Showing papers by "Nanette H. Bishopric published in 1995"

Cell-specificity and signaling pathway of endothelin-1 gene regulation by hypoxia

Abstract: Objectives: Endothelin-1 (ET-1) is a potent vasoconstrictor that is expressed in endothelial cells and in many other cells and tissues. Increased plasma levels of the peptide have been associated with ischemic heart disease, atherosclerosis, and myocardial infarction. The objectives of the current study were (1) to determine the tissue specificity for induction of the ET-1 gene by hypoxia, (2) to determine whether the hypoxia regulatory pathway is the same as that in other hypoxia regulated genes and (3) to analyze the contributions of protein kinases for basal and induced expression of ET-1. Methods: ET-1 transcript levels were measured by Northern blot and quantitative polymerase chain reaction in endothelial and non-endothelial cells following exposure to hypoxia. Regulatory steps within the pathway were identified by treating aerobic or hypoxic cultures with cycloheximide, PMA, a series of selective protein kinase inhibitors, and transition metals. The effects on ET-1 transcripts were compared with the ubiquitous hypoxia inducible pyruvate kinase gene. Results: The induction of ET-1 by hypoxia in vitro occurred exclusively in early passage endothelial cells. This induction was prevented by treatment with the protein synthesis inhibitor cycloheximide and was at least partially mimicked by treatment with transition metals. Induction by hypoxia was not effected by inhibitors of protein kinase C, protein kinase A, calcium-calmodulin dependent protein kinase, or cyclic GMP dependent protein kinase. The basal expression was decreased and hypoxic induction was eliminated by treating cells with tyrosine kinase-selective inhibitors. Conclusions: Et-1 induction by hypoxia requires endothelial cell-specific factor(s) or steps, new protein synthesis, and may involve a haeme protein-containing pathway in oxygen sensing. A protein tyrosine kinase step is implicated for both basal and induced expression of the ET-1 gene.

Tissue specific hypoxia regulated therapeutic constructs

Abstract: Methods and compositions relating to chimeric genes containing (i) a tissue-specific promoter and (ii) a hypoxia response enhancer element, both of which are operably linked to a selected gene, such as a reporter gene, therapeutic gene (e.g., bcl-2, NOS, catalase and SOD), or deleterious gene are disclosed. Expression of the selected gene is enhanced in the target tissue under hypoxic conditions, such as conditions encountered during episodes of ischemia and reperfusion. The methods and compositions may be used as therapeutics and/or diagnostics.