Depletion of peroxisome proliferator-activated receptor ␥ (PPAR␥) 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 PPAR␥ ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPAR␥ 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 PPAR␥ via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPAR␥ 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 PPAR␥ that prevents transactivation of but not binding to the PPAR response element. PPAR␥ expression also inhibits the activation markers such as the expression of ␣-smooth muscle actin, type I collagen, and transforming growth factor ␤1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPAR␥ is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPAR␥ 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 PPAR␥ is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPAR␥ reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPAR␥ and JunD.

Activation of hepatic stellate cells (HSC), a key event in liver fibrosis, is caused by diminished adipogenic transcription. This study investigated whether Wnt signaling contributes to "antiadipogenic" activation of HSC and liver fibrogenesis. Culture-activated HSC from normal rats and HSC from cholestatic rat livers were examined for expression of Wnt, Frizzled (Fz) receptors, and coreceptors by quantitative PCR. Wnt signaling was assessed by nuclear beta-catenin and T cell factor (TCF) promoter activity. Dickkopf-1 (Dkk-1), a Wnt coreceptor antagonist, was transduced by an adenoviral vector to assess the effects of Wnt antagonism on culture activation of HSC and cholestatic liver fibrosis in mice. Messenger RNA for canonical (Wnt3a and 10b) and noncanonical (Wnt4 and 5a) Wnt genes, Fz-1 and 2, and coreceptors [low-density lipoprotein-receptor-related protein (LRP)6 and Ryk] are increased approximately 3-12-fold in culture-activated HSC compared with quiescent HSC. The nuclear beta-catenin level and TCF DNA binding are markedly increased in activated HSC. TCF promoter activity is stimulated with Wnt1 but inhibited by Chibby, a protein that blocks beta-catenin interaction with TCF, and by Dkk-1. Dkk-1 enhances peroxisome proliferator-activated receptor-gamma (PPARgamma)-driven PPAR response element (PPRE) promoter activity, a key adipogenic transcriptional parameter, abrogates agonist-stimulated contraction, and restores HSC quiescence in culture. High expression of Dkk-1 increases apoptosis of cultured HSC. Expression of Wnt and Fz genes is also induced in HSC isolated from experimental cholestatic liver fibrosis, and Dkk-1 expression ameliorates this form of liver fibrosis in mice. These results demonstrate antiadipogenic Wnt signaling in HSC activation and therapeutic potential of Wnt antagonism for liver fibrosis.

In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-β signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial–mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.

Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative realtime reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TM-PRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels. (Blood. 2011;117(5): 1687-1699) IntroductionHepcidin is the key iron regulatory peptide hormone in the maintenance of iron homeostasis. It is secreted predominantly by hepatocytes. 1,2 Under physiologic conditions, its expression is regulated positively by body iron content through the bone morphogenic protein (BMP)-mediated signaling cascade. [3][4][5] In recent studies researchers have identified several proteins that can modulate BMP signaling and hepcidin expression directly or indirectly.BMP2, 4, 5, 6, 7, and 9 are cytokines of the BMP subfamily that belong to the transforming growth factor-␤ (TGF-␤) superfamily. 6 Each of these BMP ligands induces BMP signaling through receptor-activated Smad1, Smad5, and Smad8 (Smad1/5/8) and markedly increases hepcidin expression in hepatocytes. 7,8 BMP2,4,5,and 6 can also bind hemojuvelin (HJV), a BMP coreceptor, to enhance BMP signaling, resulting in an increase in hepcidin expression. 4,7 HJV is a glycosylphosphatidyl-inositol-linked membrane protein that is expressed in skeletal muscle, heart, and hepatocytes, and it plays a pivotal role in the induction of hepcidin expression. [9][10][11] Both homozygous or compound heterozygous mutations in the HJV gene, HFE2, in humans and disruption of both Hfe2 alleles in mice result in suppression of hepcidin expression and severe iron overload in the liver, pancreas, and heart. 10,12,13 In addition to BMPs, TGF-␤1 can also induce hepatic hepcidin expression. 5 BMP6 mRNA, but no other BMP mRNA, is downregulated by chronic iron depletion and up-regulated by iron loading. 3 Knockdown of the BMP6 gene in mice causes suppression of hepatic hepcidin expression. 3,14,15 These observations implicate BMP6 as a critical player in the iron-sensitive induction of hepcidin expr...

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