Normal and pathological regulation of cardiovascular cell function and growth is effected through multiple, interacting cellular signal transduction pathways. Through largely unknown mechanisms, these signals are transduced to the cell nucleus, altering the transcription and expression of specific gene networks. Identifying these genes and their associated nuclear regulatory factors is fundamental to understanding the pathogenesis f a variety of cardiovascular disorders. The (Na+,K+)- ATPase (NAKA) represents an attractive model for the study of these mechanisms. NAKA plays a central role in cell function by maintaining and regulating the Na+/K+ transmembrane electrochemical gradient essential for the function of a wide variety if ion-channel and co- and counter-transport systems. Alterations in the expression and function of this transmembrane protein complex is associated with the abnormal growth states associated with oncogenic transformation and in adaptive and pathophysiologic processes such as cardiac hypertrophy and hypertension. By unknown mechanisms, each of the three NAKA catalytic alpha isoform genes exhibits a differential pattern of expression during cardiac and vascular smooth muscle development, in the adaptive response to hypertension and hypertrophy and to several important physiologic regulators such as mineralocorticoid and thyroid hormones. Oncogene mediated cellular transformation may also alter NAKA alpha-1 gene expression and its response to mineralocorticoid hormone regulation. The proposed experimental approach is to study cellular signalling pathways regulating NAKA gene expression by focusing on their regulatory end products: the nuclear transcriptional regulatory proteins. The transcriptional promoter(s) and associated regulatory elements will be mapped from rat alpha genomic DNA clones. The cis-acting transcriptional regulatory elements and trans-acting DNA binding proteins mediating the corticosteroid hormone and tissue specific regulation of NAKA alpha-1 gene expression in normal and transformed fibroblast and post-mitotic cultured cardiac cells will be identified and characterized. Given the important relationship between hypertension, cardiovascular NAKA gene expression and the renin-angiotensin-aldosterone system this approach will be used to identify the heretofore uncharacterized mineralocorticoid response element(s) (MRE) regulating the transcription of the alpha-1 gene. The influence of a putative oncogene induced regulatory factor on MRE function will also be characterized. Cis-acting elements will be functionally mapped transfection of normal and transformed fibroblasts, and primary neonatal rat cardiocytes with reporter gene expression vectors linked to deletion and site-specific mutated alpha-1 transcriptional regulatory elements. In vitro DNA binding and protection assays will identify and characterize the interaction of nuclear binding protein(s) and hormone receptor with the cis element(s). Characterization of the structure, function and regulation of these factors is the long term goal of this proposal.
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