REVIEW ARTICLE The subtle side to hypoxia inducible factor (HIFa) regulation Rebecca L. Bilton and Grant W. Booker Department of Molecular Biosciences, The University of Adelaide, Australia The transcription factor hypoxia inducible factor a-subunit (HIFa) is pivotal in the cellular response to the stress of hypoxia. Post-translational modification of HIFa by hydroxylase enzymes has recently been identified as a key Ôoxygen sensingÕ mechanism within the cell. The absence of the substrate oxygen prevents the hydroxylases from modi- fying HIFa during hypoxia and allows dramatic up-regula- tion of both HIFa protein stability and transcriptional activation capability. In addition to this oxygen-dependent response, increased HIFa protein levels and/or enhanced transcriptional activity during normoxic conditions can be stimulated by various receptor-mediated factors such as growth-factors and cytokines (insulin, insulin-like growth factor 1 or 2, endothelial growth factor, tumour necrosis factor a, angiotensin-2). Oncogenes are also capable of HIFa activation. This induction is generally less intense than that stimulated by hypoxia and although not fully elucida- ted, appears to occur via hypoxia-independent, receptor- mediated signal pathways involving either phosphatidyl -inositol-3-kinase/Akt or mitogen activated protein kinase (MAPK) pathways, depending on the cell-type. Activation of Akt increases HIFa protein synthesis in the cell and results in increased HIFa protein and transcriptional activity. MAPK also activates HIFa protein synthesis and addi- tionally may potentiate HIF1a transcriptional activity via a separate mechanism that does not necessarily require protein stabilization. Here we review the mechanisms and function of receptor-mediated signals in the multifaceted regulation of HIFa. Keywords:HIFa; growth factor; oncogene; PI3K; MAPK. Introduction Spurred-on by the discovery of their involvement in the pathophysiology of many disease states including cerebral and pulmonary ischemia, cancer tumourigenesis and malignancy [1], the bHLH-PAS domain-containing hypoxia-inducible transcription factor (HIF) family have become a popular focus for research in the decade since the HIF1a gene was first characterized [2]. This family includes the regulatory a-subunits HIF1a and HIF2a that are both able to bind to their constitutively expressed b-subunit, ARNT, to form a functional HIF complex. The induction of HIFa by hypoxia (low physiological levels of oxygen) is dramatic and has been shown to regulate the transcription of over 40 downstream target genes, including glycolytic enzymes, glucose transporters and vascular endothelial growth factor [3]. Regulation of HIFa is complex and involves multiple mechanisms of control at the level of protein degradation and hence protein stabilization, nuclear translocation and transcriptional activation (Fig. 1). When stimulated by hypoxia, these mechanisms combine co-operatively to induce maximal HIF activation. Recently the Ôoxygen sensorsÕ monitoring this hypoxic response were identified as prolyl- and asparaginyl-hydroxylase enzymes [4–6], which during normoxia (normal physiological levels of oxygen) mediate the rapid degradation of HIFa protein and prevent transcriptional recruitment of the cofactor CBP/p300, respectively. These enzymes and the mechanisms involved in their activation of HIFa upon stimulus by hypoxia are reviewed elsewhere [7,8]. As elucidation of the hypoxic HIFa signalling pathway continues, another side to HIFa biology has quietly emerged. Zelzer and coworkers [9] were the first to demonstrate that the growth-factors insulin and insulin-like growth factor-1 (IGF-1) activate HIF1 and that this has subsequently been shown to occur through pathways separate to that employed by the classical hypoxic pathway (Fig. 1). The list of Correspondence to G. Booker, Department of Molecular Biosciences, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia. Fax: + 61 88303 4348, Tel.: + 61 88303 3090, E-mail: grant.booker@adelaide.edu.au Abbreviations: Akt, serine/threonine kinase (also known as protein kinase B); ARNT, aryl-hydrocarbon receptor nuclear translocation; bHLH-PAS, basic helix-loop-helix period-ARNT-single-minded; CBP, CREB binding protein; CO, carbon monoxide; C-TAD, C-terminal transcriptional activation domain; EGF, epidermal growth factor; eIF-4E, eukaryotic initiation factor 4E; 4E-BP1, eIF-4E binding protein 1; FGF-2, fibroblast growth factor-2; FIH-1, factor inhibiting HIF; FRAP, FKBP(FK506 binding protein) rapamycin associated-binding protein (also known as mTOR, mammalian target of rapamycin); HER2 NEU , heregulin-2 or EGF stimulated receptor tyrosine kinase; HGF, hepatocyte growth factor; HIFa,hypoxia inducible factor-1 or ) 2 a subunit; HRE, hypoxic response element; IGF-1/IGF-2, insulin-like growth factor-1 or -2; IL-1b, interleukin-1b; JNK, c-Jun amino-terminal kinase; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; NO, nitric oxide; p70 S6K , p70 S6 kinase; PDGF, platelet derived growth factor; PI3K, phosphatidyl-inositol 3-kinase; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; TGF-1b, transforming growth factor-1b;TNFa, tumour necrosis factor-a; VHL, von Hippel Lindau protein. (Received 15 October 2002, revised 6 December 2002, accepted 3 January 2003) Eur. J. Biochem. 270, 791–798 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03446.x receptor-mediated factors that stimulate HIFa currently includes many growth-factors, cytokines and circulatory factors such as PDGF, EGF, FGF-2, IGF-2, TGF-1b, HGF, TNFa,IL-1b, angiotensin-2 and thrombin [10–16]. In addition, oncogenes (HER2 NEU , Ras, v-Src) [17–19], and mutations in the tumour suppressor PTEN [20], have also been shown to affect HIF1a activity through these same signalling pathways. Other HIFa stimuli include signalling intermediates such as NO [21,22] and the in vitro phenomena of cell culture confluence [22,23]. While the degree of observable HIFa protein or tran- scriptional activation varies with each stimulus and cell-type [9,22,24], as a generalization, the magnitude of the receptor- mediated HIFa response in vitro is far less than the dramatic induction caused by hypoxia. Treatment of L8 or ARPE cells with insulin for example, results in twofold to sixfold inductions of an HRE-luciferase reporter [9,24] (Fig. 2). An exception to this trend, however, can be found in vascular smooth muscle cells, where several stimulatory factors increased the amount of protein observed to levels signifi- cantly greater than those induced by hypoxia [13]. Whilst apparently minor in comparison to the in vitro induction by hypoxia, the gene expression changes resulting from the receptor-mediated pathways are nonetheless important. These stimuli often elicit small changes in housekeeping functions that accumulate over extended periods of time [25]. Receptor-mediated HIFa regulation has been shown to occur via two well characterized signalling pathways, the Ras/MEK/MAPK and PI3K/Akt/FRAP kinase cascades [24,26,27] (Fig. 3). Although the end result is enhanced HIFa protein levels and/or transcriptional activation even under normoxic conditions, the molecular mechanisms involved must differ from those of hypoxia, as low oxygen tension and activation of the MAPK and Akt pathways can co-operate to enhance the induction of HIFa activity [20, 27–29]. There is some evidence to suggest that co-operation of these kinase pathways with hypoxia may occur via the hypoxic generation of reactive oxygen species (ROS) as an intermediate signalling step [30] (reviewed in [31]). This may also be the pathway by which some of the stimulatory factors such as thrombin, angiotensin and IL-1b influence HIFa, as their ability to activate HIF1a was blocked by ROS inhibitors and antioxidants [13,14,32]. In this way, Fig. 2. Fold induction of pTK-HRE luciferase reporter construct in stably transfected 3T3L1 adipocyte cells. Treatments include dipyridyl (100 n M ), serum free, insulin (100 n M ), IGF-1 (5 n M ) and IGF-2 (5 n M ). Data normalized using pTK-Renilla construct as a transfection control. Fig. 3. Schematic representation of the molecular interactions controlling receptor-mediated signals leading to HIFa dependent transcription of downstream target genes. Arrows indicate activating steps, truncated arrows indicate inhibitory effects and dotted lines indicate possible interactions for which only limited evidence is available. Fig. 1. Schematic overview of the receptor-mediated and hypoxic signal pathways and the mechanisms they employ to activate HIF and induce transcription of downstream target genes. The larger arrow highlights the greater magnitude of the response derived from hypoxic signals relative to receptor-mediated signals. 792 R. L. Bilton and G. W. Booker (Eur. J. Biochem. 270) Ó FEBS 2003 ROS may then recruit the MAPK or Akt pathways to activate HIFa via ligand-independent activation of various growth-factor receptors, such as the EGF receptor [33]. To add further complexity to normoxic HIFa regulation, the particular kinase pathway employed and its action upon HIFa may differ depending on cell type or specific stimulus. It is important to remember that different cell-types may express a different combination of signalling proteins and may therefore respond to the same stimuli to a lesser or greater extent. This includes the Akt and MAPK pathways, which are not always active in every cell type [34]. The use of different pathways occurs in the hepatoma cell-line HepG2 where both TNFa and IL-1b were shown to increase DNA binding by HIF, but only IL-1b was able to increase the observed HIF1a protein levels [12]. This suggests a different mechanism of action for each cytokine in this cell type. In contrast to these findings, the two cytokines were reported to act in the same manner to increase transcription of HIF1a mRNA by twofold to threefold resulting in increased protein levels in synovial fibroblasts [16]. Furthermore, the differences in stimuli-induced signalling are highlighted by the example of IGF-1 stimulation in different cell types. IGF-1 stimulation allows visualization of HIF1a protein and increases transcriptional activity of HIF1a via activa- tion of MAPK in mouse embryo fibroblasts [35], whereas in the U373 glioblastoma cell line these effects require Akt [20]. Both kinase pathways are reported to additively increase HIF1a translation and thus protein level in HCT116 colon carcinoma cells [26]. Finally, the family members HIF1a and HIF2a are both responsive to receptor-mediated stimuli, but not necessarily the same stimuli within a single cell type, even when both homologues are coexpressed [29]. Whilst confusing, this complexity and cross-talk between signalling pathways is not uncommon for growth factor stimuli. Dependant on cell type and signal intensity, stimulation of receptors by insulin can alternatively activate either MAPK or Akt, and this can result in the completely disparate outcomes of proliferation (mitogenic) or glucose uptake (metabolic) [34]. Phosphorylation Upon polyacrylamide gel electrophoresis, HIFa protein migrates as a diffuse band consistent with an approximate 20 kDa increase in molecular mass from its predicted size of 104 kDa [14,36,37,41]. This broad band contains phos- phorylated species of HIFa, as treatment with a phospha- tase returns the protein to its predicted size [41]. Several deletion studies have failed to identify specific residues, which when phosphorylated, alter hypoxic induction of HIF1a [4,5,38]. However, a report from Gradin et al.[39] suggests the presence of an oxygen-independent, ubiqui- tously phosphorylated residue that may play a role in providing structural support to the active protein. Although phosphorylation was not shown directly, mutational ana- lysis identified the threonine residue 844 of HIF2a,located near the hydroxylase targeted asparagine 851, as being important for the function of the C-terminal transcriptional activation domain (C-TAD), spanning residues 824–874, regardless of the oxygen state. Phosphorylation or intro- duction of a negatively charged residue at position 844 allows binding of CBP in a mammalian two-hybrid system [39]. It is feasible that phosphorylation of other residues also act in a similar manner to stabilize HIFa postinduction and this may efficiently maintain the structure of the newly formed active transcriptional complex. Support for this proposal is provided by the finding that HIFa is phosphor- ylated postinduction after a short lag period of up to a minute [40]. The kinase(s) and upstream regulatory signals involved in either ubiquitous or stimuli-induced phosphory- lation have not yet been fully elucidated. MAPK Direct phosphorylation of HIFa by MAPK has been reported by several groups. These researchers showed that activated recombinant or endogenous MAPK was able to phosphorylate either full-length HIFa or a C-TAD-fusion product when supplied as a substrate [27,41,42]. In those studies employing HIF1a-fusion proteins expressed in COS-7 cells, the region targeted by MAPK was shown to lie within residues 786–826 of the C-TAD [27] or residues 531–826 spanning both the inhibitory domain and C-TAD [42]. Although Sodhi et al. [42] identified up to eight serine residues within this inhibitory region that contain adjacent proline residues that may serve as putative consensus target sites for the MAPK family, the specific HIFa residue(s) that are phosphorylated by MAPK have yet to be identified. A proposed function for phosphorylation leading to increased HIF transcriptional activity is through the derepression of the inhibitory domain that lies between the two transcrip- tional activation domains of the HIF a-subunit [42]. Regions within this inhibitory domain have been shown to be important for the interaction of HIFa with factor inhibiting HIF-1 (FIH-1) [43,44] identified as the asparagine hydroxylase [45]. In the three-dimensional structure, these regions may form part of the FIH-1 recognition site. One possible explanation for the observed derepression of the HIFa inhibitory domain, is that phosphorylation of residues within this domain may prevent docking of FIH, and thus prevent the subsequent asparagine hydroxylation. This would result in a derepression of transcriptional activity, as CBP/p300 would be able to associate with HIFa. As mentioned previously, hypoxia is able to activate MAPK in some cell lines [27–29]. However, activation of MAPK in hypoxia is not necessarily required for HIF1a activation and this appears dependent on cell-type. In fibroblasts, MAPK activation was blocked by application of the MEK inhibitor PD 098059 and yet the activation of HIF1a induced by hypoxia remained unaffected [35]. In contrast, it was found that PD 098059 treatment of HT42 and Rat-1 fibroblast cell-lines moderately decreased HIF transcriptional activity in both normoxia and hypoxia [18,36]. It is possible that the reduction in HIF transcrip- tional activity in these latter studies was due to inhibition of MAPK activity that may have been stimulated by factors present in the culture media. Indeed, HIF transcriptional ability induced by receptor-mediated stimuli is impaired when the MEK chemical inhibitors PD 098059 and U0126, or dominant-negative MAPK mutants were applied to cells in culture [18,29]. Other kinase family members such as the stress kinases p38a,p38c and JNK have also been shown to be involved in signalling to HIFa in several cell-types [42]. In most reports documenting a role for MAPK in HIF Ó FEBS 2003 Kinase pathways influence HIFa (Eur. J. Biochem. 270) 793 function, no change to the observable level of HIFa protein expression, protein stability, rate of protein degradation or DNA-binding ability were observed [27,29,41]. This indi- cates that effects due to MAPK signalling, in most cell-types studied to date, do not precede HIFa protein expression or stabilization and instead improve HIF transcriptional activity. Possibilities for MAPK action upon HIF tran- scriptional ability include recruitment of cofactors to the active transcriptional complex, or a direct MAPK phos- phorylation of HIFa residue(s). Phosphorylation may improve HIF transcriptional activity by derepression of the HIFa inhibitory domain or simply favouring a confor- mation that supports the active domain. Given the varia- tions encountered so far within the characterization of the receptor-mediated signalling pathway, it is not surpri- sing that, in a few cell types, the up-regulation of HIF transcriptional activity via MAPK activity has been attri- buted to an increase in observable HIF1a protein [22,24]. In this way, MAPK may act via a similar mechanism to Akt to improve HIFa protein synthesis. The effects of MAPK on protein stability or transcriptional activation need not necessarily be mutually exclusive. Akt The serine/threonine kinase Akt has also been identified as a signalling intermediate downstream of the receptor-medi- ated factors that alter HIFa regulation. Unlike stimulation by MAPK, Akt activity increases HIF transcriptional activation by increasing the pool of available HIFa protein within the cell. The use of chemical inhibitors such as wortmannin and LY 294002 that block the phosphatidyl- inositol 3-kinase (PI3K) family of enzymes, or dominant negative mutants of the PI3K/Akt pathway were shown to inhibit factor- or hypoxia-stimulated HIF1a protein accu- mulation as detected by Western blot [10,46]. A reduction in the levels of observable HIFa protein resulted in loss of DNA binding ability of HIF and failure to up-regulate the transcription of reporter constructs or endogenous down- stream target genes [10,46]. Similarly over-expression of members of the PI3K/Akt pathway or inhibition of PTEN, a negative regulator of Akt, resulted in increased levels of HIF1a protein, DNA binding or transcriptional activity in many cell types [18,20] (Fig. 3). It was initially proposed that the observed increase in HIFa protein was due to enhanced stability, possibly through inhibition of the proteosomal degradation machinery that is active in norm- oxic conditions [20,28]. Recently several publications have clarified the role that Akt plays in HIFa biology, linking an Akt signal to an increased rate of HIFa protein synthesis [17,24,26]. When stimulated by heregulin, IGF-1 or insulin, activation of the PI3K/Akt/FRAP pathway was shown to increase de novo protein synthesis, as shown by inhibition with the transla- tion inhibitor cycloheximide and pulse chase experiments [17,24,26]. FRAP, also known as mTOR, de-represses the translational regulatory protein eIF-4E by phosphorylating and inactivating its binding protein 4E-BP1 [25]. FRAP also activates p70 S6K whichinturnisabletoactivatethe40S ribosomal protein S6 [25] (Fig. 3). Thus in a wortmannin-, LY 294002- and rapamycin-sensitive manner, activation of eIF-4E and p70 S6K results in the increased translation from HIF1a mRNA [17,24,26]. Interestingly, Fukuda et al. identified a novel role for MAPK in HCT116 cells as its activation was also shown to alter HIF1a protein synthesis [26]. Unlike other reports (see above) that document enhanced HIF transcriptional activity upon MAPK stimu- lation with no alteration to HIFa protein levels, activation ofMAPKinthesecellswasshowntoleadtoatransient activation of eIF-4E and its effects on HIF1a protein synthesis were additive to those of PI3K/Akt/FRAP [26] (Fig. 3). Enhanced levels of HIFa synthesis may explain the previous reports for which activation of MAPK resulted in an increase in observed HIFa protein levels [22,24]. The increase in HIFa protein synthesis appears to be relatively gene specific since the translation of control luciferase- reporter or ARNT mRNA was not altered [17]. In addition, over-expression of both FRAP and eIF-4E have been previously shown to disproportionately increase the trans- lation of specific target genes [25,47]. This mechanism of increased translation is in contrast to that employed by hypoxic stimuli for which it has been repeatedly shown, for most cell types, that there is no alteration of either HIFa mRNA levels or the rate of de novo protein synthesis when oxygen levels are limiting [17,48]. Increased translation of HIFa mRNA ultimately leads to an increase in the HIFa protein pool, thus explaining initial reports that observed increased HIFa protein in response to stimulatory factors. Given that the prolyl (and presumably the asparaginyl) hydroxylase enzymes are believed not to be at high concentrations within the cell [49], increasing the availability of their HIFa substrate may easily titrate them out. As well as overwhelming the HIFa degradation mechanisms, substrate saturation also relieves the transcrip- tional repression due to the asparagine hydroxylase, FIH-1. Thus it is plausible that even small increases in total HIFa protein via up-regulated translation could saturate one or both of these enzymes. Overwhelming the hydroxylase enzymes may enable a small portion of the total HIFa protein translated to escape the normoxic suppression and degradation pathways. The presence of even a small amount of active protein will result in some HIF-target gene transcription, although the level of down-stream target genes transcribed may be much less than the maximal inductions possible in hypoxia [9,14]. Although receptor- mediated signals have been shown to significantly improve the observable levels of HIFa protein and/or ability to bind DNA [13,24], this does not necessarily always correlate with large increases in transcriptional activation. In addition, over-expressed HIFa protein is not fully activated in normoxia [50]. This lack of full transcriptional activity, even in the presence of high levels of protein, could be either because cofactors are limiting in these cells, or that there are differences in the substrate saturation levels between the different hydroxylases. In concluding this section on the mechanism(s) of action of PI3K/Akt, it must be noted that although induction of the PI3K/Akt/FRAP pathway played no role in alteration of HIF1a ubiquitination, VHL interaction, proteosomal-degradation or protein sta- bility in ARPE-19, MCF-7 or HCT116 cell lines [17,24,26], up-regulated translation may not be the only mechanism for protein stabilization in all cell types. Inhibition of HIFa protein degradation, possibly by interruption of the hydroxylase function, cannot yet be ruled out as a 794 R. L. Bilton and G. W. Booker (Eur. J. Biochem. 270) Ó FEBS 2003 mechanism of action for some receptor-mediated stimuli. Recently, Chan and coworkers (2002) have shown that HIFa protein was increased by expression of the v-Src and RasG12V onco-proteins, as well as constitutively active Akt, however, there were variations in the amount of HIFa proline-hydroxylation detected for each stimulus [51]. The antibody generated during this work, which targets the hydroxylated proline 564 of HIF1a (531 of HIF2a) [51], will prove a valuable tool in delineating the exact hydroxylation status of HIFa protein during all types of stimuli. Nitric oxide, carbon monoxide and cell confluence Nitric oxide (NO) has been shown to increase HIFa protein levels, DNA binding and transcriptional activity in endo- thelial, smooth muscle, Hep3B and LLC-PK 1 cells during normoxia [21,52]. Paradoxically, it has also been reported that NO can also have the completely opposite effect of inhibition of both basal- and hypoxia-induced expression of HIF target genes in endothelial cells [53]. Inhibition of hypoxia-induced DNA-binding activity by carbon monox- ide (CO) or NO exposure was also seen in several other cell types [54,55], although reduced HIFa protein expression was only observed in one case [55]. These inhibitory effects may be stimuli specific as CO did not prevent the stabilization of HIF1a protein and transcriptional activity induced by either cobalt chloride or the iron-chelator desferrioxamine [55]. The differences in these findings indicate that cell type, concentration of NO or CO stimuli and cellular oxygen status are important experimental considerations and suggest that CO and NO may mediate their effects through multiple targets within the HIF pathway, possibly dependent on their concentration. To further complicate matters, the genes that produce these NO and CO species, inducible-nitric oxide synthase and heme oxygenase, are regulated by HIF [56,57] and the growth- factor insulin is able to stimulate NO production via a PI3K-dependant pathway [58]. Although the mechanism(s) via which NO or CO affect HIF remain unclear, one proposal is that they bind to the hydroxylase enzymes [59], and activate HIF in normoxia [53]. As analogues of molecular oxygen, they may bind to hydroxylases but not participate in the hydroxylation reaction. Thus in normal oxygen conditions, the activity of hydroxylases, and thus protein degradation may be prevented [59]. However, this cannot explain how in low oxygen concentrations, during hypoxia, NO or CO are able to prevent the hypoxic stabilization and activation of HIFa [53]. Nitric oxide was identified as a signalling intermediate between HIF and the stimulus of increased cell confluence [22]. When the density of prostate cells in culture was increased, levels of HIF1a protein increased concomitantly via a nitric oxide and Ras/MAPK dependant signalling pathway [22]. In addition, increased transcription of the HIF target gene vascular endothelial growth factor was observed in dense cultures of human glioblastoma cells (U87) and fibrosarcoma cells (HT1080) [23]. This is in contrast to several findings which document a decrease in HIF1a protein expression and consequently reduced DNA binding activity in prostate cancer cells grown at high density (90%) compared to low density (50%) during both hypoxic and normoxic conditions [10,60]. It also conflicts with the finding that stimulation of HIF1a by insulin was only possible when cells were cultured at low density [11] that suggests the capacity for induced up-regulation of HIFa is prevented at high density. Given these completely disparate results, there can be no consensus currently made as to a mechanism for cell-density mediated effects on HIFa and clearly this area requires more research. However, the phenomenon of confluence is an important consideration during in vitro cell-based assays, particularly because the effects of confluence may be due in part to localized hypoxia. Confluence should be carefully monitored during analysis of HIFa activation by each nonhypoxic stimulus so the mechanism by which that stimulus contributes to HIFa can be clearly defined. Finally, the contribution that density makes to HIFa activity in vivo within tissues is unknown. Possibly it forms part of the basal level of HIF activity and this may be different in each tissue type, depending on how tightly packed the cells are. Although HIFa is ubiquitously expressed within all cells, the level of normoxic HIFa protein observable and also the capacity for inducible up-regulation varies in different cell types [61]. A role for receptor-mediated HIFa in vivo ? HIFa is a transcription factor with a complex set of multiple regulatory mechanisms. Activation through various recep- tor-mediated pathways, to influence only a subset of these regulatory mechanisms, allows for a moderate induction of HIFa and consequently a small increase in the transcription of downstream target genes. Given the subtle effect upon HIFa, it is likely that receptor-mediated signalling during normoxia plays a secondary role to the induction of HIFa by hypoxia. It appears more than a coincidence that genes encoding a number of components of the receptor-mediated signal pathways are themselves regulated by HIFa or hypoxia. Inducible nitric oxide synthase [56] and haem oxygenase-1 [57] contain HRE within their promoters, whilst other genes, IGF-2 [11], IGF binding proteins-2 and -3 [11], PDGF [62], FGF [63] and TGF-1b [64] may be indirectly altered by hypoxia or HIF. With this complex web of autoregulatory feedback, it would seem reasonable to propose that receptor-mediated activation of HIFa has arolein vivo, and is not just an in vitro cell culture phenomenon. What is the role of receptor-mediated activation of HIFa? One proposal is that these signals may be important for stimulation of HIFa for oxygen-independent purposes and they may also act to enhance hypoxic activation in some tissue types. There are many situations where expression of HIF target genes may provide a biological advantage other than just hypoxic stress. Receptor-mediated signals may increase the transcription of only a subset of the HIF responsive genes during this time. These situations may include cell proliferation, differentiation, development or inflammatory responses. All of these biological activities would place an increased Ômetabolic loadÕ on a cell above its normal metabolic requirements, and interestingly, these are processes which are often stimulated by, or result in, the expression of growth-factors and cytokines known to activate HIFa. For example, HIF1a is one of many genes whose expression is up-regulated during adipogenesis [65] Ó FEBS 2003 Kinase pathways influence HIFa (Eur. J. Biochem. 270) 795 and IGF-2 levels are extremely high in embryogenesis [66]. Interestingly, HIF1a has been shown to be stabilized and activated by the cytokine TNFa during inflammation in normoxic wounds, allowing increased expression of the HIF target-gene vascular endothelial growth factor in order to promote wound healing [67]. It is possible that these receptor- mediated effects, particularly through HIFa protein synthe- sis, are only able to occur in active tissues, especially because the translation initiation factor eIF-4E is only abundant in nonquiescent cells [25]. Stimulation of HIFa though oxygen- independent mechanisms could increase expression of genes that promote angiogenesis, vasodilation, glucose uptake or glycolysis to provide increased nutrient supply to those tissues requiring it. Many of these HIF target genes have other regulatory elements within their promoters and their expression is a balance between converging signals. This may be the case with many of the glycolytic genes such as hexokinase that have glucose or carbohydrate responsive elements nearby to the hypoxic response elements that may combine synergistically to regulate gene expression [68,69]. 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