A23187, ionomycin and thapsigargin upregulate mRNA of HIF‐1α via endoplasmic reticulum stress rather than a rise in intracellular calcium

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Abstract
Hypoxia inducible factor 1 (HIF‐1) coordinates major responses to hypoxia, with the notion that its alpha subunit is degraded under normoxia but stable under hypoxia. Recently, calcium was shown to affect expression of HIF‐1α. While lowering cytosolic calcium accumulates HIF‐1α, a calcium increase by the ionophores A23187 or ionomycin as well as the endoplasmic reticulum (ER) Ca2+‐ATPase inhibitor thapsigargin produced inconsistent results with reports to either increase or decrease HIF‐1α protein. In the human hepatocyte cell line HepG2 only A23187, but neither ionomycin nor thapsigargin, accumulated HIF‐1α protein under normoxia. However, A23187 does so independently of a calcium increase. A23187 not only accumulated HIF‐1α protein but also mRNA of HIF‐1α, with the notion that protein but not mRNA accumulation was attenuated by blocking mitogen‐activated protein kinase (MAPK), suggesting that HIF‐1α protein accumulation is not a direct consequence of its mRNA elevation. Indeed, protein stability determinations implied that A23187 enhanced translation of HIF‐1α. Interestingly, ionomycin and thapsigargin also increased the HIF‐1α mRNA content. Although not increasing the HIF‐1α protein amount under normoxia, both compounds enhanced protein accumulation of HIF‐1α under hypoxia. Taking into account that induction of ER stress by tunicamycin and brefeldin A, without altering intracellular calcium concentrations, also increased HIF‐1α mRNA, suggests that ER stress pathways enhanced transcription of HIF‐1α mRNA. We conclude that ER stress rather than calcium fluctuations increased HIF‐1α mRNA content by established calcium liberating agents, which alone is insufficient for normoxic HIF‐1α accumulation. J. Cell. Physiol. 215: 708–714, 2008.

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