Jeongeun Hyun

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.

Potential role of Hedgehog signaling and microRNA-29 in liver fibrosis of IKKβ-deficient mouse

Recent studies have reported that NF-κB mediated down-regulation of miRNA-29 and lower expression of miRNA-29 promoted the deposition of collagens in fibrotic liver. Our previous research demonstrated that the increased Hedgehog (Hh) signaling, a key regulator for hepatic fibrogenesis, induced the severe hepatic fibrosis in the livers with impaired NF-κB signaling. These findings led us to investigate the effect of Hh and miRNA-29 on the hepatic fibrosis under dysregulated NF-κB signaling. In this study, we used IKKβF/F and IKKβ-deficient IKKβΔHEP mouse model with a defective NF-κB signaling pathway, and assessed the expression of the miRNA-29 family (miRNA-29a, miRNA-29b, and miRNA-29c), Hh, and proliferation of MF-HSCs in liver from IKKβF/F mice and IKKβΔHEP mice both before and after MCDE treatment. The activation of NF-κB was significantly increased in MCDE diet-fed IKKβF/F mice compared to IKKβΔHEP mice. Expression of miRNA-29 family was greater in MCDE diet-fed IKKβΔHEP mice than IKKβF/F mice, demonstrating that the impaired NF-κB pathway was unable to suppress the expression of miRNA-29s after injury. However, expression of the Hh signaling pathway was greatly enhanced, and activation of Hh promoted the accumulation of MF-HSCs with impaired NF-κB, eventually increasing fibrogenesis in the damaged liver of IKKβΔHEP mice. Therefore, these results demonstrated that Hh signaling regulates the proliferation of MF-HSCs irrespective of the action of miRNA-29, eventually contributing hepatic fibrosis, when the NF-κB pathway is disrupted.

MicroRNAs in liver fibrosis: Focusing on the interaction with hedgehog signaling.

Liver fibrosis is a repair process in response to damage in the liver; however, severe and chronic injury promotes the accumulation of fibrous matrix, destroying the normal functions and architecture of liver. Hepatic stellate cells (HSCs) are quiescent in normal livers, but in damaged livers, they transdifferentiate into myofibroblastic HSCs, which produce extracellular matrix proteins. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged livers and contributes to liver fibrogenesis by regulating HSC activation. MicroRNAs (miRNAs), endogenous small non-coding RNAs interfering with RNA post-transcriptionally, regulate various cellular processes in healthy organisms. The dysregulation of miRNAs is closely associated with diseases, including liver diseases. Thus, miRNAs are good targets in the diagnosis and treatment of various diseases, including liver fibrosis; however, the regulatory mechanisms of miRNAs that interact with Hh signaling in liver fibrosis remain unclear. We review growing evidence showing the association of miRNAs with Hh signaling. Recent studies suggest that Hh-regulating miRNAs induce inactivation of HSCs, leading to decreased hepatic fibrosis. Although miRNA-delivery systems and further knowledge of interacting miRNAs with Hh signaling need to be improved for the clinical usage of miRNAs, recent findings indicate that the miRNAs regulating Hh signaling are promising therapeutic agents for treating liver fibrosis.

Hedgehog-YAP Signaling Pathway Regulates Glutaminolysis to Control Activation of Hepatic Stellate Cells

BACKGROUND & AIMS Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP. METHODS Primary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis. RESULTS Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression. CONCLUSIONS Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

Potential Roles of Hedgehog and Estrogen in Regulating the Progression of Fatty Liver Disease

Non-alcoholic fatty liver disease accompanies the rise in the prevalence of obesity, diabetes and the tendency toward high-fat dietary habits. Specifically, the higher prevalence of non-alcoholic fatty liver disease in men and postmenopausal women seems to be caused by the protective effects of estrogen against liver fibrosis, or lack thereof. There are no effective preventive therapies for liver diseases because the mechanisms underlying the progression of fatty liver diseases to chronic liver diseases and the protective effects of estrogen against fibrogenesis remain unclear. Recently, it has been reported that the hedgehog signaling pathway plays an important role in the progression of chronic liver diseases. Hedgehog, a morphogen regulating embryonic liver development, is expressed in injured livers but not in adult healthy livers. The level of hedgehog expression parallels the stages of liver diseases. Hedgehog induces myofibroblast activation and hepatic progenitor cell proliferation and leads to excessive liver fibrosis, whereas estrogen inhibits the activation of hepatic stellate cells to myofibroblasts and prevents liver fibrosis. Although the mechanism underlying the opposing actions of hedgehog and estrogen on liver fibrosis remain unclear, the suppressive effects of estrogen on the expression of osteopontin, a profibrogenic extracellular matrix protein and cytokine, and the inductive effects of hedgehog on osteopontin transcription suggest that estrogen and hedgehog are associated with liver fibrosis regulation. Therefore, further research on the estrogen-mediated regulatory mechanisms underlying the hedgehog-signaling pathway can identify the mechanism underlying liver fibrogenesis and contribute to developing therapies for preventing the progression of fibrosis to chronic liver diseases.