Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose.
2-Deoxyglucose (2-DG), a nonmetabolizable glucose analogue, blocks glycolysis at the phosphohexose isomerase step and has been frequently used as a glucose starvation mimetic in studies of a wide variety of physiological dysfuctions. However, the effect of 2-DG on protein glycosylation and related signal pathways has not been investigated in depth. In HeLa, an HPV18-positive cervical carcinoma line, 2-DG treatment down-regulates human papillomavirus early gene transcription. This down-regulation was also achieved by low glucose supply or hypoxia, suggesting that this is a response commonly modulated by cellular glucose or energy level. We investigated how 2-DG and low glucose affect transcriptional activity. Human papillomavirus gene transcription was only marginally affected by the inhibition of ATP synthesis or the supplementation of pyruvate to 2-DG-treated cells, suggesting that poor ATP generation is involved only to a limited extent. 2-DG treatment also inhibited activation of p21 WAF1 promoter, which is controlled by p53 and/or Sp1. In a reporter assay using p21 WAF1 promoter constructs, 2-DG exerted a strong inhibitory effect on Sp1 activity. DNA binding activity of Sp1 in 2-DG-treated HeLa cells was intact, whereas it was severely impaired in cells incubated in a low glucose medium or in hypoxic condition. Unexpectedly, Sp1 was heavily modified with GlcNAc in 2-DG-treated cells, which is at least partially attributed to the inhibitory effect of 2-DG on N-acetyl-beta-D-glucosaminidase activity. Our results suggest that 2-DG, like low glucose or hypoxic condition, down-regulates Sp1 activity, but through hyper-GlcNAcylation instead of hypo-GlcNAcylation.
Kang
Hyun Tae
HT
Department of Life Science, University of Seoul, Seoul 130-743, Korea.
Ju
Jung Won
JW
Cho
Jin Won
JW
Hwang
Eun Seong
ES
D000117:Acetylglucosamine / Q000494:pharmacology; D000963:Antimetabolites / Q000494:pharmacology*; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004268:DNA-Binding Proteins; D003847:Deoxyglucose / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D018716:Molecular Mimicry; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D000963:Antimetabolites; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D016329:Sp1 Transcription Factor; D003847:Deoxyglucose; D000117:Acetylglucosamine