Cell type-specific effects on CTGF and PAI–1 expression when glucocorticoids meet TGF-β in primary liver cells

Aims: Glucocorticoids are involved in fundamental cellular functions such as glucose metabolism and cell differentiation. Once bound to their receptor (GR) the glucocorticoid-receptor-complex translocates to the nucleus and binds after dimerization to a cis-active response element in order to regulate target genes. A pivotal process in liver fibrosis is the activation of hepatic stellate cells (HSC) and the production of extracellular matrix (ECM), which is triggered by TGF-β. TGF-β binds and phosphorylates transmembrane receptors, which in turn propagate the signal via Smads into the nucleus in order to regulate gene expression. A variety of additional mediators is involved in HSC activation and differentiation, e.g. the plasminogen activator inhibitor type I (PAI–1) or the connective tissue growth factor (CTGF). Aim of the present investigations was to characterize the effects of glucocortiocoids on the TGF-β target genes PAI–1 and CTGF. Methods: We studied the expression of PAI–1 and CTGF after a combined hormone-cytokine stimulation of primary rat HSC and hepatocytes at the RNA level by real time PCR and at the protein level by western blot. Interaction of overexpressed GR and Smad 3 in co-transfected HEK cells was observed by life cell imaging. Results: We found an opposite regulation of PAI–1 and CTGF expression in HSC and hepatocytes. While dexamethasone acts antagonistically by inhibiting TGF-β induced PAI–1 and collagen 1 expression in HSC, it acts synergistically by enhancing TGF-β induced PAI–1 and CTGF production in hepatocytes. PAI–1 appears in the culture medium from untreated and stimulated hepatocytes and HSC already after one hour. CTGF was also fast secreted by activated HSC but selectively released into the culture medium by TGF-β stimulated hepatocytes after 6 to 20 hours. Finally, we observed that Smad 3 overexpression inhibits GR transactivation, which suggests an interaction between both transcription factors. Life cell imaging with HEK cells showed that ligand bound GR retains Smad 3 in the cytoplasm, which might be one possible mechanism for glucocorticoid mediated inhibition of TGF-β effects in HSC. Conclusions: These results indicate a cell type-specific regulation of PAI–1 and CTGF expression and secretion in HSC and hepatocytes, which might be important for the paracrine network between the liver cells.