Dual Blockade of TGF-β Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT1R/Gαq-Mediated TGF-β1 and ET-1 Signaling

Abstract

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-β1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-β1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-β1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT1R/Gαq axis in adult human cardiac fibroblasts (HCFs). Following AT1R stimulation, Gαq protein, not Gβγ subunit, was required for upregulation of TGF-β1 and ET-1. Moreover, dual inhibition of TGF-β and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT1R/Gαq cascade transduced signals to TGF-β1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-β receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-β1 and ET-1 are major effectors of AT1R/Gαq cascade, and therefore, negative regulation of TGF-β and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

New Therapeutics for Heart Failure: Focusing on cGMP Signaling

“…FFAR4 activation inhibits TGF-β mediated fibrosis [ 26 ]. Several lines of evidence have demonstrated that TGF-β signaling plays a major role in fibrosis and thus inhibition of TGF-β signaling results in the suppression of fibrosis [ 96 , 97 ]. In fibroblasts, FFAR4 stimulation activates the cGMP–PKG pathway, and thus the phosphorylation of Smad3 by PKG inhibits TGF-β-mediated fibrosis [ 26 ].…”