Myocardin‐related transcription factor A drives ROS‐fueled expansion of hepatic stellate cells by regulating p38‐MAPK signalling

Abstract

We describe in this letter a novel retrograde signalling mechanism that highlights a myofibroblast-specific role for myocardin-related transcription factor A (MRTF-A), also known as MKL1, in liver fibrosis. Liver fibrosis is considered a common pathological process in virtually all end-stage liver diseases. Absent from the liver under physiological conditions, myofibroblasts quickly emerge and expand as a result of liver injuries to mediate the pro-fibrogenic response. Hepatic stellate cells (HSCs), tucked between the liver parenchyma and the hepatic sinusoid, is considered as the predominant source from which myofibroblasts are derived regardless of aetiology.1 Reactive oxygen species (ROS) fuels HSC proliferation via a network of signalling cascades. Mitogen-activated protein kinases (MAPKs) are well-documented for their regulatory roles in ROS-driven HSC proliferation and liver fibrosis by programming transcriptional events in the nucleus. How the cytoplasm-nucleus crosstalk influences MAPK signalling and contributes to ROS-dependent HSC proliferation is not well understood. MRTF-A is a transcriptional modulator implicated in the pathogenesis of a wide range of human diseases. Despite the near-unanimous view that MRTF-A is a pivotal regulator of myofibroblast trans-differentiation,2 there is no direct genetic evidence to support or refute the hypothesis that myofibroblast-specific MRTF-A deletion is sufficient to suppress liver fibrosis in vivo due to its universal expression pattern.3 We first evaluated the possibility that the ability of MRTF-A to regulate liver fibrosis might be myofibroblast-autonomous. MRTF-Af/f mice were crossed to Postn-CreERT2 mice to generate myofibroblast-conditional MRTF-A deletion mice (MRTF-AΔMF) followed by carbon tetrachloride (CCl4) injection to induce liver fibrosis. Western blotting indicated that MRTF-A expression was markedly decreased in HSCs, but not in hepatocytes, from MRTF-AΔMF mice, compared to MRTF-Af/f mice following tamoxifen injection (Figure S1). As shown in Figure 1A,B, plasma alanine aminotransferase/aspartate aminotransferase (ALT/AST) levels were comparable between the MRTF-Af/f mice and the MRTF-AΔMF mice. Quantitative polymerase chain reaction (qPCR) analysis (Figure 1C), picrosirus red/Masson's trichrome staining (Figure 1D) and hepatic hydroxylproline quantification (Figure 1E) all supported the notion that myofibroblast-specific MRTF-A deficiency is sufficient to dampen liver fibrosis. MRTF-A deletion in myofibroblasts rendered them less capable of proliferating as evidenced by reduced expression levels of desmin (Figure 1F), a marker of HSCs. In addition, there were fewer dihydroethidium/alpha smooth muscle actin (DHE/α-SMA)-double positive cells in the MRTF-AΔMF livers than in the MRTF-Af/f livers suggesting that MRTF-A deficiency dampened ROS production in myofibroblasts (Figure 1G). Over-expression of a constitutively active (CA) MRTF-A in LX-2 cells significantly enhanced ROS production (Figure 2A) and proliferation (Figure 2B). Treatment of N-acetyl cysteine (NAC) completely blunted the pro-oxidative and the pro-proliferative effects of MRTF-A (Figure 2A,B). Similar results were obtained in primary murine HSCs (Figure 2C,D). Of interest, over-expression of MRTF-A CA markedly enhanced p38-MAPK phosphorylation (Figure 2E,F). On the contrary, MRTF-A depletion dampened p38 phosphorylation (Figure 2G,H). Treatment with a specific p38 inhibitor completely abrogated the induction of ROS production and proliferation of HSCs (Figure 2I,J). Based on prior discoveries that the integrin signalling pathway is intimately wired into the MAPK signalling pathway,4 a PCR array-based screening was performed to identify components of the integrin pathway that can potentially be regulated by MRTF-A. Using 2X-fold change as a cutoff, four genes met this criterion: Itga6 and Ilk were down-regulated, whereas Itga11 and Itgb11 were up-regulated in the MRTF-A KO HSCs, compared to the WT HSCs (Figure S2). We focused on Itga6 and Ilk for the remainder of the study because MRTF-A is primarily considered an activator of transcription. Over-expression of MRTF-A CA robustly augmented the mRNA and protein levels of ITGA6 and ILK in vitro (Figure 3A–D) and in vivo (Figure 3E,F). Further, MRTF-A over-expression stimulated the ITGA6 promoter and the ILK promoter activity (Figure 3G). Chromatin immunoprecipitation (ChIP) assay confirmed that MRTF-A was directly associated with the proximal ITGA6 promoter and the proximal ILK promoter (Figure 3H). Functionally, depletion of either Integrinα6 or ILK by siRNAs rendered the cells irresponsive to MRTF-A over-expression by dampening ROS production, suppressing proliferation and inhibiting p38 phosphorylation (Figure S3). We finally addressed the question as to whether ILK inhibition in mice could influence liver fibrosis. The mice were injected with CCl4 for 4 weeks; starting at the second week, a specific ILK inhibitor (QLT-0267) was administered peritoneally for the duration of CCl4 injection (Figure 4A). Plasma ALT (Figure 4B) and AST (Figure 4C) levels indicated that liver injury was ameliorated as a result of QLT administration. More importantly, qPCR (Figure 4D), picrosirius red/Masson's trichrome staining (Figure 4E) and hydroxylproline quantification (Figure 4F) all suggested that ILK inhibition attenuated liver fibrosis. HSC proliferation (Figure 4G) and ROS production (Figure 4H) were down-regulated by the ILK inhibitor. Western blotting confirmed that p38-MAPK phosphorylation was weakened in the ILK inhibitor injected livers, compared to the control livers (Figure 4I). In an alternative therapeutic scenario in which QLT was given to the mice with developed liver fibrosis, it was discovered that ILK inhibition partially but effectively reversed liver fibrosis (Figure S4). In summary, we describe a novel mechanism where nucleus-initiated transcriptional activation of ITGA6/ILK by MRTF-A serves to jumpstart cytoplasmic MAPK-p38 signalling contributing to ROS-fueled HSC proliferation (Figure 4J). Our data provide compelling evidence that MRTF-A functions as a pivotal link between MAPK signalling, ROS production and myofibroblast (HSC) expansion in the pathogenesis of liver fibrosis. The novel mechanistic insights provided by this study may further incentivize the screening for small-molecule compounds that target the MRTF-A-ITGA6/ILK-MPAK axis to yield therapeutic solutions against liver fibrosis. This work was supported by a grant from the National Natural Science Foundation of China (81725001). The authors declare that they have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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