RAR‐Related Orphan Receptor Gamma (ROR‐γ) Mediates Epithelial‐Mesenchymal Transition Of Hepatocytes During Hepatic Fibrosis

The epithelial‐mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR‐related orphan receptor gamma (ROR‐γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF‐β1. Expression of ROR‐γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR‐γ in hepatocyte EMT, we silenced ROR‐γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR‐γ silencing was investigated in a mouse model of TAA‐induced fibrosis by hydrodynamic injection of plasmids. ROR‐γ expression was elevated in hepatocyte cells treated with TGF‐β1, and ROR‐γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR‐γ resulted in the attenuation of TGF‐β1‐induced EMT in hepatocytes. Strikingly, ROR‐γ bound to ROR‐specific DNA response elements (ROREs) in the promoter region of TGF‐β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR‐γ. Therapeutic delivery of shRNA against ROR‐γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA‐induced liver fibrosis. Overall, our results suggest that ROR‐γ regulates TGF‐β‐induced EMT in hepatocytes during liver fibrosis. We suggest that ROR‐γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026–2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.     

MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII

Atrial fibrosis serves as an important contributor to atrial fibrillation (AF). Recent data have suggested that microRNA‐30c (miR‐30c) is involved in fibrotic remodelling and cancer development, but the specific role of miR‐30c in atrial fibrosis remains unclear. The purpose of this study was to investigate the role of miR‐30c in atrial fibrosis and its underlying mechanisms through in vivo and in vitro experiments. Our results indicate that miR‐30c is significantly down‐regulated in the rat abdominal aortic constriction (AAC) model and in the cellular model of fibrosis induced by transforming growth factor‐β1 (TGF‐β1). Overexpression of miR‐30c in cardiac fibroblasts (CFs) markedly inhibits CF proliferation, differentiation, migration and collagen production, whereas decrease in miR‐30c leads to the opposite results. Moreover, we identified TGFβRII as a target of miR‐30c. Finally, transferring adeno‐associated virus 9 (AAV9)‐miR‐30c into the inferior vena cava of rats attenuated fibrosis in the left atrium following AAC. These data indicate that miR‐30c attenuates atrial fibrosis via inhibition of CF proliferation, differentiation, migration and collagen production by targeting TGFβRII, suggesting that miR‐30c might be a novel potential therapeutic target for preventing atrial fibrosis.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.