Inhibition of Myofibroblast Differentiation and SMAD Mediated Transcription via Small Molecule Stimulation of GPR68

Abstract

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.

GPR68 inhibited nucleus or ER to exosome non-protein based circadian exercise based on Thalamus big data

“…The abnormal expression of GPR68 is related to the occurrence and development of multiple cancer [5][6][7][8][9][10][11][12][13]. The current research mainly focuses on its regulatory mechanism [14][15][16][17][18], signaling pathways [19][20][21][22][23] and potential clinical applications. GPR68 locates plasma membrane, integral component of plasma membrane, functions as protein coupled receptor activity, in ammatory response, G protein coupled receptor signaling pathway, positive regulation of insulin secretion involved in cellular response to glucose stimulus, negative regulation of monocyte differentiation, cellular response to pH, positive regulation of osteoclast development, GPCRDB class A rhodopsin like, rhodopsin like receptor activity, osteoclasts by the Database for Annotation, Visualization and Integrated Discovery (DAVID) [24,25].…”