Both the Cyclic AMP Response Element and the Activator Protein 2 Binding Site Mediate Basal and Cyclic AMP-Induced Transcription from the Dominant Promoter of the Rat α1B-Adrenergic Receptor Gene in DDT1MF-2 Cells

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Abstract
CAMP markedly increases α1B adrenergic receptor (α1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT1MF-2 cells to evaluate further the mechanisms by which cAMP stimulates α1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 μm) in the presence of isobutylmethylxanthine (0.25 mm) increased α1B-AR numbers, mRNA level, and gene transcription rate by 2.3 ± 0.2-, 2.5 ± 0.3-, and 3.5 ± 0.2-fold over control, respectively. Dibutyryl cAMP (1 mm) plus isobutylmethylxanthine (0.25 mm) also enhanced α1B-AR density by 2.7 ± 0.1-fold over control. Further experiments demonstrated that the induction of α1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT1MF-2 cells transfected with α1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat α1B-AR gene (−813 to −432) contains a cAMP response element (CRE) (−444 to −437) and an AP2 binding site (−647 to −638). Mutations in either one of these elements alone led to a decrease in both basal and cAMP-induced P2 promoter activity. Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously. Gel mobility shift and supershift assays using liver nuclear extracts from either rat liver or DDT1MF-2 cells demonstrated that the CRE in the α1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the α1B-AR gene in DDT1MF-2 cells.