AP1-mediated transcriptional enhancement of the rat tyrosine hydroxylase gene by muscarinic stimulation

We investigated transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by muscarinic stimulation in human neuroblastoma SK-N-BE(2)M17 cells. Carbachol treatment increased the levels of intracellular Ca²⁺ and inositol 1,4,5-trisphosphate (IP₃) and enhanced transcription of the TH gene. The muscarinic receptor antagonist atropine completely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic 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 bp was responsible for carbachol stimulation. But carbachol did not enhance TH gene expression in protein kinase C (PKC)-activated or down-regulated cells that had been induced by 5- min or 24-h exposure to phorbol 12-myristate 13-acetate (PMA), respectively. Thus, Ca²⁺-independent PKC may play a role in carbachol-induced TH gene expression. We demonstrated by gel retardation and competition assays that a DNA sequence containing the wild-type AP1 site formed the specific DNA- protein complex. However, treatment with carbachol or PMA did not change the amount of the specific DNA-protein complex. Our results indicate that stimulation of phospholipase C-linked muscarinic receptors leads to elevated TH gene expression via AP1-mediated enhancement in a PKC-dependent pathway.