Differential impact of MT1 and MT2 melatonin receptor deletion on biliary proliferation, senescence, and liver fibrosis during cholestatic liver injury

Senescent cholangiocytes (a key feature in primary sclerosing cholangitis, PSC) contribute to liver fibrosis through activation of hepatic stellate cells (HSCs) by autocrine/paracrine pathways. Melatonin decreases biliary hyperplasia and liver fibrosis in bile duct ligated (BDL) rats. No data exists on the role of MT1/MT2 melatonin receptors on biliary homeostasis and liver fibrosis. We studied the role of melatonin MT1/MT2 on biliary senescence, proliferation and liver fibrosis during BDL. Methods: Wild-type (WT), MT1-/- or MT2-/- mice with/without BDL for 1 wk were used. BDL WT mice were treated with Vivo-Morpholinos to silence MT1 or MT2. MT1/MT2 levels were evaluated in liver sections by IHC and in normal and PSC human liver samples by IHC and qPCR. Intrahepatic biliary mass (IBDM) and proliferation were evaluated by IHC for CK-19 and Ki67 in liver sections and qPCR/immunoblots for PCNA in cholangiocytes. SA-β-gal staining and qPCR for p18/p21 was used to evaluate biliary senescence. Liver fibrosis was measured by Sirius red staining and qPCR for fibrosis genes (TGF-β1, α-SMA, fibronectin and collagen alpha 1) in total liver, cholangiocytes and Laser Capture Microdissection (LCM)-isolated HSCs. In vitro, human HSCs (hHSCs) were treated with biliary supernatants from all groups of mice before measuring the expression of fibrosis genes by qPCR. Results: MT1/MT2 in bile ducts was increased in BDL compared to WT mice. There was increased MT1/MT2 expression in human PSC samples compared to controls Biliary senescence, proliferation and liver fibrosis were increased in BDL compared to WT mice. Biliary senescence, proliferation and liver fibrosis were increased in MT2-/- mice during BDL, but there was reduced biliary senescence, proliferation and liver fibrosis in MT1-/- mice during BDL. A similar profile was observed in BDL WT mice treated with Vivo-Morpholinos for MT1 or MT2. In LCM-HSCs from BDL mice, fibrotic gene expression was increased in MT2-/- but decreased in MT1-/- mice. In hHSCs treated with biliary supernatants from BDL WT mice, there was increased expression of fibrosis genes compared to controls. Fibrotic gene expression was reduced in hHSC treated with biliary supernatants from BDL MT1-/- mice and enhanced by supernatants from BDL MT2-/- compared to hHSC treated with supernatants from BDL WT mice. Increased hHSC fibrotic gene expression is due to senescence of MT2-/- cholangiocytes. Conclusion: Biliary senescence, proliferation and liver fibrosis are differentially regulated in MT1-/- and MT2-/- mice. Specific targeting of MT1 receptor may be a novel approach for ameliorating biliary damage and liver fibrosis during chronic liver diseases.