Paxillin regulates liver fibrosis via actin polymerization and ERK activation in hepatic stellate cells.

Liver injury leads to fibrosis and cirrhosis. The primary mechanism underlying the fibrogenic response is the activation of hepatic stellate cells (HSCs), which are 'quiescent' in normal liver but become 'activated' after injury by transdifferentiating into extracellular matrix (ECM)-secreting myofibroblasts. Given that integrins are important in HSC activation and fibrogenesis, we hypothesized that paxillin, a key downstream effector in integrin signaling, might be critical in the fibrosis pathway. Using a cell-culture-based model of HSC activation and in vivo models of liver injury, we found that paxillin is upregulated in activated HSCs and fibrotic livers. Overexpression of paxillin (both in vitro and in vivo) led to increased ECM protein expression, and depletion of paxillin in a novel conditional mouse injury model reduced fibrosis. The mechanism by which paxillin mediated this effect appeared to be through the actin cytoskeleton, which signals to the ERK pathway and induces ECM protein production. These data highlight a novel role for paxillin in HSC biology and fibrosis.

Characterization of focal adhesion proteins in rodent hepatic stellate cells.

Ongoing liver injury leads to fibrosis and ultimately cirrhosis, a leading cause of death worldwide. The primary mechanism underlying the fibrogenic response is the activation of cells known as hepatic stellate cells (HSCs) which are "quiescent" in the normal liver but become "activated" after injury by transdifferentiating into extracellular matrix-secreting myofibroblasts. Since integrins (extracellular matrix binding receptors) are important mediators of HSC activation and fibrogenesis, we hypothesized that focal adhesion (FA) proteins, which link integrins to the intracellular protein machinery, may be important in the activation process. Therefore, using both an in vitro model of activation in primary rat HSCs and an in vivo model of liver injury, we examined three FA proteins: vinculin, FAK, and talin. All three proteins were significantly upregulated during HSC activation at both the messenger RNA (mRNA) and protein levels. Confocal microscopy demonstrated that the proteins had a widespread expression throughout HSCs with prominent localization at the end of actin filaments. Finally, we stimulated HSCs with the profibrotic ligands endothelin-1 (ET-1) and transforming growth factor beta (TGF-ß) and observed an increase in the size of vinculin-containing FAs and the cell area occupied by them. The data indicate that HSCs possess a broad array of FA proteins, and given their upregulation during activation, this raises the possibility that they play a role in the fibrogenic response to injury.

Fibroblast fate determination during cardiac reprogramming by remodeling of actin filaments.

Fibroblasts can be reprogrammed into induced cardiomyocyte-like cells (iCMs) by forced expression of cardiogenic transcription factors. However, it remains unknown how fibroblasts adopt a cardiomyocyte (CM) fate during their spontaneous ongoing transdifferentiation toward myofibroblasts (MFs). By tracing fibroblast lineages following cardiac reprogramming in vitro, we found that most mature iCMs are derived directly from fibroblasts without transition through the MF state. This direct conversion is attributable to mutually exclusive induction of cardiac sarcomeres and MF cytoskeletal structures in the cytoplasm of fibroblasts during reprogramming. For direct fate switch from fibroblasts to iCMs, significant remodeling of actin isoforms occurs in fibroblasts, including induction of α-cardiac actin and decrease of the actin isoforms predominant in MFs. Accordingly, genetic or pharmacological ablation of MF-enriched actin isoforms significantly enhances cardiac reprogramming. Our results demonstrate that remodeling of actin isoforms is required for fibroblast to CM fate conversion by cardiac reprogramming.

The cellular microenvironment and cytoskeletal actin dynamics in liver fibrogenesis.

Hepatic stellate cells (HSCs) are the primary effector cells in liver fibrosis. In the normal liver, HSCs serve as the primary vitamin A storage cells in the body and retain a "quiescent" phenotype. However, after liver injury, they transdifferentiate to an "activated" myofibroblast-like phenotype, which is associated with dramatic upregulation of smooth muscle specific actin and extracellular matrix proteins. The result is a fibrotic, stiff, and dysfunctional liver. Therefore, understanding the molecular mechanisms that govern HSC function is essential for the development of anti-fibrotic medications. The actin cytoskeleton has emerged as a key component of the fibrogenic response in wound healing. Recent data indicate that the cytoskeleton receives signals from the cellular microenvironment and translates them to cellular function-in particular, increased type I collagen expression. Dynamic in nature, the actin cytoskeleton continuously polymerizes and depolymerizes in response to changes in the cellular microenvironment. In this viewpoint, we discuss the recent developments underlying cytoskeletal actin dynamics in liver fibrosis, including how the cellular microenvironment affects HSC function and the molecular mechanisms that regulate the actin-induced increase in collagen expression typical of activated HSCs.

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesis.

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.

Smooth Muscle α-Actin Deficiency Leads to Decreased Liver Fibrosis via Impaired Cytoskeletal Signaling in Hepatic Stellate Cells.

In the liver, smooth muscle α-actin (SM α-actin) is up-regulated in hepatic stellate cells (HSCs) as they transition to myofibroblasts during liver injury and the wound healing response. Whether SM α-actin has specific functional effects on cellular effectors of fibrosis such as HSC is controversial. Here, the relationship between SM α-actin and type 1 collagen expression (COL1A1), a major extracellular matrix protein important in liver fibrosis, is investigated with the results demonstrating that knockout of SM α-actin leads to reduced liver fibrosis and COL1 expression. The mechanism for the reduction in fibrogenesis in vivo is multifactorial, including not only a reduction in the number of HSCs, but also an HSC-specific reduction in COL1 expression in Acta2-deficient HSCs. Despite a compensatory increase in expression of cytoplasmic ß-actin and γ-actin isoforms in Acta2-/- HSCs, defects were identified in each transforming growth factor beta/Smad2/3 and ET-1/Erk1/2 signaling in Acta2-/- HSCs. These data not only suggest a molecular link between the SM α-actin cytoskeleton and classic fibrogenic signaling cascades, but also emphasize the relationship between SM α-actin and fibrogenesis in hepatic myofibroblasts in vivo.

Upregulation of the actin cytoskeleton via myocardin leads to increased expression of type 1 collagen.

Liver fibrosis, a model wound healing system, is characterized by excessive deposition of extracellular matrix (ECM) in the liver. Although many fibrogenic cell types may express ECM, the hepatic stellate cell (HSC) is currently considered to be the major effector. HSCs transform into myofibroblast-like cells, also known as hepatic myofibroblasts in a process known as activation; this process is characterized in particular by de novo expression of smooth muscle alpha actin (SM α-actin) and type 1 collagen. The family of actins, which form the cell's cytoskeleton, are essential in many cellular processes. ß-actin and cytoplasmic γ-actin (γ-actin) are ubiquitously expressed, whereas SM α-actin defines smooth muscle cell and myofibroblast phenotypes. Thus, SM α-actin is tightly associated with multiple functional properties. However, the regulatory mechanisms by which actin isoforms might regulate type 1 collagen remain unclear. In primary HSCs from normal and fibrotic rat liver, we demonstrate that myocardin, a canonical SRF cofactor, is upregulated in hepatic myofibroblasts and differentially regulates SM α-actin, γ-actin, and ß-actins through activation of an ATTA box in the SM α-actin and a CCAAT box in γ-actin and ß-actin promoters, respectively; moreover, myocardin differentially activated serum response factor (SRF) in CArG boxes of actin promoters. In addition, myocardin-stimulated Smad2 phosphorylation and RhoA expression, leading to increased expression of type 1 collagen in an actin cytoskeleton-dependent manner. Myocardin also directly enhanced SRF expression and stimulated collagen 1α1 and 1α2 promoter activities. In addition, overexpression of myocardin in vivo during carbon tetrachloride-induced liver injury led to increased HSC activation and fibrogenesis. In summary, our data suggest that myocardin plays a critical role in actin cytoskeletal dynamics during HSC activation, in turn, specifically regulating type I collagen expression in hepatic myofibroblasts.

The mitochondria-targeted antioxidant MitoQ attenuates liver fibrosis in mice.

Oxidative stress plays an essential role in liver fibrosis. This study investigated whether MitoQ, an orally active mitochondrial antioxidant, decreases liver fibrosis. Mice were injected with corn oil or carbon tetrachloride (CCl4, 1:3 dilution in corn oil; 1 µl/g, ip) once every 3 days for up to 6 weeks. 4-Hydroxynonenal adducts increased markedly after CCl4 treatment, indicating oxidative stress. MitoQ attenuated oxidative stress after CCl4. Collagen 1α1 mRNA and hydroxyproline increased markedly after CCl4 treatment, indicating increased collagen formation and deposition. CCl4 caused overt pericentral fibrosis as revealed by both the sirius red staining and second harmonic generation microscopy. MitoQ blunted fibrosis after CCl4. Profibrotic transforming growth factor-ß1 (TGF-ß1) mRNA and expression of smooth muscle α-actin, an indicator of hepatic stellate cell (HSC) activation, increased markedly after CCl4 treatment. Smad 2/3, the major mediator of TGF-ß fibrogenic effects, was also activated after CCl4 treatment. MitoQ blunted HSC activation, TGF-ß expression, and Smad2/3 activation after CCl4 treatment. MitoQ also decreased necrosis, apoptosis and inflammation after CCl4 treatment. In cultured HSCs, MitoQ decreased oxidative stress, inhibited HSC activation, TGF-ß1 expression, Smad2/3 activation, and extracellular signal-regulated protein kinase activation. Taken together, these data indicate that mitochondrial reactive oxygen species play an important role in liver fibrosis and that mitochondria-targeted antioxidants are promising potential therapies for prevention and treatment of liver fibrosis.

Smooth muscle α actin (Acta2) and myofibroblast function during hepatic wound healing.

Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. Liver injury, associated with cirrhosis, induces transformation of resident hepatic stellate cells into liver specific myofibroblasts, also known as activated cells. Here, we have used in vitro and in vivo wound healing models to explore the functional role of Acta2 in this transformation. Acta2 was abundant in activated cells isolated from injured livers but was undetectable in quiescent cells isolated from normal livers. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Inhibition of Acta2 using several different techniques had no effect on cytoplasmic actin isoform expression, but led to reduced cellular motility and contraction. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. The data indicate that Acta2 is important specifically in myofibroblast cell motility and contraction and raise the possibility that the Acta2 cytoskeleton, beyond its structural importance in the cell, could be important in regulating signaling processes during wound healing in vivo.

Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation.

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.

Smooth muscle α actin is specifically required for the maintenance of lactation.

Smooth muscle α-actin (Acta2) is one of six highly conserved mammalian actin isoforms that appear to exhibit functional redundancy. Nonetheless, we have postulated a specific functional role for the smooth muscle specific isoform. Here, we show that Acta2 deficient mice have a remarkable mammary phenotype such that dams lacking Acta2 are unable to nurse their offspring effectively. The phenotype was rescued in cross fostering experiments with wild type mice, excluding a developmental defect in Acta2 null pups. The mechanism for the underlying phenotype is due to myoepithelial dysfunction postpartum resulting in precocious involution. Further, we demonstrate a specific defect in myoepithelial cell contractility in Acta2 null mammary glands, despite normal expression of cytoplasmic actins. We conclude that Acta2 specifically mediates myoepithelial cell contraction during lactation and that this actin isoform therefore exhibits functional specificity.

Interferon-gamma-mediated inhibition of serum response factor-dependent smooth muscle-specific gene expression.

IFNγ exerts multiple biological effects on effector cells by regulating many downstream genes, including smooth muscle-specific genes. However, the molecular mechanisms underlying IFNγ-induced inhibition of smooth muscle-specific gene expression remain unclear. In this study, we have shown that serum response factor (SRF), a common transcriptional factor important in cell proliferation, migration, and differentiation, is targeted by IFNγ in a STAT1-dependent manner. We show that the molecular mechanism by which IFNγ regulates SRF is via activation of the 2-5A-RNase L system, which triggers SRF mRNA decay and reduced SRF expression. As a result, decreased SRF expression reduces expression of SRF target genes such as smooth muscle α-actin and smooth muscle myosin heavy chain. Additionally, IFNγ reduced p300 and acetylated histone-3 binding in both smooth muscle α-actin and SRF promoters, epigenetically decreasing smooth muscle α-actin and SRF transcriptional activation. Our data reveal that SRF is a novel IFNγ-regulated gene and further elucidate the molecular pathway between IFNγ, IFNγ-regulated genes, and SRF and its target genes.

Cell and molecular regulation of endothelin-1 production during hepatic wound healing.

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.