A23187, ionomycin and thapsigargin upregulate mRNA of HIF-1alpha via endoplasmic reticulum stress rather than a rise in intracellular calcium.
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-1alpha. While lowering cytosolic calcium accumulates HIF-1alpha, a calcium increase by the ionophores A23187 or ionomycin as well as the endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor thapsigargin produced inconsistent results with reports to either increase or decrease HIF-1alpha protein. In the human hepatocyte cell line HepG2 only A23187, but neither ionomycin nor thapsigargin, accumulated HIF-1alpha protein under normoxia. However, A23187 does so independently of a calcium increase. A23187 not only accumulated HIF-1alpha protein but also mRNA of HIF-1alpha, with the notion that protein but not mRNA accumulation was attenuated by blocking mitogen-activated protein kinase (MAPK), suggesting that HIF-1alpha protein accumulation is not a direct consequence of its mRNA elevation. Indeed, protein stability determinations implied that A23187 enhanced translation of HIF-1alpha. Interestingly, ionomycin and thapsigargin also increased the HIF-1alpha mRNA content. Although not increasing the HIF-1alpha protein amount under normoxia, both compounds enhanced protein accumulation of HIF-1alpha under hypoxia. Taking into account that induction of ER stress by tunicamycin and brefeldin A, without altering intracellular calcium concentrations, also increased HIF-1alpha mRNA, suggests that ER stress pathways enhanced transcription of HIF-1alpha mRNA. We conclude that ER stress rather than calcium fluctuations increased HIF-1alpha mRNA content by established calcium liberating agents, which alone is insufficient for normoxic HIF-1alpha accumulation.
Institute of Biochemistry I/ZAFES, Faculty of Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany.
Christian
C
Werno
Jie
J
Zhou
Bernhard
B
Brüne
D002070:Butadienes / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects*
D002070:Butadienes; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009570:Nitriles; D012333:RNA, Messenger; C113580:U 0126; D000001:Calcimycin; D015759:Ionomycin; D019284:Thapsigargin; D002118:Calcium