Sihyung Wang

MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis.

MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells.

Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.

Hedgehog Signaling Regulates the Repair Response in Mouse Liver Damaged by Irradiation

Radiotherapy is commonly used in treating many kinds of cancers that cannot be cured by other therapeutic strategies. However, radiation-induced fibrosis in the treatment of intrahepatic cancer is a major obstacle. Hedgehog pathway is known to regulate the fibrotic process and proliferation of progenitor cells. Hedgehog ligands act as a profibrotic factor and hedgehog-responsive cells undergo epithelial-to-mesenchymal transition (EMT), eventually contributing to the fibrogenic process. Herein, we investigated whether the hedgehog pathway was associated with radiation-induced hepatic fibrosis. Female mice were irradiated with a single dose of 20 Gy and were sacrificed 1 week postirradiation, to obtain the livers for biochemical and histological analysis. Hematoxylin and eosin and Sirius Red staining were used in evaluating liver morphology and fibrosis, respectively. Immunochemical staining for active caspase 3 and CD44 was used to examine the repair response of the irradiated livers. Immunoblot analysis was performed to detect the expression of hedgehog molecules and fibrogenic markers. Fat accumulation in hepatocytes and increased apoptosis were observed in liver sections from mice treated with radiation. Expression of hedgehog ligand, Indian hedgehog, and hedgehog target gene, Gli2, were significantly up-regulated in the liver of mice treated with radiation. Levels of transforming growth factor-β (inducer of fibrosis) and α-smooth muscle actin (marker of myofibroblastic hepatic stellate cells) were also greatly increased in the damaged liver compared to the normal liver. The EMT marker, laminin-β3, showed a great increase, whereas EMT inhibitor, bmp7, was significantly decreased in mouse liver postirradiation. Furthermore, CD44-positive progenitors were shown to accumulated in the injured liver. These results suggest that increased expression of hedgehog signaling promotes proliferation of myofibroblastic hepatic stellate cells and progenitors, and thereby contributes to the repair response after irradiation.

Potential role of Hedgehog pathway in liver response to radiation.

Radiation-induced fibrosis constitutes a major problem that is commonly observed in the patients undergoing radiotherapy; therefore, understanding its pathophysiological mechanism is important. The Hedgehog (Hh) pathway induces the proliferation of progenitors and myofibroblastic hepatic stellate cells (MF-HSCs) and promotes the epithelial-to-mesenchymal transition (EMT), thereby regulating the repair response in the damaged liver. We examined the response of normal liver to radiation injury. Male mice were sacrificed at 6 weeks and 10 weeks after exposure to a single dose of 6 Gy and the livers were collected for biochemical analysis. Irradiated (IR) and control mice were compared for progenitors, fibrosis, Hh pathway, and EMT at 6 and 10 weeks post irradiation. Fatty hepatocytes were observed and the expressions of Hh ligand, Indian Hh. were greater in the livers at 6 weeks, whereas expression of another Hh ligand, Sonic Hh, increased at 10 weeks post irradiation. Both Smoothened, Hh receptor, and Gli2, Hh-target gene, were up-regulated at 6 and 10 weeks after irradiation. Accumulation of progenitors (CD44, Pan-cytokeratin, and Sox9) was significant in IR livers at 6 and 10 weeks. RNA analysis showed enhanced expression of the EMT–stimulating factor, tgf-β, in the IR livers at 6 weeks and the upregulation of mesenchymal markers (α-SMA, collagen, N-cadherin, and s100a4), but down-regulation of EMT inhibitors, in IR mouse livers at 6 and 10 weeks. Increased fibrosis was observed in IR mouse livers at 10 weeks. Treatment of mice with Hh inhibitor, GDC-0449, suppressed Hh activity and block the proliferation of hepatic progenitor and expression of EMT-stimulating genes in irradiated mice. Therefore, those results demonstrated that the Hh pathway increased in response to liver injury by radiation and promoted a compensatory proliferation of MF-HSCs and progenitors, thereby regulating liver remodeling.

Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury

IntroductionTumor necrosis factor-inducible gene 6 protein (TSG-6), one of the cytokines released by human mesenchymal stem/stromal cells (hMSC), has an anti-inflammatory effect and alleviates several pathological conditions; however, the hepatoprotective potential of TSG-6 remains unclear. We investigated whether TSG-6 promoted liver regeneration in acute liver failure.MethodsThe immortalized hMSC (B10) constitutively over-expressing TSG-6 or empty plasmid (NC: Negative Control) were established, and either TSG-6 or NC-conditioned medium (CM) was intraperitoneally injected into mice with acute liver damage caused by CCl4. Mice were sacrificed at 3 days post-CM treatment.ResultsHigher expression and the immunosuppressive activity of TSG-6 were observed in CM from TSG-6-hMSC. The obvious histomorphological liver injury and increased level of liver enzymes were shown in CCl4-treated mice with or without NC-CM, whereas those observations were markedly ameliorated in TSG-6-CM-treated mice with CCl4. Ki67-positive hepatocytic cells were accumulated in the liver of the CCl4 + TSG-6 group. RNA analysis showed the decrease in both of inflammation markers, tnfα, il-1β, cxcl1 and cxcl2, and fibrotic markers, tgf-β1, α-sma and collagen α1, in the CCl4 + TSG-6 group, compared to the CCl4 or the CCl4 + NC group. Protein analysis confirmed the lower expression of TGF-β1 and α-SMA in the CCl4 + TSG-6 than the CCl4 or the CCl4 + NC group. Immunostaining for α-SMA also revealed the accumulation of the activated hepatic stellate cells in the livers of mice in the CCl4 and CCl4 + NC groups, but not in the livers of mice from the CCl4 + TSG-6 group. The cultured LX2 cells, human hepatic stellate cell line, in TSG-6-CM showed the reduced expression of fibrotic markers, tgf-β1, vimentin and collagen α1, whereas the addition of the TSG-6 antibody neutralized the inhibitory effect of TSG-6 on the activation of LX2 cells. In addition, cytoplasmic lipid drops, the marker of inactivated hepatic stellate cell, were detected in TSG-6-CM-cultured LX2 cells, only. The suppressed TSG-6 activity by TSG-6 antibody attenuated the restoration process in livers of TSG-6-CM-treated mice with CCl4.ConclusionsThese results demonstrated that TSG-6 contributed to the liver regeneration by suppressing the activation of hepatic stellate cells in CCl4-treated mice, suggesting the therapeutic potential of TSG-6 for acute liver failure.

Hepatic Stellate Cells Express Thymosin Beta 4 in Chronically Damaged Liver

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.

MicroRNA Expression Profiling in CCl4-Induced Liver Fibrosis of Mus musculus

Liver fibrosis is a major pathological feature of chronic liver diseases, including liver cancer. MicroRNAs (miRNAs), small noncoding RNAs, regulate gene expression posttranscriptionally and play important roles in various kinds of diseases; however, miRNA-associated hepatic fibrogenesis and its acting mechanisms are poorly investigated. Therefore, we performed an miRNA microarray in the fibrotic livers of Mus musculus treated with carbon-tetrachloride (CCl4) and analyzed the biological functions engaged by the target genes of differentially-expressed miRNAs through gene ontology (GO) and in-depth pathway enrichment analysis. Herein, we found that four miRNAs were upregulated and four miRNAs were downregulated more than two-fold in CCl4-treated livers compared to a control liver. Eight miRNAs were predicted to target a total of 4079 genes. GO analysis revealed that those target genes were located in various cellular compartments, including cytoplasm, nucleolus and cell surface, and they were involved in protein-protein or protein-DNA bindings, which influence the signal transductions and gene transcription. Furthermore, pathway enrichment analysis demonstrated that the 72 subspecialized signaling pathways were associated with CCl4-induced liver fibrosis and were mostly classified into metabolic function-related pathways. These results suggest that CCl4 induces liver fibrosis by disrupting the metabolic pathways. In conclusion, we presented several miRNAs and their biological processes that might be important in the progression of liver fibrosis; these findings help increase the understanding of liver fibrogenesis and provide novel ideas for further studies of the role of miRNAs in liver fibrosis.

Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling

The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by the decreased expression of both smoothened and GLI2. A TB4 CRISPR also blocked the activation of primary HSCs, with decreased expression of smoothened, GLI2 and ILK compared with cells transfected with nontargeting control CRISPR. Double immunostaining and an immunoprecipitation assay revealed that TB4 interacted with either smoothened at the cytoplasm or GLI2 at the nucleus in LX-2. Smoothened suppression in primary HSCs using a Hh antagonist or adenovirus transduction decreased TB4 expression with the reduced activation of HSCs. Tb4-overexpressing transgenic mice treated with CCl4 were susceptible to the development hepatic fibrosis with higher levels of ILK, pGSK3b, and Hh activity, as compared with wild-type mice. These findings demonstrate that TB4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting TB4 as a novel therapeutic target in liver disease.

Hedgehog Signaling is Associated with Liver Response to Fractionated Irradiation in Mice

Background/Aims: Radiation-induced liver disease (RILD) is a major obstacle in treating liver cancer; however, the mechanisms underlying RILD development remain unclear. Hedgehog (Hh) orchestrates liver response to injury. Herein, we investigated the liver response with Hh to fractionated irradiation (FI) using a small murine model for RILD. Methods: Male mice exposed to liver-targeted FI with 6Gy in 5 consecutive weekly fractions were sacrificed at one day after weekly irradiation and 6 or 10 weeks post 5th FI for the acute and late response model, respectively. Results: The levels of ALT/AST and apoptosis were elevated in all radiation groups. The expression of Hh ligand, Sonic and Indian Hh, and Hh activator, smoothened and gli2, was higher in the acute groups than the control group. Pro-fibrogenic markers were also up-regulated in this model compared with the control group. Histomorphological changes and ballooned hepatocytes were observed in the late response model. Both the expression of Hh and profibrotic genes and the fibrosis level increased in this model compared with the control groups. Conclusion: Enhanced Hedgehog signaling and liver injury with fibrosis in RILD murine model suggests hedgehog as the potential regulator in RILD progression and the suitability of this model for studying RILD.

Tumor necrosis factor-inducible gene 6 protein ameliorates chronic liver damage by promoting autophagy formation in mice

Tumor necrosis factor-inducible gene 6 protein (TSG-6) has recently been shown to protect the liver from acute damage. However, the mechanism underlying the effect of TSG-6 on the liver remains unclear. Autophagy is a catabolic process that targets cell components to lysosomes for degradation, and its functions are reported to be dysregulated in liver diseases. Here we investigate whether TSG-6 promotes liver regeneration by inducing autophagic clearance in damaged livers. Mice fed a methionine choline-deficient diet supplemented with 0.1% ethionine (MCDE) for 2 weeks were injected with TSG-6 (the M+TSG-6 group) or saline (the M+V group) and fed with MCDE for 2 additional weeks. Histomorphological evidence of injury and increased levels of liver enzymes were evident in MCDE-treated mice, whereas these symptoms were ameliorated in the M+TSG-6 group. Livers from this group contained less active caspase-3 and more Ki67-positive hepatocytic cells than the M+V group. The autophagy markers ATG3, ATG7, LC3-II, LAMP2A and RAB7 were elevated in the M+TSG-6 group compared with those in the M+V group. Immunostaining for LC3 and RAB7 and electron microscopy analysis showed the accumulation of autophagy structures in the M+TSG-6 group. TSG-6 also blocked both tunicamycin- and palmitate-induced apoptosis of hepatocytes and increased their viability by inducing autophagy formation in these cells. An autophagy inhibitor suppressed TSG-6-mediated autophagy in the injured hepatocytes and livers of MCDE-treated mice. These results therefore demonstrate that TSG-6 protects hepatocytes from damage by enhancing autophagy influx and contributes to liver regeneration, suggesting that TSG-6 has therapeutic potential for the treatment of liver diseases.