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Hypoxia Induced Redistribution of OS‐9 in Kidney Cells
Author(s) -
Harris Regine,
Stanley Corshe,
Ongeri Elimelda Moige
Publication year - 2015
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.29.1_supplement.663.6
Subject(s) - cytosol , chemistry , western blot , endoplasmic reticulum , kidney , microbiology and biotechnology , transfection , biochemistry , biology , endocrinology , enzyme , gene
OS‐9 is a component of the endoplasmic reticulum‐associated degradation machinery. OS‐9 has been shown to interact with and promote hydroxylation of the hypoxia‐inducible factor (HIF1α). Meprin metalloproteases are abundantly expressed in the brush border membranes (BBM) of proximal kidney tubules. Redistribution of meprins from the BBM to the cytosol and basolateral membranes occur in ischemia‐reperfusion induced renal injury. Meprin β interacts with and cleaves OS‐9 in an isoform‐specific manner. The present study sought to determine how hypoxia impacts OS‐9 localization in Mardin‐Darby canine kidney (MDCK) cells transfected with meprin β cDNA. The cells were depleted of oxygen by exposure to 125 µM cobalt chloride (CoCl 2 ) for 0, 0.5, 1, 2, and 3 h. Proteins were extracted from the cells and fractionated into cytosolic‐ and nuclear‐enriched fractions. Western blot analysis and optic densitometry were used to quantify the levels of OS‐9 in the cytosolic‐ and nuclear‐enriched protein fractions, and the levels of HIF1α in nuclear‐enriched protein fractions. Additional cells were cultured in slide chambers, fixed in paraformaldehyde, and probed with anti‐OS‐9 and anti‐HIF1α antibodies using immunofluorescent staining. Localization of HIF1α and OS‐9 in the cells was evaluated by confocal microscopy. Exposure to CoCl 2 resulted in increased HIFα levels in the nucleus and a time‐dependent redistribution of OS‐9 from the nucleus to the cytosol. In the meprin transfected cells, the cytosolic levels of OS‐9 subsequently decreased. The decrease in cytosolic OS‐9 levels was not observed in non‐transfected control cells, suggesting meprin mediated degradation of OS‐9. These data provide evidence that meprins may impact the response to hypoxia in part via modulation of cytosolic OS‐9 levels.