Transcriptional mechanisms linking hypoxia and expression of inositol 1,4,5-trisphosphate (IP3) receptors

Background and Purpose Glioblastoma multiforme (GBM) is the most clinically aggressive form of glioma. Hypoxic regions were observed in GBM biopsies. Hypoxia appeared to be correlated with the aggressiveness of glioma. Preliminary data showed that type 2 IP₃ receptor was downregulated following hypoxia. I hypothesised that the down-regulation is mediated through hypoxia-inducible factor (HIF)-1-mediated transcriptional effects. Experimental Approach Sub-confluent human U87 glioma cells were cultured in normoxia and treated with either vehicle control or the hypoxia-mimicking agent desferrioxamine (DFO; 0.5 mM) for 24 hours. Cells were also cultured in hypoxia and treated with either vehicle control or the HIF-1α translational inhibitor KC7F2 (40 μM). Two-step quantitative reverse transcription polymerase chain reaction was used to quantify the transcript levels of IP₃ receptor subtypes after the treatments. The levels were normalised to that of β-actin and TATA-binding protein. Key Results In contrast to previous reports, expression of β-actin was not stable in hypoxia and is on its own unsuitable to be used as the reference gene. Hypoxia did not significant alter expression of individual IP₃ receptor subtypes transcriptionally. Treatment with DFO upregulated transcript level of type 3 IP₃ receptor only (<i>p</i> = 0.0082). KC7F2 treatment reduced HIF-1α level by three-fold but did not affect the observed upregulation. Conclusions and Implications Hypoxia-mediated down-regulation of expression of type 2 IP₃ receptor does not rely on transcriptional mechanisms. Further studies of its expression on a protein level can be done to test the proposition that it is dependent on co-translational or post-translational mechanisms.