Pharma Tips

Role of Hypoxia Inducible Factors

By: Pharma Tips | Views: 5121 | Date: 16-Jun-2010

Hypoxia is a condition when the tissues are deprived of oxygen. There are four types of hypoxia that include,hypoxemic, anemic, stagnant, and histotoxic.

1)   HYPOXIA
Hypoxia is a condition when the tissues are deprived of oxygen. There are four types of hypoxia that include,hypoxemic, anemic, stagnant, and histotoxic. 

Hypoxemic hypoxia is produced when there is low partial pressure of oxygen in the air, for example at high altitudes. Also anything that would reduce the ventilation some examples include drugs like anaesthetics and analgesics. 
Anaemic hypoxia is when the concentration of hemoglobin is reduced. Anaemic hypoxia is when there are low levels of iron , vitamin B12,  and copper.
Stagnant hypoxia is when there is a low level oxygen to the tissues that is caused by reduced blood flow. 
Cytotoxic hypoxia occurs when the respiring cells in the tissues are prevented from using oxygen (6).
Hypoxia inducing factor is a specific protein involved in maintaining oxygen homeostasis and regulates hypoxia inducible genes that include human erythropoietin (EPO) gene (1).

Role of Hypoxia Inducible Factors

2 ) HYPOXIA-INDUCIBLE FACTOR:
HIF is a transcriptional factor that regulates the adaptive responses of mammalian cells to low oxygen (hypoxia).
HIF-1 is a heterodimer double helix loop and is the transcriptional factor that is needed for the activation mediated by EPO gene enhancer in hypoxic cells.
HIF complex contains  α and a ß subunit, both of which can be selected from several alternatives. They are members of a large family of transcription factors α  which contain a basic helix–loop–helix region and a PAS domain.
HIF-1 α protein is very unstable in cells exposed to oxygen, however hypoxia increases the abundance of HIF-1 alpha proteins.
The α subunit is regulatory and is unique to the hypoxic response.
HIF β subunits are constitutive and are also involved in xenobiotic responses.  
Three different genes encoding HIF -subunits are found in mammals: HIF-1 α, HIF-2 α and HIF-3 α /IPAS (IPAS is inhibitory PAS protein).
As yet, the role of HIF-3 α are unclear, but are likely to be complex since the gene produces six different transcripts, some of which encode proteins which are predicted to be oxygen responsive while others are not. 
HIF-1 consists of four chains: A, B, C, D. Chains A and B is the DNA molecule (4).

CHAIN  A :  
DNA consist of 14 bases, sequence is G-C-C-C-T-A-C-G-T-G-C-T-G-C Molecular weight is 4705 (3).
CHAIN B:
Consist of 14 bases; sequence is G-C-A-G-C-A-C-G-T-A-G-G-G-C Molecular weight 4835 (3).
CHAIN C:
Beta unit known as aryl Hydrocarbon receptor nuclear translocator. Molecular weight is 6852.
There are 59 residue (3).

CHAIN D:
Alpha unit known as hif-1 α  Highly regulated by Oxygen concentration and determine levels of hif-1 activity (2).
Molecular weight is 6959.
There are 59 residues (3).

MEMBERS
Member Gene Protein
HIF-1α   HIF1A                                hypoxia-inducible factor 1, alpha subunit
HIF-1β     ARNT  aryl hydrocarbon receptor nuclear translocator
HIF-2α             EPAS1  endothelial PAS domain protein 1

HIF-2β ARNT2  aryl-hydrocarbon receptor nuclear translocator 2
HIF-3α              HIF3A hypoxia inducible factor 3, alpha subunit
?

HIF-3β             ?                             

Function of HIF-1
HIF-1 alpha is needed to activate HIF-1. HIF-1 activates genes are erythropoietin (EPO), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), heme oxygenase 1 (OH-1), aldolase A (ALDA), enolase 1 (ENO-1), glucose transporter 1(GLUT-1), lactate dehydrogenase A (LDHA), and phosphoglycerate kinase 1 (PGK-1) (10). that can be classified into three functional groups. 
Group 1: 
Proteins escalate tissue oxygen by the formation and development of erythrocytes (red blood cells).  Examples of these particular proteins are EPO and OH-1 (8).

Group 2: 
Proteins raise oxygen delivery to tissues through blood vessel relaxation and development.  Examples of these particular proteins are iNOS and VEGF (8). 

Group 3: 
Proteins, that in contrast to the two previous groups do not alter the delivery of oxygen to tissues.  Instead, these proteins are necessary for the adaptation of cellular metabolism under conditions of low oxygen.  Examples of these proteins are GLUT-1 and most glycolytic enzymes  (8).

3) O2 HOMEOSTATIS
Hypoxia-inducible factor 1: master regulator of O2 homeostasis.
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to reduced O2 availability in mammals. Recent studies have provided insights into the O2-dependent regulation of HIF-1 expression, target genes regulated by HIF-1, and the effects of HIF-1 deficiency on cellular physiology and embryonic development.
Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1.
HIF-1 alpha is up regulated when there is lack of oxygen and heterdimerizes with the aryl hydrocarbon receptor nuclear translocator (7) also known as the HIF-1 beta. The heterodimer complex binds and activates transcription of target genes. 
HIF-1ß is a nuclear protein that is constitutively expressed and is independent of O2 tension. HIF-1 α, in contrast to HIF-1ß, is a cytoplasmic protein responsive to O2 levels. In well-oxygenated cells, HIF-1 α is continuously degraded by the ubiquitin-proteasome system (16, 17, 18). This degradation process takes place only when certain conserved prolyl residues of HIF-1 α are hydroxylated, a modification requiring O2-dependent enzyme activity. Only HIF-1 containing modified prolyl sites binds to the von Hippel-Lindau protein, which is the recognition component of an E3 ubiquitin ligase that finally targets HIF-1 for proteasomal degradation (22, 25). Under hypoxic conditions, HIF-1 α subunits translocate to the nucleus, where they heterodimerize with HIF-1ß subunits. The resultant product is an active HIF-1 protein that binds to specific hypoxic response elements present in target genes, ultimately activating transcription of these genes, which encode for erythropoietin, VEGF, various glycolytic enzymes, transferrin, and a variety of other proteins essential for systemic, local, and intracellular homeostasis. Importantly, the vast majority of these gene products are overexpressed in human tumor cells, suggesting that the HIF-dependent transcriptome changes are important in tumor pathophysiology (19,20,23). Overall, these adaptive responses to low O2 levels serve as a compensatory mechanism for increasing delivery of O2 (and nutrients) for any body cells with an inadequate O2 supply. 
However, for hypoxic tumor cells, these adaptive responses can additionally favor cell survival, further expansion, and metastasis, as outlined below.

Figure 1 Regulation of HIF-1 by cellular O2 level. O2 determines the subjection of HIF-1 to protein hydroxylation. Under normoxic conditions, ubiquitination of HIF-1α targets the subunit for proteasome degradation. Under hypoxic conditions, HIF-1ß dimerizes with HIF-1 , and the active HIF-1 dimer binds to hypoxia response elements containing the core recognition sequence 5'-RCGTC-3' and then recruits coactivator molecules, resulting in the formation of an increased transcription initiation complex and mRNA synthesis, leading ultimately to the biosynthesis of proteins that mediate responses to hypoxia.

MECHANISM
Under normoxic conditions, The alpha subunit of HIF-1 is a target for prolyl hydroxylation by HIF prolyl-hydroxylase, which makes HIF-1α a target for degradation by an E3 ubiquitin ligase, leading to quick degradation by the proteasome (12).

Under hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate.Hypoxia also results in a buildup of succinate, due to inhibition of the electron transport chain in the mitochondria. The buildup of succinate further inhibits HIF prolyl-hydroxylase action, since it is an end-product of HIF hydroxylation. In a similar manner, inhibition of electron transfer in the succinate dehydrogenase complex due to mutations in the SDHB or SDHD genes can cause a build-up of succinate that inhibits HIF prolyl-hydroxylase, stabilizing HIF-1α. This is termed pseudohypoxia.

HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low-oxygen conditions. These include glycolysis enzymes, which allow ATP synthesis in an oxygen-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. HIF-1 acts by binding to HIF-responsive elements (HREs) in promoters that contain the sequence NCGTG (13).
Transcriptional targets of HIF 
The HIF system operates on many genes besides EPO. The downstream consequences of HIF activation vary significantly from one cell type to another, which is unsurprising given that different cells and organs in vivo need to make different adaptations in the face of changes in oxygen supply. Currently there are of the order of a hundred genes that are recognized transcriptional targets of HIF. Besides erythropoiesis, the best-characterized processes that are regulated by HIF are angiogenesis, and glucose uptake and metabolism. In both cases, the homeostatic nature of the response is clear. The increased glucose uptake and expression of glycolytic enzymes increases the ability of cells to generate ATP by glycolysis, which can then compensate for impaired mitochondrial electron transport. The increase in angiogenic signalling promotes an increase in the vascular supply. Many of the other targets of HIF could be regarded as adaptive. Some interesting examples are the surface membrane carbonic anhydrases, CA IX and CA XII. These HIF targets are presumed to be important in compensating for local increases in hydrogen ion generation. Another intriguing recent discovery is that the CXC motif chemokine receptor, CXCR4, and its ligand, stromal cell-derived factor-1, are both HIF targets. This provides a means by which circulating multipotent stem cells could be guided to niches where repair is required in response to injury. Many enzymes that use molecular oxygen as cosubstrates are HIF targets, perhaps because increased expression provides an effective way for maintaining reaction rate at lower concentrations of oxygen. HIF activation is also important in decisions concerning cellular proliferation and apoptosis., there are several examples of ion channels, transporters, circulating hormones and receptors that are modulated by HIF. 
Little is known about what shapes the downstream consequences of HIF activation, but it presumably involves the range of genes in particular cells that is available for transcription, and complex interactions with other transcriptional control mechanisms. It is clear that HIF-1 α and HIF-2 α can operate selectively on downstream targets, although the way in which this is achieved is not yet under. Illustrating the potential complexity, the extent of functional overlap between HIF-1 α and HIF-2 α appears to be different from one cell type to another. 
From a physiological perspective, the important message is that HIF activation is capable of mediating a response that is precisely tailored to the requirements of the cell, tissue and organism. The range of downstream responses is very broad indeed and could include almost any pathway. 


4) HIF PROLYL HYDROXYLASES
The family of HIF prolyl hydroxylases
Epstein and colleagues identified egg-laying abnormal-9 (EGL-9) as the HIF prolyl hydroxylase in Caenorhabditis elegans and they described a family of three human and mouse PHD genes that are homologous to egl-9. Unfortunately, several acronyms were coined to describe these genes. PHD1, PHD2 and PHD3  have also been called EGL nine homologue 2 (EGLN2), EGLN1 and EGLN3, or HIF prolyl hydroxylase 3 (HPH3), HPH2 and HPH1, respectively. Each of these homologues has a conserved gene structure, which suggests duplication in the lineage leading to vertebrates.
The PHDs belong to the superfamily of iron- and 2-oxoglutarate-dependent dioxygenases. These enzymes need O2 as a co-substrate, which provides the molecular basis for their O2-sensing function. In the hydroxylation reaction, one oxygen atom is added to a peptidyl proline to form hydroxyproline, whereas the other is used in a coupled decarboxylation reaction that converts 2-oxoglutarate to succinate; thus, PHDs also require 2-oxoglutarate as a co-substrate. Likewise, PHDs use Fe2+ and ascorbate as co-factors. Fe2+ is crucial for activating O2 and as a template for the orderly binding of reactants. Ascorbate seems to act by reducing Fe3+, which binds to the active site of the enzyme after the decarboxylation reaction, and is therefore necessary for its re-activation. 

Figure 2: Catalytic function of the prolyl-hydroxylase-domain proteins. PHDs use Fe2+ and ascorbate as co-factors and require 2-oxoglutarate as a co-substrate.O2 serve as  a co-substrate .  

These enzymes need O2 and 2-oxoglutarate as co-substrates, and Fe2+ and ascorbate as co-factors. The hydroxylation reaction forms hydroxyproline and succinate. PHDs, prolyl-hydroxylase-domainproteins.

Substrate specificity. 
PHDs hydroxylate  two proline residues in a conserved LxxLAP sequence motif. In vitro studies have assigned their relative activities as PHD2>>PHD3>. However, these results are different from those of another report, which showed similar specific activities for PHD2 and PHD3, and a lower activity for PHD1
The two HIF-1α prolines are differentially hydroxylated by the PHDs All three PHDs can hydroxylate HIF-1α Pro564, but only PHD1 and PHD2 are able to hydroxylate HIF-1α Pro402. Furthermore, the PHD1 and PHD2 Km values for a Pro402-containing peptide were about 20–50 times higher than those for a Pro564 containing peptide, which suggests important differences between Pro402 and It is now well established that all three PHD proteins regulate HIF-α .However, the contribution of each PHD is dependent on its relative abundance, which supports the theory that HIF-α hydroxylation is a non-equilibrium reaction. In normoxia PHD2 has a dominant role , as it is rate limiting enzyme that sets the low steady- state levels of HIF-1α .  PHD1 and PHD3  has important role in HIF-1α degradation during long-term hypoxic stress ..
Tissue distribution and alternative splicing.
PHD1, PHD2 and PHD3 are expressed in all tissues albeit at different levels . PHD2 and PHD3 messenger RNAs are subjected to alternative splicing, although no enzyme activity has been detected for any of the alternatively spliced forms. Therefore, changes in the splicing pattern can be expected to influence the production and activity of the enzymes.
Localization.
Intracellular localization of the PHDs has been reported by using chimeric proteins fused to the green fluorescent protein. PHD1 is present exclusively in the nucleus, PHD2 is located mainly in the cytoplasm and PHD3 is distributed homogeneously in the cytoplasm and nucleus. Despite its mostly cytoplasmic localization, PHD2 is able to shuttle between the cytoplasm and the nucleus. HIF-1α can be degraded in the nucleus and cytoplasm ; therefore, PHD2 has all the attributes to target HIF-1α degradation in both cellular compartments.
Regulation of PHD expression. 
PHD expression is regulated in three ways: 
First, there is hypoxia/HIF-dependent regulation as the expression of PHD2 and PHD3 is transcriptionally induced by hypoxia, which promotes a negative feedback loop . Indeed, hypoxic induction of PHDs is HIF-1-dependent. Silencing of HIF-1α or HIF-2α results in decreased hypoxia-induced PHD3 expression. By contrast, PHD2 is not affected by HIF-2α. 
Second, there is hypoxia-dependent/HIF-independent regulation. Similar to HIF-α, PHD1 and PHD3 are also degraded by the proteasome. Degradation of PHDs by Siah 1 and 2, their specific E3 ligases, is enhanced by hypoxia and Siah 1 and 2 are transcriptionally upregulated during hypoxia in a HIF-1-independent manner. 
Third, physiological stimuli other than hypoxia can also regulate the expression of PHDs. Indeed, PHD1 mRNA is upregulated by estrogens in the ZR75-1 breast cancer cell line. SM20, the rat orthologue of human PHD3, is induced in response to p53 activation. Nerve growth factor withdrawal also induces SM20 in PC-12 cells, as do platelet-derived growth factor and angiotensin II in smooth muscle cells (14).
Regulation of PHD activity.
PHDs are effective O2 sensors. In keeping with their Km values for O2 (230–250 μM), which are slightly above the atmospheric concentration, the activity of PHDs is tightly regulated by the full range of O2 concentrations from normoxia (21% O2) to the lowest (<0.1% O2) hypoxic level. Nevertheless, as mentioned above, prolonged hypoxia can unexpectedly enhance PHD activity. Whether PHD accumulation during hypoxia and/or extra mechanisms are responsible for this re-activation is unknown. Whatever the explanation, it is noteworthy that prolonged hypoxia would limit HIF-1 activation, which supports the regulatory feedback loop.
Although the availability of O2 serves as a general determinant of PHD activity, several further parameters regulate this activity in a more complex manner to subtly adapt HIF function in a variety of dynamic microenvironments. Depletion of intracellular ascorbate and competitive inhibitors of 2-oxoglutarate lead to PHD inhibition. Similarly, normoxic stabilization of HIF-1α by some oncogenes is mediated by the inhibition of prolyl hydroxylation—such as in RasVal12 and v-Src activation. Mutations in the tumour suppressor succinate dehydrogenase (SDH) have also been linked to PHDs; succinate, which accumulates as a result of SDH mutations, inhibits PHD activity. Thus, succinate transmits an oncogenic signal from mitochondria to the cytosol. 
The role of reactive oxygen species (ROS) in the control of HIF-1α stability is also important. This role remains controversial despite the identification of PHDs as the oxygen sensors. This is mainly owing to the fact that hypoxia concomitantly inhibits PHD activity and induces the production of ROS by mitochondria. Accumulation of ROS in JunD−/− cells decreases the availability of Fe2+, which reduces the activity of PHDs . Therefore, any stress capable of inducing a persistent boost of free radicals should affect the stability of HIF-1α by indirectly inhibiting PHDs. Another source of controversy arose from blocking respiration through the inhibition of mitochondria, which prevents HIF-1α stabilization in moderate hypoxia. Redistribution of O2 occur in the cell,due to that,their is abrogation of ROS so there is  an increasing number of proteins  such as OS9 seem to bind to PHDs and to regulate their activity. Several second messengers also modify PHD activity (14).
functions of PHDs. 
PHDs are pivotal components of the signalling pathways that are elicited to assure O2 homeostasis. So far, more than 70 HIF target genes have been identified, including genes involved in the development and function of the vascular system, erythropoiesis, energy metabolism, pH regulation, cell proliferation and migration. This represents an elegantly adaptive mechanism for survival, but these target genes are also implicated in the pathogenesis of several serious diseases including myocardial ischaemia, stroke, pulmonary hypertension, pre-eclampsia, and almost every type of cancer. Therefore, PHDs, by modulating HIF-α stability and thus HIF activity, are at the heart of these pathophysiological processes.
Apart from their known function in the cellular response to low O2 availability, PHDs have a role in several other physiological and pathophysiological processes, such as the control of cell size Further analysis is necessary to explain definitively whether PHDs drive these functions through a HIF-dependent mechanism or through hydroxylation of new PHD substrates. We have shown the regulatory mechanisms and function of PHDs in Fig 3
                                                                                                                                                             Figure 3: Schematic model of prolyl-hydroxylase domain regulation and function. Several physiological stimuli, in addition to O2 availability, regulate prolyl hydroxylase domain (PHD) activity, which promotes fine-tuning adaptation to the microenvironment. PHDs, by modulating hypoxia-inducible factor-α (HIF-α) stability and thus HIF activity, are pivotal in O2 homeostasis. Nevertheless, PHDs might have a role in other physiological and pathological processes. (14)

Protein hydroxylation
Besides prolyl hydroxylation, HIF-1α is hydroxylated on an asparagine residue (Asn803)—in its carboxy-terminal transactivation domain—by factor inhibiting HIF . FIH-1, which could be subjected to the same regulatory mechanisms as the PHDs, suppresses HIF transcriptional activity under normoxic conditions by blocking its association with the coactivator p300/CBP.
In addition to HIF-α, prolyl hydroxylation has long been known to be important for collagen biosynthesis through stabilization of the triple-helical conformation of collagen fibres .. Furthermore, recent reports implicate the LxxLAP motif in the pVHL-dependent ubiquitylation of subunit 1 of RNA polymerase II, and the hydroxylation of iron regulatory protein 2 (IRP2) in its interaction with the ubiquitin machinery . With database predictions of new iron- and 2-oxoglutarate-dependent dioxygenases such as AlkB, these data hint that protein hydroxylation extends beyond the HIF system and might be widely involved in cell signalling.

  
FIGURE-4 Prolyl-hydroxylase-domain proteins regulate hypoxia inducible factor-α in response to O2 availability. 

Under aerobic conditions (blue arrows), prolyl-hydroxylase-domain proteins (PHDs) hydroxylate hypoxia inducible factor-α (HIF-α), which allows the von Hippel–Lindau protein (pVHL) to bind and thus target HIF-α to the proteasome. Likewise, by binding to PHD2 and PHD3, OS9 promotes HIF-α hydroxylation. A decrease in O2 availability (red arrows) inhibits the PHDs; HIF-α accumulates and induces the expression of target genes. In addition, Siah 1 and 2 trigger PHD1 and PHD3 degradation under hypoxic conditions. Interestingly, hypoxia controls PHD2, PHD3, OS9 and Siah 1 and 2 expression by a feedback loop mechanism. Immunofluorescence inserts show expression of HIF-1α in HeLa cells at 20% O2 (left) and 1–2% O2 (right). HRE, hypoxia response element.


5) PATHWAY
5.1) HIF-1 AND GLUCOSE METABOLISM
In contrast to normal cells, tumor cells characteristically display a relatively high glycolytic rate, even when growing in the presence of O2. Under normoxic conditions, cells generate ATP via oxidative phosphorylation.(60)

Figure 5. HIF-1-mediated switch from aerobic to anaerobic metabolism in hypoxic tumors for energy preservation. The activation of genes for glucose transporters (GLUT-1) and glycolytic enzymes results in an increased glycolytic rate. H+ ions produced are preferentially exported via a lactate–/H+ symporter and a Na+/H+ antiporter, leading to a decrease in extracellular pH. 
However, in the expanding tumor mass, which is generally characterized by a limited O2 supply and a high glucose consumption rate, anaerobic glycolysis can become the predominant pathway of ATP generation . This metabolic shift appears to be regulated by HIF-1 (Fig.4). Enzymes—including aldolase A, phosphoglycerate kinase 1, and pyruvate kinase M—are induced by HIF-1 in vitro, and lactate dehydrogenase is induced by HIF-1α in breast carcinoma lines . The efficacy of the glycolytic response is enhanced by over expression of other proteins, including glucose transporters (e.g., glucose transporter 1 [GLUT-1]), which facilitate glucose uptake by the cells, and by hexokinase, which enhances the capacity of tumor cells to catabolize glucose at higher metabolic rates, thereby increasing the production of precursors needed for cell growth and maintaining high ATP production under conditions of O2 deficiency.(61) Thus, HIF-1-induced adaptive responses not only provide for VEGF-mediated angiogenesis, but also ensure that the energy requirements of the cells are met, thereby allowing their survival in a hostile environment.
5.2) HIF-1, VEGF, AND ANGIOGENESI

Angiogenesis is the process by which new blood vessels develop from existing vasculature, thereby providing a principle mechanism for the maintenance of an adequate blood flow in expanding cell populations, including those of tumor tissue. In a rapidly growing tumor, O2 demand increases and O2 delivery decreases, primarily because of: A insufficient blood supply  increasing diffusion distances between the blood vessels and the O2-consuming cells . This leads to hypoxia in the expanding tumor mass, triggering events that stimulate angiogenesis in an effort to ameliorate the hypoxic condition. In tumor tissue, the ability to induce angiogenesis is associated with the development of an aggressive phenotype, as metastatic cells have more opportunity to enter the circulation in a well-vascularized tumor and thereby escape their hostile environment. 
One of the most potent stimulators of angiogenesis is VEGF, which is essential for the proliferation and migration of vascular endothelial cells, thereby enabling the formation of new blood vessels . Production of VEGF is driven by hypoxia via transcription activation of the VEGF gene by HIF-1.(14) 
Additionally, VEGF has been shown to stimulate migration of macrophages by activation of the VEGF receptor (Flt-1). Macrophages produce several angiogenic factors, including VEGF and tumor necrosis factor alpha (TNF-) .At the clinical level, the results of the majority of over a dozen studies comprising more than 3,500 patients generally speak in favor of an independent prognostic impact of VEGF expression regarding relapse-free and overall survival. Additionally, VEGF expression may be predictive of the anatomical site of first recurrence . 
In addition to VEGF, other angiogenesis-related gene products and receptors are regulated by HIF-1, including PDGF-B, VEGFR-1, endothelin-1, inducible nitric oxide synthetase (iNOS), monocyte chemotactic protein, adrenomedullin, and EGF. Several of these, including iNOS, endothelin-1, heme oxygenase 1, and adrenomedullin, have been shown to play roles in the regulation of local blood flow by the modulation of vascular tone . Thus, it appears that HIF-1 not only mediates angiogenesis by VEGF induction but also influences tumor blood flow by more complex mechanisms involving target genes playing a role in vessel tone. 
Yet another mechanism for stimulation of tumor angiogenesis is induction of HIF-1 and VEGF subsequent to somatic mutation. One example of this is seen in the loss of p53 tumor-suppressor activity either by direct mutational inactivation or by overexpression of mouse double minute, a ubiquitin protein ligase involved in the degradation of p53. Loss of p53 activity results in decreased hypoxia-mediated apoptosis, possibly increased HIF-1 expression , and a subsequent increase in HIF-1-mediated transactivation of VEGF and other target genes, thereby facilitating tumor angiogenesis. 
5.3) Variation in the HIF pathway in humans 
Appreciation of genetic variations in the HIF system in humans is increasing. Best characterized are individuals with von Hippel–Lindau disease, (21)caused by a germline mutation in the VHL gene. Affected families are at risk for renal cell carcinoma, haemangioblastomas of the retina and central nervous system, and pheochromocytoma (a tumour of the adrenal gland), (19,20) together with less serious clinical manifestations. All of these are associated with somatic inactivation of the normal VHL allele, explaining the variable clinical penetrance. Although von Hippel–Lindau disease is rare, it has given insight into the commonest type of kidney cancer because the great majority of cases show biallelic VHL inactivation due to two independent somatic hits. In VHL patients in the absence of this second hit, there is not an obvious cellular phenotype, implying that any effects of haploinsufficiency of VHL are limited. Interestingly, there does appear to be altered apoptosis of neutrophils in hypoxia, suggesting that there are likely to be more subtle phenotypic effects; potentially this would offer a very powerful method of determining the effect of rather minor changes in HIF in normal physiology .
 In VHL disease, the haemangioblastoma and clear cell renal cell carcinoma are both associated with constitutive HIF activation, which accounts for many of the features of these tumours, including marked angiogenesis. 
Current evidence suggests that HIF activation occurs immediately on inactivation of the second VHL allele, but has relatively little effect on the balance between cell proliferation and death. Normal renal epithelium that is hypoxic or in which VHL is inactivated expresses HIF-1 α, with little or no HIF-2 α. Evolution to cancer seems to involve a progressive switch to increased levels of HIF-2 α, which is critical for tumour growth.
Recently, investigation of familial erythrocytosis (excess red blood cell production) showed that this can be caused by homozygosity for a hypomorphic VHL allele. This shows that altered VHL function is sufficient to dysregulate erythropoiesis in humans, presumably by generating an inappropriate signal in the fibroblasts in the kidney. Importantly, these individuals have a minor defect in HIF regulation in all cells, rather than the major defect in some cells that is seen in classical VHL disease. They therefore offer a powerful resource for asking whether partial genetic activation of HIF in humans has physiological consequences. They have increased respiratory sensitivity to hypoxia, and also an increased pulmonary vascular response. 
Another approach has been to correlate more common genetic variants in the HIF system with phenotypes. Polymorphisms resulting in increased HIF activity may be associated with predisposition to cancer and also with type II diabetes. Replication of the disease associations in other populations will be of importance, since it is well recognized that population stratification can generate false positive findings in this type of study. 
Hypoxic response of cultured monocytes, such as the presence of proliferative retinopathy in diabetics, or the degree of collateral formation in patients with ischaemic heart disease . They suggest marked variation in hypoxic responses and strongly suggest that this plays an important part in determining the outcomes in ischemia. 
5.4) Hypoxia-Inducible Factor 1 as a Possible Target for Cancer Chemoprevention

Despite the intense cancer research carried out in the last 30 years, cancer therapy has not managed to decrease cancer mortality. We need new strategies to control a disease that kills over six million people worldwide every year. It is accepted that cancer chemoprevention (the use of chemicals to prevent, stop, or reverse the process of carcinogenesis) is an essential approach to controlling cancer; yet, the clinical usefulness of this strategy is very limited. 
Successful implementation of cancer chemoprevention depends on a mechanistic understanding of the carcinogenesis process. Our knowledge about this process is still limited and may therefore be preventing cancer chemoprevention from becoming a widely used anticancer tool. This report discusses recent evidence that suggests that the activation of the hypoxia-inducible factor 1 (HIF-1) is a key event in carcinogenesis and may therefore represent a key target for cancer chemoprevention. 
Based on an understanding of the mechanisms responsible for HIF-1 activation, possible general strategies for targeting HIF-1 are proposed. Successful implementation of these strategies might turn the great promise of cancer chemoprevention into a fundamental tool for reducing the burden of this disease.
During the last several decades, surgery, radiotherapy, and chemotherapy have been the useful yet unsatisfactory tools for decreasing cancer mortality. Although chemotherapy in the advanced stages of disease has been highly successful for the treatment of relatively rare cancers, the prognosis for patients with invasive and metastatic disease at the common epithelial sites remains poor (26). Because conventional strategies are not expected to increase in efficiency in the near future, (26,27) we need other approaches to decrease cancer incidence and mortality, which have virtually remained the same for the last 30 years(28,29). The process of carcinogenesis can take decades to complete, providing time and opportunity to intervene to prevent this process either before the clinical appearance of cancer or at its earliest stages. 
Thus, evidence suggests that in addition to lifestyle factors and vaccines, chemo preventive interventions hold the greatest promise for reducing cancer incidence and mortality (26, 30, 31). Cancer chemoprevention is the use of chemicals to prevent, stop, or reverse the process of carcinogenesis. It is now accepted that cancer chemoprevention is not only an essential approach to controlling cancer but also a quality-of-life issue (26). It makes more sense to prevent cancer at its earliest stages by using low-toxic chemicals than to wait until the disease has reached its final stages, where it becomes necessary to use more toxic drugs. Despite the great promise of cancer chemoprevention, this strategy is currently restricted to some areas of the head and neck, breast, and colon carcinogenesi. It is important to note that successful implementation of chemoprevention depends on a mechanistic understanding of carcinogenesis  and that, regretfully, we still do not fully understand the carcinogenesis process. Most researchers consider cancer to be a genetic disease caused by the acquisition of multiple mutations in key genes that control cell proliferation, cell death, and genetic stability . This is called the "somatic mutation theory of cancer" and has been the prevalent hypothesis to explain carcinogenesis over the last several decades.

For instance, this theory cannot explain metastasis , the key process of carcinogenesis that distinguishes a benign tumor from a malcancer. According to the most accepted theory, oncoactivation and the inactivation of tumor suppressor genes are key events in carcinogenesis. Oncogenic activation would produce increased synthesis of growth factors, increased expression of growth factor receptors, and inappropriate activation of downstream signal transduction pathways and nuclear transcription factors. Oncogenic activation would therefore stimulate cell proliferation. Tumor suppressor genes, on the other hand, would code for proteins that normally act as checkpoints for cell proliferation or cell death. Because tumor suppressor genes code for proteins that can restrain cell proliferation, the loss of such proteins would allow a cell to grow and divide in an uncontrolled fashion. Therefore, this theory suggests that cancer chemoprevention can be achieved by using chemicals to decrease oncogenic proteins or increase tumor suppressor proteins. Several years ago, it was believed that cancer could be explained by modifications in several of these cancer-related genes. To date, >100 oncogenes and >30 tumor suppressor genes have been identified. In addition to these high numbers, it has been observed that different types of cancers, and even the same cancer type from different individuals, have different genetic alterations. 
Furthermore, some of the most commonly altered cancer genes have oddly inconsistent effects. For instance, it has been observed that the much-studied oncogenes c-fos and c-erbb3 are less active (not activated) in some tumors than they are in nearby normal tissues. Likewise, the tumor suppressor gene RB has been shown to be hyperactive in some colon cancers, and it seems to protect these tumors from apoptosis (32,34,35). Chemoprevention strategies aimed at targeting specific oncogenic or tumor suppressor proteins might therefore result in unpredictable or negative outcomes. 
In other words, our limited knowledge about the process of carcinogenesis may be preventing cancer chemoprevention from becoming a widely used anticancer tool.

Figure-6 :key role of Hif-1 activation in carcinogenisis.

In addition to favoring the acquisition of several hallmarks of cancer, the activation of glycolysis seems to be critical for cancer cells to generate energy. The Nobel laureate Otto Warburg proposed several decades ago that cancer was caused by a defect in respiration (oxidative phosphorylation). This defect would produce a deficient ATP generation through oxidative phosphorylation that would be compensated by the activation of glycolysis . Although his theory has been rejected by most researchers, it is now well accepted that cancer cells have increased glycolytic rates(51). Furthermore, cancer cells depend on glycolysis for ATP generation, and that cancer cells' dependence on glycolytic energy progressively increases as malignant transformation occurs(49,50) . Because HIF-1 activation plays a key role in the activation of glycolsis(37), and because glycolysis seems necessary for cancer cells to keep adequate ATP levels , it is reasonable to think that HIF-1 activation is important for keeping ATP levels in cancer cells. Accordingly, it has been reported that hypoxia-induced glycolysis (Pasteur effect) is mediated by HIF-1 activation, and that ATP levels are dramatically reduced during hypoxia in the absence of HIF-1(48).
In brief, the most important cancer-related gene pathways seem to culminate in HIF-1 activation, and HIF-1 activation may explain all the hallmarks of cancer and be necessary for keeping adequate ATP levels in cancer cells. Furthermore, HIF-1 overexpression is observed in the most common cancer types and has been associated with increased patient mortality in several cancer types. This suggests that HIF-1 plays a key role in carcinogenesis and may therefore represent a key target for cancer chemoprevention.

Targeting HIF-1 for Cancer Chemoprevention

Inhibition of HIF-1 activity has marked effects on tumor growth(37). HIF-1 is considered a potential target for cancer therapy, and, recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories(13,). Thus, it has been found that several Food and Drug Administration–approved anticancer drugs (e.g., topotecan, imatinib mesylate, trastuzumab, NS398, celecoxib, and ibuprofen) inhibit HIF-1 activity . Several natural products (e.g., resveratrol, genistein, apigenin, and berberin) have also been found to inhibit the activity of this transcription factor . Because HIF-1 seems to have an important function in carcinogenesis, HIF-1 inhibitors may be considered a source of potential cancer chemopreventive agents. It is important to note, however, that the use of HIF-1 inhibitors in cancer chemoprevention might be associated with toxicity. An excessive inhibition of HIF-1 may produce adverse effects, as HIF-1 regulates many cellular processes under physiologic conditions(46,47). For instance, an agent that inhibits HIF-1 directly may prevent normal cells from responding to a situation of hypoxia; this may be associated with toxicity. Toxicity can be acceptable in preventing cancer in people with precancerous lesions or who are at high risk for developing cancer. 
Thus, tamoxifen is used in the chemoprevention of specific breast cancers, although it increases the rate of endometrial cancer and uterine sarcoma(45). However, any degree of toxicity is unacceptable in preventing cancer in healthy people who may never develop the disease. Therefore, although HIF-1 inhibitors may represent a useful source of chemopreventive agents, the potential toxicity associated with these agents should be considered carefully, especially when chemopreventive interventions are aimed at preventing cancer in healthy populations. 
In addition to targeting HIF-1 directly with HIF-1 inhibitors, HIF-1 can be targeted by identifying the key cellular event responsible for HIF-1 activation and developing strategies to prevent such activation. It has been proposed that cancers have increased HIF-1 because of intratumoral hypoxia and genetic alterations that activate oncogenes and inactivate tumor suppressor genes.(37) Unfortunately, intratumoral hypoxia is difficult to modulate, and, as suggested before, the genetic alterations responsible for HIF-1 activation may be too irregular and numerous to target. 
To explain HIF-1 activation under both physiologic conditions and cancer. This report discusses evidence that suggests that, although hypoxia and genetic alteration can activate HIF-1, the key cellular event involved in the activation of HIF-1 is an alteration in oxygen metabolism. (38)This alteration in O2 metabolism activates HIF-1 by increasing the cellular levels of hydrogen peroxide (H2O2) and by activating glycolysis. Therefore, in addition to targeting HIF-1 directly with HIF-1 inhibitors, HIF-1 activation may be inhibited by preventing or decreasing excessive cellular levels of H2O2 and an excessive activation of glycolysis. These general strategies may reduce the possible toxicity associated with a direct inhibition of HIF-1 induced by HIF-1 inhibitors. For instance, it is known that cancer cells have increased rates of glycolysis(44) , and that the accumulation of glucose metabolites keeps high levels of HIF-1 in cancer cells .(42,43) This suggests that a chemopreventive strategy aimed at preventing an excessive activation of glycolysis may reduce the levels of HIF-1 and might therefore produce anticancer effects. Interestingly, this strategy would allow normal cells to activate HIF-1 in response to a situation of hypoxia, as hypoxia induction of HIF-1 mainly occurs via H2O2. (40,41)
Inhibition of HIF-1 may fit into current strategies for cancer chemoprevention. For instance, it is considered that angiogenesis is a potential biomarker and target in cancer chemoprevention, and that the vascular endothelial growth factor (VEGF) is a key angiogenic factor(36). Because several angiogenic genes (including VEGF) are transcriptionally activated by HIF-1,(37) the inhibition of HIF-1 may produce angiogenesis inhibition. As mentioned above, there is evidence that H2O2 is a key activator of HIF-1(38). A reduction in the cellular levels of H2O2 would reduce HIF-1 activation and may therefore inhibit angiogenesis. overexpression of the H2O2-detoxifying enzyme catalase can reduce VEGF expression and inhibit angiogenesis .(39) 

cancer statistics remind us that new strategies are necessary for controlling this disease. Although it is accepted that cancer chemoprevention holds great promise for reducing cancer incidence and mortality, the clinical usefulness of this strategy is very limited. This restricted use may be due to our still limited knowledge of the carcinogenesis process. The present report has discussed that the activation of the transcription factor HIF-1 is a key event in carcinogenesis and may therefore represent a key target for the development of cancer chemopreventive strategies. Based on an understanding of the mechanisms responsible for HIF-1 activation, possible general strategies for targeting HIF-1 have been proposed. Successful implementation of these chemopreventive strategies may arrest or reverse the carcinogenesis process and might therefore turn the great promise of cancer chemoprevention into an essential anticancer tool.

A role for HIF-1α in myeloid cells

HIF-1α is embryonically lethal due to its essential role in the development of the vasculature. To circumvent this lethality and allow further examination of the role of HIF-1α in phagocytes,. employed a myeloid cell–specific HIF-1α–knockout approach and found that HIF-1α was an important regulator of myeloid cell metabolism (by decreasing cellular ATP levels), neutrophil bactericidal potency, and macrophage migration . myeloid cell function  is  depressed  in the absence of HIF-1α. 
HIF-1α was equivalently upregulated in WT macrophages at normoxia following exposure to Gram-positive (Streptococcus and Staphylococcus) and Gram-negative (Salmonella and Pseudomonas) bacteria. HIF-1α was activated in macrophages treated with LPS , a microbial activator of TLR-4. The 10 human TLRs are the cellular sentinels of microbial recognition. They respond to a variety of microbial products (e.g., LPS, lipoproteins, proteins, and nucleic acids) by activating signaling pathways leading to NF-κB–mediated transcriptional regulation and, in some cases, activation of Rac1 and PI3K, which may regulate more rapid cellular responses . Regardless of the exact position along their signal cascades at which the TLR and HIF-1α pathways intersect, these observations illuminate a fertile territory for further study. The recruitment of HIF-1α–/– polymorphonuclear lymphocytes (PMNs) to affected tissues was found to be defective after exposure to a chemical irritant,  found that HIF-1α–/– PMNs were recruited to sites of microbial infection . iNOS mRNA is induced by bacteria in a HIF-1α–dependent fashion and nitrite production (a measure of NO production) is decreased in HIF-1α–/– cells. NO itself acts as a microbicide, but it was involved in regulating the expression of HIF-1α. HIF-1α–/– PMNs were less able to kill Gram-positive and -negative bacteria. To examine the activity of one of the many known O2-independent antimicrobial proteins, cathelicidin-related antimicrobial peptide (CRAMP). Cathelicidins were initially thought to be stored in PMNs as inactive proforms that required the actions of elastase, proteinase 3, or gastricsin to release a cationic C-terminal peptide with antimicrobial activity . However, important innate defense activities for unprocessed cathelicidins (e.g., LPS neutralization and chemoattractant activity). HIF-1α is apparently required for the synthesis of CRAMP mRNA and protein, and deletion of vHL causes increased expression of CRAMP mRNA . The cathelicidin-activating protease neutrophil elastase was found to be similarly regulated. Not only would reduced elastase activity decrease cathelicidin processing and antimicrobial activity , but elastase itself is directly antimicrobial . While HIF-1α regulates cathelicidin expression, it has also been reported that a cathelicidin peptide, PR-39, can itself induce angiogenesis by inhibiting the degradation of HIF-1α and subsequent induction of VEGF . 


5.5) NEURONAL PROTECTION BY HIF
Hypoxia- or ischaemia-induced EPO might stimulate new vessel growth, enabling the transport of more red blood cells and thereby increasing the amount of oxygen delivered to the hypoxic tissue, which in turn counteracts the detrimental effects of hypoxia on neurones. Finally, EPO could influence neuronal survival by modulation of glial cell activation.(55,59) Thus, EPO seems to be part of an endogenous defensive system enabling the brain to counteract detrimental effects of hypoxia and ischaemia. A model of such a protective system in the brain is depicted in Fig. 6. It includes operation of various growth factors such as EPO, VEGF and others promoted by a number of stimuli via activation of several transcription factors. Activation of HIF-1 by tissue hypoxia is an obvious pathway, but many others might be involved as well. Indeed, it has been shown that hypoxia-independent activation of HIF-1 occurs, e.g. by cytokines, as well as activation of other transcription factors such as AP-1 and nuclear factor  κ  B (NF-   κB) by oxygen depletion. 


Fig.7: Hypoxia-induced neuronal protection mechanisms in the central nervous system.

Tissue hypoxia and cerebral ischaemia activate hypoxia-inducible factor-1 (HIF-1), which in turn activates gene transcription of a variety of oxygen-regulated factors, among them erythropoietin (EPO) and vascular endothelial growth factor (VEGF).(54,56) These factors, as well as HIF-1 itself, might also be activated by hypoxia-independent stimuli such as growth factors or cytokines. EPO and VEGF then confer cellular protection. The main target for EPO (indicated by a thicker arrow) is neurones, while VEGF mainly prevents apoptosis and stimulates proliferation of endothelial cells,(52) resulting in new vessel growth (angiogenesis) and ultimately better oxygenation of hypoxic tissues. However, to a lesser extent, EPO also contributes to endothelial cell proliferation, and VEGF is also a direct neuroprotective factor (indicated by thinner arrows)(57,53). In addition, both EPO and VEGF also have neurotrophic properties. Finally, as receptors for both EPO and VEGF are expressed on microglial cells and astrocytes, glial cells might be a target.(58)

Indirect neuronal protection by EPO would be achieved by affecting endothelial cell growth and survival. Hypoxia- or ischaemia-induced EPO might stimulate new vessel growth, enabling the transport of more red blood cells and thereby increasing the amount of oxygen delivered to the hypoxic tissue, which in turn counteracts the detrimental effects of hypoxia on neurones ,Finally, EPO could influence neuronal survival by modulation of glial cell activation.(55,59) Thus, EPO seems to be part of an endogenous defensive system enabling the brain to counteract detrimental effects of hypoxia and ischaemia. A model of such a protective system in the brain is depicted in Fig. 7. It includes operation of various growth factors such as EPO, VEGF and others promoted by a number of stimuli via activation of several transcription factors. Activation of HIF-1 by tissue hypoxia is an obvious pathway, but many others might be involved as well. Indeed, it has been shown that hypoxia-independent activation of HIF-1 occurs, e.g. by cytokines, as well as activation of other transcription factors such as AP-1 and nuclear factor  κB (NF-κB) by oxygen depletion.


5.6) HYPOXIA INDUCED MECHANISMS FOR CELL SURVIVAL INVASION &METASTATIS

Hypoxia (oxygen tension [pO2] <7 mmHg) can induce changes in the proteome of tumor cells that lead to impaired growth or to cell death, including cell-cycle arrest, differentiation, apoptosis, and necrosis . 
Alternatively, however, hypoxia can induce proteomic changes that allow the tumor cells to successfully adapt to or overcome their O2- and nutrient-deprived state and to survive in or escape from their hostile environment. This is accomplished through hypoxia-stimulated angiogenesis, glycolysis, inhibition of apoptosis, and upregulation of growth factors (e.g., platelet-derived growth factor-B [PDGF-B], transforming growth factor beta [TGF-ß], insulin-like growth factor-2 [IGF-2], epidermal growth factor [EGF]) and other proteins involved in tumor invasiveness (e.g., urokinase-type plasminogen activator). 
Systemic responses leading to an elevation in the hemoglobin level, and thus improvement in the O2 transport capacity of the blood, can support the local mechanisms mentioned within tumors (e.g., through activation of the genes for erythropoietin, transferrin, and transferrin receptors) . Additionally, hypoxia may induce downregulation of adhesion molecules, thereby facilitating tumor cell detachment . Many of these hypoxia-inducible genes are controlled by hypoxia-inducible factor 1 (HIF-1) .

CHANGES  IN  GENE  EXPRESSION  HIF-1 &OTHER FACTORS

Cells that are poorly oxygenated (pO2 <7 mmHg) display a series of adaptive responses that allow for survival and continued proliferation. Among these, changes in the expression of genes for erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), transferrin receptors, and other proteins allow for the development of a more effective O2 (and nutrient) supply. 
Another group of genes involved in this adaptive response controls metabolic pathways that can meet the cellular energy requirements (e.g., glycolytic enzymes and glucose transporters). Expression of the genes for most of these proteins is regulated by HIF-1 α. This transcription factor was first identified by Semenza and colleagues as a regulator of hypoxia-induced erythropoietin expression  and has since regulate the expression of more than 30 target genes. These genes also play roles in tumor progression (i.e., proliferation, invasion, and metastasis), thereby contributing to tumor aggressiveness . 
Other factors involved in the regulation of O2-dependent transcription are nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) (see below).

Adenylate kinase 3 IGF-2
Adrenergic receptor IGF binding protein 1
Adrenomedullin IGF binding protein 3
Aldolase A Lactate dehydrogenase A
Aldolase C Nitric oxide synthetase 2 (NOS 2)
Carbonic anhydrase IX p21
Carbonic anhydrase XII p35srj
Coeruloplasmin Phosphofructokinase L
Endothelin-1 (ET-1) Phosphoglycerate kinase 1
Enolase 1 (ENO1) Plasminogen activator inhibitor-1
Erythropoietin (EPO) PDGF-B
GLUT-1 Pyruvate kinase M
Glyceraldehyde phosphate 
dehydrogenase Transferrin receptor
Heme oxygenase 1 TGF-
Hexokinase 1 VEGF
Hexokinase 2 
Table 1. Known HIF-1 target genes (gene products) 

Expression of HIF-1 in human cancer: causes and consequences.

Conclusion
Hypoxia is the condition when the tissues are deprived of oxygen and disturb the O2  level in normal condition. Alterations in the cellular microenvironment are due to an inadequate oxygen (O2) supply and the resultant hypoxia or even anoxia . 
There are four types of hypoxia, hypoxemic, anemic, stagnant, hystotoxic.

Hypoxia inducible factor is a transcriptional factors that stimulated in hypoxic condition and regulate the adaptive responses of mammalian cells to the low oxygen.Members of this factors are Hif-1α ,Hif-1β, Hif-2α , Hif-2β , Hif-3α, Hif-3β .                                                                                                                                                                                         

Hif-1 consist of four chain A, B, C,D. Among them A and B have DNA molecule.

 Hif have very important role in our body like HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low-oxygen conditions. These include glycolysis enzymes, which allow ATP synthesis in an oxygen-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. Hif regulate the transcription of Erythropoietin which regulate formation of RBC in bone marrow.

Hif also important in various pathway like increase promote the Glycolysis(glucose metabolism) and increase the production of lactate and decrease the extracellular PH.

Vascular endothelial growth factor( VEGE) production is driven by hypoxia via transcription activation of VEGE gene by Hif-1 which is essential for proliferation and migration of vascular endothelial cell, thereby inducing the formation of blood vessels .hif-1 activation in carcinogenesis make it possible target to prevent the cancer chemoprevention. Hif-1 activate the gene transcription of erythropoietin and vascular endothelial growth factor which provide neuronal protection mechanism.  
 
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