Abstract
Renal-specific oxidoreductase/myo-inositol oxygenase (RSOR/MIOX) catabolizes myo-inositol and is implicated in the pathogenesis of diabetic nephropathy. How high glucose (HG) ambience up-regulates its expression and enzyme activity was investigated. MIOX up-regulation was associated with an increase in enzyme activity, which was reduced to basal levels with phosphatase treatment. Using phosphothreonine, protein kinase A (PKA), and PKC substrate antibodies, analyses of kidney lysates of diabetic animals and LLC-PK1/HK-2 cells subjected to HG ambience indicated MIOX to be a phosphoprotein. Kinase phosphorylated recombinant RSOR/MIOX proteins had increased activity confined to exons 2-5. Mutants with substituted phosphorylation sites had a minimal increase in activity. Treatment of cells with PKC, PKA, and PDK1 kinase activators increased activity, whereas inhibitors reduced it. Inhibitors also reduced the phosphorylation and activity of MIOX induced by HG. Besides HG, exposure of cells to oxidants H(2)O(2) and methylglyoxal up-regulated MIOX expression and its phosphorylation and activity, whereas antioxidants N-acetylcysteine, β-naphthoflavone, and tertiary butyl hydroquinone reduced MIOX expression. Treatment with HG or oxidants or overexpression of MIOX induced nuclear translocation of redox-sensitive transcription factor Nrf2, which binds to antioxidant response elements of various promoters. Promoter analyses revealed an increase in luciferase activity with HG and oxidants. Analyses of antioxidant response elements and carbohydrate response elements revealed an accentuation of DNA-protein interactions with oxidants and under HG ambience. ChIP-PCR and immunofluorescence studies revealed nuclear translocation of carbohydrate response element-binding protein. These findings suggest that phosphorylation of RSOR/MIOX enhances its activity, which is augmented by HG via transcriptional/translational events that are also modulated by diabetes-related pathobiological stresses.
Highlights
27594 JOURNAL OF BIOLOGICAL CHEMISTRY sion to glomerulosclerosis, tubular atrophy, and interstitial fibrosis, resulting in renal failure (1–3)
Chromatin Immunoprecipitation (ChIP)-PCR and immunofluorescence studies revealed nuclear translocation of carbohydrate response element-binding protein. These findings suggest that phosphorylation of RSOR/myo-inositol oxygenase (MIOX) enhances its activity, which is augmented by high glucose (HG) via transcriptional/translational events that are modulated by diabetes-related pathobiological stresses
The findings described include the induction of MIOX/RSOR expression and activity in a hyperglycemic state and under high glucose ambience via phosphorylation of its potential PKC, protein kinase A (PKA), and PDK1-phosphorylating sites and how hyperglycemia, glucose-derived carbonyls (MGO), and various oxidants and antioxidants affect its translational and transcriptional activities, the latter seeming to be largely influenced by unique DNA sites for the binding of certain transcription factors localized in its promoter
Summary
MIOX is highly expressed in the kidney, in the proximal tubules (26). The proximal tubular epithelial cells are believed to be involved in the pathogenesis of corticointerstitial fibrosis (27, 28), underscoring the importance in investigating the relevance of the proximal tubule metabolic enzyme MIOX in diabetic nephropathy. MIOX expression is up-regulated in diabetes mellitus (23, 28), and conceivably, its up-regulation may be responsible for the concomitant MI depletion and increased urinary excretion. The events that follow high glucose ambience or diabetic state include increased flux of glucose intermediaries into various cellular metabolic pathways, increased synthesis of advanced glycation end products, activation of signaling molecules like PKC and PKA, up-regulation of TGF-, generation of reactive oxygen species (ROS), and excessive accumulation of extracellular matrix (4 –12, 30, 31). Because MIOX is intimately involved in glucose metabolism and it catabolizes MI, which in turn modulates phosphoinositide signaling (see above), it would be of special interest to explore the mechanism(s) of its up-regulation by high glucose ambience and stresses associated with it, e.g. oxidant stress. Novo synthesis and intracellular MI turnover and to delineate the transcriptional and post-translational events that modulate the functional activity of RSOR/MIOX
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