Transcriptional enhancement of tyrosine hydroxylase by prostaglandin E(2) in SK-N-BE(2)C cells

Abstract

Transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by prostaglandin E(2) (PGE(2)) was investigated in human neuroblastoma SK-N-BE(2)C cells. Prostaglandins increased intracellular cAMP in the presence of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor. Among the prostaglandins tested for their cAMP raising property PGE(2) was the most effective. The results suggest that the cells express adenylyl cyclase-linked prostanoid receptors that have a higher affinity for PGE(2) than for any other naturally occurring prostaglandin. The treatment of cells with PGE(2) increased the TH gene expression approximately 2-fold, even though the cAMP accumulation induced by PGE(2) alone was almost negligible. Simultaneous treatment with PGE(2) and IBMX enhanced the gene expression concomitantly with a marked accumulation of cAMP. Transient transfection assays with 5' upstream serially deleted constructs of the rat TH gene promoter region fused to the chloramphenicol acetyltransferase (CAT) gene revealed that a cAMP response element (CRE) located at -45 to -38 from the start of the TH gene was essential for the enhancement of TH gene expression by PGE(2). Site-directed mutagenesis and specific deletion within the sequence of the CRE motif abolished the transcriptional enhancement by PGE(2). In addition, a protein kinase A (PKA) inhibitor, H89, specifically blocked the PGE, effect on TH gene expression. Northern blot analysis revealed that the increase in TH gene transcription with PGE(2) is associated with an elevated TH mRNA level. Gel retardation and competition assays confirmed that the binding of nuclear factors to the CRE site was sequence specific and was augmented by PGE(2). Our data indicate that PGE(2) enhances transcription of the TH gene mediated by the CRE motif through the activation of PKA. They also suggest that the signal flow from the adenylyl cyclase-linked prostanoid receptor to the nucleus is efficient although cAMP accumulation is not prominent.