Titus, A. S. | Search bioRxiv

bioRxiv2021

Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

Recent reports on the cardioprotective effects of fibronectin inhibition following myocardial injury suggest a largely unexplored role for the extracellular matrix (ECM) glycoprotein in cardiac fibroblast function. We probed the molecular regulation and functional implications of fibronectin gene expression in cardiac fibroblasts exposed to Angiotensin II, a major pro-fibrotic factor in the myocardium. Using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-stimulated transcriptional up-regulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Further, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link ECM signaling and Angiotensin II signaling in cardiac fibroblasts. Moreover, conditioned medium from DDR2- or fibronectin-silenced cardiac fibroblasts reduced AT1R expression in H9c2 cardiomyoblasts. The regulatory role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I and AT1R expression was mediated by Integrin-{beta}1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, associated with the previously reported reduction in myocardial Integrin-{beta}1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major extracellular matrix proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-{beta}1.

molecular biology↗

bioRxiv2019

Identification of a common regulatory pathway that determines cell survival and cell cycle progression in cardiac fibroblasts

Relative resistance to apoptosis and the ability to proliferate and produce a collagen-rich scar determine the critical role of cardiac fibroblasts in wound healing and tissue remodeling following myocardial injury. Identification of cardiac fibroblast-specific factors and mechanisms underlying these aspects of cardiac fibroblast function is therefore of considerable scientific and clinical interest. In the present study, gene knockdown and over-expression approaches, and promoter binding assays, showed that DDR2, a mesenchymal cell-specific collagen receptor tyrosine kinase localized predominantly in fibroblasts in the heart, acts via ERK1/2 MAPK-activated SRF transcription factor to enhance the expression of anti-apoptotic cIAP2 in cardiac fibroblasts, conferring resistance against oxidative injury. Further, DDR2 was found to act via ERK1/2 MAPK-activated SRF to transcriptionally up regulate Skp2 that in turn facilitated post-translational degradation of p27, the cyclin dependent kinase inhibitor that causes cell cycle arrest, to promote G1-S transition, as evidenced by Rb phosphorylation, increased PCNA levels and flow cytometry. DDR2-dependent ERK1/2 MAPK activation also suppressed FoxO3a-mediated transcriptional induction of p27. Inhibition of the binding of collagen type I to DDR2 using WRG-28 indicated the obligate role of collagen type I in the activation of DDR2 and its regulatory role in cell survival and cell cycle protein expression. Notably, DDR2 levels positively correlated with SRF, cIAP2 and PCNA levels in cardiac fibroblasts from Spontaneously Hypertensive Rats. To conclude, DDR2-mediated ERK1/2MAPK activation facilitates coordinated regulation of cell survival and cell cycle progression in cardiac fibroblasts via SRF. New & NoteworthyRelative resistance to apoptosis and the ability to proliferate and produce a collagen-rich scar enable cardiac fibroblasts to play a central role in myocardial response to injury. This study reports novel findings that mitogen-stimulated cardiac fibroblasts exploit a common regulatory mechanism involving collagen receptor (DDR2)-dependent activation of ERK1/2 MAPK and SRF to achieve coordinated regulation of apoptosis resistance and cell cycle progression, which could facilitate their survival and function in the injured myocardium.

molecular biology↗