An analysis of KCNN4 promoter elements underlying expression in vascular smooth muscle cells

• Main Author: Mcardle, Matthew Charles
• Published: University of Leeds 2012
• Subjects: 616.13
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590310

The intermediate-conductance calcium-activated potassium channel, KCa3.1 , allows the efflux of intracellular potassium and is activated by increased intracellular calcium. This plays an important role in vascular physiology and pathophysiology. Expression of KCNN4, the gene encoding this channel, is upregulated in proliferative vascular smooth muscle cells (VSMC) and enhances cell proliferation through increased extracellular calcium influx. Due to its clinical importance, we investigated the regulation of KCNN4 in VSMC. Bioinformatic analysis of the KCNN4 gene was used to determine potential regulatory regions and transcription factor binding sites. Analysis of these sites involved a series of reporter constructs driven by the KCNN4 promoter sequence in promoter deletion assays within both cultured and primary VSMC. This detected potential repressor regions containing putative NFAT, CREB and KLF sites. As both NFAT activity and KCa3.1 expression are perturbed in diabetes mellitus, which is frequently associated with cardiovascular disease, we focused on KCNN4 regulation in response to high extracellular glucose. Whilst KCNN4 mRNA levels were decreased in response to 7.5mM and 11mM glucose, this was not due to NFAT activity at the KCNN4 promoter. In silico identification of CREB and KLF sites within a novel repressor reg ion of the KCNN4 promoter was complemented with electrophoretic mobility shift analysis demonstrating protein-DNA interactions. Mutation of either putative CREB or KLF sites and over-expression of constitutively active or dominant negative CREB proteins had no effect on KCNN4 mRNA levels or promoter activity. However, over-expression of KLF15 reduced both KCNN4 mRNA levels and promoter activity. In addition , stimulation of cAMP production in VSMC caused an 80% reduction in KCNN4 mRNA levels and repressed promoter activity. These data provide insights into the molecular mechanisms controlling KCNN4 expression in VSMC through mechanisms involving KLF15, glucose and cAMP-induced regulatory factors that potentially present novel targets for the therapeutic intervention of vascular 'disease.