Cloning, cell-type specificity, and regulatory function of the mouse α(1B)-adrenergic receptor promoter

The functionality of a 3422-base pair promoter fragment from the mouse α(1B)-adrenergic receptor (α(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat α(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known α(1B)AR-expressing BC₃H1, NB41A3, and DDT₁MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known {1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that α₁ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be {1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis- acting elements, RAT-1, R3T3, NB41A3, BC₃H1, and DDT₁ MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue- specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb Pvull fragment of the murine α(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.