Characterization of the α 1B -adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle

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Abstract
We previously demonstrated that alpha1B-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O2 exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that alpha1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb alpha1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The alpha1BAR 5' flanking region was sequenced to -2,460 (relative to ATG +1). Transient transfection experiments identify the minimal promoter region between -270 and -143 and sequence between -270 and -248 that are required for transcription of the alpha1BAR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT1MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately -160 in aorta SMC. Sequence between -837 and -273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the cis-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the alpha1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the alpha1BAR gene also were identified that may confer the differential hypoxic increase in alpha1BAR gene transcription in aorta, but not in vena cava SMC.