The transition from HIF-1 to HIF-2 during prolonged hypoxia results from reactivation of PHDs and HIF1A mRNA instability

Abstract

The hypoxia-inducible factors (HIF) are transcription factors that activate the adaptive hypoxic response when oxygen levels are low. The HIF transcriptional program increases oxygen delivery by inducing angiogenesis and by promoting metabolic reprograming that favors glycolysis. The two major HIFs, HIF-1 and HIF-2, mediate this response during prolonged hypoxia in an overlapping and sequential fashion that is referred to as the HIF switch. Both HIF proteins consist of an unstable alpha chain and a stable beta chain. The instability of the alpha chains is mediated by prolyl hydroxylase (PHD) activity during normoxic conditions, which leads to ubiquitination and proteasomal degradation of the alpha chains. During normoxic conditions, very little HIF-1 or HIF-2 alpha–beta dimers are present because of PHD activity. During hypoxia, however, PHD activity is suppressed, and HIF dimers are stable. Here we demonstrate that HIF-1 expression is maximal after 4 h of hypoxia in primary endothelial cells and then is dramatically reduced by 8 h. In contrast, HIF-2 is maximal at 8 h and remains elevated up to 24 h. There are differences in the HIF-1 and HIF-2 transcriptional profiles, and therefore understanding how the transition between them occurs is important and not clearly understood. Here we demonstrate that the HIF-1 to HIF-2 transition during prolonged hypoxia is mediated by two mechanisms: (1) the HIF-1 driven increase in the glycolytic pathways that reactivates PHD activity and (2) the much less stable mRNA levels of HIF-1α (HIF1A) compared to HIF-2α (EPAS1) mRNA. We also demonstrate that the alpha mRNA levels directly correlate to the relative alpha protein levels, and therefore to the more stable HIF-2 expression during prolonged hypoxia.

A novel OSA-related model of intermittent hypoxia in endothelial cells under flow reveals pronounced inflammatory pathway activation

“…In contrast, a 4 hour incubation with permanent hypoxia of 1% of oxygen clearly induced HIF stabilization. Our data are in line with different publications showing that various other cell types similarly require lower oxygen concentrations or prolonged exposure in order to mediate HIF1α and HIF2α accumulation in a time-dependent manner ( Bracken et al, 2006 ; Bartoszewski et al, 2019 ; Jaskiewicz et al, 2022 ). From a translational perspective, it needs to be considered that coronary artery endothelial cells are not exposed to such low oxygen concentrations during OSA desaturations further underlining the need for more physiological OSA models.…”

Hypoxia‐Inducible Factor‐2α Signaling in the Skeletal System

“…( 54 ) In this study, Hif2a mRNA levels were twofold higher than Hif1a under normoxia and were fivefold higher during chronic hypoxia (24 hours). ( 54 ) Time‐course mRNA studies on human endothelial cells under hypoxia revealed that differences in Hif1a and Hif2a mRNA levels were the result of microRNA destabilization of the adenylate‐uridylate‐rich element (ARE) at the 3' end of Hif1a and Hif2a transcript untranslated region. The Hif1a 3' UTR is highly sensitive to ARE‐dependent destabilization compared with the 3' UTR of Hif2a .…”

“…( 52 , 53 ) In human umbilical vein endothelial cells (HUVECs) exposed to hypoxia, HIF‐1α rapidly accumulates during acute hypoxia (4 hours), reduces during the acute‐chronic hypoxia transition (8 hours), and drastically reduces thereafter during chronic hypoxia exposure (24 hours). ( 54 , 55 ) HIF‐2α levels reach a peak during the acute‐chronic hypoxia transition (8 hours) and are sustained during chronic hypoxia exposure (24 hours). ( 54 , 55 ) Hif1a mRNA levels were reduced post‐HIF‐1α peak (at 8 hours), whereas Hif2a mRNA levels were sustained at higher levels throughout the time course.…”

“…( 54 , 55 ) HIF‐2α levels reach a peak during the acute‐chronic hypoxia transition (8 hours) and are sustained during chronic hypoxia exposure (24 hours). ( 54 , 55 ) Hif1a mRNA levels were reduced post‐HIF‐1α peak (at 8 hours), whereas Hif2a mRNA levels were sustained at higher levels throughout the time course. ( 54 ) In this study, Hif2a mRNA levels were twofold higher than Hif1a under normoxia and were fivefold higher during chronic hypoxia (24 hours).…”

“…( 54 , 55 ) Hif1a mRNA levels were reduced post‐HIF‐1α peak (at 8 hours), whereas Hif2a mRNA levels were sustained at higher levels throughout the time course. ( 54 ) In this study, Hif2a mRNA levels were twofold higher than Hif1a under normoxia and were fivefold higher during chronic hypoxia (24 hours). ( 54 ) Time‐course mRNA studies on human endothelial cells under hypoxia revealed that differences in Hif1a and Hif2a mRNA levels were the result of microRNA destabilization of the adenylate‐uridylate‐rich element (ARE) at the 3' end of Hif1a and Hif2a transcript untranslated region.…”