Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

Abstract

Background: α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP −/− ) mouse model. Methods: α-CGRP −/− and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). Results: There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP-receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP −/− and BDL α-CGRP −/− mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP −/− mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

The CGRP receptor component RAMP1 links sensory innervation with YAP activity in the regenerating liver

“…To elucidate whether RAMP1 may have a more general role for regulating the expression and/or activity of YAP/TAZ during liver regeneration, chronic liver injury was induced by biweekly injections of the hepatocellular toxin CCl 4 for 4 weeks. The CCl 4 treatment caused a significant upregulation of αCGRP and RAMP1 mRNA levels, but did not affect expression of βCGRP and CLR (Figure S3), confirming earlier studies where increased αCGRP or RAMP1 mRNA expression levels were observed upon bile duct ligation or CCl 4 ‐induced liver cirrhosis 37‐39 . In addition, expression of Gα11, but not of Gαq and Gαs was significantly increased after CCl4 treatment of mice (Figure S3).…”

Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2−/− mouse model of primary sclerosing cholangitis (PSC)

“…Previous studies have shown that quiescent hepatic stellate cells (HSCs) are capable of transdifferentiation into myofibroblastic HSCs during the progression of liver fibrosis and gain mesenchymal phenotype [26,27]. We also evaluated the expression change of E-cadherin and vimentin in HSCs by co-staining with desmin, the HSC marker [13,23,28]. With the treatment of vimentin Vivo-Morpholino, we observed less co-expression of desmin/vimentin and increased co-localization of desmin/E-cadherin compared to Mdr2 −/− mice, indicating the HSCs are less myofibroblastic with the knockdown of vimentin (Supplementary Fig.…”

“…Cell viability (>97%) was assessed by trypan blue exclusion. Mouse HSCs were isolated by laser capture microdissection (LCM) using an antibody against desmin (marker of HSCs) [12,22,23].…”

Knockout of the Tachykinin Receptor 1 in the Mdr2−/− (Abcb4−/−) Mouse Model of Primary Sclerosing Cholangitis Reduces Biliary Damage and Liver Fibrosis

“…8e11 Several studies have demonstrated that during the progression of cholestatic liver diseases, cholangiocytes acquire neuroendocrine phenotypes and secrete and respond to several neuropeptides, including a-calcitonin gene related peptide (a-CGRP) and substance P (SP). 9,12 Both a-CGRP and SP are secreted by neuronal and nonneuronal cells, including macrophages, eosinophils, and cholangiocytes. 9,12e15 There is also growing information regarding the role of neurotransmitters and sensory neuropeptides in the regulation of biliary damage and liver fibrosis.…”

“…9,12e15 There is also growing information regarding the role of neurotransmitters and sensory neuropeptides in the regulation of biliary damage and liver fibrosis. 9,12 For example, the a1-adrenergic receptor agonist, phenylephrine, stimulates biliary proliferation via Ca 2þ -dependent activation of nuclear factor of activated T cells 2 and specificity protein 1. 16 In addition, parasympathetic innervation has been shown to modulate biliary proliferation and apoptosis in the bile duct ligated (BDL) rat model.…”