Activation of TDAG8 Prevents Myofibroblast Differentiation via Inhibition of RhoA Signaling

Abstract

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.