Abstract
Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.
Highlights
Liver fibrosis is a wound healing response to liver injury and is characterized by excessive synthesis and accumulation of extracellular matrix (ECM) in the liver [13, 24]
Since hepatic stellate cells (HSCs) activation is a central event in liver fibrogenesis [40], we first examined whether myocardin and myocardin-related transcription factor-A (MRTF-A) are involved in HSC activation
To verify the inhibitory specificity of CCG-203971 on myocardin and MRTF-A mediated actin cytoskeleton dynamics, we overexpressed myocardin or MRTF-A in HSCs via adenovirus (Ad) approach. We found that both myocardin and MRTF-A stimulated actin polymerization as increased F/globular actin (G-actin) ratios (Ad-control vs. Ad-flag-MRTF-A: 1.50 vs. 1.74; Ad-control vs. Ad-HA-myocd: 1.50 vs. 2.60)
Summary
Liver fibrosis is a wound healing response to liver injury and is characterized by excessive synthesis and accumulation of extracellular matrix (ECM) in the liver [13, 24]. Advanced fibrosis leads to cirrhosis, which, in turn, is associated with devastating clinical sequelae and even death [24]. Hepatic stellate cells (HSCs) are viewed to be the primary effectors of matrix production during liver injury in a process known as activation [3, 16]. At the molecular level, activated HSCs exhibit a robust actin cytoskeleton [e.g., smooth muscle (SM) ␣-actin], which assigns them to the myofibroblast lineage, and express abundant ECM proteins, in particular, interstitial collagens, such as type 1 collagen (COL.1) [24, 33]. Targeting HSC activation is an attractive approach to suppress liver fibrogenesis [31]
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