Influence of hypoxia inducible factor (HIF)-1α on TLR6 expression during hypoxia.

<p><i>A</i>, Map of TLR6 promoter region showing positions of the putative HIF binding sites and the binding site for NFκB relative to the transcription start site (TSS). <i>B</i>, Stable transfected HMEC-1 monolayers containing either HIF-1α siRNA or control-siRNA were exposed to normoxia or hypoxia for indicated time points. Total RNA was isolated, and 1 µg of RNA was transcribed into first strand cDNA. Relative expressional levels of TLR6 transcripts were compared to normoxic controls by real-time RT-PCR. Data were calculated relative to internal control gene (ß-actin), and are expressed as fold change over normoxia±SEM, *, significant differences from normoxia and control cells. Results are derived from three different experiments in each condition. <i>C</i>, Total RNA of normoxic monolayers of either wildtype (WT) or oxygen-stable HIF-1α expressing (HIF^+/+) HMEC-1 cells was isolated and realt-time RT-PCR was performed as described above. *, significant differences from wildtype cells. <i>D</i>, Western blot analysis of TLR6 protein of normoxic HMEC-1 wildtype (WT) and oxygen-stable HIF-1α expressing (HIF+/+) cells. The same blot was probed for ß-actin expression as a control for protein loading. <i>E</i>, HMEC-1 monolayers were treated with 1mM of dimethyloxalylglycine (DMOG) for 24 hours. Afterwards transcript levels of TLR6 where quantified by real-time RT-PCR as described above. *, significant differences from untreated cells. <i>F</i>, ChIP assay was utilized to examine HIF-1α binding to the TLR6 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific igG monoclonal antibody (IgG) and PCR performed using HMEC-1 DNA (input). An example of three experiments is shown.