Search results for: VDR Overexpression Lysate
#29357419 
2018/01/10
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Modulation of apolipoprotein L1-microRNA-193a axis prevents podocyte dedifferentiation in high-glucose milieu.
The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms' tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.Abheepsa Mishra, Kamesh Ayasolla, Vinod Kumar, Xiqian Lan, Himanshu Vashistha, Rukhsana Aslam, Ali Hussain, Sheetal Chowdhary, Shadafarin Marashi Shoshtari, Nitpriya Paliwal, Waldemar Popik, Moin A Saleem, Ashwani Malhotra, Leonard G Meggs, Karl Skorecki, Pravin C Singhal
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100 μg400Tests96 wells (1 kit)100 μg96 wells (1 kit)
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Prohibitin is a novel target gene of vitamin D involved in its antiproliferative action in breast cancer cells.
Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.Xinjian Peng, Rajeshwari Mehta, Sheng Wang, Srikumar Chellappan, Rajendra G Mehta