The CSRP2-Promoter: A Tool for Transcriptional Targeting of Hepatic Stellate Cells During Liver Fibrogenesis?

Introduction: An initial key event in liver fibrogenesis is the activation of quiescent hepatic stellate cells (HSC) and their transdifferentiation into a proliferative, myofibroblast like cell type (MFB). During this process several genes are differentially expressed and their regulatory units may be useful tools for antifibrotic strategies, which target HSC specifically. The LIM-domain protein cyteine- and glycine-rich protein 2 encoding gene (CSRP2) is within rat liver cell subpopulations solely expressed in activated HSC and downregulated in MFB. The aim of this study was the proof of the ability of human (h) CSRP2 promoter fragments to express transgenes in culture-activated rat HSC, but not in MFB or hepatocytes. Material/Methods: Culture-activated HSC from rats were infected with adenoviral vectors (Ad5) harbouring p53 or Bax encoding cDNAs under transcriptional control of a 0.9 kb hCSRP2 promoter fragment or the constitutive active CMV promoter to induce apoptosis in infected cells. Apoptosis was examined by Annexin-V-FLUOS-assay, Caspase 3-assay and cell death-ELISA. Additional reporter gene-assays were performed in the CFSC cell line, rat HSC and MFB as well as human MFB. Results: In activated HSC we found no significant p53 or Bax induced apoptosis directed by the 0.9 kb hCSRP2 promoter compared to CMV promoter driven expression of p53. Luciferase constructs transfected into CFSC showed that the activity of the CSRP2 promoter fragment is repressed by Bax, which is upregulated by p53. Beside these findings a comparison between the proximal human and rat CSRP2 promoter sequences showed only 30% homology indicating a different regulation in these species. Indeed, transgene expression after adenoviral infection of rat and human MFB with a GFP reporter gene driven by a 4.2 kb hCSRP2 promoter fragment was found to be much higher in the human system. Conclusions/Discussion: We tested a human CSRP2 promoter fragment to express apoptosis inducing proteins in rat HSC and found that overexpression of these effectors resulted in a self limitation of the chimeric expression construct. Therefore, usage of CSRP2-promoter fragments to express apoptotic acting proteins is questionable, but possible for other antifibrotic effectors. In further experiments it is important, regarding the limited homology of human and rat CSRP2 promoters, to deal with species-specific components. Therefore, we now clone rat promoter fragments to direct transgene expression.

Key words

CSRP2-promoter - apoptosis - gene therapy - liver fibrosis