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Research ArticleArticle

Cloning, Cell-Type Specificity, and Regulatory Function of the Mouse α1B-Adrenergic Receptor Promoter

Michael J. Zuscik, Michael T. Piascik and Dianne M. Perez
Molecular Pharmacology December 1999, 56 (6) 1288-1297; DOI: https://doi.org/10.1124/mol.56.6.1288
Michael J. Zuscik
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Michael T. Piascik
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Dianne M. Perez
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Abstract

The functionality of a 3422-base pair promoter fragment from the mouse α1B-adrenergic receptor (α1BAR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat α1BAR 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 α1BAR-expressing BC3H1, NB41A3, and DDT1MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known α1BAR-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 α1BAR negative, showed minimal CAT expression. Indicating regulatory function throughcis-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 α1BAR 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.

Footnotes

    • Received August 12, 1999.
    • Accepted September 21, 1999.
  • Send reprint requests to: Prof. Dianne M. Perez, Department of Molecular Cardiology, NB5, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. E-mail: perezd{at}ccf.org

  • This work was supported by National Institutes of Health Grants RO1HL52544 (D.M.P.), RO1HL61438 (D.M.P.), RO1HL31820 (M.T.P.), and F-32-HL-10004–02 (M.J.Z.); and an unrestricted grant from Glaxo Wellcome (D.M.P.). The work was performed under the tenureship of an Established Investigator Award from the American Heart Association (D.M.P.). The sequence reported in this article has been deposited in the GenBank database (accession no. AF116943).

  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 56 (6)
Molecular Pharmacology
Vol. 56, Issue 6
1 Dec 1999
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Research ArticleArticle

Cloning, Cell-Type Specificity, and Regulatory Function of the Mouse α1B-Adrenergic Receptor Promoter

Michael J. Zuscik, Michael T. Piascik and Dianne M. Perez
Molecular Pharmacology December 1, 1999, 56 (6) 1288-1297; DOI: https://doi.org/10.1124/mol.56.6.1288

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Research ArticleArticle

Cloning, Cell-Type Specificity, and Regulatory Function of the Mouse α1B-Adrenergic Receptor Promoter

Michael J. Zuscik, Michael T. Piascik and Dianne M. Perez
Molecular Pharmacology December 1, 1999, 56 (6) 1288-1297; DOI: https://doi.org/10.1124/mol.56.6.1288
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