Stretch‐Induced Transcriptional Regulation of the Large Conductance Calcium‐Activated Potassium Channel (BK)‐β4 Subunit in Human Mesangial Cells (MC)

Abstract

Renal nephropathy is a complication of Type 2 Diabetes. In hyperinsulinemic mice, hyperfiltration transmits stretch to MC. A stretch‐induced increase in [Ca2+]i can activate the calcineurin/ Nuclear Factor of activated T cells (NFAT), a transcription factor that associates with GATA in smooth muscle. In physiological conditions, the pore‐forming BK‐α interacts with the β1 to regulate mesangial contractile tone. However, the mouse glomerulus also contains the β4 and only the glomerular β4 is up‐regulated (immunohistochemical staining) in hyperinsulinemic mice. Our underlying hypothesis is that stretch due to hyperfiltration increases the expression of β4 and that BK‐α /β4 causes a sustained elevation in [Ca2+], causing MC expansion. Using nested RT PCR, following 10% cyclical stretching (24 h; Flexercell), β4 expression was 6‐fold > (p< 0.05) and β1 was 50% < control. β4 protein level doubled after 2 hours of stretch. Using the Transcription Element Search System of the University of Pennsylvania, (TESS) we identified 6 GATA and 12 NFAT sites on the β4 promoter. Through ChIP, we confirmed that NFATc3 and GATA4 bind to the β4 promoter 1760 bases upstream of the transcription start site, yielding a 175 bp fragment identified by sequencing as β4.Conclusion(1) Stretching MC results in transcriptional up‐regulation of β4 and down‐regulation of β1 and (2) NFATc3 and GATA4 bind β4 at a consensus promoter site.