This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca 2C. We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca 2C influx through L-or Ttype voltage-gated Ca 2C channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetinplus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca 2C efflux by sodium orthovanadate, a Ca 2C -pump inhibitor, blocked quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electron microscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.