Endoplasmic reticulum stress and autophagy are involved in adipocyte-induced fibrosis in hepatic stellate cells

Liver fibrosis, with the characterization of progressive accumulation of extracellular matrix (ECM), is the common pathologic feature in the process of chronic liver disease. Hepatic stellate cells (HSCs) which are activated and differentiate into proliferative and contractile myofibroblasts are recognized as the main drivers of fibrosis. Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression, but the direct fibrogenic effect of mature adipocytes on HSCs has been rarely reported. Therefore, the purpose of this study was to explore the fibrogenic effect of adipocyte 3T3-L1 cells on hepatic stellate LX-2 cells. The results showed that incubating LX-2 cells with the supernatant of 3T3-L1 adipocytes triggered the expression of ECM related proteins, such as α-smooth muscle actin (α-SMA), type I collagen (CO-I), and activated TGF β/Smad2/3 signaling pathway in LX-2 cells. In addition, 3T3-L1 cells inhibited insulin sensitivity, activated endoplasmic reticulum stress and autophagy to promote the development of fibrosis. These results supported the notion that mature adipocytes can directly activate hepatic stellate cells, and the establishment of an in vitro model of adipocytes on HSCs provides an insight into screening of drugs for liver diseases, such as nonalcoholic fatty liver disease.

     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.     

Loss of TGF-beta dependent growth control during HSC transdifferentiation

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-beta inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-beta function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-alpha enhanced proliferation inhibition of TGF-beta, whereas interleukin-6, oncostatin M, interleukin-1alpha, and interleukin-1beta did not. Hepatocyte growth factor (HGF) counteracted TGF-beta dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-beta contributes to loss of TGF-beta dependent inhibition of DNA synthesis in HSCs.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.     

Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling

BACKGROUND/AIMS: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce. METHODS: Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting. RESULTS: Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. CONCLUSIONS: Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.     

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.     

Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.     

Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells

Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.     

TLR4 enhances TGF-beta signaling and hepatic fibrosis

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

Astragaloside IV synergizes with ferulic acid to suppress hepatic stellate cells activation in vitro

Because hepatic fibrosis usually involves more than one pathological process, combination therapy with modalities that target aberrant signaling cascade in activated hepatic stellate cells (HSCs) represents an alternative strategy. This study evaluates the hypothesis that astragaloside IV (AS-IV) and ferulic acid (FA) synergize to inhibit HSCs activation via simultaneous activating nuclear factor erythroid-2-related factor-2 (Nrf2) and blocking transforming growth factor-β (TGF-β) pathways. The combination of FA and AS-IV, hereafter referred to as the AS-IV/FA, at suboptimal concentrations synergistically inhibited HSCs activation, as measured by expressions of α-smooth muscle actin (α-SMA), collagen α type I (Col I) and fibronectin. Nrf2 nuclear accumulation, glutathione (GSH) increase, and reactive oxygen species (ROS) reduction by AS-IV were not potentiated by co-treatment with FA. Similarly, inhibition of TGF-β1 secretion and Smad activity by FA also was not enhanced by combined treatment with AS-IV. AS-IV/FA synergistically suppresses the p38 mitogen-activated protein kinase (MAPK) activity. Inhibition of HSCs activation by AS-IV/FA could be completely blocked by TGF-βs-neutralizing antibody plus shRNA-mediated knockdown of Nrf2. Dual blockade of the TGF-β1/Smad pathway by FA and activation of Nrf2/ARE pathway by AS-IV contributed to the synergistic effects of this combination treatment. These results suggest that combinatorial treatments that target different pathway may afford a more effective strategy to inhibit HSC activation.