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

We previously demonstrated that a1B-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 a1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb a1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The a1BAR 5* flanking region was sequenced to 22,460 (relative to ATG 11). Transient transfection experiments identify the minimal promoter region between 2270 and 2143 and sequence between 2270 and 2248 that are required for transcription of the a1BAR 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 2160 in aorta SMC. Sequence between 2837 and 2273 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 a1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the a1BAR gene also were identified that may confer the differential hypoxic increase in a1BAR gene transcription in aorta, but not in vena cava SMC. Three subtypes of the G protein-coupled a1-adrenergic receptors (AR) designated as the a1A, a1B, and a1D have been identified (1). Native and cultured rat aorta and vena cava smooth muscle cells (SMC) express both the a1BAR and a1DAR subtypes (2, 3). Among a1AR subtypes, a1DAR mediates constriction of the rat aorta (4, 5), iliac artery (6), and arterioles in skeletal muscle (7), whereas the a1BAR signals constriction of the vena cava (8) and skeletal muscle venules (7). Sympathetic constriction of arterioles and venules mediated by these vascular a1AR shows a prominent difference in sensitivity to inhibition by metabolic signals including hypoxia and acidosis (a1DAR sensitive, a1BAR insensitive), which we have proposed serves to simultaneously maximize autoregulation and reflex control of venous return (9). Vascular a1AR also have been shown to couple to additional cellular functions, including SMC hypertrophy signaled by the a1DAR (10) and modulation of glucose metabolism in liver and heart mediated by a1BAR (11, 12). Consistent with this association of the a1BAR with altered metabolic states, hypoxia (3% O2) transcriptionally up-regulates a1BAR, but not a1DAR, gene expression in aorta, but not in vena cava SMC, examined in vitro and in vivo (3). However, it is not known if hypoxic induction of a1BAR expression is shared by other nonvascular tissues. Although nothing is known concerning how oxygen levels regulate transcription of the a1BAR gene, control depends on trans-acting factors binding to the transcription promoter region and regulatory elements within the DNA. To begin to examine transcriptional control, knowledge of gene sequence upstream to the translation start is required. However, from the coding region of the rat a1BAR upstream to 2610 (translation start 11) there is 98% similarity (13–15) but upstream of 2611 to the end of the reported sequences [GenBank accession L28752 (14) and D32045 (15)] there is no similarity. Thus, functional analysis of a1BAR promoter regulation requires resequencing the 59 f lanking region. There is also significant disagreement in the location of the site of transcription initiation of the a1BAR in rat cells (14, 15). Kanasaki and coworkers (15) describe a single transcription start site at 2173 similar to that reported for the human a1BAR (16). In contrast, Gao and Kunos (14) proposed the existence of three different transcription initiation sites in rat liver. Certain hypoxia-induced genes, under the control of multiple promoters, have preference for a particular promoter andyor transcription start site during hypoxia (17). Therefore, it is important to determine a1BAR sequence required for transcription initiation in both aorta and vena cava SMC, and to identify whether this changes during hypoxia. Selection of an alternative promoter for the a1BAR during hypoxia in aorta, but not in vena cava, could explain differential induction of transcription by reduced oxygen (3). Erythropoietin, the primary humoral regulator of increased erythropoiesis during chronic hypoxemia, is the best characterized mammalian gene for hypoxic regulation of expression (18). Several cis-elements and their binding factors have been shown to be essential for hypoxic increase in erythropoietin transcription in in vitro model systems, notably hypoxiainducible factor 1 (HIF-1) (18). The consensus HIF-1 binding site is present in the a1BAR gene (3). However, it is not clear whether HIF-1 is involved in the hypoxic up-regulation of a1BAR in aorta SMC. Although specific and hypoxia-induced nuclear protein binding to HIF-1 has been demonstrated in all cell types tested (19), whether HIF-1 or other hypoxiainducible enhancers are capable of mediating hypoxic regulation of a1BAR in SMC remains to be determined. MATERIALS AND METHODS In Vivo Hypoxia and Cell Culture. Adult rats were exposed to 10% inspired O2 environmental hypoxia for 8 hr as described previously (3). Preparation and culture of adult rat thoracic aorta and vena cava SMC primary cultures were performed as described (2, 3). For hypoxic exposure, cells were placed in an O2-regulated incubator (Forma Scientific, Marietta, OH) at 1.5% or 3% O2, 5% CO2, balance N2 (3). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y949487-6$2.00y0 PNAS is available online at http:yywww.pnas. org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: SMC, smooth muscle cell; AR, adrenergic receptor; RPA, RNase protection assay; HIF-1, hypoxia-inducible factor 1. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. U83985). *To whom reprint requests should be addressed at: Department of Physiology, 474 MSRB, CB #7545, University of North Carolina, Chapel Hill, NC 27599-7545. e-mail: jefaber@med.unc. edu.