Gastroenterology

Gastroenterology

Volume 137, Issue 4, October 2009, Pages 1478-1488.e8
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease

https://doi.org/10.1053/j.gastro.2009.06.051Get rights and content

Background & Aims

Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors.

Methods

Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc+/−) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD.

Results

In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc+/− mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD.

Conclusions

Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

Section snippets

Animals

C57BL/6 Patched-deficient (Ptc+/−) mice were obtained from P. A. Beachy (Johns Hopkins University, Baltimore, MD), and wild-type mice (WT) were obtained from Jackson Laboratories (Bar Harbor, ME). Ptc+/− mice have only 1 copy of patched, a Hh-pathway repressor. Therefore, these mice are unable to silence Hh signaling and exhibit excessive Hh-pathway activity. WT and Ptc+/− mice were fed the methionine-choline- deficient (MCD) diet (n = 4/group) to induce nonalcoholic steatohepatitis (NASH) and

Exogenous Shh Promotes EMT in Liver Progenitors

To examine the direct effects of Hh-pathway activation on progenitor cell EMT, immature ductular cells (603B cells) were treated with N-terminal Shh ligand (0–1000 ng/mL), and RNA was analyzed by QRT-PCR (Figure 1A). Experiments were repeated by treating cells with Shh (100 ng/mL) with or without cyclopamine 3 μmol/L (a specific Hh-pathway antagonist) or tomatidine 3 μmol/L (an inactive cyclopamine analog), and cellular RNA and protein were obtained for analysis (Figure 1B and Supplementary

Discussion

Hepatocyte injury and death are much more extensive in NASH than in simple steatosis, and this is thought to explain why individuals with NASH are significantly more likely to develop cirrhosis.6, 7 However, the fact that many patients with NASH do not become cirrhotic37 suggests that interindividual differences in the response to liver injury also play an important role in defining the ultimate outcomes of NASH. Results of the present studies demonstrate that, in both mice and humans, the Hh

Acknowledgments

The authors thank Dr Jiawen Huang for his assistance with animal care and Carl Stone for his administrative efforts; Dr P. A. Beachy (Johns Hopkins University, Baltimore, MD) for providing the Patched-deficient (Ptc+/−) mice; Dr G. J. Gores (Mayo Clinic, Rochester, MN) and Yoshiyuki Ueno (Tohoku University, Sendai, Japan) for providing the murine immature ductular cell line (603B); and N. LaRusso (Mayo Clinic, Rochester, MN) for providing the normal rat cholangiocyte line.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by RO1 DK077794 and RO1 DK053792 (to A.M.D.).

    W.-K.S. and Y.J. contributed equally to the work.

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