Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF.

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Transforming growth factor beta (TGF-β) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis and other fibrotic lung diseases. Canonical intracellular TGF-β signaling is hallmarked by phosphorylation, nuclear localization, and chromatin binding of SMAD proteins. Our lab has previously demonstrated that stimulation of the proton sensing G-protein coupled receptor (GPCR) GPR68 (OGR1) with the small molecule Ogerin inhibits and partially reverses TGF-β induced myofibroblast differentiation. Additionally, we have highlighted evidence that Ogerin induces Gα s signaling pathways. We thus sought to determine the intersection of these signaling cascades with TGF-β signaling to elucidate the mechanisms through which Ogerin inhibits myofibroblast differentiation. Methods: Primary human lung fibroblasts (PHLFs) derived from the lung tissue of healthy donors and donors with IPF were stimulated with 1ng/mL TGF-β and/or 150μM Ogerin for 40 minutes and 24 hours. Western Blots for total and phosphorylated SMAD proteins were performed on whole cell lysates and subcellular fractionation lysates. A SMAD reporter cell line (SB10) was also utilized to evaluate SMAD mediated gene transcription. SB10 cells, kindly provided by Dr. Collynn Woeller, contain a luciferase which is controlled by upstream SMAD binding elements. SB10 cells were treated with TGF-β and/or Ogerin for 24 hours and luciferase activity was measured using a luminometer. To determine whether Gα s -PKA mediated CREB phosphorylation was necessary for Ogerin to inhibit TGF-β signaling, PHLFs and SB10 cells were pretreated with the PKA inhibitors H-89 (30μM) or the myristoylated PKA inhibitory peptide 14-22 (10μM) and myofibroblast differentiation, SMAD phosphorylation, and SMAD luciferase activity were assessed as above. Results: Ogerin partially inhibited TGF-β induced pSMAD phosphorylation but did not substantially inhibit SMAD nuclear localization or chromatin binding at 40 minute or 24-hour timepoints. However, Ogerin significantly inhibited TGF-β induced SMAD gene transcription in a dose dependent manner in SB10 cells. Pre-treatment with either of the PKA inhibitors did not attenuate the ability of Ogerin to inhibit myofibroblast differentiation or inhibit transcription in the SB10 SMAD reporter cell line. Conclusions: These data suggest that Ogerin inhibits myofibroblast differentiation via inhibition of SMAD mediated gene transcription. Additionally, although Ogerin does induce of PKA mediated CREB phosphorylation, inhibition of PKA does not attenuate the ability of Ogerin to inhibit myofibroblast differentiation. Therefore, we hypothesize that is inhibiting the induction, nuclear localization, and/or association of pSMADs and transcriptional co-factors.

Rationale: Respiratory diseases are leading causes of mortality worldwide. Integral to all tissue repair is the maintenance of pH homeostasis. Within the interstitium of the lung, pH may decline in the setting of infection, tissue injury or ischemia and fibrosis. In animal models of lung fibrosis, the pH of lung tissue drops as low as 6.5 in fibrotic lesions. However, it is largely unknown how alterations in interstitial pH may contribute to disease pathology. Our lab is interested in the mechanisms by which fibroblasts sense and respond to changes in extracellular pH. Four G-protein coupled receptors (GPCRs) have been identified as pH sensors: G2A, GPR4, GPR65 (T-cell Death Associated Gene 8 (TDAG8)) and GPR68 (Ovarian Cancer Gene 1 (OGR1)). Lung fibroblasts express all of these receptors, though little is known about their function(s). We have recently generated data suggesting that activation of GPR65 by the exogenous small molecule ligand BTB inhibits TGF-β induced myofibroblast differentiation. Our data suggest activation of TDAG8 with BTB blocks myofibroblast differentiation via disruption of the TGF-β induced ROCK/Rho signaling pathway. We therefore hypothesize that small molecule activators of TDAG8 may represent novel anti-fibrotic therapeutics. Methods: Primary human lung fibroblasts, isolated from patients with and without pulmonary fibrosis were treated with TGFβ (1 ng/mL) and/or BTB (50 μM). Protein was harvested at the indicated timepoints to assess markers of myofibroblast differentiation (alpha smooth muscle actin (αSMA)) and extracellular matrix production (fibronectin and collagen 1). To examine the potential mechanism(s) by which BTB inhibits myofibroblast differentiation, we measured expression of proteins in the ROCK/Rho pathway including the key effectors of RhoA (ROCK1, mDia and profilin) and performed a RhoA activation assay. RhoA activity was assessed by incubating protein lysates with rhotekin-Rho binding domain coated beads to selectively isolate active GTP-bound RhoA which was then subsequently measured via Western blot. Results: Fibroblasts treated with BTB exhibited decreased baseline protein expression of collagen 1 and αSMA. BTB also blocked TGF-β induced αSMA, Collagen 1 and fibronectin protein expression. BTB inhibited ROCK1 protein expression and RhoA activity levels. Interrogation of other downstream effectors of RhoA revealed that BTB also decreased profilin protein, an important mediator of intracellular actin dynamics previously unstudied in relation to myofibroblast differentiation. Conclusions: Our data demonstrate that BTB blocks myofibroblast differentiation. Previous work underscores the importance of ROCK1 in myofibroblast differentiation. Our data suggest BTB may block myofibroblast differentiation via disruption of ROCK/Rho signaling.

Transforming Growth Factor Beta (TGF-β) is a crucial cytokine that promotes the development of pulmonary fibrosis by inducing expression of extracellular matrix proteins. For example, TGF-β has been shown to stabilize elastin mRNA. Although the concentration of elastin is increased, less mature elastin is deposited in the extracellular matrix. Similarly, TGF-β has been shown to increase gene expression of alpha 1 anti-trypsin (A1AT), but its activity is less than control conditions. We are currently studying a family of proton sensing G-protein coupled receptors, which includes the Ovarian Cancer G-Protein Coupled Receptor 1 (OGR1). We have previously shown that OGR1 antagonizes TGF-β mediated signaling. We therefore hypothesize that OGR1 mitigates TGF-β-induced disruption of elastin homeostasis. Methods: Healthy human lung fibroblasts were obtained in accordance with an IRB-approved protocol at URMC. Briefly, fibroblasts were cultured Modified Eagle Media (MEM) with 10% fetal bovine serum. After plating, fibroblasts were treated with either control plasmid, OGR1 plasmid, non-targeting siRNA or OGR siRNA. Twenty four hours later, cells were treated with either volume matched DMSO or TGF-β (1 ng/mL) for an additional 24 hours. Cells were then harvested and qualitative real time PCR was performed. Statistical analysis involved either ANOVA or student t-test and statistical significance was considered to be present when p < 0.05. Results: In healthy human fibroblasts, treatment with TGF-β induced a significant increase in elastin and A1AT gene expression. However, changes in gene expression were reduced to control levels when OGR1 was overexpressed in the presence of TGF-β. In contrast, there was an enhancement of TGF-β-induced elastin and A1AT expression when OGR expression was decreased by targeted siRNA. Conclusions: TGF-β signaling is complex and it is unclear how competing signals like increased elastin mRNA stabilization and A1AT expression lead to increased elastin degradation. It is known that neutrophil elastase activity is increased in fibrosis and can lead to latent TGF-β activation. It is likely that TGF-β also induces elastase activity, which degrades elastin into elastin degradation products before mature elastin fibers can form. In addition, TGF-β-induced A1AT is less active, and is thus unable to prevent degradation of pre-existing mature elastin. Therefore, it appears that in a fibrotic environment, elastin degradation rather than elastin maturation and deposition, is likely to be favored by TGF-β mediated signaling. This preliminary data suggests that OGR1 inhibits TGF-β-induced disruption of elastin homeostasis, though the mechanism by which this occurs requires additional study.