Neuropeptide Calcitonin Gene-related Peptide Signaling via RAMP1 Stimulates Liver Fibrosis and Regulates Yes-associated Protein Activity during Chronic Liver Damage

Background & aims: Chronic liver damage can ultimately lead to liver cirrhosis. Sensory nerves innervating the liver secrete the neuropeptide calcitonin gene-related peptide (CGRP), which binds to receptor activity-modifying protein (RAMP)1. We investigated whether the loss of RAMP1 affects liver fibrosis in chronic liver inflammation. Methods: Wild-type and RAMP1-deficient mice were injected with carbon tetrachloride (CCl4) to induce liver injury. CGRP/RAMP1 levels in CCl4-treated livers were determined by RT-PCR. Fibrosis was assessed by Sirius red staining and Western blot (α-SMA and collagen). The influence of CGRP/RAMP1 on the Hippo signaling pathway was assessed by Western blot. In addition, we stimulated the human hepatic stellate cell line (LX-2) in vitro with CGRP. The expression of α-SMA, collagen and YAP proteins was quantified by Western blot. Results: Chronic liver injury resulted in upregulation of hepatic CGRP/RAMP1 mRNA expression. In the absence of RAMP1, murine livers exhibited less fibrosis. In addition, expression of markers for hepatocyte proliferation was decreased. Also, the expression of YAP was diminished in RAMP1-deficient mice. At the same time, phosphorylation of YAP on Ser127, which promotes inactivation of YAP, was increased in RAMP1-deficient livers. In vitro, stimulation of LX-2 cells with CGRP promoted hepatic stellate cell activation. Moreover, treatment with CGRP resulted in inactivation of YAP, confirming our in vivo results. Conclusion: RAMP1/CGRP signaling promotes liver fibrosis and controls YAP activity, as well as the activation of LX-2 cells in vitro. The CGRP receptor RAMP1 may serve as a potential target of antifibrotic therapy in liver disease.