Involvement of Net and Hif1α in Distinct yet Intricately Linked Hypoxia-induced Signaling Pathways

Abstract

The present study compares negative Ets transcription factor (Net) and hypoxia-inducible factor 1␣ (HIF1␣) regulation by hypoxia. Their protein stabilities are differently regulated by hypoxia, defining three periods in the kinetics: normoxia (high Net levels and low HIF1␣ levels), early hypoxia (high levels of Net and HIF1␣), and late hypoxia (degradation of Net and HIF1␣). Modulators of prolyl hydroxylase domain protein (PHD) activity induce a mobility shift of Net, similar to HIF1␣, suggesting that post-translational modifications of both factors depend on PHD activity. The three PHDs have different roles in the regulation of Net protein levels; PHD1 and PHD3 are involved in the stabilization of Net, whereas PHD2 controls its degradation in late hypoxia. Net physically interacts with PHD2 in hypoxia, whereas PHD1 and PHD3 bind to Net in normoxia and hypoxia. Under the same conditions, PHD2 and PHD3 regulate both HIF1␣ stabilization in early hypoxia and its degradation at late hypoxia, whereas PHD1 is involved in HIF1␣ degradation in late hypoxia. We describe interconnections between the regulation of both Net and HIF1␣ at the protein level. Evidence is provided for a direct physical interaction between Net and HIF1␣ and indirect transcriptional regulation loops that involve the PHDs. Taken together our results indicate that Net and HIF1␣ are components of distinct signaling pathways that are intricately linked.

Hypoxia-inducible factors and the prevention of acute organ injury

“…HIF stimulation is not all-protective. The wide range of HIF-dependent genes, and its tight cross-communication with other key regulators of gene expression [ 13 , 58 , 59 ] raise concern regarding concomitant non-selective activation of protective as well as harmful systems. Among potential unwanted outcomes is the enhancement of tumor growth [ 60 ], promotion of fibrosis [ 61 ] or the induction of pre-eclampsia in pregnant women [ 62 ].…”

Hypoxia-inducible Factors and the Prevention of Acute Organ Injury

“…Th e wide range of HIF-dependent genes, and its tight cross-communication with other key regulators of gene expression [13,58,59] raise concern regarding concomitant non-selective activation of protective as well as harmful systems. Among potential unwanted outcomes is the enhancement of tumor growth [60], promotion of fi brosis [61] or the induction of pre-eclampsia in pregnant women [62].…”

The Oncogenic MicroRNA Hsa-miR-155-5p Targets the Transcription Factor ELK3 and Links It to the Hypoxia Response

“…Hsa-miR-155-5p is a hypoxamir [19] , [20] , raising the possibility that it could regulate ELK3 in hypoxia. Although ELK3 mRNA is stable in 1% oxygen hypoxia for 24 h in SEND endothelial cells [5] , [14] , we have evidence for specific degradation of ELK3 mRNA in more extreme conditions (anoxia, 0% oxygen) in these cells ( Figure S2A–B ). Therefore, we examined the effects of very low oxygen in HUVEC.…”

“…Immunoblotting was performed as described in Serchov et al [14] . The antibody used and dilutions were as follows: rabbit anti-human ELK3-2005 (generated in-house using a peptide fragment from the C-terminus - HMPVPIPSLDRAASPVLLSSNSQKS) 1/1000, mouse anti-human TBP 1/1500 (clone 3TF1/3G3– Active Motif), HIF1α 1/1000 (NB100-134, Novus Biologicals).…”

“…Subsequently, its importance in the hypoxic response was revealed by a micro-array study, which showed that the majority of the genes that required HIF1α for their regulation under hypoxia also required ELK3 [6] . ELK3 and HIF1α have been proposed to be involved in distinct yet intricately linked hypoxia-induced signalling pathways [14] , yet the ways they interact to regulate gene expression is still not fully understood.…”