Hepatic stellate cells (HSCs) constitute the major mesenchymal cell type of the liver and play pivotal roles in a wound response. Upon injury, HSCs undergo myofibroblastic trans- differentiation (MTD) to participate in scar formation and cause liver fibrosis and cirrhosis if injury is sustained. Our research to date demonstrates this MTD is caused by re-activation of morphogens such as Necdin, Wnt, and delta-like homolog (DLK1). Necdin directly upregulates Wnt10b via its binding to a putative Necdin site (GN box) of the canonical Wnt10b promoter. Wnt10b and DLK1 positively cross-regulate each other and epigenetically repress the HSC quiescence gene Ppar? to drive HSC MTD. This epigenetic regulation is caused by increased levels of the methyl-CpG binding protein MeCP2 which binds to the Ppar? promoter to recruit HDAC and the co-repressor HP-1? while stimulating expression of EZH2 for consequent H3K27 di- or tri-methylation at the 3'exons to silence the gene. Our results also suggest that canonical Wnt pathway increases MeCP2 protein via suppression of miR-212 and miR-132 which normally block MeCP2 translation. We also demonstrate HSCs transiently activated in liver regeneration after partial hepatectomy express DLK1 which stimulates hepatocyte DLK1 expression via a paracrine manner to support early liver growth. This notion of HSC-hepatocyte crosstalk with DLK1 is also supported by our preliminary results in acute CCl4 injury model indicating initial DLK1 induction by activated HSCs is followed by DLK1 induction in regenerating hepatocytes. To extend our research, we will test the hypotheses: 1) canonical Wnt pathway activates HSCs via downregulation of miR-212 and miR-132 and consequent MeCP2-mediated Ppar? epigenetic repression and 2) HSC-derived DLK1 is a novel mitogen which initiates liver regeneration via HSC-hepatocyte crosstalk. We will test the causality of reduced miR-212/132 in Wnt-mediated MeCP2 induction by expressing miR-212/132 mimics or anti-miR-212/132 under canonical Wnt antagonism or activation in HSCs (Aim 1a and b). Once the causality is established, we will determine whether or how canonical Wnt pathway epigenetically or transcriptionally repress miR-212/132 (Aim 1-c and d). To test the novel mitogenic role of DLK1, acute and chronic CCl4 mouse models will be used to determine temporal and cell-type specific expression of DLK1 by immunostaining and analysis of isolated HSCs vs. hepatocytes and to test the effects of anti-DLK1 antibody administration on liver fibrosis and regeneration (Aim 2-a and b). To determine respective roles of DLK expressed by activated HSCs vs. hepatocytes, we will render acute and chronic CCl4 hepatotoxicity to compound mice generated from Dlk1+(m)/flox(p) mice crossed with ?1(I) collagen promoter-Cre or Albumin-Cre mice to determine the effects of respective conditional knockout on liver fibrosis and regeneration. Collectively, these efforts will define upstream mechanisms of MeCP2 upregulation responsible for Ppar? repression in HSC MTD and the role of HSC-derived DLK1 in liver fibrosis and regeneration, and provide new insights into potential therapeutic targets for chronic liver disease.

Public Health Relevance

Hepatic stellate cells (HSCs) constitute the major mesenchymal cell type of the liver and play pivotal roles in a wound response. Upon injury; HSCs undergo myofibroblastic trans- differentiation (MTD) to participate in scar formation and cause liver fibrosis and cirrhosis if injury is sustained. Our research to date demonstrates this MTD is caused by re-activation of morphogens such as Necdin; Wnt; and delta-like homolog (DLK1). Necdin directly upregulates Wnt10b via its binding to a putative Necdin site (GN box) of the canonical Wnt10b promoter. Wnt10b and DLK1 positively cross-regulate each other and epigenetically repress the HSC quiescence gene Ppar to drive HSC MTD. This epigenetic regulation is caused by increased levels of the methyl-CpG binding protein MeCP2 which binds to the Ppar promoter to recruit HDAC and the co-repressor HP-1 while stimulating expression of EZH2 for consequent H3K27 di- or tri-methylation at the 3' exons to silence the gene. Our results also suggest that canonicalWnt pathway increases MeCP2 protein via suppression of miR-212 and miR-132 which normally block MeCP2 translation. We also demonstrate HSCs transiently activated in liver regeneration after partial hepatectomy express DLK1 which stimulates hepatocyte DLK1 expression via a paracrine manner to support early liver growth. This notion of HSC-hepatocyte crosstalk with DLK1 is also supported by our preliminary results in acute CCl4 injury model indicating initial DLK1 induction by activated HSCs is followed by DLK1 induction in regenerating hepatocytes. To extend our research; we will test the hypotheses: 1) canonical Wnt pathway activates HSCs via downregulation of miR-212 and miR-132 and consequent MeCP2-mediated Ppar epigenetic repression and 2) HSC-derived DLK1 is a novel mitogen which initiates liver regeneration via HSC-hepatocyte crosstalk. We will test the causality of reduced miR-212/132 in Wnt-mediated MeCP2 induction by expressing miR-212/132 mimics or anti-miR-212/132 under canonical Wnt antagonism or activation in HSCs (Aim 1a and b). Once the causality is established; we will determine whether or how canonical Wnt pathway epigenetically or transcriptionally repress miR-212/132 (Aim 1-c and d). To test the novel mitogenic role of DLK1; acute and chronic CCl4 mouse models will be used to determine temporal and cell-type specific expression of DLK1 by immunostaining and analysis of isolated HSCs vs. hepatocytes and to test the effects of anti-DLK1 antibody administration on liver fibrosis and regeneration (Aim 2-a and b). To determine respective roles of DLK expressed by activated HSCs vs. hepatocytes; we will render acute and chronic CCl4 hepatotoxicity to compound mice generated from Dlk1+(m)/flox(p) mice crossed with 1(I) collagen promoter-Cre or Albumin-Cre mice to determine the effects of respective conditional knockout on liver fibrosis and regeneration. Collectively; these efforts will define upstream mechanisms of MeCP2 upregulation responsible for Ppar repression in HSC MTD and the role of HSC-derived DLK1 in liver fibrosis and regeneration; and provide new insights into potential therapeutic targets forchronic liver disease.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX001991-01A1
Application #
8540076
Study Section
Gastroenterology (GAST)
Project Start
2013-10-01
Project End
2017-09-30
Budget Start
2013-10-01
Budget End
2014-09-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
VA Greater Los Angels Healthcare System
Department
Type
DUNS #
066689118
City
Los Angeles
State
CA
Country
United States
Zip Code
90073