/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7321140/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12931-025-03387-3","type":"published","date":"2025-11-22T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89659851,"identity":"3f4915fe-2822-4352-a329-81735f332d4f","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":707381,"visible":true,"origin":"","legend":"\u003cp\u003eTreprostinil inhibits TGF-β-induced collagen 1A1 and PAI-1 expression in non-fibrotic and fibrotic HLF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A): Measurement of collagen 1A1 and PAI-1 expression (n=10) in TGF-β (5 ng/ml for 24 hr) stimulated HLF from non-fibrotic donors with and without treprostinil pretreatment (30 min, 1 nM-1 µM)\u0026nbsp;using immunoblot analysis.\u003c/p\u003e\n\u003cp\u003e(B): Measurement of collagen 1A1 and PAI-1 expression (n=5) in TGF-β (5 ng/ml for 24 hr) stimulated HLF from fibrotic donors with and without treprostinil pretreatment (30 min, 1 nM-1 µM)\u0026nbsp;using immunoblot analysis.\u003c/p\u003e\n\u003cp\u003eData represent means ± SEM of biological replicates. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, \u003csup\u003e**** \u003c/sup\u003ep\u0026lt;0.0001.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/e13fa707e0b22c9832291413.png"},{"id":89659848,"identity":"9ae28674-0321-4bb9-bd6c-e0ed84fc299b","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":949126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the inhibitory effects of treprostinil, isoproterenol, and prostaglandin E2 on TGF-β-induced expression of collagen 1A1 and PAI-1.\u003c/strong\u003e\u0026nbsp; Measurements of intracellular cAMP levels induced by cAMP-mobilizing agents were determined by ELISA and by real-time nuclear cAMP production with treprostinil or isoproterenol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A): Comparison of the inhibitory action of treprostinil (30 min preincubation, 10 nM) and isoproterenol (n=6) (30 min preincubation, 1 nM to 1 μM) and prostaglandin E2 (n=5) (30 min preincubation, 10 nM to 10 μM) on TGF-β-induced (5 ng/ml, 24 hr) collagen 1A1 and PAI-1 expression as assessed by immunoblotting.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(B): ELISA quantification of intracellular cAMP in HLF following stimulation with treprostinil (5 min, 10 nM – 1 µM) or isoproterenol (5 min, 10 nM – 1 µM) or prostaglandin E2 (5 min, 10 nM – 1 µM) (n=5). Treprostinil induces comparable levels of intracellular cAMP to those induced by isoproterenol and prostaglandin E2.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(C): Comparison of nuclear cAMP production induced by treprostinil (10 nM and 100 nM) or isoproterenol (100 nM and 1 μM) (n=4) over the course of 240 seconds was assessed by cADDIS assay.\u0026nbsp;Treprostinil (10 nM) induced lower levels of nuclear cAMP compared to isoproterenol (1 μM), yet resulted in significantly greater inhibition of TGF-β–induced PAI-1 and collagen 1A1 expression.\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM from biological replicates. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, \u003csup\u003e**** \u003c/sup\u003ep\u0026lt;0.0001.\u0026nbsp;Data is presented as a nonlinear regression and the product of the constant K*plateau for each dose.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/c3be09f8e010f56333d56e1d.png"},{"id":89659852,"identity":"65ba755c-54cc-4b2d-b601-3f7331dfb5a9","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":788662,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of treprostinil and prostaglandin E2 inhibition of TGF-β induced collagen 1A1 and PAI-1 expression in a G\u003csub\u003eαs\u003c/sub\u003e HLF knock-down model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A): Measurement of residual G\u003csub\u003eαs\u003c/sub\u003e expression in a G\u003csub\u003eαs\u003c/sub\u003e HLF knockdown model (n=9). G\u003csub\u003eαs\u003c/sub\u003e HLF knockdown inhibits HSP20 phosphorylation (a known G\u003csub\u003eαs\u003c/sub\u003e -dependent phosphorylation event) by treprostinil and prostaglandin E2 (10 nM, 1 hr) in HLF (n=5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(B): Measurement of treprostinil and prostaglandin E2 (pre-treatment 30 min, 10 nM) inhibition of TGF-β-induced (5 ng/ml for 24 hr) collagen 1A1 and PAI1 expression in HLF and after G\u003csub\u003eαs\u003c/sub\u003e HLF knockdown (n=7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(C):\u0026nbsp; Representative immunoblot of treprostinil and prostaglandin E2 phosphorylation of HSP20 and inhibition of TGF-β-induced collagen 1A1 and PAI-1 expression in G\u003csub\u003eαs\u003c/sub\u003e knockdown of HLF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM from biological replicates. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001,\u003csup\u003e **** \u003c/sup\u003ep\u0026lt;0.0001.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/b0f86b8a98033b56dd4cba06.png"},{"id":89659853,"identity":"41e63e9a-4da5-4571-9ba6-98d9d896465a","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1691596,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of treprostinil and prostaglandin E2 on TGF-β-induced collagen 1A1 and PAI-1 expression in HLF models with IPR blockade, EP2R knockdown, DP1R blockade, and EP4R blockade.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A): Measurement of treprostinil and prostaglandin E2 (pre-treatment 30 min, 10 nM) inhibition of TGF-β-induced (5 ng/ml for 24 hr) collagen 1A1 and PAI1 expression in HLF with and without IPR blockade (cay10441, pre-treatment 30 min, 1 µM) (n=5-6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(B): Measurement of treprostinil and prostaglandin E2 (pre-treatment 30 min, 10 nM) inhibition of TGF-β-induced (5 ng/ml for 24 hours) collagen 1A1 and PAI1 expression in a EP2R HLF knockdown model (n=5-6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(C): Measurement of treprostinil (pre-treatment 30 min, 10 nM) inhibition of TGF-β-induced (5 ng/ml for 24 hr) collagen 1A1 and PAI1 expression in HLF with and without DP1R (Laropiprant) (pre-treatment 30 min, 10 nM to 10 μM) (n=5).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(D) Measurement of treprostinil (pre-treatment 30 min, 10 nM) inhibition of TGF-β-induced (5 ng/ml for 24 hr) collagen 1A1 and PAI1 expression in HLF with and without EP4R (ONOAE3208) blockade (pre-treatment 30 min, 10 nM to 1 μM) (n=5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(E): IPR blockade and EP2R HLF knockdown models, inhibit HSP20 phosphorylation by treprostinil and prostaglandin E2 (30 min, 10 nM).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(F): Representative immunoblot showing that IPR blockade reverses treprostinil inhibition of TGF-β-induced collagen 1A1 and PAI-1 expression. In contrast, the EP2R HLF knockdown model reverses treprostinil and prostaglandin E2 inhibition of TGF-β-induced collagen 1A1 but not PAI-1.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM in biological replicates. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, \u003csup\u003e**** \u003c/sup\u003ep\u0026lt;0.0001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/c22c8e6a2e2ee5f912f9cbef.png"},{"id":96651054,"identity":"e9dcb3e2-3e19-461d-a0de-4336aa518603","added_by":"auto","created_at":"2025-11-24 16:13:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5289660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/121fcbac-b5d0-418b-b623-5afe2a6df805.pdf"},{"id":89659846,"identity":"a60aa2a4-a9ac-4d6a-bfc8-4e2e6c7f5561","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementalfigurelegeds.docx","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/f6d5efb30b5fc740f9f9de68.docx"},{"id":89659850,"identity":"557465dd-edcb-4c94-87c5-679e2aa0a564","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2144438,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures\u003c/p\u003e","description":"","filename":"Orfanosrespresearch.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/d2ca8103122a14d028122fcb.pptx"},{"id":89659854,"identity":"3142527c-c728-4311-9d0f-711c1a60146a","added_by":"auto","created_at":"2025-08-22 10:57:50","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1780987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"uncroppedblotsRR.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7321140/v1/e142e9fbb335090755508fce.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: Insights into therapeutic targets for lung fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntracellular cyclic adenosine monophosphate (cAMP) can play a significant role in modulating fibrosis, specifically by influencing fibroblast activity and the production of extracellular matrix (ECM) (1, 2). While cAMP can be beneficial in some contexts by inhibiting fibrosis, its effects can vary depending on the specific tissue and signaling pathways involved (3). \u0026nbsp;Concerning lung fibrosis, idiopathic pulmonary fibrosis (IPF) represents a devastating, progressive disease with limited therapeutic options. Despite recent efforts to advance the field, the prognosis remains bleak, with a median survival of 3.8 years in a cohort of patients over 65 years old (4, 5). Fibrotic diseases in other organs can be partly due to aberrant injury-repair response driven by TGF-\u0026beta; effects on mesenchymal-derived cells such as fibroblasts and myofibroblasts (6).\u003c/p\u003e\n\u003cp\u003eCurrent IPF therapies, including nintedanib and pirfenidone, are encouraging; however, despite slowing the decline in the forced vital capacity (FVC), no benefit in survival was reported (7, 8). Only lung transplantation in IPF improves survival, yet many IPF patients are ineligible for lung transplants due to advanced age and contraindications for immunosuppressive therapies. Recently, treprostinil, a therapeutic used to treat pulmonary arterial hypertension, improved lung function in a small cohort of patients with pulmonary hypertension secondary to IPF (9, 10). Motivated by these findings, clinical trials are now examining the efficacy of treprostinil in IPF patients; the precise antifibrotic mechanisms of action of treprostinil and other cAMP-mobilizing agents remain unclear. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreprostinil activates several G protein-coupled receptors (GPCR), including the IP (Prostacyclin), EP2 (Prostaglandin E2), DP1 (Prostaglandin D2 receptor 1), and EP4 (Prostaglandin E2 receptor 4) receptors (11). As an agonist for GPCRs coupled to G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e, treprostinil increases intracellular cAMP ([cAMP]\u003csub\u003eI\u003c/sub\u003e) by activating adenylyl cyclase. Increases in [cAMP]\u003csub\u003eI\u003c/sub\u003e stimulate protein kinase A (PKA), whose substrates modulate gene expression. Some G\u003csub\u003e\u0026alpha;s-\u003c/sub\u003ecoupled GPCRs co-localize with specific adenylyl cyclase isoforms (12-14). Adenylyl cyclase then forms a complex with surrounding effector molecules, including the tetrameric PKA unit, the A-kinase anchoring proteins (AKAPs), and the exchange protein activated by cAMP (EPAC). These mechanisms may contribute to the precise spatial and temporal regulation of cAMP signaling within cells (15-20). Whether the anti-fibrotic effects of treprostinil are cAMP-dependent and -independent, and whether specific receptors mediate treprostinil effects, remains unclear (21, 22).\u003c/p\u003e\n\u003cp\u003eIn this study, we posit that the anti-fibrotic effects observed with treprostinil and other cAMP-mobilizing agents are mediated through the activation of receptors coupled to the G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e subunit and can be attributed, in part, to selective downstream cAMP signaling rather than solely relying on the magnitude of agonist-induced intracellular cAMP levels. Understanding how treprostinil compares with other cAMP-mobilizing agents in mediating their anti-fibrotic effects can provide insight into the development of novel targeted therapies for fibrosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials:\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSDS PAGE/immunoblotting supplies were purchased from Life Technologies (Grand Island, NY). Odyssey blocking buffer and secondary antibodies were purchased from Li-Cor (Lincoln, NE). cAMP ELISA kit was purchased from Applied Biosystems, Thermo Fisher Scientific (Bedford, MA). Green nucleus-targeted cADDis cAMP was obtained from Montana Molecular (Bozeman, MT). Trichostatin A was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for detection of collagen 1A1, vimentin and S100A4 were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies for detection of recombinant anti-Smad3 (phospho S423 + S425), phospho-HSP20, PTGER2 and PAI-1 were obtained from Abcam (Waltham, MA). Antibodies for detection of G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ewere purchased from Santacruz Biotechnologies (Santa Cruz, CA). Antibodies for detection of SMA (actin \u0026alpha;) were purchased from Sigma (Burlington, MA). PTGER2 silencer siRNA was obtained from Invitrogen, Thermo Fisher Scientific (Waltham, MA). GNAS silencer siRNA was purchased from Dharmacon (Lafayette, CO). \u0026nbsp;Treprostinil and prostaglandin E2 were procured from Cayman chemical (Ann Arbor, MI), isoproterenol from Sigma Aldrich (St. Louis, MO) and TGF-\u0026beta; from R\u0026amp;D Systems (Minneapolis, MN). The IP antagonist CAY10441 was purchased from Cayman Chemical (Ann Arbor, MI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman lung fibroblasts (HLF) were isolated and characterized from lung parenchyma tissue:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDe-identified human lungs were obtained from the National Disease Research Interchange (Philadelphia, PA), the International Institute for the Advancement of Medicine (New York, NY), or BioIVT (Gladstone, NJ). Since the tissues are de-identified, Rutgers deemed the tissue procurement as non-human subject research and exempted it from IRB approval. \u0026nbsp;All fibroblast cells were isolated from these tissues as previously described by our laboratory (23). In some experiments, the antifibrotic effects of cAMP-mobilizing agents were compared between HLF derived from donors with non-lung fibrosis and those with lung fibrosis. The clinical characteristics of these donors are shown in Supplement Figure 2. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBriefly, HLF isolation was obtained after all vessels and bronchioles were removed from the tissue, the tissue was then cut into 1x1mm or smaller pieces. The tissue was transferred to a 30 ml digestion solution containing collagenase D (1.5mg/ml, Roche\u003cem\u003e)\u003c/em\u003e in F12 medium (Invitrogen, Waltham, MA), supplemented with 2mM calcium chloride, and incubated for 60 minutes.\u003cem\u003e\u0026nbsp;\u003c/em\u003e At the end of digestion, 20 ml 1X PBS was added and the solution was vortexed for 1min, then filtered through a 70 \u0026mu;m cell strainer. The flow-through was centrifuged at 1500 rpm for 5min, and the supernatant was discarded. Cells were washed with 50 ml of 1X PBS and centrifuged. Cells were seeded into T75 flasks and cultured in F-12 medium with 10% FBS for 7-10 days before passage (23).\u003c/p\u003e\n\u003cp\u003eOur laboratory has extensive experience in characterizing HLF cell lines as previously described (23). Briefly, HLF were characterized using vimentin and S100A4 immunocytochemistry. HLF were S100A4 (fibroblast-specific protein 1) (+) and vimentin (+) whereas smooth muscle cells were vimentin (-) and S100A4 (-). At confluence, cultured HLF monolayers revealed that over 98% of the cells were S100A4 (+), vimentin (+), SMA (smooth muscle actin) (-) or weak (+), and EpCAM (epithelial cell adhesion molecule) (-) (Supplemental Figure 1). HLF cells at passage numbers 2-4 were used for all experiments (23). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of HLF:\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCells were pretreated with varying doses of isoproterenol (1 nM,10 nM, 100 nM, 1\u0026micro;M), treprostinil (1nM, 10 nM, 100 nM, 1\u0026micro;M), or prostaglandin E2 (10nM, 100 nM, 1\u0026micro;M, 10 \u0026micro;M) for 30 minutes, then TGF-\u0026beta; (5 ng/ml) was added. Levels of collagen 1A1 and PAI-1 were measured at baseline and after 24 hr of TGF-\u0026beta; stimulation. Collagen 1A1 and PAI-1 are proteins associated with lung fibrosis, and accordingly, we used these proteins as markers of a fibrotic signature (23-29). Measurements were performed in the presence and absence of the IP receptor antagonist CAY10441, as well as with the G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e (\u003cem\u003eGNAS\u003c/em\u003e) or \u003cem\u003ePTGER2\u003c/em\u003e (EP2 receptor) siRNA. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting:\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter treatment, the cell monolayers were collected by adding 0.1% perchloric acid. Cells were pelleted, lysed, and incubated with NuPage\u0026reg; reducing agent and sample buffer. Proteins were separated using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with the primary antibodies described in the reagents section. Protein bands were detected using near-infrared conjugated secondary antibodies with Li-Cor Odyssey CLX, and the intensity of the protein bands was calculated using Image Studio v. 5.2 (30). \u0026nbsp; The collagen 1A1, PAI-1, SMAD3, pHSP20, and G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e were normalized to tubulin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecAMP ELISA assay:\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSub-confluent HLF (grown to 50-70% confluence) were treated for 5 min with varying concentrations of treprostinil, isoproterenol, and prostaglandin E2. Cells were lysed, and total cAMP was measured using the Applied Biosystems cAMP Screen Immunoassay System\u0026reg; (Thermo Fisher Scientific). Measurements were performed using a luminometer (BMG Labtech CLARIOstar microplate reader, Cary, NC) (31). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive cell cAMP cADDis assay:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHLF were seeded in a 4-well chamber slide (50,000 cells/chamber). HLF were then incubated with the BacMam\u0026reg; viral vector with a promoter expressing the green nucleus-targeted cAMP sensor and trichostatin A. Under live microscopy (Image Pro Plus 6.0), cells were stimulated with treprostinil (10nM) or isoproterenol (100nM), and sequential images every 2 sec were captured. Nuclei were selected prospectively, and a decrease in the fluorescence of the cADDis\u0026reg; vectors, which correlates with increasing [cAMP]\u003csub\u003eI\u0026nbsp;\u003c/sub\u003e\u003cem\u003ein vivo\u003c/em\u003e, was measured using Image J (32). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in four or more unique cell lines (Supplemental Fig. 2). GraphPad Prism software (version 10, Boston, MA) was used for data analysis. Statistical significance was determined using the Wilcoxon\u0026rsquo;s paired test or Friedman\u0026rsquo;s test for multiple comparisons (30). Nonlinear regression was used to quantify the nuclear cAMP response. Statistical significance was as follows: NS: not significant, * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001; the data presentation represents means and SEM from biological and technical replicates. \u0026nbsp;\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTreprostinil decreases TGF-\u0026beta;-induced extracellular matrix markers in HLF.\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the effects of treprostinil on TGF-\u0026beta;\u0026ndash;induced extracellular matrix protein expression in HLF derived from non-fibrosis (Fig. 1A) and fibrosis donors (Fig. 1B), we performed immunoblot analysis for collagen 1A1 (Col1A1) and plasminogen activator inhibitor-1 (PAI-1). Stimulation with TGF-\u0026beta; (5 ng/mL, 24 hours) increased Col1A1 and PAI-1 expression in HLF. Although a slight variation in baseline Col1A1 and PAI-1 expression was observed in HLF from non-fibrosis donors, substantial heterogeneity in response to TGF-\u0026beta; stimulation was seen. Despite this variation, treprostinil inhibited TGF-\u0026beta;\u0026ndash;induced expression in a dose-dependent manner, with 10 nM identified as the lowest effective concentration (Fig. 1). In HLF from donors with fibrosis, there was an increase in baseline Col1A1 but not PAI-1 compared with HLF from non-fibrosis donors. \u0026nbsp;However, the magnitude of TGF-\u0026beta;-induced Col 1A1 and PAI-1 expression was comparable, as was the inhibitory response to treprostinil. In separate experiments, we investigated whether treprostinil inhibited TGF-\u0026beta;-induced SMAD phosphorylation (pSMAD), a necessary signaling event in regulating TGF-\u0026beta;-induced cellular function. Treprostinil had little effect on TGF-\u0026beta;-induced pSMAD (Supplemental Fig. 3). The dose and time interval of TGF-\u0026beta; exposure of HLF are those experimentally determined to be physiologically and pharmacologically relevant, as previously described (23-29)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLevels of [cAMP]\u003csub\u003eI\u003c/sub\u003e induced by treprostinil, PGE\u003csub\u003e2\u003c/sub\u003e, or isoproterenol failed to predict their ability to inhibit TGF-\u0026beta;-induced extracellular matrix.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe reasoned that agonists that increase [cAMP]\u003csub\u003ei\u003c/sub\u003e by activating GPCRs coupled to G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ewould have comparable inhibitory effects on TGF-\u0026beta;-induced Col1A1 and PAI-1 expression. We compared the lowest effective inhibitory dose of treprostinil (10 nM) on TGF-\u0026beta;-induced Col1A1 and PAI-1 expression with increasing doses of isoproterenol (1 nM to 1 \u0026micro;M) and PGE\u003csub\u003e2\u003c/sub\u003e (10 nM to 10 \u0026micro;M). Isoproterenol had little inhibitory effect on TGF-\u0026beta;-induced Col1A1 and PAI-1 expression. Although PGE₂ reduced TGF-\u0026beta;-induced Col1A1 and PAI-1 expression, this inhibition was realized only at the highest dose tested (10 \u0026micro;M) (Fig. 2A). The lack of an inhibitory effect by isoproterenol, a well-known inducer of intracellular cAMP, suggests that the inhibition of TGF-\u0026beta;\u0026ndash;induced extracellular matrix protein expression is unlikely to be mediated solely by increased [cAMP]\u003csub\u003eI\u003c/sub\u003e. Comparing [cAMP]\u003csub\u003eI\u003c/sub\u003e induced by treprostinil, isoproterenol, and PGE\u003csub\u003e2\u003c/sub\u003e, all agents increased [cAMP]\u003csub\u003eI\u0026nbsp;\u003c/sub\u003e(Fig. 2B). Treprostinil and PGE\u003csub\u003e2\u003c/sub\u003e, arithmetically induced higher levels of [cAMP]\u003csub\u003eI\u003c/sub\u003e compared to isoproterenol; however, this difference was not statistically significant. Since nuclear cAMP levels can impact gene expression, we also assessed whether treprostinil and isoproterenol manifested differential effects on nuclear cAMP levels. Interestingly, isoproterenol, which has little impact on TGF-\u0026beta;-induced extracellular matrix expression, induced comparable levels of nuclear cAMP to that induced by 10 nM treprostinil (Fig. 2C). Collectively, these data suggest that the activation of GPCR differentially modulates TGF-\u0026beta;-induced Col1A1 and PAI-1 expression, and that levels of [cAMP]\u003csub\u003eI\u003c/sub\u003e and nuclear cAMP failed to predict the inhibitory effects of the agonists on TGF-\u0026beta;-induced extracellular matrix responses. \u0026nbsp;Collectively, our data suggest that the robust inhibitory impact of treprostinil on TGF-\u0026beta;-induced Col1A1 and PAI-1 expression can be mediated by activating a G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-independent signaling pathway and a G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-selective response through treprostinil\u0026rsquo;s activation of its multiple cognate GPCRs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003csub\u003e\u0026alpha;s\u003c/sub\u003e partially inhibits TGF-\u0026beta;-induced collagen 1A1 by treprostinil and PGE\u003csub\u003e2\u003c/sub\u003e. However, the inhibition of TGF-\u0026beta;-induced PAI-1 by treprostinil and PGE\u003csub\u003e2\u003c/sub\u003e in part is G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-independent.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e is required to inhibit TGF-\u0026beta;-induced collagen 1A1 and PAI-1 by GPCR agonists, G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e expression was decreased using siRNA, and the effects of agonists on inhibition of TGF-\u0026beta;-induced protein expression were assessed (Fig. 3). In these experiments, the G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003eknockdown efficiency was greater than 98%. The physiological effects of the knockdown were confirmed by the inability of treprostinil and PGE\u003csub\u003e2\u003c/sub\u003e to activate phosphorylation of HSP20, a known signaling event that is G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-, cAMP-, and protein kinase A-dependent (Fig. 3A). Inhibition of G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ereversed treprostinil inhibition of TGF-\u0026beta;-induced collagen 1A1; the reversal of PGE\u003csub\u003e2\u003c/sub\u003e inhibition was modest. G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003eknockdown had far less effect on treprostinil- or PGE\u003csub\u003e2\u003c/sub\u003e-induced inhibition of TGF-\u0026beta;-induced PAI-1 expression (Fig. 3B and C). These data suggest that treprostinil and PGE₂ differentially activate G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-coupled GPCRs and their downstream signaling pathways to inhibit TGF-\u0026beta;-induced collagen 1A1 and PAI-1 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreprostinil inhibits TGF-\u0026beta;-induced protein expression by activating the prostacyclin (IP) receptor.\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreprostinil activates several GPCRs, including the prostacyclin (IP) receptor, prostaglandin D2 receptor 1 (DP1), prostaglandin E2 receptor 2 (EP2), and the receptor 4 (EP4). To address the receptor specificity mediating treprostinil\u0026rsquo;s inhibitory effects on TGF-\u0026beta;-induced protein expression, HLF were stimulated with TGF-\u0026beta; in the presence and absence of treprostinil, soluble IP, DP1, and EP4 receptor antagonists, as well as siRNA to the gene encoding the prostaglandin E receptor 2 (PTGER2). The IP receptor antagonist (CAY1044) profoundly reversed treprostinil-mediated inhibition of TGF-\u0026beta;-induced collagen 1A1 and PAI-1 expression (supplemental Fig. 4, Fig. 4A). As expected, the IP receptor antagonist had little effect on the inhibition of TGF-\u0026beta;-induced collagen 1A1 and PAI-1 expression induced by prostaglandin E2 (Fig. 4A). After the knockdown of the EP2 receptor, the inhibition of TGF-\u0026beta;- induced collagen 1A1 by treprostinil or prostaglandin E2 was partially reversed. However, EP2 knockdown had little impact on treprostinil, and prostaglandin E2-mediated inhibition of PAI-1 expression induced by TGF-\u0026beta; was also unaffected (Fig. 4B). Similarly, EP4 and DP1 receptor antagonists had little effect on treprostinil-mediated inhibition of PAI-1 and collagen 1A1 expression (Fig. 4C and 4D). \u0026nbsp;IP antagonism and EP2 knockdown were confirmed by measuring treprostinil- and PGE\u003csub\u003e2\u003c/sub\u003e-induced phosphorylation of HSP20, which was profoundly decreased, suggesting inhibition of the cognate receptor activation of G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e (Fig. 4E and 4F). These data suggest that the activation of the IP receptor plays a key role in inhibiting TGF-\u0026beta;-induced collagen 1A1 and PAI1 expression. In contrast, the EP2 receptor can partially block TGF-\u0026beta;-induced collagen 1A1 expression. \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite agonists inducing comparable levels of [cAMP]\u003csub\u003eI\u003c/sub\u003e or nuclear cAMP, treprostinil and PGE\u003csub\u003e2\u003c/sub\u003e, but not isoproterenol, decreased TGF-\u0026beta;-induced extracellular matrix protein. These findings support our hypothesis that G\u003csub\u003eas\u003c/sub\u003e-coupled\u003csub\u003e\u0026nbsp;\u003c/sub\u003eGPCRs selectively modulate TGF-\u0026beta;-induced collagen and PAI-1 in HLF. Treprostinil activation of the IP receptor-linked G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003esubunit robustly inhibits TGF-\u0026beta;-induced extracellular matrix production. This was specific to the IP receptor, while a modest inhibitory effect was observed with treprostinil activation of the EP2 receptor.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concept that GPCRs can induce selective cAMP responses through a spatial colocalization of their linked G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003esubunit with specific adenylyl cyclase complexes has been suggested (33-35). \u0026nbsp;Evidence suggests that overexpression of adenylyl cyclase 6 decreased collagen 1A1 synthesis and bleomycin-induced fibrosis through specific colocalization of adenylyl cyclase 6 with \u0026beta;1, \u0026beta;2 and IP receptors but not with EP2 or EP4 receptors (33-35). These data support our findings that G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e subunits can evoke differential downstream signaling pathway that modulate cellular function.\u003c/p\u003e\n\u003cp\u003eGPCR- G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003einteractions vary among different GPCRs and can modulate differential cellular functions. Notably, the EP2 receptor-coupled G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ecomplex differs from the \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR-coupled G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ecomplex regarding transmembrane domain distance and alignment. Such signaling events can be explained by differential coupling to the same G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003esubunit, potentially modulating specific TGF-\u0026beta; responses, as observed in our study (36). The dynamic spatial localization and the conformational state of the activated IP receptor coupled G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003e may also serve as an initial step in generating selective cAMP responses, activating receptor-associated independent cAMP nanodomains and ultimately contributing to the anti-fibrotic effects of treprostinil (37, 38).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dissociation among the antifibrotic function of GPCRs and the agonist-induced levels of intracellular cAMP was described by Roberts \u003cem\u003eet al\u003c/em\u003e, who suggested the possibility of nuclear cAMP compartmentalization. \u0026nbsp;However, our data did not support differential increases in nuclear cAMP levels by G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-coupled receptor agonists (39, 40).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eActivation of IPR coupled-G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003ewas the primary mechanism by which treprostinil inhibited TGF-\u0026beta;-induced collagen 1A1. However, our findings suggest this is not the sole pathway involved, particularly in inhibiting TGF-\u0026beta;-induced PAI1 expression. Therefore, exploring other G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e -independent hypotheses that may also mediate the anti-fibrotic function of treprostinil is warranted, particularly concerning the involvement of additional G\u003csub\u003e\u0026alpha;\u003c/sub\u003e protein subunits. This includes G\u003csub\u003e\u0026alpha;q\u003c/sub\u003e activation of phospholipase C, which generates inositol triphosphate and diacylglycerol, and G\u003csub\u003e\u0026alpha;12/13\u0026nbsp;\u003c/sub\u003eactivation of Rho guanine exchange factors. Additionally, the potential role of the G\u003csub\u003e\u0026beta;/\u0026gamma;\u0026nbsp;\u003c/sub\u003esubunit is particularly intriguing and warrants further investigation. G\u003csub\u003e\u0026beta;/\u0026gamma;\u0026nbsp;\u003c/sub\u003eregulates adenylyl cyclase, phospholipase C, and ion channels. G\u003csub\u003e\u0026beta;/\u0026gamma;\u0026nbsp;\u003c/sub\u003ecomplex is involved in cardiac fibroblasts activation and fibrosis (41). \u0026nbsp;Therefore, an interesting next step would be to determine if treprostinil is a biased agonist decreasing the recruitment of the G\u003csub\u003e\u0026beta;/\u0026gamma;\u0026nbsp;\u003c/sub\u003ecomplex.\u003c/p\u003e\n\u003cp\u003eThe existence of \u0026ldquo;functional selectivity\u0026rdquo; in certain GPCR/ligand pairs, characterized by an imbalance between G-protein signaling and \u0026beta;-arrestin pathways, poses an alternative hypothesis. Treprostinil and prostaglandin E2, despite both targeting the same EP2 receptor, may act as biased ligands. This bias could promote distinct conformational changes in \u0026beta;-arrestin, creating an imbalance between the \u0026beta;-arrestin and G\u003csub\u003e\u0026alpha;s\u0026nbsp;\u003c/sub\u003epathway (42, 43). Highlighting the ability of treprostinil to influence a SMAD-independent TGF-\u0026beta;-induced fibrosis pathway could offer novel therapeutic options for treating IPF (Supplemental Fig. 4). Further investigation is needed into how treprostinil can serve as a co-agonist via non-canonical GPCR pathways and through GPCR cross-talk (44, 45). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on our data, we propose that treprostinil inhibits TGF-\u0026beta;-induced extracellular matrix production through a selective cAMP response mediated by the recruitment of IP receptor\u0026ndash;linked G\u0026alpha;s. This effect is not replicated by activation of EP2-linked G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e. Additionally, treprostinil exerts anti-fibrotic effects through a G\u003csub\u003e\u0026alpha;s\u003c/sub\u003e-independent mechanism, particularly by inhibiting TGF-\u0026beta;-induced PAI-1 expression. Potentially, the anti-fibrotic action of treprostinil is mediated by activation of the IP receptor, coupled to G\u0026alpha;s, and by modulating canonical and non-canonical pathways of GPCR signaling. A better understanding of these pathways may lead to the identification of novel therapeutic targets for treating IPF and fibrosis.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003ecAMP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCyclic adenosine monophosphate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEP2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProstaglandin E2 receptor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG protein coupled receptor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHLF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman lung fibroblasts\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSP20\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeat shock protein 20\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProstacyclin receptor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIPF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdiopathic pulmonary fibrosis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePAI1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasminogen activator inhibitor 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePGE\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProstaglandin E2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePKA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein kinase A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF-\u0026beta;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Transforming growth factor beta\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: Not applicable.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: SO, BTD, GC, NK, AR, CKW, QI, RO and RAP designed the experiments. SO, BTD, GC, NK and AR performed the experiments and analyzed the data. SO, BTD, GC drafted the manuscript. CKW, QI, RO, RAP reviewed and edited manuscript. All authors approved the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDelaunay M, Osman H, Kaiser S, Diviani D. The Role of Cyclic AMP Signaling in Cardiac Fibrosis. Cells. 2019;9(1).\u003c/li\u003e\n\u003cli\u003eInsel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, et al. cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol. 2012;166(2):447-56.\u003c/li\u003e\n\u003cli\u003eLv T, Du Y, Cao N, Zhang S, Gong Y, Bai Y, et al. Proliferation in cardiac fibroblasts induced by \u0026beta;1-adrenoceptor autoantibody and the underlying mechanisms. Sci Rep. 2016;6:32430.\u003c/li\u003e\n\u003cli\u003eEsposito DB, Lanes S, Donneyong M, Holick CN, Lasky JA, Lederer D, et al. 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Treprostinil inhibits proliferation and extracellular matrix deposition by fibroblasts through cAMP activation. Sci Rep. 2018;8(1):1087.\u003c/li\u003e\n\u003cli\u003eNikam VS, Wecker G, Schermuly R, Rapp U, Szelepusa K, Seeger W, et al. Treprostinil Inhibits the Adhesion and Differentiation of Fibrocytes via the Cyclic Adenosine Monophosphate\u0026ndash;Dependent and Ras-Proximate Protein\u0026ndash;Dependent Inactivation of Extracellular Regulated Kinase. American Journal of Respiratory Cell and Molecular Biology. 2011;45(4):692-703.\u003c/li\u003e\n\u003cli\u003eYang Z, Cao G, Tan X, Orfanos S, Jude J, Barbet G, et al. Distinct mural cells and fibroblasts promote pathogenic plasma cell accumulation in idiopathic pulmonary fibrosis. Eur Respir J. 2025.\u003c/li\u003e\n\u003cli\u003eGuidi R, Xu D, Choy DF, Ramalingam TR, Lee WP, Modrusan Z, et al. Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma. Sci Transl Med. 2022;14(641):eabl8146.\u003c/li\u003e\n\u003cli\u003eXie Y, Jiang H, Zhang Q, Mehrotra S, Abel PW, Toews ML, et al. Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis. Respir Res. 2016;17(1):103.\u003c/li\u003e\n\u003cli\u003eXu E, Cao G, Yang Z, Zhang Y, Si Y, Singh K, et al. Adventitial stromal cells and myofibroblasts recruit pro- and anti-inflammatory immune cells in allergic airway inflammation. Allergy. 2023;78(11):2994-7.\u003c/li\u003e\n\u003cli\u003eYan H, Deshpande DA, Misior AM, Miles MC, Saxena H, Riemer EC, et al. Anti-mitogenic effects of \u0026beta;-agonists and PGE2 on airway smooth muscle are PKA dependent. Faseb j. 2011;25(1):389-97.\u003c/li\u003e\n\u003cli\u003eGoncharova EA, Goncharov DA, Damera G, Tliba O, Amrani Y, Panettieri RA, Jr., et al. 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Am J Respir Cell Mol Biol. 2022;66(1):96-106.\u003c/li\u003e\n\u003cli\u003eNu\u0026ntilde;ez FJ, Johnstone TB, Corpuz ML, Kazarian AG, Mohajer NN, Tliba O, et al. Glucocorticoids rapidly activate cAMP production via G(\u0026alpha;s) to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects. Faseb j. 2020;34(2):2882-95.\u003c/li\u003e\n\u003cli\u003eOstrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, Insel PA. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem. 2001;276(45):42063-9.\u003c/li\u003e\n\u003cli\u003eLiu X, Li F, Sun SQ, Thangavel M, Kaminsky J, Balazs L, et al. Fibroblast-specific expression of AC6 enhances beta-adrenergic and prostacyclin signaling and blunts bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2010;298(6):L819-29.\u003c/li\u003e\n\u003cli\u003eLiu X, Ostrom RS, Insel PA. cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts. Am J Physiol Cell Physiol. 2004;286(5):C1089-99.\u003c/li\u003e\n\u003cli\u003eQu C, Mao C, Xiao P, Shen Q, Zhong YN, Yang F, et al. Ligand recognition, unconventional activation, and G protein coupling of the prostaglandin E(2) receptor EP2 subtype. Sci Adv. 2021;7(14).\u003c/li\u003e\n\u003cli\u003eBuyanov I, Popov P. Characterizing conformational states in GPCR structures using machine learning. Sci Rep. 2024;14(1):1098.\u003c/li\u003e\n\u003cli\u003eAnton SE, Kayser C, Maiellaro I, Nemec K, M\u0026ouml;ller J, Koschinski A, et al. Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling. Cell. 2022;185(7):1130-42.e11.\u003c/li\u003e\n\u003cli\u003eRoberts MJ, Broome RE, Kent TC, Charlton SJ, Rosethorne EM. 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Proc Natl Acad Sci U S A. 2008;105(29):9988-93.\u003c/li\u003e\n\u003cli\u003eMasuho I, Kise R, Gainza P, Von Moo E, Li X, Tany R, et al. Rules and mechanisms governing G protein coupling selectivity of GPCRs. Cell Rep. 2023;42(10):113173.\u003c/li\u003e\n\u003cli\u003eCrudden C, Shibano T, Song D, Suleymanova N, Girnita A, Girnita L. Blurring Boundaries: Receptor Tyrosine Kinases as functional G Protein-Coupled Receptors. Int Rev Cell Mol Biol. 2018;339:1-40.\u003c/li\u003e\n\u003cli\u003eN\u0026uuml;rnberg B, Beer-Hammer S, Reisinger E, Leiss V. Non-canonical G protein signaling. Pharmacology \u0026amp; Therapeutics. 2024;255:108589.\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":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"treprostinil, idiopathic pulmonary fibrosis, human lung fibroblasts, cAMP signaling, prostacyclin receptor, GPCR, Gαs subunit","lastPublishedDoi":"10.21203/rs.3.rs-7321140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7321140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u0026nbsp;\u003c/strong\u003eInjury-repair responses typically induce tissue or organ scarring. Generally, cAMP-mobilizing agents inhibit the activation of fibroblasts and deposition of extracellular matrix. Some but not all cAMP-mobilizing agents inhibit fibrosis. Evidence suggests that inhaled treprostinil, a prostacyclin (IP) analog, increases intracellular cAMP levels [cAMP]\u003csub\u003eI\u003c/sub\u003e, and slows the decline in pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). \u0026nbsp;However, the molecular mechanisms by which cAMP-mobilizing agents, including treprostinil, alter the expression of matrix proteins in human lung fibroblasts (HLF) remain unclear. Unlike other G\u003csub\u003eαs\u003c/sub\u003e\u0026nbsp;-coupled receptors, we posit that the antifibrotic properties of treprostinil are driven by cAMP-mobilizing-dependent and -independent responses mediated by the IP receptor activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u0026nbsp;As a model of lung fibrosis, primary HLF derived from non-IPF and IPF donors were stimulated with TGF-β; collagen 1A1 and plasminogen activator inhibitor-1 (PAI) expression were then measured in the presence and absence of cAMP mobilizing agents. The necessity of receptor activation for inhibiting TGF-β-induced markers of fibrosis was determined by using soluble receptor inhibitors and decreasing receptor expression with siRNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u0026nbsp;Treprostinil decreased TGF-β-induced extracellular matrix production by HLF, and the magnitude of the inhibition was greater than that of other cAMP-mobilizing GPCR agonists despite these agents comparably increasing cAMP levels. There was no difference in the sensitivity and magnitude of the treprostinil inhibition in HLF derived from non-fibrosis and lung fibrosis donors. \u0026nbsp;Treprostinil inhibition of TGF-β-induced collagen 1A1and PAI-1 was mediated through the activation of the IP receptor. The activation of the EP2 receptor, in part, inhibited TGF-β-induced collagen 1A1 expression by treprostinil or prostaglandin E2. \u0026nbsp;β2 agonists had little effect on TGF-β-induced expression of collagen 1A1 and PAI-1. The inhibitory effects of treprostinil on TGF-β-induced collagen 1A1 expression required G\u003csub\u003eαs\u0026nbsp;\u003c/sub\u003eactivation, while G\u003csub\u003eαs\u0026nbsp;\u003c/sub\u003eonly partially mediated treprostinil inhibition of PAI-1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u0026nbsp;The anti-fibrotic properties of treprostinil are primarily mediated by the IP receptor, acting through both G\u003csub\u003eαs\u003c/sub\u003e-dependent and -independent pathways. \u0026nbsp;Understanding the differential effects of cAMP-mobilizing pathways on HLF fibrotic signatures can provide insight into developing novel targets to manage IPF. \u0026nbsp;\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: Insights into therapeutic targets for lung fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 10:57:45","doi":"10.21203/rs.3.rs-7321140/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-06T19:16:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T18:34:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T12:33:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T16:49:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93716751379700472092084863440093100075","date":"2025-08-20T16:52:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8883359689281133862992388166500461397","date":"2025-08-16T16:54:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274142760805918040000096091414732447827","date":"2025-08-15T13:25:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-15T13:22:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T17:58:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-14T06:25:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Respiratory Research","date":"2025-08-07T17:38:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54b07043-5417-4404-94c1-673048899722","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:11:13+00:00","versionOfRecord":{"articleIdentity":"rs-7321140","link":"https://doi.org/10.1186/s12931-025-03387-3","journal":{"identity":"respiratory-research","isVorOnly":false,"title":"Respiratory Research"},"publishedOn":"2025-11-22 15:58:12","publishedOnDateReadable":"November 22nd, 2025"},"versionCreatedAt":"2025-08-22 10:57:45","video":"","vorDoi":"10.1186/s12931-025-03387-3","vorDoiUrl":"https://doi.org/10.1186/s12931-025-03387-3","workflowStages":[]},"version":"v1","identity":"rs-7321140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7321140","identity":"rs-7321140","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}