Upstream Stimulatory Factors (USF-1/USF-2) Regulate Human cGMP-dependent Protein Kinase I Gene Expression in Vascular Smooth Muscle Cells

Abstract

Cyclic GMP-dependent protein kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent protein kinase gene expression. The present work was undertaken to characterize the mechanisms controlling kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent protein kinase I 5-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between ؊472 and ؊591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor proteins and decreased the activity of the cGMPdependent protein kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent protein kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent protein kinase I promoter is regulated by tandem Eboxes that bind upstream stimulatory factors.The serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), 1 belongs to the large protein kinase family and mediates the actions of nitric oxide (NO) and natriuretic peptides in target cells (1-3). The effects of PKG activation in target cells include smooth muscle relaxation, inhibition of platelet aggregation, calcium homeostasis, intestinal secretion, cell growth, differentiation, and gene regulation (4, 5).Two genes encoding mammalian PKG have been identified, type I and type II (6 -8). The type I PKG (PKG-I) is expressed as two isoforms created by alternative mRNA splicing of the two first coding exons, and these two isoforms are referred to as type I␣ and type I␤. Because these two isoforms differ only in the initial coding region, they contain an identical catalytic domain with identical substrate specificity. Although the two isoforms of PKG-I are rather widely expressed in mammalian tissues, three cell types, smooth muscle, cerebellum Purkinje cells, and platelets, contain the highest levels of the enzyme. The type II PKG is expressed most abundantly in intestinal epithelium, chondrocytes, and certain regions of the brain but is not expressed in smooth muscle (9 -12).In vascular SMC, both PKG-I␣ and -I␤ isoforms are abundant, but expression is highly variable. For instance, upon subc...

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