Structure and transcriptional function of the 5'-flanking region of rat thromboxane receptor gene

We cloned a cDNA for rat TX receptor, and observed its expression in the kidney, including vascular smooth muscle. The aim of the present study was to clone the 5'-flanking region (5'-FL) of rat TX receptor gene, and to examine its transcriptional gene expression regulation. The 5'-FL was cloned by a PCR method, and the nucleic acid structure of 5'-FL (approximately 1 Kb) was disclosed. The transcription initiation site was shown to be 63 bases upstream of the 5' end of the cDNA by the primer extension. In the 5'-FL, putative AP-1 binding sites, glucocorticoid-responsive elements, NF-kappa B binding sites, GATA box, and shear stress-responsive elements were identified. The 5'-FL was then fused upstream of firefly luciferase cDNA in an expression vector, and we examined its transcriptional activity in transiently transfected cultured vascular smooth muscle cells (VSMC). Luciferase expression was dependent on the length of 5'-FL, and it was significantly stimulated by phorbol 12-myristate 13-acetate (PMA), dexamethasone (Dex), tumor necrosis factor-alpha, and interleukin (IL). By a semi-quantitative RT-PCR method, TX receptor mRNA was shown to be induced by Dex, IL-6, and PMA in cultured VSMC. In conclusion, we have revealed the structure of transcription regulatory region of TX receptor. Expression of TX receptor gene is possibly up-regulated by activation of protein kinase C, glucocorticoid excess, and IL-6, in vascular smooth muscle.