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. 2010 Oct 1;285(40):30463-71.
doi: 10.1074/jbc.M110.156703. Epub 2010 Jul 27.

The Necdin-Wnt pathway causes epigenetic peroxisome proliferator-activated receptor gamma repression in hepatic stellate cells

Nian-Ling Zhu  1 , Jiaohong Wang, Hidekazu Tsukamoto
Affiliations

The Necdin-Wnt pathway causes epigenetic peroxisome proliferator-activated receptor gamma repression in hepatic stellate cells

Nian-Ling Zhu et al. J Biol Chem. .

Abstract

Hepatic stellate cells (HSCs), vitamin A-storing liver pericytes, undergo myofibroblastic trans-differentiation or "activation" to participate in liver wound healing. This cellular process involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Necdin, a melanoma antigen family protein, promotes neuronal and myogenic differentiation while inhibiting adipogenesis. The present study demonstrates that necdin is selectively expressed in HSCs among different liver cell types and induced during their activation in vitro and in vivo. Silencing of necdin with adenovirally expressed shRNA, reverses activated HSCs to quiescent cells in a manner dependent on PPARγ and suppressed canonical Wnt signaling. Promoter analysis, site-directed mutagenesis, and chromatin immunoprecipitation demonstrate that Wnt10b, a canonical Wnt induced in activated HSCs, is a direct target of necdin. Necdin silencing abrogates three epigenetic signatures implicated in repression of PPARγ: increased MeCP2 (methyl CpG binding protein 2) and HP-1α co-repressor recruitments to Pparγ promoter and enhanced H3K27 dimethylation at the exon 5 locus, again in a manner dependent on suppressed canonical Wnt. These epigenetic effects are reproduced by antagonism of canonical Wnt signaling with Dikkopf-1. Our results demonstrate a novel necdin-Wnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPARγ.

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Figures

FIGURE 1.
FIGURE 1.
Necdin mRNA (A) and protein (B) are selectively expressed in HSCs among different liver cell types. KC, Kupffer cells; SEC, sinusoidal endothelial cells; Hep, hepatocytes.
FIGURE 2.
FIGURE 2.
Necdin expression is induced in activated HSCs. A, note that a time-dependent increase in necdin mRNA in cultured HSCs on plastic concomitant with induction of the HSC activation marker α1(I) procollagen (α1(I)Procoll) and suppression of the HSC quiescence marker PPARγ. B, necdin protein level is conspicuously increased in HSCs cultured for 7 days as compared with quiescent day 1 cells in a manner identical to a change in adipocytes (3T3L1 cells treated with the adipocyte differentiation mixture MDI) versus preadipicytes (3T3L1 treated with the solvent DMSO). C, necdin induction also is confirmed in “in vivo activated” HSCs isolated from fibrotic rat livers induced by BDL or repetitive carbon tetrachloride injection (CCl4), again concomitant with induction of α1(I) procollagen and down-regulation of PPARγ. D, necdin protein level is increased in activated HSCs from BDL- or CCl4-induced liver fibrosis as compared with respective controls. *, p < 0.05; **, p < 0.01 compared to Day 1 or controls. Error bars are standard deviations.
FIGURE 3.
FIGURE 3.
Necdin silencing reverses activated HSCs to quiescent cells in a manner dependent on PPARγ. A, nuclear and cytosolic necdin levels were effectively reduced by adenovirally expressed shRNA against necdin (Ad.Necdin.shRNA) in culture-activated HSCs as compared with the cells infected with adenovirus expressing shRNA against LacZ (Ad.LacZ.shRNA). B, necdin silencing with Ad.Necdin.shRNA at 100 MOI reverts activated HSCs to the cells with morphology of quiescent HSCs (Phase) and increases intracellular vitamin A content as assessed by UV-excited autofluorescence (UV) and intracellular lipid content as stained with oil red O (Oil Red). C, these effects are accompanied by reduced expression of α1(I) procollagen (α1(I)Coll). D, necdin silencing does not affect the expression of neuronal (left) or muscle cell (right) markers. E, necdin silencing in culture-activated HSC increases the expression of adipogenic transcription factors (Pparγ, C/ebpα, and Lxrα) and their target lipogenic genes, Acc and Fas (left). As these genes are all down-regulated in culture-activated cells (Day 7 HSC) compared with quiescent day 1 cells (right), the inductions seen after necdin silencing is considered as restoration of normal expressions. F, morphologic reversal of activated HSCs with Ad.Necdin.shRNA is completely abrogated by concomitant expression of dominant-negative PPARγ (+Ad.dnPPARγ) as assessed by phase contrast microscopy and UV-excited autofluorescence. Sma, smooth muscle actin. *, p < 0.05; **, p < 0.01; ***, p < 0.005 as compared to respective controls. Error bars are standard deviations.
FIGURE 4.
FIGURE 4.
Necdin silencing reduces the expression of canonical (Wnt10b and Wnt3a) and noncanonical (Wnt4) Wnts (A) and nuclear level of β-catenin (B) in culture-activated HSCs. C, morphologic reversal to the quiescent state achieved by necdin silencing is prevented by addition of recombinant Wnt3a (100 ng/ml). D, restored PPARγ mRNA expression by necdin silencing is abrogated by Wnt3a. *, p < 0.05 compared to LacZ.shRNA; †, p < 0.05 compared to Necdin.shRNA without Wnt3a.
FIGURE 5.
FIGURE 5.
A, necdin silencing with Ad.Necdin.shRNA at 50 ∼ 200 MOI inhibits Wnt10b proximal promoter (−705/+30) activity in the hepatic stellate cell line (BSC). B, ChIP analysis detects recruitment of necdin to two most proximal ChIP sites (−143/+42 and −268/−119) labeled 1 and 2 within the Wnt10b promoter in day 7 culture-activated HSC infected with Ad.LacZ.shRNA. These binding are abrogated by necdin silencing with Ad.Necdin.shRNA. Day 1 quiescent HSCs show minimal necdin binding to these sites. ChIP data are expressed as relative to maximal recruitment at the most proximal site seen in day 7 HSC tansduced with LacZ.shRNA. C, mutations of a potential necdin binding GN box at −108/−96: GG(T)GTGG(T)GGTGG(T)GG within the Wnt10b proximal promoter, abrogate necdin-inducible promoter activity in Huh7 cells. *, p < 0.05 compared to Ad.LacZ.shRNA.
FIGURE 6.
FIGURE 6.
Necdin silencing reduces epigenetic PPARγ repression. Necdin shRNA reduces MeCP2 (A) and HP-1α (B) binding to Pparγ promoter and H3K27me2 at the exon 5 locus (C), and these effects were abrogated by recombinant Wnt3a (A–C). Adenovirally expressed Dkk-1 largely attenuates MeCP2 enrichment in the Pparγ promoter and H3K27me2 enrichment at exon 5 (D) and restored PPARγ expression (E). *, p < 0.05 compared to LacZ.shRNA or Day 1 Ad.GFP; † compared to Necdin.shRNA without Wnt3a or Day 7 Ad.GFP.
FIGURE 7.
FIGURE 7.
The necdin-Wnt10b pathway activates HSCs via epigenetic repression of PPARγ involving MeCP2 and H3K27me2. This schematic diagram summarizes our finding of necdin-mediated Wnt10b up-regulation causing increased MeCP2 recruitment to the Pparγ promoter and increased H3K27 dimethylation at the Pparγ locus with consequent PPARγ repression and HSC activation.
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