Cloning and promoter activity of rat Smad1 5'-flanking region in rat hepatic stellate cells

Abstract

Smad1 is an important signaling molecule for members of transforming growth factor-beta (TGF-beta) superfamily. Increased expression of Smad1 in activated hepatic stellate cells (HSCs) indicates a role of Smad1 in liver fibrosis. Therefore, understanding of Smad1 gene expression could be important to control the activation of HSCs. Current study reports the cloning and characterizing rat Smad1 5'-flanking region in liver cells. Rat Smad1 5'-flanking region was cloned by PCR method. Promoter deletional analysis and electrophoretic mobility shift assay (EMSA) were examined in hepatocyte and HSCs cell line (CFSC-8B cells), respectively. Results indicated that rat Smad1 used GC-box as its promoter and there was a transcriptional regulatory element located at the region of -163 to -56bp. EMSA demonstrated two bands on Smad1 promoter region. Smad1 promoter activity was higher in CFSC-8B cells cultured on uncoated plastic dish than that of CFSC-8B cells cultured on Matrigel-coated plastic dish. In conclusion, rat Smad1 promoter was cloned and characterized in hepatocyte and HSC cell line (CFSC-8B cells) at different culture conditions.