Hypoxic stabilization of mRNA is HIF-independent but requires mtROS

Grey W Fortenbery,Kyle D Mansfield,Kristen R Carraway,Brinda Sarathy

doi:10.1186/s11658-018-0112-2

Abstract

BackgroundTissue ischemia can arise in response to numerous physiologic and pathologic conditions. The cellular response to decreased perfusion, most notably a decrease in glucose and oxygen, is important for cellular survival. In response to oxygen deprivation or hypoxia, one of the key response elements is hypoxia inducible factor (HIF) and a key protein induced by hypoxia is vascular endothelial growth factor (VEGF). Under hypoxia, we and others have reported an increase in the half-life of VEGF and other hypoxia related mRNAs including MYC and CYR61; however, the mediator of this response has yet to be identified. For this study, we sought to determine if HIF-mediated transcriptional activity is involved in the mRNA stabilization induced by hypoxia.MethodsHEK293T or C6 cells were cultured in either normoxic or hypoxic (1% oxygen) conditions in the presence of 1 g/L glucose for all experiments. Pharmacological treatments were used to mimic hypoxia (desferroxamine, dimethyloxaloglutamate, CoCl2), inhibit mitochondrial respiration (rotenone, myxothiazol), scavenge reactive oxygen species (ROS; ebselen), or generate mitochondrial ROS (antimycin A). siRNAs were used to knock down components of the HIF transcriptional apparatus. mRNA half-life was determined via actinomycin D decay and real time PCR and western blotting was used to determine mRNA and protein levels respectively.ResultsTreatment of HEK293T or C6 cells with hypoxic mimetics, desferroxamine, dimethyloxaloglutamate, or CoCl2 showed similar induction of HIF compared to hypoxia treatment, however, in contrast to hypoxia, the mimetics caused no significant increase in VEGF, MYC or CYR61 mRNA half-life. Knockdown of HIF-alpha or ARNT via siRNA also had no effect on hypoxic mRNA stabilization. Interestingly, treatment of HEK293T cells with the mitochondrial inhibitors rotenone and myxothiazol, or the glutathione peroxidase mimetic ebselen did prevent the hypoxic stabilization of VEGF, MYC, and CYR61, suggesting a role for mtROS in the process. Additionally, treatment with antimycin A, which has been shown to generate mtROS, was able to drive the normoxic stabilization of these mRNAs.ConclusionOverall these data suggest that hypoxic mRNA stabilization is independent of HIF transcriptional activity but requires mtROS.

Highlights

Tissue ischemia can arise in response to numerous physiologic and pathologic conditions
Activation of the hypoxia inducible factor (HIF) pathway is not sufficient to stabilize mRNA In our previous report, we showed that high glucose (4 g/L) could prevent the hypoxic stabilization of many mRNAs including vascular endothelial growth factor (VEGF), MYC, and CYR61 in both HEK293T and rat C6 cells [25]
To investigate whether induction of the HIF pathway is involved in the mRNA stabilization response, we used the hypoxic mimetics desferroxamine (DFX), dimethyloxalylglycine, (DMOG), and cobalt chloride (CoCl2) to induce stabilization of HIF-α protein independent of the oxygen concentrations

Summary

Introduction

Tissue ischemia can arise in response to numerous physiologic and pathologic conditions. We sought to determine if HIF-mediated transcriptional activity is involved in the mRNA stabilization induced by hypoxia. Ischemia results in the deprivation of numerous nutrients, the deprivation of oxygen and glucose arguably have the greatest impact on cellular metabolism, and as such, appropriate adaptation is critical to cellular survival during an ischemic event [1, 2]. There, along with its heterologous binding partner Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT, HIF-β), it binds Hypoxia Response Elements (HREs) within promoters and activates transcription of target genes, such as VEGF and GLUT1, that are necessary for adaptation to low oxygen levels

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Conclusion