Activating Transcription Factor-1-mediated Hepatocyte Growth Factor-induced Down-regulation of Thrombospondin-1 Expression Leads to Thyroid Cancer Cell Invasion

Abstract

Hepatocyte growth factor (HGF) plays a major role in the pathogenesis of a variety of human epithelial tumors including papillary carcinoma of the thyroid. Previous reports demonstrated that HGF, acting through the Met receptor, repressed thrombospondin-1 (TSP-1) expression. To study the mechanisms by which HGF down-regulated TSP-1 expression, we transiently transfected a panel of deleted human TSP-1 promoter reporter plasmids into papillary thyroid carcinoma cells. We identified a region between -1210 and -1123 bp relative to the transcription start site that is responsive to HGF treatment and harbors a cAMP-responsive element (CRE) at position -1199 (TGACGTCC). Overexpression of various members of the CRE-binding protein family identified activating transcription factor-1 (ATF-1) as the transcription factor responsible for HGF-induced repression of TSP-1 promoter activity. This inhibition was associated with a concomitant increase in the abundance of nuclear ATF-1 protein. Gel shift and antibody supershift studies indicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site. Finally, we utilized small hairpin RNA to target ATF-1 and showed that these small interfering RNA constructs significantly inhibited ATF-1 expression at both the RNA and the protein level. ATF-1 knockdown prevented HGF-induced down-regulation of TSP-1 promoter activity and protein expression and also reduced HGF-dependent tumor cell invasion. Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by the interaction of ATF-1 with the CRE binding site in the TSP-1 promoter and that this transcription factor plays a crucial role for tumor invasiveness in papillary carcinoma of the thyroid triggered by HGF.