LIM-domain protein CRP2 is upregulated in activated hepatic stellate cells by TGFβ1

Background/Aim: Hepatic stellate cells (HSC) are prominent cellular effectors of liver fibrosis, which become activated during liver injury and transdifferentiate from quiescent, fat storing cells into a proliferative myofibroblast-like cell type (MFB) 1,2. This process is accompanied by modulations of gene expression. We examined the expression of the LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) during transdifferentiation, a protein which in smooth muscle cells is associated with the cell skeleton as well as located in the nucleus 3,4. Results: An increase of CRP2 was detectable by Western Blot in lysates from liver samples of bile duct ligated rats, an animal model of liver fibrosis. Analysis of different liver cell subpopulations revealed that activated HSC are the source of this upregulation. Cell culture experiments with primary rat HSC, which become activated by contact with uncoated plastic dishes, as well as CFSC, a rat cell line derived from MFB, and A10 cells, a rat smooth muscle cell line, demonstrated that CSRP2 (gene name of CRP2) is upregulated by TGFβ1. To localize promoter regions responsible for induction of gene expression, we generated luciferase constructs with different fragments of the rat CSRP2 promoter. Transfection experiments in CFSC and A10 cells showed that potential binding sites for Smad factors within the proximal 1100 nucleotides of the promoter are not relevant for TGFβ1 induction. Promoter region –1100 to –1600 basepairs was shown to be responsible for serum induction of reporter genes in A10 cells. Future experiments will be performed to identify promoter regions, which are necessary for response to TGFβ signals. Conclusion: Our data demonstrate that CRP2 is increased in activated HSC and that TGFβ1 is involved in upregulation of CSRP2 gene expression. These findings are similar to other known smooth muscle marker like α-smooth muscle actin or SM22α, which are upregulated in activated HSC and associated with the cell skeleton. In contrast, CRP2 is also found in the nucleus, thereby demonstrating an additional function in transition of HSC to MFB in liver fibrosis. Knowledge of CSRP2 regulation and function may help to extend our understanding of molecular mechanisms involved in hepatic fibrogenesis.