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Promoting ubiquitination‐mediated degradation of Orai1 protein by high glucose in glomerular mesangial cells
Author(s) -
Chaudhari Sarika,
Hui Jiang,
Zou Shubiao,
Huang Linjing,
Ma Rong
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.620.4
Subject(s) - orai1 , mg132 , cycloheximide , chemistry , protein degradation , western blot , ubiquitin , medicine , endocrinology , pathogenesis , protein turnover , microbiology and biotechnology , proteasome , endoplasmic reticulum , protein biosynthesis , proteasome inhibitor , biology , stim1 , biochemistry , gene
Glomerular mesangial cells (MCs) are an important target of metabolic abnormalities in a diabetic environment. The Orai1‐mediated store‐operated calcium entry (SOCE) is associated with many physiological processes in a variety of cells, including MCs. However, whether SOCE in MCs is involved in diabetic kidney disease is not clear. High glucose (HG) is the principal cause of MC pathogenesis in diabetes and altered MC function by HG is central to the pathogenesis of progressive diabetic glomerulopathy. The present study was carried out to determine if HG treatment altered the protein content of Orai1 and the protein‐mediated SOCE in MCs. Western blot was conducted to estimate abundance of Orai1 protein and Fura‐2 fluorescence ratiometry was used to analyze SOCE. We found that treatment of rat MCs with HG (25 mM) for time periods ranging from 2 hours to 24 hours decreased the abundance of Orai1 protein. A significant decrease was observed at the time point of 8 hours, which sustained at least for additional 16 hours. Consistently, HG treatment for 8 hours significantly reduced SOCE. HG treatment for the same time periods did not alter the level of Orai1 transcript. In the presence of cycloheximide, a protein synthesis inhibitor, the HG effects on the level of Orai1 protein still existed, suggesting posttranslational mechanisms involved. Furthermore, both MG132 (the ubiquitin‐proteasome inhibitor) and NH 4 Cl (the lysosomal pathway inhibitor) significantly attenuated the HG‐induced reduction of Orai1 protein abundance. Moreover, HG treatment for 8 hours stimulated ubiquitination of Orai1 protein. We further found that HG treatment elevated the level of cellular hydrogen peroxide (H 2 O 2 ) in a time‐dependent manner. Treatment of cells with PEG‐catalase significantly blunted the HG‐induced reduction of Orai1 protein abundance. In addition, H 2 O 2 itself also significantly decreased the abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Taking together, these results suggest that HG treatment for a short‐term decreased abundance of Orai1 protein in MCs by promoting its degradation through the ubiquitination‐proteosome and –lysosome mechanisms. This HG‐stimulated posttranslational regulation of Orai1 protein was mediated by H 2 O 2 . Support or Funding Information 1) NIH/NIDDK: R56 DK108761; 2) NIH/NIDDK: 1RO1DK115424‐01; 3) AHA Southwest Affiliate: 16GRNT27780043; 4) The Harry S. Moss Heart Trust. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .