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

The functionality of a 3422-base pair promoter fragment from the mouse alph a(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, c loned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the con sensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fid elity of the murine promoter, robust in vitro expression of a chloramphenic ol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expre ssing BC(3)H1, NB41A3, and DDT1MF-2 cells transiently transfected with a pr omoter-CAT construct. Conversely, minimal CAT expression was detected in kn own alpha(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 alpha(1)ARs are differentially exp ressed in the smooth muscle of the vasculature, primary cultures of superio r mesenteric and renal artery vascular smooth muscle cells showed significa ntly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-form ing cells, which are known to be alpha(1B)AR negative, showed minimal CAT e xpression. Indicating regulatory function through cis-acting elements, RAT- 1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter -CAT construct all showed increased CAT production when challenged with for skolin or hypoxic conditions. Additionally, tissue-specific regulation of t he promoter was observed when cells were simultaneously challenged with bot h forskolin and hypoxia. These results collectively demonstrate that a 3.4- kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tis sue-specific production of a CAT reporter in both clonal and primary cell l ines; and 2) confer tissue-specific regulation of that CAT reporter when in duced by challenge with forskolin and/or hypoxic conditions.