Transcriptional Regulation of Hypertrophy –Sensitive Genes by an Inducible Calcineurin Expression System

Yun, Bai, TRANSCRIPTIONAL REGULATION OF HYPERTROPHY –SENSITIVE GENES BY AN INDUCIBLE CALCINEURIN EXPRESSION SYSTEM. Master of Science (Biomedical Science), December, 1998, 86pp., 1 table, 16 illustrations, Bibliography, 83 titles. Calcium/calmodulin-dependent protein phosphatase, calcineurin, has recently been found to play a principal role in activating hypertrophy-sensitive gene expression upon the initiation of cardiac hypertrophy. In order to further quantitatively characterize the roles of calcineurin, we have established an inducible calcineurin expression system in which the expression of constitutively active form of calcineurin is under the control of a tetracycline-dependent transactivator (tTA). By using a reporter gene as a tTA target,we confirm that expression of the target gene can be dramatically induced by tT A in primary cardiomyocytes. In consistent with the results from calcineurin transgenic study,our transfection studies have indicated that the transcription of hypertrophy-sensitive genes, including cardiac a-actin, ANF. and skeletal a-actin gene, was obviously upregulated by induced expression of active calcineurin in cardiomyocytes. The transcription activation of three hypertrophy response genes mediated by active calcineurin is both active calcineurin plasmid DNA dose and time-dependent. Furthermore, the calcineurin induction of hypertrophy-sensitive genes can be significantly blocked by a specific calcineurin inhibitor, cyclosporin A (CsA), and the inhibition of hypertrophy response gene expression is CsA concentration-dependent. More importantly, the transcription of hypertrophy-sensitive genes mediated by active calcineurin can be regulated reversibly by CsA manipulation. In addition, the expression of tT A target gene can also be inhibited by effector doxcycline, but doxcycline inhibition is not complete. Based on these results. we conclude that transcription of hypertrophysensitive genes can be regulated conveniently by this inducible system. Therefore, the experimental manipulation of temporal and quantitative expression of calcineurin would be useful for further elucidating precise molecular. mechanisms for calcineurin-dependent signaling transduction during the onset of cardiac hypettrophy.