PROMOTER ELEMENTS AND 2ND MESSENGER PATHWAYS INVOLVED IN TRANSCRIPTIONAL ACTIVATION OF TYROSINE-HYDROXYLASE BY IONOMYCIN

Membrane depolarization, or agents which increase intracellular calciu m, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PC12 cells were transiently transfec ted with plasmids containing wild type or mutated 5' flanking sequence s of the rat TH gene, fused to bacterial chloramphenicol acetyl transf erase (CAT). Point mutations introduced into the consensus cAMP-regula tory element (CRE) abolished the induction of CAT by ionomycin indicat ing that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity und er the control of TH promoter by ionomycin were similar in PC12 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylatio n of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca2+/calmodulin dependent (CaM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.