Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor

Hirsilä, Maija; Koivunen, Peppi; Günzler, Volkmar; Kivirikko, Kari I.; Myllyharju, Johanna

doi:10.1074/jbc.m304982200

Cited by 721 publications

(794 citation statements)

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“…The estimated K m values of FIH-1 and the PHDs for molecular oxygen were first reported to be approximately 90 and 250 mM, respectively. 29,30 Although these values were determined in vitro using relatively short peptides as substrates, they do provide a guide as to the oxygen sensitivity of these enzymes within cells. Given that physiological oxygen concentrations are around 4-40 mM and below the estimated K m , and the enzymes are present within cells in limiting amounts, this implies that the hydroxylases are poised to respond to physiologically relevant changes in intracellular oxygen concentrations, with any decrease in oxygen tension likely to impart a direct reduction in the catalytic rate of the enzymes (reviewed in Schofield and Ratcliffe 31 and Fandrey et al 32 ).…”

Section: Oxygen-dependent Regulation Of Hif-cad By Fih-1mentioning

confidence: 99%

Turn me on: regulating HIF transcriptional activity

2008

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The hypoxia-inducible factors (HIFs) are critical for cellular adaptation to limiting oxygen and regulate a wide array of genes when cued by cellular oxygen-sensing mechanisms. HIF is able to direct transcription from either of two transactivation domains, each of which is regulated by distinct mechanisms. The oxygen-dependent asparaginyl hydroxylase factor-inhibiting HIF-1a (FIH-1) is a key regulator of the HIF C-terminal transactivation domain, and provides a direct link between oxygen sensation and HIFmediated transcription. Additionally, there are phosphorylation and nitrosylation events reported to modulate HIF transcriptional activity, as well as numerous transcriptional coactivators and other interacting proteins that together provide cell and tissue specificity of HIF target gene regulation. The maintenance of oxygen homeostasis is a crucial physiological requirement that involves coordinated regulation of a plethora of genes. The hypoxia-inducible transcription factors (HIFs) are responsible for a major genomic response to hypoxia, where cellular oxygen demand exceeds supply. The HIFs directly regulate the transcription of more than 70 genes involved in cellular processes that act to directly address this deficit by decreasing oxygen dependence and consumption by cells, and by increasing the efficiency of oxygen delivery to cells. These processes include vasculogenesis and angiogenesis, metabolism, vasodilation, cell migration, signalling and cell fate decisions. As such, the HIFs are fundamental to embryonic development and the pathophysiology of many serious human diseases, and are therefore subject to strict regulatory mechanisms that act to limit transcriptional activity to periods of cellular oxygen stress. The HIF proteins are subject to oxygen-dependent regulation, both at the level of protein stability (reviewed in this issue by R Bruick) and transcriptional activity, rendering them almost inactive at normoxia, but potently inducible in hypoxia. The regulation of the transcriptional activity of the HIF proteins, both via oxygen-dependent and oxygen-independent mechanisms, will be discussed in this review. The HIFsHIF is assembled from a-and b-subunits to become a transcriptionally active heterodimer. 1 Both subunits are members of the bHLH/PAS (basic helix-loop-helix/Per-ArntSim homology) family of transcription factors, and both contain transactivation domains (Figure 1). 2,3 Whereas HIF1b, also known as the aryl hydrocarbon receptor nuclear translocator (Arnt1), is a constitutively expressed nuclear protein, the a-subunit is strictly regulated in an oxygendependent manner. There are three paralogues of the HIF-a subunit, HIF-1a, HIF-2a and HIF-3a, and three paralogues of HIF-1b (Arnt1, Arnt2 and Arnt3), with either HIF-1a or HIF-2a being able to heterodimerize with HIF-1b to form the functional HIF transcription factor complexes responsible for the hypoxic shift in gene expression. HIF-3a has no known role as an active transcription factor, and is instead postulated to behave as a negative reg...

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Section: Oxygen-dependent Regulation Of Hif-cad By Fih-1mentioning

confidence: 99%

Turn me on: regulating HIF transcriptional activity

2008

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“…To date, expression has only been studied at the mRNA level of whole-organ homogenates [24,31]. These studies emphasized organ expression in adult rat, although different human tissues including lung were shown to express HIF-3α mRNA [32,33,34]. This study demonstrates that when cells were exposed to 1% oxygen for 2 h, a major increase in HIF-3α and HIF-1α protein was observed, whereas HIF-1α protein levels decreased from 8 to 16 h. In addition to the decrease in HIF-1α protein, hypoxia also induced a decrease in HIF-1α mRNA.…”

Section: Discussionmentioning

confidence: 99%

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

et al. 2006

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The role of the hypoxia-inducible factor (HIF) subunits 1α and 2α in response to hypoxia is well established in lung epithelial cells, whereas little is known about HIF-3α with respect to transcriptional and translational regulation by hypoxia. HIF-3α and HIF-1α are two similar but distinct basic helix-loop-helix-PAS proteins, which have been postulated to activate hypoxia responsive genes in response to hypoxia. Here, we used quantitative real time RT-PCR and immunoblotting to determine the activation of HIF-3α vs. HIF-1α by hypoxia. HIF-3α was strongly induced by hypoxia (1% O 2 ) both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation, whereas HIF-1α protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stability in A549 cells, as measured on mRNA and protein levels. Interestingly, HIF-3α and HIF-1α exhibited strikingly similar responses to a variety of activating or inhibitory pharmacological agents. These results demonstrate that HIF-3α is expressed abundantly in lung epithelial cells, and that the transcriptional induction of HIF-3α plays an important role in the response to hypoxia in vitro. Our findings suggest that HIF-3α, as a member of the HIF system, is complementary rather than redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells.

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“…Techniques such as sensitivity analysis allow us to do just that. 7 We first theoretically vary the parameter controlling PHD2 mediated feedback strength and simulate the corresponding HIF-1 response to hypoxia. The model predicts that strong feedback induces not only a lower steady-state level of HIF-1 response, but also a lower peak amplitude and shorter peak duration ( Figure 2B).…”

Section: Which Phd Isoforms Control the Transient Dynamics Of Hif-1?mentioning

confidence: 99%

“…To date, three HIF-regulating prolyl-4-hydroxylases (PHD1, -2 and -3) have been identified in mammalian cells [6]. They utilize molecular oxygen and the Krebs cycle (TCA cycle) intermediate -ketoglutarate as co-substrates to hydroxylate HIF- subunits at conserved prolyl residues [7,8]. This increases the  subunit's affinity for the von Hippel-Lindau protein (pVHL), a member of an E3 ubiquitin ligase complex [9].…”

Section: Introductionmentioning

confidence: 99%