TGF-β induced upregulation of CRP2 and SRF in activated hepatic stellate cells–Involvement in smooth muscle gene expression during liver fibrogenesis?

Introduction: Activation of hepatic stellate cells (HSC), mainly triggert by transforming growth factor-β (TGF-β), is a key event in liver fibrogenesis accompanied by modulations of gene expression e.g. upregulation of contractile proteins known as markers of smooth muscle cell (SMC) differentiation. In SMCs the marker genes are principally regulated by binding of serum response factor (SRF) to unique sequences in their promoters, called CArG-boxes. In early chick embryonic cardiovascular development a functional co-association between SRF and the potent SMC marker cystein- and glycine-rich protein 2 (CRP2) was seen in the nucleus of proepicardial cells followed by a transition of CRP2 to the cytoskeleton during differentiation into SMCs [1]. Aim: We analysed the regulation and cellular distribution of CRP2 in activated HSC, which in liver is exclusively expressed in this cell type, as well as the presence of SRF in these cells. Methods: Endogenous CRP2 and SRF contents were detected in cell lysates from cultured rat HSC or cell lines (A10 cells, CFSC) by Western Blot analysis. Cells were treated with soluble TGF-β type II receptor (STR), TGF-β1, or different serum contents in medium. Regulation of CSRP2 (gene name of CRP2) was investigated by reporter gene constructs. Cellular distribution of CRP2 was detected by separation of activated HSC into 4 fractions (membrane, cytoplasm, nucleus, cytoskeleton). Results: CRP2 is upregulated by TGF-β in activated HSC and accumulates in the nucleus. These findings were confirmed by TGF-β mediated rapid increase of CRP2 content in vascular SMCs. Reporter gene constructs pointed out that evolutionary conserved CAGA-sites in between the proximal 1.2 kbp rat CSRP2 promoter are not relevant for this effect. Upregulation of CRP2 is also serum inducible, which was shown in transfection experiments in A10 cells. Another finding was a TGF-β regulated expression of SRF in activated HSC. Discussion: We found CRP2 and SRF increased in activated HSC by TGF-β. While SRF is an established transcription factor especially involved in SMC differentiation, the role and function of CRP2 in SMC or activated HSC is largely unknown. To date it is well accepted that CRP2 is associated to the cytoskeleton of SMC [2]. The localisation and accumulation of CRP2 in the nucleus of HSC during fibrogenesis indicates an additional function in gene regulation of SMC marker genes maybe together with SRF as described by Chang et al. [1].

Literatur: [1] Chang DF et al.(2003), Developmental Cell 4: 107-118 [2] Grubinger M and Gimona M (2004), FEBS Letters 557: 88-92

Schlüsselwörter

CRP2 - Fibrosis - HSC - SRF - TGF-beta - gene regulation