Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ...ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.
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•Inhibition of EGLN1 locally or at a distance protects the heart against I/R injury•Remote ischemic protection after EGLN1 loss is mediated by a humoral factor•Diversion of the EGLN1 co-substrate αKG stimulates hepatic kynurenic acid production
Cardiac protection from ischemia can be provided remotely by skeletal muscle inhibition of the oxygen sensor EGLN1, which leads to accumulation of its co-substrate α-ketoglutarate in the blood and increased liver production of the tryptophan metabolite kynurenic acid.
The EGLN (also called PHD) prolyl hydroxylase enzymes and their canonical targets, the HIFα subunits, represent the core of an ancient oxygen-monitoring machinery used by metazoans. In this review, ...we highlight recent progress in understanding the overlapping versus specific roles of EGLN enzymes and HIF isoforms and discuss how feedback loops based on recently identified noncoding RNAs introduce additional layers of complexity to the hypoxic response. Based on novel interactions identified upstream and downstream of EGLNs, an integrated network connecting oxygen-sensing functions to metabolic and signaling pathways is gradually emerging with broad therapeutic implications.
This review from Mircea and Kealin provides a historical perspective on prolyl hydroxylases and their HIF targets, and delves into the more complex role that feedback loops and noncoding RNAs play in sensing and regulating response to hypoxia.
Germline von Hippel-Lindau tumour suppressor gene (VHL) mutations cause renal cell carcinomas, haemangioblastomas and phaeochromocytomas in humans. Mutations in VHL also occur in sporadic renal cell ...carcinomas. The protein encoded by VHL, VHL, is part of the ubiquitin ligase that downregulates the heterodimeric transcription factor Hif under well-oxygenated conditions. Here we show that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo. This phenotype was independent of p53 and Hif but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a Hif-independent fashion. Our results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.
Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone ...methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. Here we report that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes.
RBP2−/− mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of
SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylated H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4.
Aberrations in epigenetic processes, such as histone methylation, can cause cancer. Retinoblastoma binding protein 2 (RBP2; also called JARID1A or KDM5A) can demethylate tri- and dimethylated lysine ...4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by the retinoblastoma protein (pRB). Here, we show that genetic ablation of Rbp2 decreases tumor formation and prolongs survival in Rb1+/– mice and Men1-defective mice. These studies link RBP2 histone demethylase activity to tumorigenesis and nominate RBP2 as a potential target for cancer therapy.
The EglN2/PHD1 prolyl hydroxylase is an important oxygen sensor contributing to breast tumorigenesis. Emerging studies suggest that there is functional cross talk between oxygen sensing and ...mitochondrial function, both of which play an essential role for sustained tumor growth. However, the potential link between EglN2 and mitochondrial function remains largely undefined. Here, we show that EglN2 depletion decreases mitochondrial respiration in breast cancer under normoxia and hypoxia, which correlates with decreased mitochondrial DNA in a HIF1/2α‐independent manner. Integrative analyses of gene expression profile and genomewide binding of EglN2 under hypoxic conditions reveal nuclear respiratory factor 1 (NRF1) motif enrichment in EglN2‐activated genes, suggesting NRF1 as an EglN2 binding partner. Mechanistically, by forming an activator complex with PGC1α and NRF1 on chromatin, EglN2 promotes the transcription of ferridoxin reductase (FDXR) and maintains mitochondrial function. In addition, FDXR, as one of effectors for EglN2, contributes to breast tumorigenesis in vitro and in vivo. Our findings suggest that EglN2 regulates mitochondrial function in ERα‐positive breast cancer.
Synopsis
While prolyl hydroxylase EglN2/PHD1 is well known for its role in oxygen‐dependent degradation of HIF1α, this work reports a HIF1‐independent role for EglN2 in regulating mitochondrial function via the formation of a transcription regulatory complex with PGC1α and NRF1.
EglN2/PHD1 regulates mitochondrial function in ERα‐positive breast cancer under normoxia and hypoxia.
The effect of EglN2 on mitochondrial function is HIF1/2α independent.
EglN2 is an NRF1 binding partner that promotes transcription activation.
EglN2 forms an activator complex with PGC1α and NRF1 on chromatin and promotes FDXR transcription.
FDXR regulates mitochondrial function and contributes to breast tumorigenesis in vitro and in vivo.
While prolyl hydroxylase EglN2/PHD1 is well known for its role in oxygen‐dependent degradation of HIF1α, this work reports a HIF1‐independent role for EglN2 in regulating mitochondrial function via the formation of a transcription regulatory complex with PGC1α and NRF1.
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other ...abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.
IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to ...transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.
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•(R)-2-hydroxyglutarate, produced by IDH1/2 mutants, inhibits the BCAT transaminases•IDH mutant gliomas display a transamination-dependent glutamate biosynthesis defect•BCAT loss increases reliance on glutaminase for glutamate and glutathione synthesis•Mutant IDH and glutaminase inhibition are synthetic lethal under oxidative stress
Gliomas with IDH mutations show increased sensitivity to radiation in concert with glutaminase inhibition, offering a new approach to treating these tumors.
Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, ...pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL-/- renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for "hits" identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL-/- cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL-/- renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.
Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While ...energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.
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