HIF-1α的可逆性SUMO化修饰

低氧诱导因子1(hypoxia inducible factor-1, HIF-1)是参与调节机体氧平衡的重要转录因子,在细胞低氧应答反应中起核心作用,能调节100多种涉及低氧应激下细胞适应和存活的靶基因．HIF-1由氧敏感的α亚基和在细胞内稳定表达的β亚基组成．其中α亚基可受到多种翻译后化学修饰作用,如在常氧下,HIF-1α通过泛素化蛋白酶修饰并导致其快速降解．最近几年发现的泛素样蛋白家族成员小泛素蛋白样修饰蛋白（SUMO）也能与HIF-1α共价结合．SUMO是一种分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在．SUMO可共价结合许多靶底物蛋白,并对其进行翻译后修饰,该过程称为SUMO化．与泛素化蛋白酶体途径不同的是,SUMO化修饰能在常氧和相对低氧的条件下调节HIF-1α蛋白的稳定性,从而改变其转录活性．SUMO化是一个可逆的动态过程,可被特异性蛋白酶ULP/SENP将其从底物上去除．本文主要就HIF-1α的可逆性SUMO化修饰作一综述．     

Oxygen treatment after experimental hypoxia-ischemia in neonatal rats alters the expression of HIF-1alpha and its downstream target genes.

Recently, mounting evidence has emerged to suggest that hyperbaric oxygenation (HBOT)-induced neuroprotection after experimental global ischemia and subarachnoid hemorrhage entails a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Therefore, the purpose of this study was to test the hypothesis that oxygen-induced neuroprotection after neonatal hypoxia-ischemia involves alterations in the expression of HIF-1alpha. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O(2) at 37 degrees C). Pups were then treated with HBOT (2.5 ATA) or normobaric oxygenation treatment (NBOT) for 2 h. The expression and phosphorylation status of HIF-1alpha was evaluated at intervals up to 24 h after the insult, as was the expression of glucose transporter (GLUT)-1, GLUT-3, lactate dehydrogenase (LDH), aldolase (Ald), and p53. The protein-protein interaction of HIF-1alpha and p53 was also examined. An elevated expression of HIF-1alpha, GLUT-1, GLUT-3, Ald, and LDH was observed after the insult. An increase in the dephosphorylated form of HIF-1alpha was followed by an increase in the association of HIF-1alpha with p53 and an increase in p53 levels. Both HBOT and NBOT reduced the elevated expression of HIF-1alpha and decreased its dephosphorylated form. Furthermore, both treatments promoted a transient increase in the expression of GLUT-1, GLUT-3, LDH, and Ald, while decreasing the HIF-1alpha-p53 interaction and decreasing the expression of p53. Therefore, the alteration of the HIF-1alpha phenotype by a single oxygen treatment may be one of the underlying mechanisms for the observed oxygen-induced neuroprotection seen when oxygen is administered after a neonatal hypoxic-ischemic insult.     

Mitochondrial Bioenergetics of Metastatic Breast Cancer Cells in Response to Dynamic Changes in Oxygen Tension: Effects of HIF-1α

Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.     

Activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway

Accumulation of HIF-1alpha during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF-1alpha protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF-1alpha, obtained under normoxia, has been underestimated. In this study, we have systematically compared the related pools of HIF-1alpha stabilized in normoxia by high cell density to those obtained at low density in hypoxia. At first glance, these two stimuli appear to have similar outcomes: HIF-1alpha stabilization and induction of HIF-1-dependent genes. However, upon careful analysis, we observed that molecular mechanisms involved are different. We clearly demonstrate that density-dependant HIF-1alpha accumulation during normoxia is due to the cells high consumption of oxygen, as demonstrated by using a respiration inhibitor (oligomycin) and respiratory-defective mutant cells (GSK3). Finally and most importantly, our data indicate that a decrease in AKT activity followed by a total decrease in p70(S6K) phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O(2) levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF-1alpha activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF-1alpha stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF-target genes.     

pPKCα mediated-HIF-1α activation related to the morphological modifications occurring in neonatal myocardial tissue in response to severe and mild hyperoxia

In premature babies birth an high oxygen level exposure can occur and newborn hyperoxia exposure can be associated with free radical oxygen release with impairment of myocardial function, while in adult animal models short exposure to hyperoxia seems to protect heart against ischemic injury. Thus, the mechanisms and consequences which take place after hyperoxia exposure are different and related to animals age. The aim of our work has been to analyze the role played by HIF-1α in the occurrence of the morphological modifications upon hyperoxia exposure in neonatal rat heart. Hyperoxia exposure induces connective compartment increase which seems to allow enhanced blood vessels growth. An increased hypoxia inducible factor-1α (HIF-1α) translocation and vascular endothelial growth factor (VEGF) expression has been found upon 95% oxygen exposure to induce morphological modifications. Upstream pPKC-α expression increase in newborn rats exposed to 95% oxygen can suggest PKC involvement in HIF-1α activation. Since nitric oxide synthase (NOS) are involved in heart vascular regulation, endothelial NOS (e-NOS) and inducible NOS (i-NOS) expression has been investigated: a lower eNOS and an higher iNOS expression has been found in newborn rats exposed to 95% oxygen related to the evidence that hyperoxia provokes a systemic vasoconstriction and to the iNOS pro-apoptotic action, respectively. The occurrence of apoptotic events, evaluated by TUNEL and Bax expression analyses, seems more evident in sample exposed to severe hyperoxia. All in all such results suggest that in newborn rats hyperoxia can trigger oxygen free radical mediated membrane injury through a pPKCα mediated HIF-1α signalling system, even though specificity of such response could be obtained by in vivo administration to the rats of specific inhibitors of PKCα. This intracellular signalling can switch molecular events leading to blood vessels development in parallel to pro-apoptotic events due to an immature anti-oxidant defensive system in newborn rat hearts.