Calreticulin regulates hepatic stellate cell activation through modulating TGF-beta-induced Smad signaling

Abstract

Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) as a wound healing process. Activated hepatic stellate cells (HpSCs) are the major producer of the ECM and play a central role in liver fibrogenesis. It has been widely accepted that elimination of activated HpSCs or reversion to a quiescent state can be a feasible strategy for resolving the disease, further highlighting the urgent need for novel therapeutic targets. Calreticulin (CRT) is a molecular chaperone that normally resides in the endoplasmic reticulum (ER), important in protein folding and trafficking through the secretory pathway. CRT also plays a critical role in calcium (Ca²⁺) homeostasis, with its Ca²⁺ storage capacity. In the current study, we aimed to demonstrate its function in directing HpSC activation. In a mouse liver injury model, CRT was up-regulated in HpSCs. In cellular experiments, we further showed that this activation was through modulating the canonical TGF-β signaling. As down-regulation of CRT in HpSCs elevated intracellular Ca²⁺ levels through a form of Ca²⁺ influx, named store-operated Ca²⁺ entry (SOCE), we examined whether moderating SOCE affected TGF-β signaling. Interestingly, blocking SOCE had little effect on TGF-β-induced gene expression. In contrast, inhibition of ER Ca²⁺ release using the inositol trisphosphate receptor inhibitor 2-APB increased TGF-β signaling. Treatment with 2-APB did not alter SOCE but decreased intracellular Ca²⁺ at the basal level. Indeed, adjusting Ca²⁺ concentrations by EGTA or BAPTA-AM chelation further enhanced TGF-β-induced signaling. Our results suggest a crucial role of CRT in the liver fibrogenic process through modulating Ca²⁺ concentrations and TGF-β signaling in HpSCs, which may provide new information and help advance the current discoveries for liver fibrosis.