Date Published: March 15, 2016
Publisher: JKL International LLC
Author(s): Antero Salminen, Kai Kaarniranta, Anu Kauppinen.
Hypoxia is an environmental stress at high altitude and underground conditions but it is also present in many chronic age-related diseases, where blood flow into tissues is impaired. The oxygen-sensing system stimulates gene expression protecting tissues against hypoxic insults. Hypoxia stabilizes the expression of hypoxia-inducible transcription factor-1α (HIF-1α), which controls the expression of hundreds of survival genes related to e.g. enhanced energy metabolism and autophagy. Moreover, many stress-related signaling mechanisms, such as oxidative stress and energy metabolic disturbances, as well as the signaling cascades via ceramide, mTOR, NF-κB, and TGF-β pathways, can also induce the expression of HIF-1α protein to facilitate cell survival in normoxia. Hypoxia is linked to prominent epigenetic changes in chromatin landscape. Screening studies have indicated that the stabilization of HIF-1α increases the expression of distinct histone lysine demethylases (KDM). HIF-1α stimulates the expression of KDM3A, KDM4B, KDM4C, and KDM6B, which enhance gene transcription by demethylating H3K9 and H3K27 sites (repressive epigenetic marks). In addition, HIF-1α induces the expression of KDM2B and KDM5B, which repress transcription by demethylating H3K4me2,3 sites (activating marks). Hypoxia-inducible KDMs support locally the gene transcription induced by HIF-1α, although they can also control genome-wide chromatin landscape, especially KDMs which demethylate H3K9 and H3K27 sites. These epigenetic marks have important role in the control of heterochromatin segments and 3D folding of chromosomes, as well as the genetic loci regulating cell type commitment, proliferation, and cellular senescence, e.g. the INK4 box. A chronic stimulation of HIF-1α can provoke tissue fibrosis and cellular senescence, which both are increasingly present with aging and age-related diseases. We will review the regulation of HIF-1α-dependent induction of KDMs and clarify their role in pathological processes emphasizing that long-term stress-related insults can impair the maintenance of chromatin landscape and provoke cellular senescence and tissue fibrosis associated with aging and age-related diseases.
Hypoxia, i.e. a decline in the oxygen partial pressure, is an environmental stress but hypoxia is also commonly present in many pathological conditions in which blood flow into tissues is impaired, e.g. in ischemia/stroke, arteriosclerosis, and inflammatory disorders. Organisms effectively respond to hypoxia, e.g. switching their energy production from oxidative to glycolytic pathway [4, 5]. Moreover, animals can adapt to hypoxic conditions by generating hypoxia resistance [6, 7]. Early 1990, Gregg Semenza and his collaborators discovered that hypoxia induced a nuclear accumulation of DNA-binding factor, which was de novo synthesized in several cell lines [8-11]. They called that protein hypoxia-inducible factor-1 (HIF-1). HIF-1 protein was able to bind to the promoter of human Erythropoietin (EPO) gene, which is a well-known hypoxia-inducible gene. They also observed that the binding of HIF-1 protein induced the transactivation of EPO gene in hypoxia. Subsequently, they cloned the HIF-1α gene and identified the HIF-1α protein . At the same time, Peter Ratcliffe and his team clarified the oxygen-regulated, cis-acting enhancer sequences in target genes, such as EPO and LDH-A genes [12-14]. HIF-1α protein can bind to a specific DNA sequence as a heterodimer with HIF-1β protein, also known as Aryl hydrocarbon receptor nuclear translocator (ARNT). This binding site was termed the hypoxia response element (HRE), present at the promoters of hypoxia-inducible genes. Wang et al.  observed that HIF-1α protein was post-translationally modified in hypoxia and its post-hypoxic decay was very rapid indicating that HIF-1α protein is unstable in normoxia. In 2001, several studies demonstrated that HIF-1α was hydroxylated by specific prolyl-4-hydroxylases (PHDs) and subsequently ubiquitylated by von Hippel-Lindau E3 ligase (pVHL) [15-17]. This ubiquitylation directed HIF-1α protein to proteasomal degradation. Interestingly, the PHD1-3 enzymes, also called EGLN1-3, are 2-oxoglutarate-dependent dioxygenases (2-OGDOs), which require O2 for a substrate and thus they are inactive in hypoxia [5, 17, 18]. This dependency on oxygen prevents the hydroxylation of HIF-1α protein in hypoxia and thus enhances its stabilization and subsequent transactivation of target genes. The 2-OGDO enzymes are not only sensors for oxygen availability but also for the presence of 2-oxoglutarate, a Krebs cycle intermediate, and iron homeostasis [19, 20]. In addition to PHD1-3, the 2-OGDO family includes both DNA and histone demethylases [21, 22], as discussed later. We have recently reviewed the potential role of 2-OGDO enzymes in the control of aging process .
Reduced oxygen availability is not the only way to stimulate the HIF-1α signaling. The activation of HIF-1α and subsequent target gene expression under normal oxygen pressure is called pseudohypoxic response (Fig. 1). There are several mechanisms which can induce the HIF-1α signaling in normoxia by e.g. (i) suppressing the activity of PHDs with mitochondrial metabolites, such as succinate and fumarate, as well as nitric oxide (NO) and iron chelators, (ii) inhibiting the VHL ligase-induced ubiquitination of HIF-1α, (iii) stabilizing HIF-1α expression through post-translational modifications, (iv) enhancing the transcription and transcriptional activity of HIF-1α. In 2005, Selak et al.  observed that the inhibition of succinate dehydrogenase (SDH) in normoxia increased the accumulation of succinate, which consequently augmented the expression of HIF-1α in human embryonic kidney cells. They demonstrated that succinate inhibited the activity of PHD in vitro and thus might have stabilized HIF-1α protein. Consequently, Koivunen et al.  revealed that fumarate and succinate were potent inhibitors of all three PHDs and they also increased the expression of HIF-1α and its target gene VEGF in cultured cells. Pollard et al.  reported that the germline mutations of fumarate hydratase (FH) and SDHB, -C, and -D genes provoked the accumulation of fumarate and succinate into cultured cells and induced the over-expression of HIF-1α, which promoted the appearance of different types of cancers. These studies clearly indicated that fumarate and succinate were competitive inhibitors of Fe2+/2-oxoglutarate-dependent PHDs and thus stabilized HIF-1α expression in normoxia. Moreover, several studies have revealed that iron chelators are potent inducers of HIF-1α expression, such as 1,10-phenanthroline and flavonoid quercetin [37, 38]. Given that hypoxia tolerance can provide protection against ischemic and inflammatory diseases, many drug discovery studies have been launched to develop effective inhibitors to PHD enzymes .
DNA and histone methylation are the two major epigenetic mechanisms which regulate gene expression. The methylation status of histones is controlled by histone methyltransferases and histone demethylases . The methylation of histone C-terminal lysines can either activate or repress gene expression, i.e. typical activating marks are histone 3 di- or trimethylated lysine 4 (H3K4me2,3) and H3K36me2,3, whereas H3K9me1,2 and H3K27me2,3 are common repressive sites (Table 1). There are six Jumonji C domain containing histone lysine demethylases (KDM2-7), which can remove both activating and repressing methyl groups in an enzyme specific manner [21, 80, 81]. For instance, the demethylases of KDM3 and KDM6 subgroups are the potent activators of gene expression erasing the methyl groups from the repressive H3K9 and H3K27 sites, respectively. The Jumonji-type of KDMs contain different protein-protein binding domains through which they can specifically interact with distinct chromatin proteins, such as histone deacetylases, transcription factors and other chromatin proteins [21, 80]. All Jumonji-type of histone demethylases are the members of 2-oxoglutarate-dependent dioxygenase family (2-OGDO); this means that they are dependent on the availability of oxygen and 2-oxoglutarate, a Krebs cycle intermediate, similarly to PHDs (see above). However, they are not as sensitive oxygen sensors as PHDs [82-84]. In 2008, Pollard et al.  screened the effects of hypoxia on the expression levels of several Jumonji-type of histone demethylases in many cultured cell lines. They observed that the expression of KDM3A and KDM4B mRNAs but not that of several other demethylases were significantly induced in U2OS, MCF7, HeLa, IMR32 and HL60 cells cultured in 0.5% O2 pressure. They also revealed that the promoters of KDM3A and KDM4B genes contained three putative HRE binding sites, which were not present in the promoters of the non-responsive demethylases. Moreover, they reported that HIF-1α proteins could directly bind to these sites at the promoters of KDM3A and KDM4B genes. Beyer et al.  also demonstrated that hypoxia stimulated the HIF-1α-dependent expression of KDM3A and KDM4B mRNAs and proteins in cultured cells (Table 1). Moreover, they observed that hypoxia did not affect the global levels of di- and trimethylated H3K9 indicating that KDM3A and KDM4B proteins have specific binding partners and distinct targets in the genome. Krieg et al.  reported that only a few of the hypoxia-inducible genes are dependent on the presence of KDM3A (53 out of 821) in RCC4 cells. This subset included e.g. adrenomedullin (ADM), heme oxygenase 1 (HMOX1), and SERPINE1. The KDM3A-induced increase in the expression of these genes was associated with a decrease in the level of repressive H3K9me2 marks at the target promoters. Many other studies have also reported that hypoxia stimulated the expression of KDM3A and KDM4B genes in different cellular contexts [88-92] (Table 1). Interestingly, Wellmann et al.  demonstrated that the normobaric hypoxia (8% O2) of rats robustly increased the expression of KDM3A in all tissues studied, e.g. brain, heart, and liver. Given that KDM3A and KDM4B are the major histone demethylases which remove the repressive H3K9 sites, their role as transcriptional cofactors seems to be important in the activation of HIF-1α signaling.
Molecular oxygen is an obligatory co-substrate for the Jumonji-type histone demethylases  as well as for other 2-OGDO enzymes, e.g. PHD1-3 . However, there is a great variation for the requirement of O2 tension in the catalytic mechanism of different 2-OGDO enzymes [68, 83, 86, 91, 97]. PHDs are clearly more sensitive hypoxic sensors than Jumonji-type histone demethylases. For instance, Beyer et al.  demonstrated that the 1% O2 tension did not affect the activity of transfected KDM3A and KDM4B, whereas the reduction of O2 pressure down to 0.2% compromised the activity of KDM3A and KDM4B. In contrast, it is known that a modest decrease in O2 level can stimulate a robust hypoxic response, e.g. in mouse kidney with a chronic treatment at 11% O2 pressure  or disturb coronary artery development in a HIF-1α-dependent manner at 15% O2 tension .
Several studies have revealed that hypoxia provokes epigenetic changes in the chromatin landscape, which consequently affect the transcriptional profiles of tissues [28, 103-105]. However, hypoxia is a complex subject since results are dependent on many details in hypoxic treatments, e.g. the type of model (acute/chronic or constant/intermittent), oxygen level during exposure, and post-hypoxia timing, even transgenerational changes. Moreover, hypoxic training can induce hypoxic/ischemic tolerance , which involves an appearance of specific epigenetic signature . Hypoxia tolerance is also associated with increased lifespan, e.g. in the case of naked mole rats . Moreover, hypoxia is present in the pathogenesis of many age-related diseases, such as cardiovascular diseases and Alzheimer’s disease, which involve substantial epigenetic alterations in the chromatin landscape [109, 110]. Currently, it is not known how hypoxia generates chromatin changes, which can be either protective enhancing hypoxic tolerance or detrimental provoking pathological changes. However, it is clear that epigenetic alterations, such as the stimulation of distinct KDMs, are crucial mechanisms in the induction of HIF-1α-mediated hypoxic response  (Fig. 1).
Jumonji histone demethylases have many important functions in addition to the gene-specific activation or repression of HIF-1α-dependent transcription. KDM proteins contain specific binding domains, which can mediate interactions between KDMs and several chromatin proteins as well as some DNA loci. This means that hypoxia and pseudohypoxia affect not only gene expression but also chromatin structures, e.g. the maintenance of heterochromatin and polycomb complexes which can silence large genome sequences and preserve chromosome configuration. Both the constitutive and facultative heterochromatin involve DNA regions with increased histone methylation of H3K9, H3K27, and H3K36 sites, which are assembled by heterochromatin protein 1 (HP1) and histone methyltransferases, such as those in polycomb complexes [124-126]. Heterochromatin is a platform for the binding of chromatin effector proteins participating in e.g. gene silencing and the formation of 3D higher-order chromosome foldings . However, heterochromatin regions, especially facultative segments, are dynamic structures since HP1 proteins can recruit Jumonji histone demethylases, which activate the transcription in heterochromatin repeats [127-129]. In particular, the hypoxia-inducible H3K9 and H3K27 demethylases have a key role in the dissociation of heterochromatin segments. Transient hypoxia typically causes a short-term induction of HIF-1α signaling and changes in gene expression, whereas chronic exposure to hypoxia can have detrimental effects on heterochromatin maintenance and DNA integrity (Fig. 1).
Currently, it seems that HIF-1α signaling can have positive and negative effects on the regulation of longevity . There are some dramatic examples that the living in hypoxic conditions is associated with a significant increase in the lifespan of animals, e.g. subterranean blind mole rats can live for 30 years  and ocean quahog Arctica islandica for hundreds of years . Shams et al.  demonstrated that the expression of HIF-1α was not only higher in normoxic kidney of Spalax compared to Rattus norvegicus but the induction of HIF-1α expression was especially enhanced in hypoxia, being 6-fold higher in Spalax than Rattus norvegicus. However, it is not known whether increased hypoxic stress tolerance is related to longer lifespan. There are many reports indicating that the stimulation of HIF-1α expression in hypoxia is declined with aging [172-174]. Interestingly, Ndubuizu et al.  revealed that the age-dependent decline in hypoxia-inducible expression of HIF-1α was correlated with an increased expression of PHD1 in rat brain. Rohrbach et al.  reported that the expression of PHD3 was significantly increased in rat heart, liver, and skeletal muscle with aging. An increased expression of PHDs can be a negative feedback response to increased HIF-1α expression, since some studies have demonstrated that HIF-1α induces the transcription of PHDs and thus protects against the chronic stimulation of HIF-1α [25, 176].
The HIF-1α signaling is a principal survival mechanism in acute hypoxic insults, where hypoxia-inducible KDMs are confined to support the transcription of genes comprising hypoxia response. However, it can be converted to detrimental response in some host defence condition, e.g. in chronic hypoxia and long-term pathological stresses, as soon as the activation of KDMs disturbs the maintenance of chromatin structures, e.g. heterochromatin repressed DNA segments and 3D folding of chromosomes, or stimulates embryonal processes, such as the EMT fibrosis. It seems that the HIF-1α-inducible KDMs have a crucial role in the activation of genes in the INK4 box as well as the RB-stimulated tumor suppressor genes, which both provoke a cell fate called cellular senescence. Senescent cells are deleterious for neighbouring cells since they secrete inflammatory mediators and thus they can activate signaling pathways known to stabilize the HIF-1α signaling. Given that cellular senescence is an irreversible alteration, it provides an inflammatory milieu which activates the HIF-1α-inducible KDMs and thus jeopardizes the nearby cells. This vicious cycle is increasingly present in the aging process and especially in the age-related diseases.