The differential effects of cAMP mobilizing agents on inhibition of TGF-β-induced extracellular matrix and growth factor expression in human lung fibroblasts.

The differential effects of cAMP mobilizing agents on inhibition of TGF-β-induced extracellular matrix and growth factor expression in human lung fibroblasts. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 1 February 2025 V1 Latest version Share on The differential effects of cAMP mobilizing agents on inhibition of TGF-β-induced extracellular matrix and growth factor expression in human lung fibroblasts. Authors : sarah orfanos , Brian Deeney , Gaoyuan Cao , Nikhil Karmacharya , Anjani Ravi , Cindy Koziol-White , Rennolds Ostrom , and Reynold Panettieri, Jr. 0000-0003-4127-6313 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173840072.27477554/v1 386 views 233 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract not-yet-known not-yet-known not-yet-known unknown Background and purpose: Idiopathic pulmonary fibrosis (IPF), evokes scarring of the lung due to extracellular matrix deposition by fibroblasts. Evidence suggests that inhaled treprostinil slows the decline in pulmonary function in IPF. The molecular mechanisms by which cAMP mobilizing agents, alter human lung fibroblast (HLF) expression of matrix proteins remain unclear. We posit that the antifibrotic properties of treprostinil are driven by differential cAMP mobilizing responses mediated by the IP receptor. Experimental approach: As a model of lung fibrosis, TGF-β-stimulated primary HLF were used to measure collagen 1A1, PAI1, and growth factor levels in the presence and absence of cAMP mobilizing agents. The necessity of receptor activation in inhibiting TGF-β-induced fibrosis was determined using soluble receptor inhibitors and selective inhibition of receptor expression with siRNA. Key results: Treprostinil decreased TGF-β-induced extracellular matrix and growth factors production by HLF, the magnitude of the inhibition was greater than other cAMP mobilizing GPCR agonists despite comparable increases in cAMP levels. Treprostinil inhibition of TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 was mediated through the activation of the IP receptor. The EP2 receptor was partially involved in the inhibition of TGF-β-induced collagen 1A1 by treprostinil or prostaglandin E2. β2 agonists had little effect on TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 expression. The inhibitory effects on TGF-β-induced matrix expression by treprostinil required Gαs activation. Treprostinil inhibition of TGF-β-induced IGFBP3 expression also correlated with the inhibition of TGF-β-induced phosphorylation of AKT. Conclusion and implications: Understanding the differential effects of cAMP mobilizing pathways on HLF fibrotic signatures can provide insight into developing novel targets to manage IPF. The differential effects of cAMP mobilizing agents on inhibition of TGF-β-induced extracellular matrix and growth factor expression in human lung fibroblasts. Sarah Orfanos 1 , Brian T. Deeney 1 , Gaoyuan Cao 1 , Nikhil Karmacharya 1 , Anjani Ravi 1 , Cynthia J. Koziol-White 1 , Rennolds S. Ostrom 2 , Reynold A. Panettieri, Jr. 1 1: Department of Medicine, Rutgers Institute for Translational Medicine and Science, Robert Wood Johnson School of Medicine, New Brunswick, NJ, USA. 2: Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, USA. Corresponding author: Reynold A. Panettieri, Jr, M.D. (732)235-6404 [email protected] Author contributions: SO, BTD, GC, NK, AR, CKW, 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, RO, RAP reviewed and edited manuscript. All authors approved the manuscript. Abstract: Background and purpose: Idiopathic pulmonary fibrosis (IPF), evokes scarring of the lung due to extracellular matrix deposition by fibroblasts. Evidence suggests that inhaled treprostinil slows the decline in pulmonary function in IPF. The molecular mechanisms by which cAMP mobilizing agents, alter human lung fibroblast (HLF) expression of matrix proteins remain unclear. We posit that the antifibrotic properties of treprostinil are driven by differential cAMP mobilizing responses mediated by the IP receptor. Experimental approach: As a model of lung fibrosis, TGF-β-stimulated primary HLF were used to measure collagen 1A1, PAI1, and growth factor levels in the presence and absence of cAMP mobilizing agents. The necessity of receptor activation in inhibiting TGF-β-induced fibrosis was determined using soluble receptor inhibitors and selective inhibition of receptor expression with siRNA. Key results: Treprostinil decreased TGF-β-induced extracellular matrix and growth factors production by HLF, the magnitude of the inhibition was greater than other cAMP mobilizing GPCR agonists despite comparable increases in cAMP levels. Treprostinil inhibition of TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 was mediated through the activation of the IP receptor. The EP2 receptor was partially involved in the inhibition of TGF-β-induced collagen 1A1 by treprostinil or prostaglandin E2. β2 agonists had little effect on TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 expression. The inhibitory effects on TGF-β-induced matrix expression by treprostinil required G αs activation. Treprostinil inhibition of TGF-β-induced IGFBP3 expression also correlated with the inhibition of TGF-β-induced phosphorylation of AKT. Conclusion and implications: Understanding the differential effects of cAMP mobilizing pathways on HLF fibrotic signatures can provide insight into developing novel targets to manage IPF. Keywords: treprostinil, idiopathic pulmonary fibrosis, human lung fibroblasts, cAMP signaling, prostacyclin receptor, GPCR, G αs subunit. Abbreviations: Akt Protein kinase B cAMP Cyclic adenosine monophosphate EP2 Prostaglandin E2 receptor 2 GPCR G protein coupled receptor HLF Human lung fibroblasts HSP20 Heat shock protein 20 IGFBP3 Insulin-like growth factor-binding protein 3 IP Prostacyclin receptor IPF Idiopathic pulmonary fibrosis MEK Mitogen-activated protein kinase enzyme PAI1 Plasminogen activator inhibitor 1 not-yet-known not-yet-known not-yet-known unknown PGE2 Prostaglandin E2 PI3K8 Phosphoinositide 3-kinase PKA Protein kinase A TGF-β Transforming growth factor beta What is already known? Treprostinil has potential as an antifibrotic agent in idiopathic pulmonary fibrosis. What does this study add? Treprostinil inhibits growth factor production including IGFBP3 in human lung fibroblasts Treprostinil’s antifibrotic function on collagen 1A1, PAI1 and IGFBP3 is specific to the IP receptor activation of the G αs subunit with minimal involvement of the EP2 receptor. Not all GPCRs coupled to G αs that increase cAMP levels inhibit TGF-β mediated fibrotic signals in HLF. The magnitude of the cAMP mobilizing inhibitory effects on TGF-β induced growth factors is associated with the inhibition of pAKT stimulated by TGF-β. What is the clinical significance? Understanding the differential mechanisms by which ligand/GPCR/ G αs alter cAMP-dependent inhibitory responses in matrix deposition can facilitate the development of new IPF and fibrotic disease therapies. Introduction: Idiopathic pulmonary fibrosis (IPF) has a prevalence of 58.7 per 100,000 persons in adults over 50 years old to 494.5 per 100,000 persons in patients 65 and older in the US (1, 2). Despite recent efforts to advance the field, the prognosis remains poor, with a median survival of 3.8 years in the same study population of patients 65 and older. Fibrotic diseases in many organs, including the lung, can be partly due to aberrant injury-repair response driven by TGF-β effects on mesenchymal-derived cells such as fibroblasts and myofibroblasts (3). The advent of specific IPF therapies, including nintedanib and pirfenidone, was welcomed however, despite slowing the decline in the forced vital capacity (FVC), as of today, no benefit in survival has been realized (4, 5). Only lung transplants have shown efficacy in prolonging life, 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 WHO group III pulmonary hypertension secondary to IPF (6, 7). Motivated by these findings, ongoing clinical trials will address the efficacy of treprostinil in a larger population of 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 (8). 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. Whether the anti-fibrotic effects of treprostinil are cAMP-dependent and whether specific receptors mediate treprostinil effects remain unclear (9, 10). Understanding how treprostinil mediates its anti-fibrotic effects can provide insight into developing novel targeted therapies for fibrosis. Treprostinil’s mechanism of action may lie in a more nuanced explanation, potentially involving differential activation of cAMP signaling pathways to cell proliferation, growth factor secretion, extracellular matrix production, and the differentiation of fibroblasts into myofibroblasts. Differential activation of cAMP-dependent signaling pathways can potentially address why treprostinil generates anti-fibrotic signals compared to other G αs coupled GPCRs. Some G αs coupled GPCRs co-localize with specific adenylyl cyclase isoforms (11-13). Adenylyl cyclase then forms a complex with surrounding effector molecules, including the tetrameric protein kinase A unit (PKA), 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 (14-19). In this study, we posit that the anti-fibrotic effects observed with treprostinil, and other cAMP mobilizing agents are primarily mediated through activation of receptors coupled to the G αs subunit and can be attributed, at least in part, to selective downstream cAMP signaling rather than solely relying on the magnitude of agonist-induced intracellular cAMP levels. Materials and methods: Materials: All 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 purchased from Montana Molecular (Bozeman, MT). Trichostatin A was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for detection of collagen 1A1, IGFBP3, phospho-Akt (Ser 473), total Akt, 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 purchased 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 purchased from Invitrogen, Thermo Fisher Scientific (Waltham, MA). GNAS silencer siRNA was purchased from Dharmacon (Lafayette, CO). Treprostinil and prostaglandin E2 were purchased from Cayman chemical (Ann Arbor, MI), isoproterenol from Sigma Aldrich (St. Louis, MO) and TGF-β from R&D Systems (Minneapolis, MN). The MEK inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Selleckchem (Radnor, PA). The IP antagonist CAY10441 was purchased from Cayman Chemical (Ann Arbor, MI). Human Growth Factor Antibody Array was purchased from Abcam (Waltham, MA). 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 have deemed them exempt from IRB approval. All fibroblast cells were isolated from these tissues. All vessels and bronchioles were removed from the tissue, and the tissue was cut into 1x1mm or smaller pieces. Tissue was transferred to 30ml 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 20ml 1X PBS was added and the solution was vortexed for 1min then filtered through a 70um cell strainer. The flow-through was centrifuged at 1500 rpm for 5min, and supernatant discarded. Cells were washed with 50ml 1X PBS and centrifuged. Cells were seeded into T75 flasks and culture in F-12 medium with 10% FBS for 7-10days before passage. To assess the purity of the HLF culture, we performed vimentin and S100A4 immunohistochemistry of lung tissue slices. HLF were S100A4 (+) and vimentin (+) whereas smooth muscle cells were vimentin (-) and S100A4 (-). Subsequently, immunofluorescence staining of the HLF culture revealed that over 95% of the cells were S100A4 (+), vimentin (+), SMA (-) or weak (+), and EpCAM (-) (supplemental figure 1). Cells at passage numbers 2-4 were used for all experiments. Treatment of HLF: Cells were pretreated with varying doses of isoproterenol (10nM, 100 nM, 1µM), treprostinil (1nM, 10nM, 100 nM, 1µM) or prostaglandin E2 (10nM, 100 nM, 1µM) for 30 minutes then TGF-β (5 ng/ml) was added. Levels of collagen 1A1, IGFBP3 and PAI-1 were measured at baseline and after 24 hr of TGF-β stimulation. Levels of Akt and SMAD3 phosphorylation were measured at baseline and after 1 hr of TGF-β stimulation. Measurements were done in the presence and absence of the IP receptor antagonist CAY10441, the GNAS siRNA or PTGER2 (EP2 receptor) siRNA. Immunoblotting: After treatment of HLF, the cell monolayers were collected after adding 0.1% final concentration of 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. The collagen 1A1, IGFBP3, PAI-1, SMAD3, G αs were normalized to tubulin, and the phosphorylation of Akt and HSP20 were normalized to total Akt and HSP20, respectively. Growth Factor Antibody array: Cells were grown in 6 well plates until confluent. Cells were serum deprived, then incubated with treprostinil (10nM, 30 min) before TGF-β was added (5ng/mL, overnight). The media was collected at 24 hr, and membranes were placed in provided 8-well plate and blocked. After blocking, 2mL of collected sample was incubated with the blots overnight. The samples were then aspirated, and blots were washed. The blots were sequentially incubated in 1x Biotin-Conjugated Anti-Cytokines, then 1x HRP-Conjugated, and finally Detection Buffer C and D. Imaging was done using chemiluminescence detection method exposing for 5 sec, 10 sec, 30 sec, 45 sec, 1 min and 2 min. cAMP ELISA assay: Sub-confluent HLF (grown to 50-70% confluence) were treated for 5 min with different doses of treprostinil, isoproterenol and prostaglandin E2. Cells were lysed and total cAMP was measured using the Applied Biosystems cAMP screen immunoassay system (ThermoFisher Scientific). Measurements were done on a luminometer (BMG Labtech CLARIOstar microplate reader, Cary, NC). 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 taken. Nuclei were selected preemptively, and a decrease in the fluorescence of the cADDis vectors in the sequential images was measured using Image J. Statistical analysis: All experiments were performed in 4 or more unique cell lines (supplemental fig. 2). GraphPad Prism software (v10, Boston, MA) was used for analysis of data. Normality was assessed using the Shapiro-Wilk normality test. Statistical significance was determined using the Student’s paired two-tail t-test or an analysis of variance (ANOVA) for multiple comparisons. Nonlinear regression was used to quantify the nuclear cAMP response. Statistical significance was as follow: NS: not significant, * p<0.05, ** p<0.01, *** p<0.001. Data represented in figures are in mean and SEM. Results: Treprostinil decreases TGF-β induced extracellular matrix markers and insulin-like growth factor-binding protein 3 (IGFBP3) in HLF. To determine the effects of treprostinil on TGF-β stimulated expression of growth factors, we stimulated HLF with TGF-β (5 ng/ml, 24hr) with and without treprostinil (10nM, 30 min pre-treatment). Using a protein array, treprostinil selectively decreased TGF-β-induced IGFBP3 (supplemental fig. 3). Interestingly, IGFBP3 levels have been shown to be increased in fibroblasts isolated from IPF lungs (20). Next, using immunoblot analysis, we examined whether treprostinil inhibited TGF-β-induced extracellular matrix proteins associated with IPF, including collagen 1A1 (Col1A1), plasminogen activator inhibitor-1 (PAI-1) and IGFBP3. TGF-β (5 ng/ml, 24hr) increased Col1A1, PAI-1 and IGFBP3 expression in HLF (Fig. 1A), while treprostinil inhibited TGF-β-induced Col1A1, PAI-1, and IGFBP3 (Fig. 1B). Levels of [cAMP] i induced by treprostinil, PGE 2 or isoproterenol failed to predict their ability to inhibit TGF-β induced extracellular matrix and growth factor expression. We reasoned that agonists that increase [cAMP] i by activating GPCRs coupled to G αs would have comparable inhibitory effects on TGF-β-induced extracellular matrix and growth factor expression. As shown in Fig 2A, treprostinil, PGE 2 , and isoproterenol increased [cAMP] i ; however, treprostinil and PGE 2 were more potent as compared to isoproterenol. Comparable levels of [cAMP] i were generated by 10nM treprostinil and PGE 2 , and 100nM isoproterenol. Using these concentrations, we next examined the inhibitory effects of agonists on TGF-β-induced extracellular matrix and growth factor expression. Although all agonists induced similar levels of [cAMP] i , isoproterenol had little impact on TGF-β-induced Col1A1, PAI-1 and IGFBP3 expression in HLF. In contrast, treprostinil had a marked inhibitory response, and PGE 2 had a modest effect on PAI-1, Col1A1 and IGFBP3 expression (Fig. 2B). Since nuclear cAMP levels can impact on gene expression, we assessed whether treprostinil and isoproterenol manifested differential effects on nuclear levels on cAMP. Interestingly, 100nM isoproterenol, which had little impact on TGF-β-induced protein expression, exhibited higher levels of nuclear cAMP than 10nM treprostinil (supplemental fig 4). Collectively, these data suggest that activation of GPCR coupled to G αs differentially modulates TGF-β-induced extracellular matrix and growth factor expression, and that levels of [cAMP] i and nuclear cAMP fail to predict the inhibitory effects of the agonists on TGF-β-induced responses. G αs mediates the inhibition of TGF-β-induced collagen 1A1 by treprostinil and prostaglandin E2. However, the inhibition of TGF-β-induced PAI1 and IGFBP3 by treprostinil is partially G αs -dependent, while that of PGE 2 is G αs -independent. To determine whether G αs is required to inhibit TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 by GPCR agonists, G αs expression was decreased using siRNA, and the effects of agonists on modulation of TGF-β-induced protein expression was determined. After inhibiting expression of G αs , inhibition of TGF-β-induced collagen 1A1 by treprostinil and PGE 2 was markedly reversed. G αs knock-down also attenuated treprostinil-mediated inhibition of TGF-β-induced expression of IGFBP3 and, to a lesser extent, PAI-1. Following G αs knock-down, inhibition of TGF-β-induced PAI-1 and IGFBP3 by treprostinil was reduced to levels comparable to those seen with PGE 2 . G αs knock-down had little effect on TGF-β-induced PAI-1 and IGFBP3 inhibition by PGE 2 (Fig 3B and C). In these experiments, the G αs knockdown efficiency was greater than 88%, and the physiological effects of the knockdown were confirmed by the inability of treprostinil and PGE 2 to activate protein kinase A to phosphorylate HSP20, a process that is G αs - and cAMP-dependent (Fig 3A). These data suggest that treprostinil and PGE 2 differentially activate G αs -coupled GPCRs and their downstream signaling pathways to inhibit TGF-β-induced protein 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/or the receptor 4 (EP4). To address the receptor specificity mediating treprostinil inhibitory effects on TGF-β-induced protein expression, HLF were stimulated with TGF-β in the presence and absence of soluble IP, DP1, and EP4 receptor antagonists before treprostinil stimulation. The IP receptor antagonist (CAY1044) profoundly reversed treprostinil-mediated inhibition of TGF-β-induced collagen 1A1, PAI-1, and IGFBP3 expression (supplemental Fig. 5, Fig 4A). As expected, due to its high specificity for the IP receptor, the IP receptor antagonist had little effect on the inhibition of TGF-β-induced collagen 1A1, PAI1, and IGFBP3 expression induced by prostaglandin E2 (Fig 4A). After knocking down the EP2 receptor, the inhibition of TGF-β induced collagen 1A1 by both treprostinil and prostaglandin E2 was partially reversed. However, EP2 knockdown had little impact on treprostinil and prostaglandin E2-mediated inhibition of PAI-1 and IGFBP3 expression induced by TGF-β (Fig 4B). EP4 and DP1 receptor antagonists also had little effect on treprostinil-mediated inhibition of PAI1 and collagen 1A1 expression (Fig. 4C). IP antagonism and EP2 knock-down were confirmed by measuring treprostinil- and PGE 2 -induced phosphorylation of HSP20 that was profoundly decreased, suggesting inhibition of the cognate receptor activation of G αs (Fig4D). These data suggest that activation of the IP receptor plays a key role in inhibiting TGF-β-induced collagen 1A1, PAI1, and IGFBP3 expression. In contrast, the EP2 receptor can solely partially block TGF-β-induced collagen 1A1 expression. Treprostinil inhibits TGF-β-induced phosphorylation of Akt and expression of IGFBP3 in an IP receptor-dependent and SMAD-independent manner. To further explore the cytosolic mechanisms by which cAMP mobilizing agents inhibit TGF-β-induced protein expression, we investigated whether activation of G αs -coupled GPCRs inhibits TGF-β-induced SMAD and PI3K activation. In pulmonary fibrosis, SMAD and PI3K activation reportedly are required to mediate TGF-β effects in fibrosis (21) (22). Treprostinil, however, had little impact on TGF-β -induced Smad3 phosphorylation (Fig 5A). To address whether treprostinil modulates TGF-β-induced PI3K, we measured phosphorylation of Akt, a substrate of PI3K. HLF were treated with TGF-β (5ng/ml, 1hr) in the presence and absence of treprostinil (10nM, 30 min pre-treatment) or PGE 2 .(10nM, 30 min pre-treatment). Treprostinil decreased TGF-β-induced phosphorylation of Akt to a greater extent than that of PGE 2 (Fig. 5A). The inhibition of TGF-β-induced Akt phosphorylation by treprostinil was G αs -dependent since G αs knockdown reversed the inhibitory effects of treprostinil on TGF-β-induced AKT phosphorylation (Fig 5B). Interestingly, the inhibitory effects of PGE2 on TGF-β-induced Akt phosphorylation were unaffected by G αs - knockdown (Fig. 5B). Moreover, IP receptor antagonists completely reversed treprostinil-mediated inhibition of TGF-β-induced Akt phosphorylation whereas EP2 knockdown had little impact (Fig 5C). These data suggest that IP coupled to G αs decreases TGF-β induced phosphorylation of Akt by activating IP receptor. In parallel, we also addressed whether PI3K modulates TGF-β induced pAkt. HLF were pretreated with LY 294002 (a PI3 Kinase p110 subunit inhibitor) and measured TGF-β-induced phosphorylation of Akt (supplemental Fig 6). Inhibition of PI3K abrogated TGF-β-induced phosphorylation of Akt dose-dependently and selectively inhibited TGF-β-induced IGFBP3 expression. At the same time, TGF-β-induced col1A1 and PAI-1 were unaffected (Fig. 5D). Collectively, these data suggest that treprostinil activates IP-coupled G αs but not EP2 to decrease TGF-β-induced phosphorylation of Akt and reduce IGFBP3 levels. Discussion: Despite agonists inducing comparable levels of [cAMP] i or nuclear cAMP, treprostinil and to a lesser extent PGE 2 , but not isoproterenol, decreased TGF-β-induced extracellular matrix protein and growth factor expression. These findings support the hypothesis that G as -coupled GPCRs selectively modulate TGF-β-induced collagen, PAI-1 and IGFBP3 in HLF. The IP receptor linked G αs subunit played a major role in mediating the inhibitory effects of treprostinil on TGF-β-induced extracellular matrix production and growth factors expression. This was specific to the IP receptor and to a modest extent with activation of the EP2 receptor that inhibited TGF-β-induced collagen 1A1 but not PAI1 or IGFBP3 expression. 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 (23-25). These investigators found that overexpression of adenylyl cyclase 6 decreased collagen 1A1 synthesis and bleomycin-induced fibrosis. As well as demonstrated a specific colocalization of this adenylyl cyclase isoform with β1, β2 and IP receptors but not with EP2 or EP4 receptors. This is concordant with our findings that G αs subunits lead to differential downstream pathway depending on their coupled GPCR. Evidence suggests that the GPCR- G αs interactions vary among different GPCRs. 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 evoking differential effects on TGF-β responses, as observed in our study (26). 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 (RAINS) and ultimately contributing to the anti-fibrotic effects of treprostinil (27, 28). 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 (29, 30). Activation of IPR coupled-G αs was the primary mechanism by which treprostinil inhibited TGF-β-induced collagen 1A1 and IGFBP3. 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. Notably, G β/γ regulates adenylyl cyclase, phospholipase C, and ion channels, as well as the PI3 kinase and Akt/ERK pathways. G β/γ complex is involved in cardiac fibroblasts activation and fibrosis (31). 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 (32, 33). The functional selectivity of the treprostinil IPR binding towards G αs , along with reduced recruitment of Gβ/γ, is particularly intriguing in the context of the PI3K/Akt pathway. Our data suggests that treprostinil inhibits Akt phosphorylation in a manner dependent on the IPR and G αs . G αs can suppress the PI3K/Akt pathway, while G β/γ is an activator (34, 35). Treprostinil inhibition of the IGFBP3/Akt axis, a pathway that has been shown to be implicated in cancer cell growth, may be due to selective G αs recruitment over G β/γ (36). Our findings suggest that treprostinil targets the PI3K/Akt/mTOR pathway, which plays a significant role in SMAD-independent TGF-β-induced fibrosis and which is a key focus in cancer therapy (37-40). Highlighting the ability of treprostinil to influence a SMAD-independent TGF-β-induced fibrosis pathway could offer novel therapeutic options for treating IPF. Further investigation is needed into how treprostinil can serve as a co-agonist via non-canonical GPCR pathways and through GPCR cross-talk, particularly concerning growth factors receptors and receptor tyrosine kinases (41, 42). Conclusion: Based on our data, we posit that the pathway by which treprostinil inhibits TGF-β-induced extracellular matrix and growth factor production requires a selective cAMP response initiated by the recruitment of IP receptor-linked G αs . This effect is particularly significant for PAI1 and IGFBP3 and is not observed when EP2-linked G αs is activated (Fig. 6). Potentially, the anti-fibrotic action of treprostinil is mediated by activation of the IP receptor-coupled to G αs and by the interplay of canonical and non-canonical pathways of GPCR signaling. A better understanding of these pathways may identify novel therapeutic targets to treat IPF. References: 1. 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Pharmacology & Therapeutics 2024; 255: 108589. Data and materials availability: All data to evaluate conclusions in the paper are present in the paper or the supplementary materials. All materials in the manuscript are available from the corresponding author. Figure legends: Figure 1: Treprostinil inhibits TGF-β-induced collagen 1A1, PAI-1 and IGFBP3 expression in HLF. (A): Measurement of collagen 1A1 (n=5); PAI-1 (n=4) and IGFBP3 (n=6) levels after 24 hr treatment with TGF-β (5 ng/ml) by immunoblot analysis. (B): Measurement of collagen 1A1 (n=5), PAI-1 (n=4) and IGFBP3 (n=6) levels in TGF-β (5 ng/ml for 24 hr) stimulated HLF with and without treprostinil pretreatment (30 min, 1 nM-1 µM) by immunoblot analysis. Data are represented as mean ± SEM of biological replicates. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Figure 2: Measurement of intracellular cAMP levels induced by GPCR agonists: treprostinil, isoproterenol and prostaglandin E2. Comparison of the inhibitory effects of these agonists on TGF-β-induced expression of collagen 1A1, PAI-1 and IGFBP3 at doses that elicit comparable intracellular cAMP levels. (A): 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) (n=6) or prostaglandin E2 (5 min, 10 nM – 1 µM) (n=5). Treprostinil (10 nM) induces comparative levels of intracellular cAMP as isoproterenol (100 nM) and prostaglandin E2 (10 nM). (B): Comparison of the inhibitory action of treprostinil (30 min preincubation, 10 nM) and isoproterenol (30 min preincubation, 100 nM) and prostaglandin E2 (30 min preincubation, 10 nM) on TGF-β-induced (5 ng/ml, 24 hr) collagen 1A1 (n=6), PAI-1 (n=4) and IGFBP3 (n=4-6) expression as assessed by immunoblotting. Data are represented as mean ± SEM from biological replicates. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Figure 3: Measurement of treprostinil and prostaglandin E2 inhibition of TGF-β induced collagen 1A1, PAI-1 and IGFBP3 expression in a G αs HLF knock-down model. (A): Measurement of residual G αs expression in a G αs HLF knockdown model (n=9). G αs HLF knockdown inhibits HSP20 phosphorylation (a known G αs -dependent phosphorylation event) by treprostinil and prostaglandin E2 (10 nM, 1 hr) in HLF (n=4). (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, PAI1 and IGFBP3 expression in HLF and after G αs HLF knockdown (n=7-9). (C): Representative immunoblot showing that treprostinil and prostaglandin E2 fail to phosphorylate HSP20 or reverse TGF-β-induced collagen 1A1, PAI-1 and IGFBP3 expression in G αs HLF knockdown. Data are represented as mean ± SEM from biological replicates. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Figure 4 : Effect of treprostinil and prostaglandin E2 on TGF-β-induced collagen 1A1, PAI-1 and IGFBP3 expression in HLF models with IPR blockade, EP2R knockdown, DP1R blockade, and EP4R blockade. (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, PAI1 and IGFBP3 expression in HLF with and without IPR blockade (cay10441, pre-treatment 30 min, 1 µM) (n=4-6). (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, PAI1 and IGFBP3 expression in a EP2R HLF knockdown model (n=5-8). (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 and in HLF with and without DP1R (Laropiprant) and EP4R (ONOAE3208) blockade (pre-treatment 30 min, 1 nM to 100 nM) (n=3-5). (D): IPR blockade and EP2R HLF knockdown models, inhibit HSP20 phosphorylation by treprostinil and prostaglandin E2 (30 min, 10 nM). Representative immunoblot showing that IPR blockade reverses treprostinil inhibition of TGF-β-induced collagen 1A1, PAI-1 and IGFBP3 expression. In contrast, the EP2R HLF knockdown model reverses treprostinil and prostaglandin E2 inhibition of TGF-β-induced collagen 1A1. Data are represented as mean ± SEM in biological replicates. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Figure 5: Measurement of treprostinil inhibition of TGF-β-induced phosphorylation of Akt via the IP/ G αs , and its effect on TGF-β- induced IGFBP3 expression. (A): Measurement of TGF-β-induced (5 ng/ml, 1 hr) phosphorylation of SMAD3 and Akt in HLF preincubated with treprostinil or prostaglandin E2 (30 min, 10 nM) (n=4-5). (B): Measurement of TGF-β-induced (5 ng/ml, 1 hr) phosphorylation of Akt in non-targeted and G αs HLF knockdown model preincubated with treprostinil or prostaglandin E2 (30 min, 10 nM) (n=4). (C): Measurement of TGF-β-induced (5 ng/ml, 1 hr) phosphorylation of Akt in IPR blockade (cay10441, pre-treatment 30 min, 1 µM) and after EP2R knockdown preincubated with treprostinil or prostaglandin E2 (30 min, 10 nM) (n=4-5). (D): Measurement of TGF-β-induced (5 ng/ml, 1 hr) collagen 1A1, PAI-1 and IGFBP3 expression in HLF pretreated with LY294002 (a pan-PI3K p110 inhibitor) (30 min, 100 nM-10 µM). Data are represented as mean ± SEM in biological replicates. * p<0.05, ** p<0.01, *** p<0.001 Figure 6: A model of treprostinil inhibition of TGF-β-induced collagen 1A1, PAI-1 and IGFBP3 expression. Treprostinil recruits both the IPR/ G αs and EP2R/ G αs to decrease TGF-β-induced collagen 1A1 expression. The IPR/ G αs recruitment by treprostinil increases cAMP levels that are associated with reduced TGF-β-induced PAI-1 levels and decreased Akt phosphorylation, which in turn lowers IGFBP3 expression. In contrast, activation of EP2R/ G αs has little effect on TGF-β-induced PAI-1 levels and Akt phosphorylation. Supplemental figure 1: Immunohistochemistry of lung slices and immunofluorescence of HLF culture A: Immunohistochemistry of lung slice (20X) demonstrating vimentin positive fibroblasts ( * ) and vimentin negative airway smooth muscle cells (SM) B: Immunohistochemistry of lung slice (20X) demonstrating S100A4 positive fibroblasts ( * ) and S100A4 negative airway smooth muscle cells (SM) C: Immunofluorescence of HLF culture (Vimentin: green, SMA: red) D: Immunofluorescence of HLF culture (S100A4: green, SMA: red) Supplemental figure 2 : Demographic donor data of HLF cell lines that were studied. Supplemental figure 3 : Screening of 41 growth factors using the growth factor antibody array The image shows a significant decrease in TGF-β- induced IGFBP3 expression (red rectangle), quantified as relative light unit (RLU). Data are represented as mean ± SEM. *** p<0.001 Supplemental figure 4: Real-time nuclear cAMP production with treprostinil or isoproterenol acute treatment of HLF. Comparison of nuclear cAMP production induced by treprostinil (10 nM) or isoproterenol (100 nM) (n=3-4) over the course of 240 seconds was assessed by cADDIS assay. Data is presented as nonlinear regression and the product of the constant K*plateau for each dose. Supplemental figure 5: Dose response of IPR blocker on treprostinil phosphorylation of HSP20 HLF were pretreated with an IPR blocker (CAY10441 – 30 min, 10 nM-10 µM), treated with treprostinil (30 min, 10 nM), then stimulated with TGF-β (5 ng/ml, 1 hr) and phosphorylation of HSP20 was assessed. Supplemental figure 6: Dose response of LY294002 (a pan-PI3K p110 inhibitor) on TGF-β-induced Akt phosphorylation. HLF were pretreated with LY294002 (30 min, 100 nM- 10 µM), then stimulated with TGF-β (5ng/ml, 1 hr). Phosphorylation of Akt was assessed. Supplementary Material File (figures bjp final (1).pdf) Download 1.32 MB Information & Authors Information Version history V1 Version 1 01 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords gpcr intracellular signaling respiratory pharmacology Authors Affiliations sarah orfanos Rutgers Robert Wood Johnson Medical School View all articles by this author Brian Deeney Rutgers Biomedical and Health Sciences View all articles by this author Gaoyuan Cao Rutgers Biomedical and Health Sciences View all articles by this author Nikhil Karmacharya Rutgers Biomedical and Health Sciences View all articles by this author Anjani Ravi Rutgers Biomedical and Health Sciences View all articles by this author Cindy Koziol-White Rutgers The State University of New Jersey View all articles by this author Rennolds Ostrom Chapman University View all articles by this author Reynold Panettieri, Jr. 0000-0003-4127-6313 [email protected] Rutgers The State University of New Jersey View all articles by this author Metrics & Citations Metrics Article Usage 386 views 233 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation sarah orfanos, Brian Deeney, Gaoyuan Cao, et al. 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