Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells

Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) accompanies myofibroblastic transdifferentiation of hepatic stellate cells (HSC), the primary cellular event underlying liver fibrogenesis. The treatment of activated HSC in vitro or in vivo with synthetic PPARgamma ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPARgamma is indeed a molecular target of this effect, because the ligands are also known to have receptor-independent actions. To test this question, the present study examined the effects of forced expression of PPARgamma via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPARgamma itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype with retracted cytoplasm, prominent dendritic processes, reduced stress fibers, and accumulation of retinyl palmitate. These effects are abrogated by concomitant expression of a dominant negative mutant of PPARgamma that prevents transactivation of but not binding to the PPAR response element. PPARgamma expression also inhibits the activation markers such as the expression of alpha-smooth muscle actin, type I collagen, and transforming growth factor beta1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPARgamma is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPARgamma interaction as demonstrated by co-immunoprecipitation and glutathione S-transferase pull-down analysis. Further, the use of deletion constructs reveals that the DNA binding region of PPARgamma is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPARgamma reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPARgamma and JunD.     

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARγ) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARγ ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1α(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARγ through an adenovirus-expressing PPARγ (Ad-PPARγ), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARγ is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARγ in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARγ in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARγ. It is likely that PPARγ reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.     

Hepatic stellate cell-derived delta-like homolog 1 (DLK1) protein in liver regeneration

Hepatic stellate cells (HSCs) undergo myofibroblastic activation in liver fibrosis and regeneration. This phenotypic switch is mechanistically similar to dedifferentiation of adipocytes as such the necdin-Wnt pathway causes epigenetic repression of the master adipogenic gene Pparγ, to activate HSCs. Now we report that delta-like 1 homolog (DLK1) is expressed selectively in HSCs in the adult rodent liver and induced in liver fibrosis and regeneration. Dlk1 knockdown in activated HSCs, causes suppression of necdin and Wnt, epigenetic derepression of Pparγ, and morphologic and functional reversal to quiescent cells. Hepatic Dlk1 expression is induced 40-fold at 24 h after partial hepatectomy (PH) in mice. HSCs and hepatocytes (HCs) isolated from the regenerating liver show Dlk1 induction in both cell types. In HC and HSC co-culture, increased proliferation and Dlk1 expression by HCs from PH are abrogated with anti-DLK1 antibody (Ab). Dlk1 and Wnt10b expression by Sham HCs are increased by co-culture with PH HSCs, and these effects are abolished with anti-DLK Ab. A tail vein injection of anti-DLK1 Ab at 6 h after PH reduces early HC proliferation and liver growth, accompanied by decreased Wnt10b, nonphosphorylated β-catenin, p-β-catenin (Ser-552), cyclins (cyclin D and cyclin A), cyclin-dependent kinases (CDK4, and CDK1/2), p-ERK1/2, and p-AKT. In the mouse developing liver, HSC precursors and HSCs express high levels of Dlk1, concomitant with Dlk1 expression by hepatoblasts. These results suggest novel roles of HSC-derived DLK1 in activating HSCs via epigenetic Pparγ repression and participating in liver regeneration and development in a manner involving the mesenchymal-epithelial interaction.