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 (p = 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.