AP1-MEDIATED TRANSCRIPTIONAL ENHANCEMENT OF THE RAT - TYROSINE-HYDROXYLASE GENE BY MUSCARINIC STIMULATION

We investigated transcriptional regulation of the rat tyrosine hydroxy lase (TH) gene by muscarinic stimulation in human neuroblastoma SK-N-B E(2)M17 cells. Carbachol treatment increased the levels of intracellul ar Ca2+ and inositol 1,4,5-trisphosphate (IP3) and enhanced transcript ion of the TH gene, The muscarinic receptor antagonist atropine comple tely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 mu M ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraa cetic acid/acetoxymethyl ester (BAPTA/AM) to chelate intracellular Ca2 +, carbachol still raised intracellular IP3 level and enhanced TH gene expression. Transient transfection analysis of the 5' upstream region of TH gene revealed that the AP1 cis-acting element at -205 to -199 b p was responsible for carbachol stimulation. But carbachol did not enh ance TH gene expression in protein kinase C (PKC)-activated or downreg ulated cells that had been induced by 5-min or 24-h exposure to phorbo l 12-myristate 13-acetate (PMA), respectively. Thus, Ca2+-independent PKC may play a role in carbachol-induced TH gene expression. We demons trated by gel retardation and competition assays that a DNA sequence c ontaining the wild-type AP1 site formed the specific DNA-protein compl ex. However, treatment with carbachol or PMA did not change the amount of the specific DNA-protein complex. Our results indicate that stimul ation of phospholipase C-linked muscarinic receptors leads to elevated TH gene expression via AP1-mediated enhancement in a PKC-dependent pa thway.