Glycyrrhizin and its metabolite inhibit Smad3-mediated type I collagen gene transcription and suppress experimental murine liver fibrosis.

AIMS: Glycyrrhizin has been widely used for the treatment of chronic hepatitis C. It decreases the serum levels of aminotransferases, and suppresses progression of liver fibrosis as well as subsequent occurrence of hepatocellular carcinoma. Although previous studies have shown that glycyrrhizin and its metabolite inhibit collagen gene expression, its underlying mechanisms are virtually unknown. This study was aimed to explore molecular mechanisms responsible for the inhibitory effect of glycyrrhizin on type I collagen gene transcription. MAIN METHODS: Effects of glycyrrhizin and its metabolite, glycyrrhetinic acid, on collagen promoter activity were examined by using transgenic reporter mice harboring alpha2(I) collagen gene (COL1A2) promoter. Their effects on the TGF-beta/Smad signaling pathway were studied by cell transfection assays and immunofluorescence studies using cultured hepatic stellate cells. KEY FINDINGS: Administration of glycyrrhizin or its metabolite, glycyrrhetinic acid, significantly suppressed COL1A2 promoter activation and progression of liver fibrosis induced by repeated carbon tetrachloride injections. In cultured hepatic stellate cells, glycyrrhetinic acid, but not glycyrrhizin, inhibited type I collagen synthesis mostly at the level of gene transcription. This inhibitory effect of glycyrrhetinic acid was abolished by a mutation introduced into a Smad3-binding region within the COL1A2 promoter. Glycyrrhetinic acid did not affect gene expression of TGF-beta receptors or Smad proteins, but inhibited nuclear accumulation of Smad3 in activated hepatic stellate cells. In addition to those direct inhibitory effects on COL1A2 transcription, glycyrrhetinic acid also suppressed activation of quiescent hepatic stellate cells in primary culture. SIGNIFICANCE: The results provide a molecular basis for the anti-fibrotic effect of glycyrrhizin treatment.

Hepatocyte growth factor suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7.

BACKGROUND & AIMS: Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS: Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS: Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS: These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.

UNLABELLED: Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)-derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)-expressing BM cells. The distribution and characteristics of EGFP-positive (EGFP(+)) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP(+) cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP-negative (EGFP(-)) liver resident cells, expressed matrix metalloproteinase (MMP)-13 and MMP-9. Whereas MMP-13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP-9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony-stimulating factor (G-CSF) treatment significantly enhanced migration of BM-derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G-CSF and HGF synergistically increased MMP-9 expression along the fibrous septa. CONCLUSION: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis.

Allogeneic MHC gene transfer enhances antitumor activity of allogeneic hematopoietic stem cell transplantation without exacerbating graft-versus-host disease.

Enhancement of the specific antitumor activity of allogeneic hematopoietic stem cell transplantation (alloHSCT) against solid cancers is a major issue in the clinical oncology. In this study, we examined whether intratumoral allogeneic MHC (alloMHC) gene transfer can enhance the recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. In minor histocompatibility antigen-mismatched alloHSCT (DBA/2-->BALB/c: H-2(d)) recipients, alloMHC gene (H-2K(b)) was transduced directly into a s.c. tumor of CT26 colon cancer cells. Because CT26 cells have an aggressive tumorigenicity in syngeneic BALB/c mice, an H-2K(b) gene transfer provides only a limited antitumor effect after syngeneic (BALB/c-->BALB/c) HSCT. By contrast, the H-2K(b) gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors without gene transduction. In vitro cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to the H-2K(b) gene transfer. Graft-versus-host disease was not exacerbated serologically or clinically in the treated mice, demonstrating that alloMHC gene transfer enhances the antitumor effects of alloHSCT without exacerbating graft-versus-host disease. This combination strategy has important implications for the development of therapies for human solid cancers.

Cell type-specific intervention of transforming growth factor beta/Smad signaling suppresses collagen gene expression and hepatic fibrosis in mice.

BACKGROUND & AIMS: Transforming growth factor beta and its intracellular mediators, Smad proteins, play important roles in stimulating collagen gene transcription and, thus, could be the targets for treating hepatic fibrosis. However, intervention of transforming growth factor beta/Smad signaling affects physiological signal transduction as well and may cause serious adverse effects on clinical application. Here we have attempted to suppress hepatic fibrosis by expressing a transforming growth factor beta/Smad antagonist selectively in collagen-producing cells only in the fibrotic liver. METHODS: Recombinant adenoviruses expressing either green fluorescent protein or a transforming growth factor beta/Smad signal repressor, YB-1, were injected into mice untreated or treated with carbon tetrachloride. Green fluorescent protein expression was analyzed under a confocal laser scanning microscope. Antifibrotic effects of YB-1 overexpression were examined by luciferase assays and histological examination with transgenic reporter mice. RESULTS: When the CAG expression unit was used as a control, green fluorescent protein was strongly expressed in a large number of hepatocytes in both normal and carbon tetrachloride-treated liver. In contrast, green fluorescent protein expression driven by a tissue-specific enhancer of the mouse alpha2(I) collagen gene ( COL1A2 ) was detected in activated hepatic stellate cells in carbon tetrachloride-induced fibrotic liver, but not in untreated normal liver. No green fluorescent protein fluorescence was observed in any other organs when the COL1A2 enhancer was used. Adenovirus-mediated YB-1 expression under the control of the COL1A2 enhancer significantly decreased COL1A2 promoter activity after carbon tetrachloride injection and subsequently suppressed the progression of hepatic fibrosis. CONCLUSIONS: These results validate a new concept of the therapy for hepatic fibrosis to achieve cell type-specific gene expression only in the fibrotic liver, with little damage to other organs.

c-Jun NH2-terminal kinase pathway is involved in constitutive matrix metalloproteinase-1 expression in a hepatocellular carcinoma-derived cell line.

Transcription factor c-Jun serves for cellular proliferation, survival, differentiation and transformation and is recognized as an important factor in cancer development, including hepatocellular carcinoma (HCC). The purpose of present study is to determine the involvement of c-Jun in matrix metalloproteinase-1 (MMP-1) expression, which is previously reported by us to be expressed only in the early stage of human HCC showing stromal invasion. Of 5 human HCC cell lines examined, only HLE cells revealed mRNA and protein expression as well as enzymatic activity of MMP-1. Transient transfection of an MMP-1 promoter/luciferase construct (including 4.4 kb full promoter region) into HLE and HCC-T cells (MMP-1 nonproducer) showed that high promoter activity was observed only in HLE cells without inducers, and that this promoter activity was still observed when a shorter 0.6 kb proximal promoter construct was transfected. The 0.6 kb promoter region contained 3 AP-1 sites, and c-jun mRNA was constitutively expressed in HLE cells without inducers. Furthermore, phosphorylated c-Jun and c-Jun NH2-terminal kinase (JNK) were detected in HLE cells. Promoter activity of the 0.6 kb construct was suppressed with SP600125, a potent inhibitor of JNK, but not with PD98059 and SB203580, potent inhibitors of MEK1/2 and p38, respectively. The inhibitory effect of SP600125 was also observed at protein expression level and in enzymatic activity of MMP-1. Taken together, this study suggests that the JNK pathway is involved in the expression of MMP-1 in HCC cells and may represent a new functional role of c-Jun for HCC development.

Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice.

The equilibrium between the production and degradation of collagen is rigorously controlled by a number of growth factors and cytokines. Interferon alfa (IFN-alpha) is now widely used for the treatment of chronic hepatitis C, which can improve serum levels of fibrotic markers and the degree of hepatic fibrosis, not only in patients who responded to therapy but also in those in whom it is ineffective. These findings may suggest that IFN-alpha possesses direct antifibrotic effects in addition to its antiviral activity. However, in contrast to IFN-gamma, which has been shown to suppress collagen gene transcription, little is known about the mechanisms responsible for the antifibrotic effects of IFN-alpha. Here, we report that IFN-alpha, when administered into transgenic mice harboring the alpha2(I) collagen gene (COL1A2) promoter sequence, significantly repressed promoter activation and prevented the progression of hepatic fibrosis induced by carbon tetrachloride injection. Transient transfection assays indicated that IFN-alpha decreased the steady-state levels of COL1A2 messenger RNA (mRNA) and inhibited basal and TGF-beta/Smad3-stimulated COL1A2 transcription in activated hepatic stellate cells (HSC). These inhibitory effects of IFN-alpha on COL1A2 transcription were exerted through the interaction between phosphorylated Stat1 and p300. Blocking of the IFN-alpha signal by overexpressing the intracellular domain-deleted IFN receptor increased basal COL1A2 transcription and abolished the inhibitory effects of IFN-alpha. In conclusion, our results indicate that IFN-alpha antagonizes the TGF-beta/Smad3-stimulated COL1A2 transcription in vitro and suppresses COL1A2 promoter activation in vivo, providing a molecular basis for antifibrotic effects of IFN-alpha.