title: High Glucose-Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase: Implication in the Pathobiology of Diabetic Tubulopathy.
abstract: The catabolic enzyme myo-inositol oxygenase (MIOX) is expressed in proximal tubules and up-regulated in the diabetic state. Previously, we reported its transcriptional and translation regulation by high glucose (HG), osmolytes, and fatty acids. However, its epigenetic regulation is unknown. Bisulfite sequencing revealed that both human and mouse MIOX promoters, enriched with CpG sites, are hypomethylated and unmethylated under HG ambience and hyperglycemic states associated with increased MIOX expression. Eletrophoretic mobility shift assays revealed increased binding of unmethylated oligos with nucleoproteins of cells maintained under HG. In addition, a strong binding of specificity protein (Sp)-1 transcription factor with MIOX promoter was observed under HG, especially with unmethylated Sp-1 oligo. Specificity of binding was established by supershift assays and treatment with the Sp-1 inhibitor mithramycin. Promoter analysis revealed an increase in luciferase activity under HG, which was reduced after mutation of the Sp-1-binding site. Sp1 siRNA treatment reduced mRNA and protein expression of Sp-1 and MIOX and generation of reactive oxygen species derived from NADPH oxidase (NOX)-4 and mitochondrial sources. In addition, there was reduced expression of hypoxia-inducible factor-1α relevant in the pathogenesis of diabetic nephropathy. Sp1 siRNA treatment reduced fibronectin expression, an extracellular matrix protein that is increased in diabetic nephropathy and tubulopathy. HG-induced MIOX expression was also reduced with the treatment of apelin-13, which deacetylates histones. Overall, these findings highlight the epigenetic regulation of MIOX in the pathogenesis of diabetic tubulopathy.
authors.affiliation: Department of Pathology, Northwestern University, Chicago, Illinois.
authors.forename: Isha
authors.initials: I
authors.lastname: Sharma
authors.affiliation: Department of Pathology, Northwestern University, Chicago, Illinois.
authors.forename: Rajesh K
authors.initials: RK
authors.lastname: Dutta
authors.affiliation: Department of Pathology, Northwestern University, Chicago, Illinois.
authors.forename: Neel K
authors.initials: NK
authors.lastname: Singh
authors.affiliation: Department of Pathology, Northwestern University, Chicago, Illinois; Department of Medicine, Northwestern University, Chicago, Illinois. Electronic address: y-kanwar@northwestern.edu.
authors.forename: Yashpal S
authors.initials: YS
authors.lastname: Kanwar
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D003928:Diabetic Nephropathies / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D005353:Fibronectins / Q000378:metabolism; D017353:Gene Deletion; D005947:Glucose / Q000633:toxicity*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D050562:Inositol Oxygenase / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*
chemical_list: D005353:Fibronectins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D036341:Intercellular Signaling Peptides and Proteins; D009698:Nucleoproteins; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; C495199:apelin-13 peptide; D050562:Inositol Oxygenase; D005947:Glucose