title: Basal transcription is regulated by lipopolysaccharide and glucosamine via the regulation of DNA binding of RNA polymerase II in RAW264.7 cells.
abstract: OBJECTIVE
  The objective of this study is to investigate glucosamine (GlcN) as a transcriptional regulator of iNOS and other genes in association with the dynamic O-GlcNAcylation of RNA polymerase II (RNAPII).
  
  
  METHODS
  The LPS- and/or GlcN-stimulated transcriptional activities of various Gal4-binding site/TATA-box-containing reporter constructs were measured.
  
  
  RESULTS
  Basal transcriptional activities of nuclear factor-κB (NF-κB) and nitric oxide synthase (iNOS) reporter plasmids are inhibited by GlcN in RAW264.7 cells. Furthermore, GlcN suppressed whereas lipopolysaccharide (LPS) stimulated the basal activity of Gal4-binding site/TATA-box-containing reporter constructs. LPS reduced the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of RNAPII, but enhanced the binding of this enzyme to the iNOS promoter. In contrast, GlcN enhanced RNAPII O-GlcNAcylation, but inhibited iNOS promoter binding. Furthermore, the basal activities of reporter plasmids containing activator protein 1 (AP1), E2F, or cyclic AMP response element (CRE) binding sites were consistently inhibited by GlcN in a dose-dependent manner. However, GlcN did not inhibit the phorbol 12-myristate 13-acetate- (PMA-) or forskolin-induced transcriptional activities of AP1 and CRE. The transcriptional activity of transforming growth factor alpha (TGF-α) was slightly increased by both LPS and GlcN.
  
  
  CONCLUSIONS
  In conclusion, our data demonstrate that LPS activates, whereas GlcN suppresses, basal activities of transcription through the regulation of RNAPII O-GlcNAcylation and DNA binding.
authors: Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon, Republic of Korea.
authors: Ji-Sun
authors: JS
authors: Hwang
authors: Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon, Republic of Korea.
authors: So-Young
authors: SY
authors: Hwang
authors: Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon, Republic of Korea. Electronic address: iohan@inha.ac.kr.
authors: Inn-Oc
authors: IO
authors: Han
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D050684:E2F Transcription Factors / Q000378:metabolism; D005700:Galactosyltransferases; D005786:Gene Expression Regulation / Q000502:physiology*; D005944:Glucosamine / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000378:metabolism*; D008156:Luciferases; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D050436:Regulatory Elements, Transcriptional / Q000235:genetics / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014909:Wheat Germ Agglutinins
chemical_list: D050684:E2F Transcription Factors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014909:Wheat Germ Agglutinins; D004247:DNA; D008156:Luciferases; D019001:Nitric Oxide Synthase; D005700:Galactosyltransferases; D012319:RNA Polymerase II; D005944:Glucosamine
keywords: Glucosamine; O-GlcNAc; RNAPII