Diabetologie und Stoffwechsel 2006; 1 - A85
DOI: 10.1055/s-2006-943810

Sp1 O-glycosylation leads to glucose-dependent NF-κBp65 activation

Y Seregin 1, O Voron'ko 1, Z Djuric 1, PM Humpert 1, G Rudofsky 1, I Konrade 1, M Morcos 1, PP Nawroth 1, A Bierhaus 1
  • 1University of Heidelberg, Department of Medicine I, Heidelberg, Germany

Introduction: The transcription factor NF-κB plays an important role in diabetes. The molecular mechanisms underlying the observed long-term activation of NF-κB p65 mRNA transcription in diabetes are yet unknown. Characteristics of the p65 promoter shows three binding sites for the transcription factors Sp1/Sp3, implying that these proteins might act as transactivators of p65 de novo synthesis under hyperglycemic conditions.

Aims: We studied the influence of glucose-mediated posttranslational modifications of Sp1 on NF-κB p65 gene transcription to characterize the molecular mechanisms underlying p65 de novo synthesis in diabetes.

Methods: Binding activity of Sp1/Sp3 was measured in THP-1 and BAEC cells using electrophoretic mobility shift assay (EMSA). Full-length p65 promoter and its deletion mutants were cloned into luciferase reporter vectors and transfected into THP-1 and BAEC cells. Posttranslation modifications of Sp1 (O-glycosylation, phosphorylation and nitrotyrosine residues) were characterised by immunoprecipitation followed by immunoblotting and chromatin precipitation (ChIP)-experiments. NF-κBp65 mRNA was determined by RT-PCR.

Results: High glucose (30 mM) induced a time- and dose-dependent activation of Sp1 binding to p65-specific Sp1 sequences. The binding is functionally active as stimulation with 30mM glucose was followed by induction of p65 transcription. Sp1-antisense blocked p65 mRNA synthesis. Transient transfection of p65 promoter constructs indicated that deletion of every Sp1 binding site significantly impaired p65 expression. Sp1 is constitutively phosphorylated and O-glycosylated, however, O-glycosylation of Sp1 was increased in high glucose. Selective down-regulation of O-glycosylation by blocking the hexosamine pathway or up-regulation by blocking the enzyme O-GlcNAcase resulted in a significant reduction of Sp1 binding activity and p65 mRNA.

Conclusion: High glucose induces O-glycosylation of Sp1, which results in increased Sp1 binding to the NF-κB p65 promoter and activation of p65 de novo synthesis.