Impairment of hepatic nuclear factor-4α binding to theStim1promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells

Abstract

Wang Y, Chaudhari S, Ren Y, Ma R. Impairment of hepatic nuclear factor 4␣ binding to the Stim1 promoter contributes to high glucoseinduced upregulation of STIM1 expression in glomerular mesangial cells. Am J Physiol Renal Physiol 308: F1135-F1145, 2015. First published March 18, 2015 doi:10.1152/ajprenal.00563.2014.-The present study was carried out to investigate if hepatic nuclear factor (HNF)4␣ contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4␣ expression in MCs. Knockdown of HNF4␣ using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4␣ reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4␣ protein in MCs but significantly attenuated HNF4␣ binding activity to the Stim1 promoter. Moreover, knockdown of HNF4␣ significantly augmented store-operated Ca 2ϩ entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4␣ significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4␣ negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4␣ binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4␣ repression. Since the STIM1-gated storeoperated Ca 2ϩ entry pathway in MCs has an antifibrotic effect, inhibition of HNF4␣ in MCs might be a potential therapeutic option for diabetic kidney disease. hepatic nuclear factor 4␣; stromal interacting molecule 1; storeoperated Ca 2ϩ entry; mesangial cells; high glucose; diabetic nephropathy STORE-OPERATED Ca 2ϩ entry (SOCE) via store-operated Ca 2ϩ channels (SOCs) is a ubiquitous signaling mechanism in both nonexcitable and excitable cells. This Ca 2ϩ signaling regulates diverse cellular functions ranging from cell proliferation and gene expression to cell contraction and secretion (35). Although SOCE was originally described over two decades ago, its molecular mediators were unknown until recently. By high-throughput RNA inhibition screening, two protein families, stromal interacting molecule (STIM) (27, 36) and Orai (13, 48, 59), were identified as required components of SOCE. STIM1 is a single-pass transmembrane protein located primarily in the endoplasmic reticulum (ER) membrane and functions as an ER Ca 2ϩ sensor to sense the ER luminal Ca 2ϩ concentration. Orai1 is a small plasma membrane protein and is believed to be the pore-forming unit of SOCs. Upon depletion of ER Ca 2ϩ , STIM1 aggregates and translocates to ER-plasma membrane junctions, where it physically associates and subsequently activates Orai1 and causes Ca 2ϩ entry into the cytosol (...

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