Transcriptional Targeting of Activated Hepatic Stellate Cells by Modular Promoter Fragments

Transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells is a key event in liver fibrogenesis accompanied by differential gene regulation. Antifibrotic strategies focus on manipulation of this relevant cell type, e.g. induction of HSC apoptosis, but to date a specific and easy-to-handle targeting of activated HSC is not reported. As demonstrated by Lemken et al. [1] a targeting of reporter genes to liver using enhancer elements of hepatocyte specific genes fused to a minimal promoter was successful. Therefore, we proofed if short promoter fragments with essential cis-acting regulatory elements from genes upregulated during transdifferentiation are able to direct HSC specific reporter gene expression. Four different DNA-fragments, each ˜120 bp in length, were amplified from the promoters of rat α-smooth muscle actin and SM22α genes by PCR (‘modulesrsquor;) and cloned into the pGL3 promoter vector. Transcriptional efficiency and specificity of 20 different module combinations were determined by luciferase expression levels after transfection into CFSC and HepG2 cells and selected combinations were further verified by transfection into primary rat HSC and hepatocytes. We could select 3 constructs that were able to direct high reporter gene activity in CFSC as well as in HSC. Two of these constructs contain modules of the SM22α gene promoter in different combinations with a TCE element or SP1 binding site, respectively. The most promising module combination (SP1+TCE) mediated a 10-fold higher luciferase expression in CFSC compared to HepG2, while the other combinations revealed an expression level that was high in both cell lines. Principally, we could demonstrate that combination of small promoter modules with known regulatory elements from genes upregulated during transdifferentiation enables the targeting of HSC. This allows the easy-to-handle expression of diverse transgenes in contrast to a recent publication [2] showing that a targeting of HSC was only successfully by delivery of 2 different adenoviral constructs. We think that our approach implicate the potential to enhance expression efficiency and specificity by easy recombination with additional promoter modules together with an exchange of the used SV40 minimal promoter with a more HSC related proximal gene promoter, e.g. TIMP–1.