Transcriptional and post-translational modulation of myo-inositol oxygenase by high glucose and related pathobiological stresses.

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.