Down-regulation of Sp1 Activity through Modulation of O-Glycosylation by Treatment with a Low Glucose Mimetic, 2-Deoxyglucose

Hyun Tae Kang,Jung Won Ju,Jin Won Cho,Eun Seong Hwang

doi:10.1074/jbc.m307332200

Hyun Tae Kang, Jung Won Ju + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m307332200

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Abstract

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.

Highlights

For most tumor cells and some rapidly proliferating cells, poor blood supply would impose an environmental stress such as nutritional starvation and hypoxia, which leads to
Through investigation on the possible regulatory events associated with the glucose-linked modulation of human papillomavirus (HPV) upstream regulatory region (URR) and p21 WAF1 transcription, we found that Sp1 activity is inhibited and that its glycosylation status is severely altered by the treatment of 2-DG
Our results indicate that 2-DG causes repression of certain promoters by inducing hyper-O-GlcNAcylation of Sp1

Summary

Introduction

For most tumor cells and some rapidly proliferating cells, poor blood supply would impose an environmental stress such as nutritional starvation and hypoxia, which leads to. There might be several different routes through which 2-DG causes repression of HPV gene expression: inhibition of glycolysis and ATP production, interference in protein glycosylation and the resulting endoplasmic reticulum stress, and attenuation of activities of certain transcription factors through alterations in its O-GlcNAcylation level.

Results

Conclusion