Transcriptional activation of the Hepatocyte Growth Factor receptor (c-met) gene by its ligand (Hepatocyte Growth Factor) is mediated through AP-1

Abstract

Hepatocyte Growth Factor (HGF) exerts its biological effects via binding and activating a transmembrane protein tyrosine kinase receptor known as c-Met. Previous studies from our laboratory demonstrated that c-met gene expression is inducible by its own ligand (HGF). However, the molecular mechanism(s) involved in this process are unknown. The present study was carried out to address this question. Transfection of various c-met-CAT promoter constructs into the mouse hepatocellular carcinoma cell line Hepa 1-6 in combination with electrophoretic mobility shift assays (EMSA) identified the responsive element as an activated protein-1 (AP-1) binding site (TGAGTCA) within the c-met core promoter region at position −158 to −152. The c-met AP-1 element binds specifically to AP-1 protein as verified by supershift assays. EMSA studies and mutational analyses of the promoter region also revealed that the members of the Sp family of transcription factors (Sp-1 and Sp-3) bind to the c-met Sp-1 element (located at position −124) which is adjacent to the AP-1 site. We show that Sp binding dampens binding of AP-1 to its cognate site in the c-met promoter region. Stimulation of Hepa 1-6 cells with HGF resulted in a rapid and dramatic enhancement of the AP-1 binding activity as well as an overall increase in the level of AP-1 protein. Cotransfection of AP-1 expression vectors (c-Fos plus c-Jun) with c-met promoter constructs resulted in stimulation of c-met promoter activity. We found that transactivation of the c-met promoter by AP-1 can be blocked by Curcumin, an inhibitor of AP-1. Moreover, we found that the induction of the endogenous c-met gene by HGF is inhibited by the addition of Curcumin. The results demonstrate that the HGF-induced transcription of the c-met gene by HGF is, at least in part, due to activation of the AP-1 pathway.