CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells , resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position -45 to -38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cA MP responsive element binding protein (CREB) in regulating TH gene expressi on. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA level s were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell l ines. PKA activity in the CREB-deficient cell lines was comparable to the a ctivity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most signif icantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenou s gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Fur thermore, our studies indicate that these CREB-deficient PC12 cells are exc ellent tools to study the participation of CREB in gene regulation. (C) 199 9 Elsevier Science B.V. All rights reserved.