Lipopolysaccharide and hypoxia-induced HIF-1 activation in human gingival fibroblasts

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.

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.

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.

LPS induces the nuclear accumulation of HIF-1α in HGFs and induces HIF-1 biologic activity under normoxia or hypoxia possibly through TLR4.