MiR-200b modulates angiogenesis and liver fibrosis in the Mdr2-/- model of primary sclerosing cholangitis

Biliary hyperplasia and liver fibrosis are hallmarks of chronic cholestatic liver diseases such as primary sclerosing cholangitis (PSC). Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone (synthesized from GnRH hypothalamic neurons as well as cholangiocytes) that regulates proliferative capacity in many cells by interaction with GnRHR1 and GnRHR2. We have shown that both GnRH (by interaction with GnRHR1) and VEGF (by interaction with its receptors, VEGFR-2/3) stimulate biliary proliferation during cholestatic liver injury. miR-200b regulates angiogenic signaling through VEGF and its receptors. Thus, in the Mdr2-/- PSC mouse model, we evaluated the molecular mechanisms by which GnRH regulates liver fibrosis and angiogenesis. Methods: Wild-type (WT) mice were treated with saline or GnRH (1.0 mg/day), while Mdr2-/- mice were treated with scrambled Morpholino or GnRH Morpholino sequences (1.0 mg/Kg BW/day to reduce the hepatic GnRH expression) for 1 wk before evaluating serum GnRH levels and liver fibrosis (by Sirius red staining) and the expression of GnRHR1 (by immunoblots), fibrosis (fibronectin, collagen type I alpha 1, and α-smooth muscle actin) and angiogenesis (VEGF-A/C and angiopoietin 1 and 2) genes and miR-200b in total liver and cholangiocytes by qPCR. In early stage human PSC samples (serum and total liver), we measured serum GnRH levels and the expression of GnRH and GnRHR1 by qPCR. In vitro, cholangiocytes or human hepatic stellate cell lines (hHSC) (expressing GnRH receptors) were treated for 24 hr with GnRH (100 nM) with/without GnRHR antagonist (cetrorelix acetate, 50 nM) and cholangiocytes were also treated with miRNA 200b inhibitor before evaluating cell proliferation and the expression of fibrosis and angiogenesis genes. Results: Treatment of WT mice with GnRH increased liver fibrosis and expression of fibrosis and angiogenesis genes. In Mdr2-/- mice, there was enhanced GnRH serum levels and liver fibrosis in liver sections, and increased expression of fibrosis and angiogenic genes and miR-200b compared to WT mice; all of which were blocked by administration of GnRH Morpholino. The expression of GnRH and its receptors increases in Mdr2-/- and PSC samples compared to controls. In vitro, GnRH increased proliferation and expression of fibrosis and angiogenesis genes of cholangiocytes and hHSC, which was blocked by GnRHR antagonist. Inhibition of miR-200b prevented the fibrotic and angiogenic reaction in cholangiocytes. Conclusions: Our findings unravel a novel function for GnRH in maintaining the angiogenic phenotype and further indicate that this effect is mediated through the regulation of miR-200b during biliary fibrosis. Furthermore, these results suggest a plausible etiologic explanation for progressive cholestatic liver injury and suggest the potential therapeutic use of inhibitors for GnRH signaling and miR-200b