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. 2009 Oct;137(4):1478-1488.e8.
doi: 10.1053/j.gastro.2009.06.051. Epub 2009 Jul 3.

Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease

Wing-Kin Syn  1 , Youngmi Jung, Alessia Omenetti, Manal Abdelmalek, Cynthia D Guy, Liu Yang, Jiangbo Wang, Rafal P Witek, Caitlin M Fearing, Thiago A Pereira, Vanessa Teaberry, Steve S Choi, J Conde-Vancells, Gamze F Karaca, Anna Mae Diehl
Affiliations

Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease

Wing-Kin Syn et al. Gastroenterology. 2009 Oct.

Abstract

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.

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Figures

Figure 1
Figure 1. Treatment with Shh ligand induces Hh-_target genes and EMT-related genes in liver-progenitors
Immature, murine ductular-type progenitor cells (603B) were maintained in standard culture conditions. Recombinant Sonic hedgehog (Shh) protein (0, 100, 1000 ng/ml) was added for 24 hours; cells were harvested, and changes in gene expression were assessed by QRT-PCR analysis. (a) gli1, α-sma, bmp7, inhibitor of differentiation (id)2, keratin-7 and e-cadherin. Results are expressed as fold change relative to vehicle-treated control cultures. Mean ± SEM of duplicate experiments are graphed. To determine if the effects of Shh were directly attributable to Hh-signaling, 603B cells were cultured in the presence of Shh (100 ng/ml) with 3uM cyclopamine (a specific hedgehog-pathway antagonist) or 3uM tomatidine (an inactive cyclopamine analogue) for 24 hours; protein was harvested and analyzed by Western-blot. (b) Quantitative data for these studies are shown in Supplemental Fig 1. *P < 0.05 or ** P< 0.005 vs 0 ng/ml Shh
Figure 2
Figure 2. Rapid induction of EMT-related genes in mice treated with MCDE diets to induce progenitor cell-dependent liver regeneration
C57BL/6 mice were fed control diet or MCD diet + 0.1% ethionine (in drinking water) for 1 week (MCDE diet; n=4 / group). At the end of the treatment period, total RNA was examined by QRT-PCR. (a) mpk, (b) gli2, (c) tgfβ, (d) s100A4, (e) vimentin, and (f) bmp7. Results are expressed as fold change relative to chow-fed controls. Mean ± SEM are graphed. * P < 0.05 or **P < 0.005 vs control
Figure 3
Figure 3. Increased induction of Hh-_target genes and liver fibrosis during diet-induced NASH in Ptc+/- mice with an overly-active Hh-pathway
Ptc+/- mice and wild type control littermates (WT) were fed regular chow or methionine-choline deficient (MCD) diets for 4 weeks. At the end of the treatment period, mice (n = 4/group) were sacrificed. (a) QRT-PCR analysis of gli2 mRNA; (b) Accumulation of Gli-2-positive ductular cells (white bars) and Gli2-positive hepatocytes (solid bars) in WT and Ptc+/- groups. Results are expressed as fold change relative to the respective chow-fed controls and mean ± SEM are graphed; (c-d) immunohistochemistry for Gli2 in representative MCD diet fed-WT (c) and Ptc+/- (d) mice - small inserts in each photomicrograph display Gli2 staining in the respective chow-fed control; (e) collagen mRNA levels and (f) hepatic hydroxyproline content at the end of the treatment period. Results are expressed as fold change relative to the respective chow-fed control group. Mean ± SEM are graphed. * P <0.05 or ** P < 0.005 vs WT groups
Figure 4
Figure 4. Enhanced alterations in expression of EMT-related genes during diet-induced NASH in Ptc+/- mice
QRT-PCR analysis was done to assess changes in expression of several EMT-related genes in total liver RNA from the mice described in the legend to Figure 3 (a) tgfβ, (b) α sma, (c) mmp9, (d) timp1, (e) bmp7, (f) keratin-7. Results are expressed as fold change relative to the respective chow-fed control group and graphed as mean ± SEM. *P < 0.05 vs WT group
Figure 5
Figure 5. Inhibition of Hh-signaling attenuates EMT-associated fibrogenesis in vivo
WT mice were fed either normal chow (n= 4) or MCDE diet (n = 8) for 1 week with or without cyclopamine (n= 4/group; 0.6mg per mouse per day; i.p.). At the end of treatment, total liver RNA was harvested for QRT-PCR (a) gli2, (b) tgf-β, (c) bmp7, (d) vimentin, (e) mpk. Results are expressed as fold change relative to the respective chow-fed control group and graphed as mean ± SEM. * P < 0.05 vs control or MCDE group; **P < 0.005 vs control
Figure 6
Figure 6. Hedgehog-pathway activation in NAFLD patients
Coded liver sections from 16 patients with well-characterized NAFL (n = 5), NASH (n = 5), and NASH-related cirrhosis (n=6) were stained for the Hh-ligand, Shh and the Hh-_target gene, Gli2. Shh staining was analyzed by computer-assisted morphometry. Photomicrographs are from representative patients with (a) NASH (small insert displays Shh staining in NAFL) and (b) NASH-related cirrhosis. (c) Quantitative analysis of Shh in all patients. Amount of Shh is expressed as % of stained cells per high-powered field (Magnification X400). The number of Gli2-stained cells was determined in 10 high-power fields / section on liver sections by blinded observers. Gli2 staining in (d) NASH (small insert displays Gli2 staining in NAFL), and (e) NASH-related cirrhosis. (f) Quantitative analysis of Gli2 in all patients. Data are graphed as mean ± numbers of Gli2 cells/high-powered field (Magnification X400). *P< 0.05 or **P < 0.005 vs NAFL
Figure 7
Figure 7. Evidence for EMT in patients with NAFL, NASH and NASH-related cirrhosis
Coded liver sections from the patients described in Figure 6 legend were stained for S100A4, a marker of fibroblasts derived from epithelial cells. S100A4-staining was analyzed by computer-assisted morphometry. Photomicrographs are from representative patients with (a) NAFL, (b) NASH, and (c) NASH-related cirrhosis (Magnification X400). (d) Low-power view of S100A4 staining in a representative patient with NAFLD-related cirrhosis (Magnification x100). (e) Quantitative analysis of S100A4 in all patients. Amount of S100A4 is expressed as % of stained cells per high-powered field. *P < 0.05 or ** P < 0.005 vs NAFL. (f) Representative Gli2 (brown) and Vimentin (blue) - double-immuno-staining in NASH-related cirrhosis. Small Insert displays Gli2 and vimentin-double staining in human healthy liver (Magnification x630).
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