The Necdin-Wnt Pathway Causes Epigenetic Peroxisome Proliferator-activated Receptor γ Repression in Hepatic Stellate Cells

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 ␥ (PPAR␥). 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 PPAR␥ 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 PPAR␥: increased MeCP2 (methyl CpG binding protein 2) and HP-1␣ co-repressor recruitments to Ppar␥ 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 necdinWnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPAR␥. Hepatic stellate cells (HSCs)2 are desmin-positive mesenchymal cells located in the subendothelial (perisinusoidal) space of hepatic sinusoids. They store vitamin A, function as hepatic pericytes, communicate with hepatocytes via a gap junction and the release of soluble factors, and produce the components of the normal matrix milieu of the perisinusoidal space. HSCs also actively participate in matrix remodeling and wound healing through their production of matrix metalloproteinases and extracellular matrix proteins and are considered as one of the principal cell types responsible for the genesis of liver fibrosis (1). The most unique feature of the manner in which HSCs participate in liver fibrogenesis is their myofibroblastic transdifferentiation (activation) characterized by depletion of vitamin A storage, cell proliferation, induction of matrix genes, and acquisition of the myofibroblastic phenotype via induction of ␣-smooth muscle actin. Although major classes of mediators are identified for HSC trans-differentiation, including soluble factors (cytokines, hormones, and lipid mediators), reactive oxygen species, and altered extracellular matrix milieu (1), the fundamental understanding of cell lineage and cell fate regulation of HSCs is elusive. In this regard, Asahina et al. (2) has demonstrated recently the mesenchymal origin of fetal HSCs and "submesothelial cell" as a potential precursor for HSCs. Intriguingly, HSCs express markers for cell types derived from multipotent mesenchymal progenitor cells such as neural cells, chondrocyt...

Aortic carboxypeptidase–like protein, a WNT ligand, exacerbates nonalcoholic steatohepatitis

“…Immunofluorescence doublestaining of ACLP and β-catenin in these specimens demonstrated a significant increase in ACLP/β-catenin double-positive cells as compared with normal livers, and β-catenin was upregulated specifically in ACLP-positive cells in NASH ( Figure 3D and Supplemental Figure 13). As for the mRNA expression levels of Wnt1, Wnt3a, Wnt5a, and Wnt10b, which are reported to be correlated with the canonical WNT pathway in liver pathology (12,16,20,21), murine NAFLD livers and HSCs as well as human NAFLD liver samples demonstrated no changes when compared with their respective controls (Supplemental Figure 14). The above results indicated that in the progression of NASH, HSC-derived ACLP acts on HSCs themselves, thereby activating the HSCs and advancing liver fibrosis.…”

“…Because ACLP/canonical WNT pathway activation triggered HSC activation, we examined this pathological mechanism further. It has been recently reported that activation of canonical WNT signaling represses PPARγ activity in HSCs, thereby promoting their activation (12,16). Therefore, we examined PPARγ activity upon the addition of rACLP to HSCs.…”

“…HSC activation has recently been reported to correlate with canonical WNT pathway activation (12,16). Therefore, we examined mRNA expression of Axin2, Myc, and Ccnd1, which are downstream targets of this pathway, in HSCs from NASH model mice.…”

Epigenetic Modifications in Hepatic Stellate Cells Contribute to Liver Fibrosis

“…Necdin protein can prevent HSCs from developing adipogenic characters, while sustain the forming of myogenic characters which are favorable to the fibrogenesis (Tseng et al 2005;Zhu et al 2010). Canonical wingless-related MMTV integration site 10b (Wnt10b), downstream of necdin, has a "GN" box in its promoter.…”

Necdin enhances muscle reconstitution of dystrophic muscle by vessel-associated progenitors, by promoting cell survival and myogenic differentiation

“…Interestingly, time course analysis of adipogenic markers shows that overexpression of necdin decreases their levels of expression. Necdin inhibition of adipogenic differentiation of pre-adipocytes has been previously described, 26,27 but no data are available on the co-factor that may cooperate with necdin in this process, and more experiments will be required to get more insight into this issue. There is evidence that in the aged or dystrophic muscle mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in degenerating skeletal muscle, in aging or dystrophy.…”