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Vol. 56, Issue 6, 1288-1297, December 1999
1B-Adrenergic Receptor Promoter
Department of Molecular Cardiology, The Lerner Research Institute,
The Cleveland Clinic Foundation, Cleveland, Ohio (M.J.Z., D.M.P.); and
Department of Pharmacology and the Vascular Biology Research Group,
University of Kentucky College of Medicine, Lexington, Kentucky
(M.T.P.)
The functionality of a 3422-base pair promoter fragment from the mouse
1B-adrenergic receptor (1BAR) gene was
examined. This fragment, cloned from a mouse genomic library,
was found to have significant sequence homology to the known human and
rat 1BAR 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 1BAR-expressing BC3H1, NB41A3, and DDT1MF-2 cells transiently transfected with a
promoter-CAT construct. Conversely, minimal CAT expression was detected
in known 1BAR-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 1ARs 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 1BAR negative,
showed minimal CAT expression. Indicating regulatory function through
cis-acting elements, RAT-1, R3T3, NB41A3,
BC3H1, and DDT1MF2 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 PvuII
fragment of the murine 1BAR 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.
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