BOVINE TYROSINE-HYDROXYLASE GENE-PROMOTER REGIONS INVOLVED IN BASAL AND ANGIOTENSIN II-STIMULATED EXPRESSION IN NONTRANSFORMED ADRENAL-MEDULLARY CELLS

The tyrosine hydroxylase-gene is expressed specifically in catecholami nergic cells, and its activity is regulated by afferent stimuli. To ch aracterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase g ene promoters (wild-type or deletion mutants) and a luciferase reporte r gene. The basal expression of these genes and their regulation by an giotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasm id DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of th e tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletio n of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and ot her putative regulatory elements increased luciferase expression fivef old. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE) -like site, reduced promoter activity. These results indicate the pres ence of negatively and positively acting regions in the bovine tyrosin e hydroxylase gene promoter controlling basal promoter activity in adr enal medullary cells. Angiotensin II stimulated the expression of endo genous tyrosine hydroxylase gene and pTHgoodLUC approximately threefol d without affecting the expression of pOLUC. A comparable threefold st imulation was observed following the deletion of the -194/-54-bp promo ter region, despite the increase in basal promoter activity. Additiona l deletion of the -269/-194-bp promoter fragment reduced stimulation b y angiotensin II to 1.5-fold. These results indicate that the angioten sin II receptor-responsive element is located in the -269/-194-bp prom oter region containing the TRE-like site. Additional angiotensin II-re sponsive site(s) may be present outside this region. Gel mobility shif t assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein co mplexes were displaced with c-Fos antibodies. The results suggest that c-fos-related antigens support basal promoter activity and mediate ac tivation of tyrosine hydroxylase by angiotensin II receptor.