Molecular cloning and functional characterization of the mouse hepatocyte growth factor receptor gene (c-met) promoter

Upon binding Hepatocyte Growth Factor (HGF), the HGF receptor (c-Met) transmits multiple biological signals such as mitogenic, motogenic, morphogenic, anti-tumor and anti-apoptosis activities. The HGF receptor gene (c-met) is expressed in a cell-type-specific manner. Expression of the c-met gene is induced by various growth factors and cytokines. To decipher the molecular mechanisms that regulate HGF receptor gene expression at the transcriptional level, I cloned and characterized the mouse HGF receptor gene promoter. The following results were obtained from my studies:;I. Nucleotide sequence analysis of the mouse HGF receptor gene promoter revealed that this promoter lacks a TATA box, and is similar to the housekeeping and constitutively expressed gene promoters with a high GC-content and several Sp1 binding sites. In addition to several Sp1 binding sites, multiple potential regulatory elements for various transcription factors such as AP-1, p53, C/EBP, Myb, Myc and WT1 were identified in the promoter. Sequence comparison of the mouse and human c-met promoters revealed, that numerous regions are evolutionarily conserved in the regions containing the potential regulatory elements including Myc, Ap-1 and Sp1 consensus binding sequences. In the functional characterization of the promoter, two regulatory regions were identified; a region (;II. Analysis of the promoter identified Sp1 protein binding to two cognate response elements at ;III. In addition to Sp1, functional analysis also identified that p53 regulates the HGF receptor gene promoter. In cotransfection analysis, wild-type p53 but not various tumor-derived mutant forms of p53 stimulated HGF receptor gene promoter in a dosage-dependent manner. Nucleotide sequence analysis identified a p53 binding motif (;IV. Functional analysis demonstrated that growth factors including EGF stimulate the HGF receptor gene promoter. Subsequent analysis revealed that AP-1 protein specifically binds to this region, and that binding is significantly increased by EGF. Moreover, overexpression of AP-1 protein (Jun/Fos) stimulated HGF receptor promoter activity, and its stimulatory activity depended upon the identified AP-1 binding site. These results indicate that induction of HGF receptor gene expression by EGF is mediated by AP-1.