title: Lipopolysaccharide and hypoxia-induced HIF-1 activation in human gingival fibroblasts.
abstract: BACKGROUND
  We previously reported that chronic periodontal inflammation causes the accumulation of the transcriptional activator hypoxia-inducible factor-1α (HIF-1α) in human gingival fibroblasts (HGFs) in vivo. Here, evidence is provided that bacterial lipopolysaccharides (LPS) and cellular hypoxia, both associated with periodontitis, can individually, or in combination, lead to the accumulation and activation of HIF-1 in HGF in vitro.
  
  
  METHODS
  Primary gingival fibroblasts were cultured from human gingival biopsies. HIF-1α peptide from HGFs treated with Escherichia coli LPS under normoxia or hypoxia was detected by nuclear protein extraction, immunoprecipitation, immunoblotting, and immunocytofluorescence. HIF-1α transcripts were detected using reverse transcription polymerase chain reaction (PCR). The transcript expression levels of vascular endothelial growth factor-A (VEGF-A), a downstream gene of HIF-1α, were assessed by quantitative real-time PCR.
  
  
  RESULTS
  Two HIF-1α splicing transcription variants were found to be constitutively expressed in HGFs. E. coli LPS induced a dose- and time-dependent nuclear accumulation of HIF-1α peptide in HGFs. This accumulation could be attenuated by treatment with a Toll-like receptor 4 (TLR4)-neutralizing antibody. Under hypoxia, LPS further increased HIF-1α accumulation in HGFs. VEGF-A transcript expression was upregulated by LPS under both normoxia and hypoxia but was downregulated by pretreatment with TLR4-neutralizing antibody or the specific HIF-1α inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole.
  
  
  CONCLUSIONS
  LPS induces the nuclear accumulation of HIF-1α in HGFs and induces HIF-1 biologic activity under normoxia or hypoxia possibly through TLR4.
authors.affiliation: Faculty of Dentistry, The University of Hong Kong, 34 Hospital Rd., Hong Kong, China.
authors.forename: Jing-Ping
authors.initials: JP
authors.lastname: Li
authors.forename: François Y L
authors.initials: FY
authors.lastname: Li
authors.forename: Aimin
authors.initials: A
authors.lastname: Xu
authors.forename: Bin
authors.initials: B
authors.lastname: Cheng
authors.forename: Sai Wah
authors.initials: SW
authors.lastname: Tsao
authors.forename: Man-Lung
authors.initials: ML
authors.lastname: Fung
authors.forename: W Keung
authors.initials: WK
authors.lastname: Leung
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D018929:Cell Culture Techniques; D015687:Cell Hypoxia / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology; D004926:Escherichia coli; D005091:Exons / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D014644:Genetic Variation / Q000235:genetics; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006162:Guanylate Cyclase / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000235:genetics; D015151:Immunoblotting; D047468:Immunoprecipitation; D007191:Indazoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000502:physiology; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects / Q000235:genetics
chemical_list: D057134:Antibodies, Neutralizing; D020536:Enzyme Activators; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007191:Indazoles; D008070:Lipopolysaccharides; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D006162:Guanylate Cyclase