- Email: [email protected]
Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation
Free Radical Biology & Medicine, Vol. 31, No. 7, pp. 847– 855, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(01)00657-8
Review Article TRANSDUCTION PATHWAYS INVOLVED IN HYPOXIA-INDUCIBLE FACTOR-1 PHOSPHORYLATION AND ACTIVATION E. MINET, G. MICHEL, D. MOTTET, M. RAES,
and
C. MICHIELS
Laboratoire de Biochimie et Biologie Cellulaire, Faculte´s Universitaires Notre-Dame de la Paix, Namur, Belgium (Received 13 February 2001; Accepted 3 July 2001)
Abstract—Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor which is activated by hypoxia and involved in the adaptative response of the cell to oxygen deprivation. During hypoxic stress, HIF-1 triggers the overexpression of genes coding for glycolytic enzymes and angiogenic factors. To be active HIF-1 must be phosphorylated. HIF-1 is a substrate for various kinase pathways including PI-3K and the MAP kinases ERK and p38. Several transduction pathways have been proposed which act downstream of putative oxygen sensors and lead to the activation of these kinases. In this review, we summarize some of the latest advances describing the possible signaling pathways leading to HIF-1 phosphorylation and subsequent activation. The physiological relevance of these regulations is also discussed. © 2001 Elsevier Science Inc. Keywords— Hypoxia, Hypoxia-inducible factor-1, Mitogen-activated protein kinases, Phosphoinositide-3 kinase, Signaling, Tumorigenesis, Free radicals
INTRODUCTION
cerebral strokes, as well as thrombosis [1]. Mammalian cells have developed adaptative systems allowing them to survive to moderate or even severe hypoxia. This process involves an increase in the expression of genes coding for proteins responsible for the anaerobic production of ATP [2–5], namely aldolase A, enolase-␣, lactate dehydrogenase, pyruvate kinase, and glucose transporter-1. Hypoxic cells also secrete vascular endothelial growth factor (VEGF) [4,6], an angiogenic growth factor allowing neovascularization of the hypoxic tissue. Several transcription factors have been reported to be involved in the response to hypoxic stress (AP-1, NF-B, HIF-1) [7]. Among these transcription factors HypoxiaInducible Factor-1 (HIF-1) is the most potent inducer of the expression of genes such as those coding for glycolytic enzymes, VEGF and erythropoietin (EPO) [2–5]. HIF-1 is a heterodimer composed of the HIF-1␣ and ARNT-1 subunits [8]. Both subunits belong to the basic Helix-Loop-Helix-Per/ARNT/AhR/Sim (bHLH-PAS) transcription factor family [8]. HIF-1␣ as well as ARNT-1 are constitutively expressed [9]. However, HIF-1␣ appears to be the HIF-1 subunit regulated by hypoxia as under this condition, HIF-1␣ degradation is inhibited [10 –12], whereas in contrast, it is rapidly degraded by the ubiquitin-proteasome system under nor-
Vascular injuries can lead to an inadequate flow of the blood, which results in a decrease in the delivery of oxygen and nutrients to the tissues [1]. These ischemic conditions can lead to tissue necrosis, cardiovascular and Emmanuel Minet has obtained his Ph.D. thesis at the University of Namur. This work was focused on the transcriptional and post-translational regulation of HIF-1. He is now a post-doctoral fellow at the Dana Farber Cancer Institute in Boston. Gaetan Michel is a Ph.D. student in chemistry. He has developed a 3D model of the DNA-binding domain of HIF-1 when bound to HRE. He is now studying the effect of mutations within the DNA-binding domain of HIF-1␣. Denis Mottet is a Ph.D. student in Biology. His research is aimed at identifying new proteins interacting with HIF-1. Martine Raes obtained her doctorate in biological sciences in 1983 at the University of Namur, where she is now in charge of the Laboratory of Biochemistry and Cellular Biology. Her research is focused on signal transduction in inflammatory conditions and during the development of atherosclerosis. Carine Michiels was awarded her doctorate on the protective effect of antioxidant enzymes against oxidative stress in 1989. Since then, her research interests include understanding the effects of hypoxia on cells at the transcriptional as well as on biochemical levels. She is also currently engaged in the development of new anti-ischemic molecules. Address correspondence to: Dr. Carine Michiels, Laboratoire de Biochimie et Biologie Cellulaite, Faculte´s Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, 5000 Namur; Tel: ⫹32 (81) 72 4321; Fax: ⫹32 (81) 72 41 35; E-Mail: [email protected]. 847
848
E. MINET et al. Table 1. Examples of Kinases Activated Under Hypoxia and/or Involved in the Activation of HIF-1 in Different Cell Types
Cell type
Kinase and stimulus for kinase activation
Transcription factor phosphorylated
Target gene studied
References
PC12
p38␣, p38␥ (hypoxia) ERK (hypoxia) ERK (hypoxia) ERK (hypoxia) p38 ␣, p38 ␥ (GPCR transformation) ERK (GPCR transformation) PI-3K (hypoxia ⫹ Ha-Ras transformation) JNK1 (hypoxia)
AP-1 HIF-2 elk-1 HIF-1 HIF-1 HIF-1 HIF-1 c-jun
TH HRE-luc reporter c-fos HRE-luc reporter VEGF VEGF VEGF bFGF
[22] [21] [26] [28] [32] [32] [30] [31]
HeLa HMEC-1 NIH3T3 NIH3T3 MCF-7
moxic conditions [10 –12]. Under hypoxia, HIF-1␣ is seen to dissociate from the chaperone protein heat shock protein 90 (Hsp90) [13] and then translocates into the nucleus [14], where it dimerizes with the nuclear protein ARNT-1 to form the HIF-1 complex. To be fully activated, HIF-1 requires suitable redox conditions [15] as well as interaction with coactivators, namely CRB binding protein/p300 (CBP/p300) and steroid receptor coactivator-1 (SRC-1) [14]. The first evidence indicating that HIF-1 is a phosphoprotein was produced by Wang and Semenza [16]. Using electrophoretic mobility shift assay (EMSA) they showed that when nuclear extracts of hypoxic Hep3B cells were treated with phosphatase, the HIF-1/DNA complex is disrupted [16]. They went on to demonstrate that the serine/threonine kinase inhibitor 2-aminopurine, the tyrosine kinase inhibitor genistein, as well as the serine threonine phosphatase inhibitor NaF, are able to inhibit HIF-1-DNA-binding activity and HIF-1␣ stabilization in under hypoxic conditions [17]. Moreover, the MAP kinase inhibitor, PD98059, is able to inhibit HIF-1 transcriptional activity [18]. These data suggest that phosphorylation as well as dephosphorylation could both be involved in HIF-1 activation. Similar results have already been described for c-jun, which needs to be dephosphorylated in its DNA-binding domain and phosphorylated in its transactivation domain to be active [19]. Several kinases are known to be activated under hypoxia and are therefore possible candidates for HIF-1 activation. This is the case for some of the MAP kinase family members and for PI-3 kinase.
KINASES ACTIVATED DURING HYPOXIA
Under hypoxic conditions, the activation of several MAP kinases has been demonstrated in different cell types and is associated with the activation of transcription factors such as AP-1 (activated protein-1, c-Jun, c-Jos, Fra-1, ATF-2), elk-1, and nuclear factor-B (NFB) [7]. However, all the kinases and the transcription factors that are involved, as well as the target genes that
are over-expressed, could differ according to the cell type investigated (Table 1). One of the most comprehensive studies of MAP kinase activation in hypoxic cells has been performed by Conrad et al. in PC12 cells [20,21]. PC12 cells are rat pheocytochroma cells that can be differentiated into neurones following NGF stimulation. Using Western blots and in vitro kinase assays, it was demonstrated that a moderate hypoxia (6% O2) strongly activates both the p38␣ and p38␥ isoforms of the p38/SAP kinase 2 pathway in these cells [20]. Under the same conditions, ERK are also activated but to a lesser extent; however, JNK activity does not increase [20]. When PC12 cells are incubated for 6 h under more severe hypoxic conditions (1% O2), ERK1 activity is strongly increased [21]. In addition, exposure of PC12 cells to hypoxia causes membrane depolarization and extracellular calcium influx as assessed by Fura-2 fluorescence imaging [22]. The calcium influx observed in the early stage of hypoxia is mediated by L-type Ca⫹⫹ channels and is involved in the activation of downstream effectors such as several calmodulin kinases and cAMP responsive element binding protein (CREB) [23]. However, whether hypoxia activates voltage-gated channels is controversial. We should consider that in other cell types such as carotid body glomus cells, hypoxia inhibits the calcium current [23]. However, it could be possible that different L-type Ca⫹⫹ channel subunits are expressed as a function of cell type, and trigger a cell-typespecific response stimulating different downstream effectors. When pheocytochroma PC12 cells are incubated in a calcium-free medium and placed under hypoxic conditions, p38␥ is not activated [22,24]. p38␥ activation is still observed in PKA-deficient PC12 cells, which implies that p38␥ activation is not dependent on PKA activity [24]. The hypoxic activation of ERK also seems to result from the calcium influx. Interestingly, W13 and calmidazolium chloride, two calcium-calmodulin pathway inhibitors, inhibit ERK activation in hypoxic PC12 cells, as demonstrated by Western blot detection of the phosphorylated form of ERK [21]. However, the exact
Transduction pathways involved in HIF-1
mechanism linking calcium-calmodulin and HIF-1 activation is not known. Under hypoxia, the tyrosine hydroxylase (TH) gene is over-expressed [22,25]. TH participates in the synthesis of catecholamines in the carotid body, hence triggering an adaptative respiratory response. This gene is a target gene for AP-1 and HIF-1, two transcription factors activated by hypoxia [7,22]. However, when hypoxic PC12 cells are incubated in calcium-free medium, TH gene induction is not observed [22]. This indicates that in PC12 cells, calcium influx contributes to the transcriptional response to hypoxia, probably through pathways involving p38 and ERK activation. However, the link between the increase in calcium and the activation of MAP kinases is still unknown. In HeLa cells, the activation of the ERK pathway is involved in the induction of the c-fos gene transcription in hypoxic conditions [26]. Indeed, it has been demonstrated that in hypoxic HeLa cells, PD98059 inhibits the c-fos gene induction and that ERK2 is hyperphosphorylated [26]. Furthermore, in vitro kinase assays showed that immunoprecipitated ERK from hypoxic HeLa cells are able to phosphorylate the transcription factor elk-1, which triggers the hypoxic induction of the c-fos gene transcription [26]. The hypoxic activation of the ERK pathway has also been observed in pulmonary artery fibroblasts and in human microcirculation endothelial cells-1 (HMEC-1) [27,28]. The Raf-1/ERK pathway has also been associated with the hypoxic induction of the VEGF gene [29]. In hypoxic NIH3T3 cells expressing a dominant negative form of c-src or of Raf-1, VEGF hypoxic induction is observed to be inhibited. VEGF gene induction is also inhibited in the presence of 6-thioguanine, an ERK pathway inhibitor [29]. In contrast, in NIH3T3R, a Ha-Ras transformed NIH3T3 cell line, the use of ERK dominant negative mutants does not inhibit the VEGF overexpression under hypoxia [30]. In this particular cell line, wortmannin, a PI-3K/Akt pathway inhibitor, is able to inhibit the hypoxic induction of VEGF gene transcription. This was confirmed by the use of dominant negative mutants for the p85 subunit (⌬p85) of PI-3K or for Akt [HA-Akt(K119M)] [30]. Both ⌬p85 and HAAkt(K119M) mutants were able to inhibit the hypoxic over-expression of the VEGF gene [30]. Taken together, these results indicate that hypoxia triggers ERK and PI-3K activation in several cell lines (Table 1). However, it is not clear whether the ERK pathway is also involved in the increase in the transcription of the VEGF gene during hypoxia in the NIH3T3R cells. The kinases of the JNK pathway are not activated by hypoxia in PC12 cells [20]. However, they are involved in the activation of AP-1 and in the phosphorylation of c-Jun in hypoxic MCF-7 cells [31]. An increase in JNK1
849
phosphorylation and activity was detected by Western blot and kinase assays in hypoxic MCF-7 cells incubated under severe hypoxia (0.1% O2). In these cells, JNK1 activation is concomitant with the phosphorylation of c-Jun [31]. Additional evidence recently indicated that p38 can also regulate HIF-1 [32]. HIF-1␣ phosphorylation by p38 could be involved in the inhibition of the inhibitory domain located within the carboxy-terminal region of HIF-1␣ [32]. However, it is currently unclear whether this pathway is involved in hypoxia-induced apoptosis or cell survival. MAP and many other kinases are potentially activated by hypoxia but the responses vary according to the cell type. Thus, depending on the particular set of kinases activated in a given cell line, it is possible that the cellular response to hypoxia could be either adaptative or apoptotic. Accordingly, kinase activation under hypoxic conditions could activate different sets of transcription factors orienting the transcriptional response in different directions, depending on the tissues and on the severity of the hypoxic stress. HIF-1 ACTIVATION DEPENDS ON SEVERAL KINASE PATHWAYS
As previously described, HIF-1 is a phosphorylated protein and its phosphorylation is involved in HIF-1␣ subunit stabilization as well as in the regulation of HIF-1 transcriptional activity [16 –18]. Here, we describe the role played by PI-3K/Akt and ERK in HIF-1 stabilization and activation under hypoxic conditions.
HIF-1 stabilization is dependent on the PI-3 kinase/Akt pathway The hypoxic expression of VEGF is regulated by HIF-1. During hypoxia, HIF-1 is activated and binds several HIF-1 Responsive Elements (HRE) located within the VEGF gene promoter and 5⬘UTR [4,6]. The use of dominant negative mutants for the PI-3 kinase or for the Akt kinase inhibits the hypoxia-induced overexpression of VEGF, suggesting that the PI-3K/Akt pathway is involved in HIF-1 activation [30]. The co-transfection of a HIF-1 reporter plasmid either with the p85 or with the Akt dominant negative vector demonstrated that the disruption of the PI-3K/Akt pathway impaired the activation of HIF-1 as well as VEGF gene transcription in hypoxic NIH3T3R cells [30,33] (Table 1). This result indicates that HIF-1 is a protein regulated by the PI-3K/ Akt pathway (Fig. 1). Moreover, this pathway is downregulated by the tumor suppressor factor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [33,34], which is a phosphatidylinositol 3,4,5-triphos-
850
E. MINET et al.
Fig. 1. Putative role of the PI-3K/Akt pathway in hypoxia-induced stabilization of HIF-1␣ (1). Under normoxia, the HIF-1␣ sub-unit is degraded by the ubiquitin-proteasome system. Under hypoxia, HIF-1␣ degradation is inhibited through a PI-3K/Akt pathway. PI-3K could be activated by mitochondrial ROS (2) or by an NADPH-oxidase-like oxygen sensor associated to Rac1, a small G protein (3). PIP3 activates Akt, which is then involved in the HIF-1␣ stabilization process (4). When PIP3 degradation by PTEN is inhibited either due to PTEN mutation(s) or possibly under hypoxia, the stabilization of HIF-1␣ is enhanced (5).
phate phosphatase [35] (Fig. 2). Mutations within the PTEN catalytic domain increase the phosphorylation and activation of Akt [35]. In glioblastoma-derived cell lines lacking functional PTEN, VEGF expression is high even under normoxia [33]. Restoration of wild-type PTEN expression suppresses VEGF expression in normoxic cells [33,34]. Moreover, expression of a constitutively active Akt as well as the absence of functional PTEN leads to constitutive stabilization of the HIF-1␣ subunit [33]. Finally, inhibitors of PI-3K such as wortmannin and LY294002 are able to inhibit the stabilization of the HIF-1␣ subunit in hypoxic Hep3B cells as demonstrated by Western blotting [33,34]. These results indicate that HIF-1␣ stabilization under hypoxia is dependent on a PI-3K/Akt pathway [30,33,34] (Fig. 2). However, the mechanisms of PI-3K activation by hypoxia remain unknown. One possible hypothesis is that ROS generation in hypoxic cells is increased [36] and that ROS activate the PI-3K/Akt pathway [37] (Fig. 2). Similarly, the activation of Akt following angiotensin II stimulation in vascular smooth muscle cells (VSMC) has been shown to be dependent on reactive oxygen species (ROS) production [38]. Indeed, in angiotensin II-stimulated VSMC, catalase overexpression or DPI incubation inhibits Akt activation [38]. Chandel et al. have
shown that hypoxia triggers higher mitochondrial ROS production in Hep3B cells when compared to Hep3B-° cells, which are deplete of mitochondrial DNA and incapable of mitochondrial respiration [36,37]. It is therefore possible that mitochondrial ROS are involved in Akt activation and HIF-1␣ stabilization (Fig. 2). However, even if HIF-1␣ stabilization was observed in VSMC stimulated with angiotensin II [39], the use of a PI-3K/ Akt inhibitor (LY294002) in these cells failed to inhibit HIF-1␣ stabilization [39] indicating that other pathways are involved in HIF-1␣ stabilization, depending on the stimulus. HIF-1 transcriptional activity is partially dependent on the ERK pathway Because the hypoxic induction of the VEGF gene was inhibited in NIH3T3 cells expressing inactive c-src or Raf-1 proteins [29], it has been hypothesized that the Raf-1/MEK1/ERK pathway and the modulation of Raf-1 activity by c-src could be involved in HIF-1 activation. However, kinase assay experiments performed in embryonic fibroblasts failed to detect c-src activation in hypoxic conditions (1% O2) [40], while after 1 h of anoxic incubation, a significant activation of c-src was observed.
Transduction pathways involved in HIF-1
851
Fig. 2. Schematic representation of putative upstream activators of the ERK pathway that could be involved in the regulation of HIF-1␣ phosphorylation and HIF-1 transcriptionnal activity. Under hypoxia, HIF-1␣ is stabilized and translocated into the nucleus. In hypoxic cells (HeLa, HMEC-1, PC12), or in growth factor or Ang II-stimulated cells, the ERK pathway is activated and ERK phosphorylate HIF-1␣. This phosphorylation enhances HIF-1 transcriptional activity. The hypoxic activation of the ERK pathway may be due to calcium influx triggered by a glucose/sodium symport or by membrane depolarization. Mitochondrial ROS or NADPH-oxidase associated to Rac1 are putative activators of the ERK pathway. Finally, the hypoxia-induced VEGF production as well as other growth factors and oncogenes are ERK activators that can participate in enhancing HIF-1 activity.
Similarly, no increase in c-src activity was detected in hypoxic Hep3B cells [40]. In c-SRC⫺/⫺ fibroblasts as well as in Hep3B cells expressing a dominant negative form of c-src, no inhibition of HIF-1 DNA-binding activity as well as of the overexpression of HIF-1-regulated genes (GLUT-1, VEGF) was observed [40]. This indicates that c-src is unlikely to be part of the HIF-1 activation pathway under hypoxia. Even if the c-src kinase is not the upstream kinase in the process of HIF-1 activation, this does not exclude that the ERK pathway plays a role in HIF-1 activity. Figure 2 summarizes possible ways to activate ERK under hypoxia, resulting in HIF-1 activation. Salceda et al. showed by reporter gene assays and EMSA experiments that in hypoxic Hep3B cells the MEK1 inhibitor PD98059 was able to block the transcriptional activity of HIF-1 without affecting its DNA-binding activity [18]. This suggested that the ERK pathway plays a role in regulating the transcriptional activity of HIF-1 and is not involved in HIF-1 stabilization. More recently, Richard et al. demonstrated in an in vitro kinase assay that the
HIF-1␣ subunit was phosphorylated by either active ERK1 or ERK2, but not by active JNK or p38 SAP kinases [41]. However, when measuring ERK1 or ERK2 activity in serum-deprived CCL39 cells incubated under hypoxia, no activation of these kinases was detected [41]. The same authors also used the Raf-1:ER cell line, a clonal cell line derived from CCL39 cells, where the ERK pathway can be selectively activated by estradiol. When these cells are stimulated with estradiol under normoxia, the expression of a HIF-1 reporter gene is strongly induced indicating that ERK can trigger HIF-1 transcriptional activity. It must be noted that in this case, there is no increase in HIF-1␣ protein level [41]. Consistent with the results obtained by Salceda et al. in Hep3B cells [18], Muller et al. in HeLa cells [26], and Conrad et al. in PC12 cells [21], we demonstrated that in HMEC-1 cells, an endothelial cell line, hypoxia moderately activates ERK [28]. Using PD98059 or ERK dominant negative mutants together with a HIF-1 reporter gene, we showed that in hypoxic HMEC-1, the transcriptional activity of HIF-1 is dependent on the MEK1/ERK
852
E. MINET et al.
transduction pathway. Using an in vitro kinase assay, the carboxy-terminal domain of HIF-1␣, which encompasses the transactivation domains of the protein, was demonstrated to be directly phosphorylated by ERK1 [28]. Taken together, these results indicate that the activation of the ERK pathway could directly participate in the activation of HIF-1 transcriptional activity. Several hypotheses could explain how ERK are activated under hypoxia. Firstly, hypoxia-induced calcium influx has been proposed to trigger the activation of ERK in hypoxic PC12 cells [21]. Calcium influx has also been observed in endothelial cells incubated under hypoxia [42]. In endothelial cells, under hypoxia, more glucose is used for the anaerobic production of ATP through glycolysis and the activity of the glucose/sodium symport is increased [42]. As in the case of PC12 cells, calcium could thus be the messenger leading to ERK activation in hypoxic endothelial cells. Secondly, hypoxia is known to induce an increase in mitochondrial ROS production in Hep3 cells in comparison to Hep3B-° cells depleted of mitochondrial DNA and incapable of mitochondrial respiration [36,37]. ERK1 and ERK2 are activated by ROS in different cell types including JB6 (mouse epidermal cells) [43], MCT (mouse proximal tubular cells) [44], and rat endothelial cells [45]. Therefore, ROS production could be another way to activate the ERK pathway (Fig. 2). However, other studies have demonstrated that the ROS production decreases in hypoxic cells and consequently other mechanisms were proposed for the activation of HIF-1 [46]. For example, an NADPH-oxidaselike oxygen sensor could be the upstream activator of the transduction pathways leading to HIF-1 phosphorylation and activation [47]. Moreover, it was recently reported that in endothelial cells and in hepatocytes, the overexpression of a Rac1 dominant negative mutant was able to inhibit hypoxia-induced VEGF overexpression [48]. Rac1 is an NADPH-oxidase-associated small G protein belonging to the Rho family. The Rho GTP-binding proteins are mainly involved in the SAP kinase transduction pathway, but it was demonstrated in NIH3T3 and in HEK 293 cells that Rac was able to activate elk-1 and the c-fos gene transcription through a Raf-1/MEK-1/ERKdependent pathway; indicating that Rac can be a ERK activator [49] (Fig. 2). Finally, VEGF and other growth factors are activators of the ERK pathway [50]. Thus, the secreted VEGF present in hypoxic tissues could be involved in an autoregulatory feedback loop leading to ERK activation and HIF-1 phosphorylation (Fig. 2). As described here, several possible pathways leading to ERK activation have been proposed on the basis of results obtained using different cell types. It still remains to be determined whether all or only some of these
pathways are required working cooperatively or whether HIF-1 activation relies on specific mechanisms depending on the cell type. PHYSIOLOGICAL RELEVANCE OF HIF-1 PHOSPHORYLATION
Although other transcription factors such as AP-1, Egr-1, or NF-B mediate the hypoxia-inducible expression of specific genes in specific cell types [9], HIF-1 appears to be unique with respect to its function as a global regulator of oxygen homeostasis. The increase in the expression of glycolytic enzymes, the glucose transporter GLUT-1, or VEGF all contribute towards the survival of cells and tissues undergoing oxygen deficiency [51]. In the process of HIF-1 activation by hypoxia, phosphorylation of the HIF-1␣ subunit is thus crucial for this adaptative response. Despites its utility, HIF-1 has a downside because it plays an important role in tumorigenesis. It is known that the loss of HIF-1␣ in embryonic stem cells dramatically retards solid tumor growth [4,52]. In addition, HIF-1␣ upregulation is observed in most human cancers [53] and has been proposed as a marker for unfavorable prognosis in early stage invasive cervical cancer [54]. Tumor cells respond to hypoxia developing in growing tumors by HIF-1-mediated VEGF expression. Increased VEGF expression triggers neoangiogenesis that will sustain tumor growth. Some oncoproteins such as src and abl form complexes with PI-3K leading to cell transformation [55]. It has been demonstrated that the loss of src or abl ability to form complexes with PI-3K results in a significant decrease in their transforming activity [55]. Constitutive activation of the PI-3K/Akt pathway by interference with oncoproteins or following inactivation of antioncogenes such as PTEN lead to the stabilization of HIF-1 [33] and hence may be involved in tumor vascularization. PTEN loss of function has been correlated with angiogenesis and advanced tumor stage in human prostate cancer [56,57]. It has recently been demonstrated that even under normoxic conditions, NIH3T3 cells transformation by viral oncogenes such as the G protein-coupled receptor (GPCR) from the Kaposi’s sarcoma-associated herpes virus, can lead to the activation of HIF-1, VEGF overexpression, and possibly, to Kaposi’s sarcoma angiogenesis. In these cells, the GPCR protein is able to induce the activation of both p38 and ERK pathways that trigger HIF-1 activation [32]. Oncogenic ras mutations are also very common in human cancer and can lead to VEGF expression via either the PI-3K or MAPK pathway depending upon the cell type [58]. The induction of VEGF in Ha-Ras-transformed NIH3T3 cells [30] and of Glut-1 in ras-overex-
Transduction pathways involved in HIF-1
pressing rat fibroblasts [59] is dependent on PI-3K and HIF-1. One needs to emphasize that tumor cells subjected to hypoxia must not only overexpress VEGF to sustain angiogenesis but also undergo metabolic adaptation to survive. Upregulating GLUT-1 and glycolytic enzyme expression through HIF-1 is part of this adaptation as exemplified by the work of Chen et al. [59]. Tumor growth is the result of the imbalance between cell proliferation and cell death. The role of HIF-1 in apoptosis is highly controversial. HIF-1␣ is known to stabilize the proapoptotic protein p53 under hypoxia [60]. HIF-1␣ and p53 then conspire to promote hypoxiainduced apoptosis as shown in ES cells [61] and in neurones [62,63]. p53 inhibits HIF-1 activity and induces mdm-2-mediated degradation of HIF-1␣ [61,64]. Amplification of normal HIF-1-dependent responses to hypoxia via p53 loss of function contributes to the angiogenic switch during tumorigenesis and this, in addition to the inhibition of apoptosis, will favor tumor growth [64]. However, other recent data indicate that HIF-1 may be protective against apoptosis. VEGF has been shown to protect against apoptosis in neurons [65] and in hematopoietic cells [66], this effect depends on the PI-3K/Akt signaling pathway initiated at the VEGF receptor. A positive protective feedback loop has been suggested by the work of Baek et al. [67] because hypoxia-induced VEGF enhances viability via suppression of serum deprivation-induced apoptosis. The ERK pathway plays an important role in this regulation. Overexpression of Glut-1, another HIF-1 target gene, also protects cells against apoptosis [68]. Moreover, activation of HIF-1 and of its target genes has been shown to be involved in the anti-apoptotic effect of iron chelator in cultured neurons [69]. However, the role of kinases in this protective effect has yet to be studied. CONCLUSION
The activation of HIF-1 is a multi-step process requiring HIF-1␣ subunit stabilization [10 –12], redox-regulation [15], phosphorylation, and interaction with Hsp90 [13] and coactivator(s) [14]. Here, we present some of the recent data highlighting the activation of several kinases in hypoxic cells and their possible role in the activation of the HIF-1 transcription factor. Firstly, the PI-3 kinase/Akt pathway is involved in HIF-1␣ stabilization under hypoxia [30,33,34,37], but it must be noted that HIF-1␣ stabilization induced by growth factors does not seem to be dependent on the Akt pathway [39]. Secondly, the ERK pathway is involved in the activation of the transcriptional activity of HIF-1, under normoxic and/or hypoxic conditions, depending on the cell type and on the stimuli [18,28,41]. Thirdly, p38-mediated
853
regulation of HIF-1 has recently been suggested by Sodhi et al. [32]. The upstream activators of these kinases have yet to be identified, but mitochondrial ROS generated during hypoxia [36,37,45] or an NADPH-like oxidase associated with signal transducers are good candidates [47]. Finally, the precise role of calcium in several transduction pathways involved in the hypoxic response [21,22,24,42] remains to be elucidated. Acknowledgements — C. Michiels is Research Associate and E. Minet Research Assistant of the National Funds for Scientific Research (FNRS, Belgium). G. Michel and D. Mottet are fellows of FRIA (Fonds pour la Recherche dans I’Industrie et I’Agriculture, Belgium). This text presents results of the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsability is assumed by its authors. This work was partly supported by FRRC and SSTC.
REFERENCES [1] Semenza, G. L. HIF-1 and human disease: one highly involved factor. Genes Dev. 14:1983–1991; 2000. [2] Semenza, G. L.; Jiang, B. H.; Passantino, S. W.; Condorcet, J. P.; Maire, P.; Giallongo, A. Hypoxia response element in the aldolase A, enolase 1, and lactate deshydrogenase A promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 271:32529 –32537; 1996. [3] Semenza, G. L. HIF-1: mediator of physiological and pathological responses to hypoxia. J. Appl. Physiol. 88:1474 –1480; 2000. [4] Ryan, H. E.; Lo, J.; Johnson, R. S. HIF-1a is required for solid tumor formation and embryonic vascularization. EMBO J. 17: 3005–3015; 1998. [5] Iyer, N. V.; Kotch, L. E.; Agani, F.; Leung, S. W.; Laughner, E.; Wenger, R. H.; Gassmann, M.; Gearhart, J. D.; Lawler, A. M.; Yu, A. Y.; Semenza, G. L. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 12:149 –162; 1998. [6] Levy, A. P.; Levy, N. S.; Wegner, S.; Goldberg, M. A. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J. Biol. Chem. 270:13333–13340; 1995. [7] Faller, D. Endothelial cell responses to hypoxic stress. Clin. Exp. Pharmacol. Physiol. 26:74 – 84; 1999. [8] Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. HypoxiaInducible Factor-1 is a basic-helix-loop-helix-PAS hetrodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92:5510 –5514; 1995. [9] Semenza, G. L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell. Dev. Biol. 15:551– 578; 1999. [10] Salceda, S.; Caro, J. Hypoxia-inducible factor 1␣ (HIF-1␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. J. Biol. Chem. 272:22642–22647; 1997. [11] Huang, L. E.; Gu, J.; Schau, M.; Bunn, H. F. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 95:7987–7992; 1998. [12] Kallio, P. J.; Wilson, W. J.; O’Brien, S.; Makino, Y.; Poellinger, L. Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J. Biol. Chem. 274:6519 – 6525; 1999. [13] Minet, E.; Motte, D.; Michel, G.; Roland, I.; Raes, M.; Remacle, J.; Michiels, C. Hypoxia-induced activation of HIF-1 : role of HIF-1alpha-Hsp90 interaction. FEBS Lett. 460:251–256; 1999. [14] Kallio, P. J.; Okamoto, K.; O’Brien, S.; Carrero, P.; Makino, Y.; Tanaka, H.; Poellinger, L. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17:6573– 6586; 1998.
854
E. MINET et al.
[15] Lando, D.; Pongratz, I.; Poellinger, L.; Whitelaw, M. L. A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor. J. Biol. Chem. 275:4618 – 4627; 2000. [16] Wang, G. L.; Semenza, G. L. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 268:21513–21518; 1993. [17] Wang, G. L.; Jiang, B. J.; Semenza, G. L. Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1. Biochem. Biophys. Res. Commun. 212:550 –556; 1995. [18] Salceda, S.; Beck, I.; Srinivas, V.; Caro, J. Complex role of protein phosphorylation in gene activation by hypoxia. Kidney Int. 51:556 –559; 1997. [19] Boyle, W. J.; Smeal, T.; Defize, L. H.; Angel, P.; Woodgett, J. R.; Karin, M.; Hunter, T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64:573–584; 1991. [20] Conrad, P. W.; Rust, R. T.; Han, J.; Millhorn, D. E.; BeitnerJohnson, D. Selective activation of p38alpha and p38gamma by hypoxia. J. Biol. Chem. 274:23570 –23576; 1999. [21] Conrad, P. W.; Freeman, T. L.; Beitner-Johnson, D.; Millhorn, D. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK. J. Biol. Chem. 274:33709 –33713; 1999. [22] Raymond, R.; Millhorn, D. Regulation of tyrosine hydroxylase gene expression during hypoxia: role of Ca2⫹ and PKC. Kidney Int. 51:536 –541; 1997. [23] Premkumar, D. R.; Mishra, R. R.; Overholt, J. L.; Simonson, M. S.; Cherniack, N. S.; Prabhakar, N. R. L-type Ca(2⫹) channel activation regulates induction of c-fos transcription by hypoxia. J. Appl. Physiol. 88:1898 –1906; 2000. [24] Conrad, P. W.; Millhorn, D. E.; Beitner-Johnson, D. Hypoxia differentially regulates the mitogen- and stress-activated protein kinases. Role of Ca2⫹/CaM in the activation of MAPK and p38 gamma. Adv. Exp. Med. Biol. 475:293–302; 2000. [25] Norris, M. L.; Millhorn, D. E. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. J. Biol. Chem. 270:23774 –23779; 1995. [26] Muller, J.; Krauss, B.; Kaltschmidt, C.; Baeuerle, P.; Rupec, R. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway. J. Biol. Chem. 272:23435–23439; 1997. [27] Scott, P. H.; Paul, A.; Belham, C. M.; Peacock, A. J.; Wadsworth, R. M.; Gould, G. W.; Welsh, D.; Plevin, R. Hypoxic stimulation of the stress-activated protein kinases in pulmonary fibroblasts. Am. J. Respir. Crit. Care. Med. 158:958 –962; 1998. [28] Minet, E.; Arnould, T.; Michel, G.; Roland, I.; Mottet, D.; Raes, M.; Remacle, J.; Michiels, C. ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett. 468:53–58; 2000. [29] Mukhopadhyay, D.; Tsiokas, L.; Zhou, X. M.; Foster, D.; Brugge, J. S.; Sukhatme, V. P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 375:577–591; 1995. [30] Mazure, N.; Chen, Y.; Laderoute, K.; Giaccia, A. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-rastransformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 90:3322–3331; 1997. [31] Le, Y. J.; Corry, P. M. Hypoxia-induced bFGF gene expression is mediated through the JNK signal transduction pathway. Mol. Cell. Biochem. 202:1– 8; 1999. [32] Sodhi, A.; Montaner, S.; Patel, V.; Zohar, M.; Bais, C.; Mesri, E. A.; Gutkind, J. S. The Kaposi’s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogenactivated protein kinase and p38 pathways acting on hypoxiainducible factor 1 alpha. Cancer Res. 60:4873– 4880; 2000. [33] Zundel, W.; Schindler, C.; Haas-Kogan, D.; Koong, A.; Kaper, F.; Chen, E.; Gottschalk, A. R.; Ryan, H. E.; Johnson, R. S.; Jefferson, A. B.; Stokoe, D.; Giaccia, A. J. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev. 14:391–396; 2000.
[34] Zhong, H.; Chiles, K.; Feldser, D.; Laughner, E.; Hanrahan, C.; Georgescu, M. M.; Simons, J. W.; Semenza, G. L. Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60:1541–1545; 2000. [35] Sun, H.; Lesche, R.; Li, D. M.; Liliental, J.; Zhang, H.; Gao, J.; Gavrilova, N.; Mueller, B.; Liu, X.; Wu, H. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 96:6199 – 6204; 1999. [36] Chandel, N.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA 95:11715–11720; 1998. [37] Chandel, N. S.; McClintock, D. S.; Feliciano, C. E.; Wood, T. M.; Melendez, A.; Rodriguez, A. M.; Schumacker, P. T. Reactive oxygen species generated at mitochondrial complex III stabilize HIF-1 alpha during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275:25130 –25138; 2000. [38] Ushio-Fukai, M.; Alexander, R. W.; Akers, M.; Yin, Q.; Fujio, Y.; Walsh, K.; Griendling, K. K. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J. Biol. Chem. 274:22699 –22704; 1999. [39] Richard, D. E.; Edurne, B.; Pouysse´ gur, J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor-1 alpha in vascular smooth muscle cells. J. Biol. Chem. 275:26765–26771; 2000. [40] Gleadle, J.; Ratckliffe, P. Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase. Blood 89:503–509; 1997. [41] Richard, D. E.; Berra, E.; Gothie, E.; Roux, D.; Pouyssegur, J. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1 alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. J. Biol. Chem. 274:32631–32637; 1999. [42] Michiels, C.; Arnould, T.; Remacle, J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions. Biochim. Biophys. Acta 1497:1–10; 2000. [43] Bae, G. U.; Seo, D. W.; Kwon, H. K.; Lee, H. Y.; Hong, S.; Lee, Z. W.; Ha, K. S.; Lee, H. W.; Han, J. W. Hydrogen peroxide activates p70(S6k) signaling pathway. J. Biol. Chem. 274:32596 – 32602; 1999. [44] Hannken, T.; Schroeder, R.; Zahner, G.; Stahl, R. A.; Wolf, G. Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells. J. Am. Soc. Nephrol. 11: 1387–1397; 2000. [45] Natarajan, V.; Scribner, W. M.; al-Hassani, M.; Vepa, S. Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells. Environ. Health Perspect. 106(Suppl. 5):1205–1212; 1998. [46] Zulueta, J. J.; Yu, F. S.; Hertig, I. A.; Thannickal, V. J.; Hassoun, P. M. Release of hydrogen peroxide in response to hypoxiareoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. Am. J. Respir. Cell. Mol. Biol. 12:41– 49; 1995. [47] Bunn, H. F.; Poyton, R. O. Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76:839 – 885; 1996. [48] Gorlach, A.; Acker, H.; Cool, R. H.; Frandrey, J.; Schini-Kerth, V.; Busse, R.; Kietzmann, T. Rac1 modulates activation of hypoxia-inducible factor-1. Hypoxia and its role in angiogenesis. Ascona, Switzerland 2000. [49] Frost, J. A.; Steen, H.; Shapiro, P.; Lewis, T.; Ahn, N.; Shaw, P. E.; Cobb, M. H. Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. EMBO J. 16:6426 – 6438; 1997. [50] Gupta, K.; Kshirsagar, S.; Li, W.; Gui, L.; Ramakrishnan, S.; Gupta, P.; Law, P. Y.; Hebbel, R. P. VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on
Transduction pathways involved in HIF-1
[51] [52]
[53]
[54]
[55] [56] [57]
[58]
[59]
[60] [61]
[62] [63] [64]
MAPK/ERK and SAPK/JNK signaling. Exp. Cell Res. 247:495– 504; 1999. Semenza, G. L. Surviving ischemia: adaptive responses mediated by hypoxia-inducible factor 1. J. Clin. Invest. 106:809 – 812; 2000. Ryan, H. E.; Poloni, M.; McNulty, W.; Elson, D.; Gassmann, M.; Arbeit, J. M.; Johnson, R. S. Hypoxia-inducible factor-1 alpha is a positive factor in solid tumor growth. Cancer Res. 60:4010 – 4015; 2000. Zhong, H.; De Marzo, A. M.; Laughner, E.; Lim, M.; Hilton, D. A.; Zagzag, D.; Buechler, P.; Isaacs, W. B.; Semenza, G. L.; Simons, J. W. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res. 59:5830 –5835; 1999. Birner, P.; Schindl, M.; Obermair, A.; Plank, C.; Breitenecker, G.; Oberhuber, G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res. 60:4693– 4696; 2000. Krasilnikov, M. A. Phosphatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry 65:59 – 67; 2000. Giri, D.; Ittmann, M. Inactivation of the PTEN tumor suppressor gene is associated with increased angiogenesis in clinically localized prostate carcinoma. Hum. Pathol. 30:419 – 424; 1999. McMenamin, M. E.; Soung, P.; Perera, S.; Kaplan, I.; Loda, M.; Sellers, W. R. Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res. 59:4291– 4296; 1999. Rak, J.; Mitsuhashi, Y.; Sheehan, C.; Tamir, A.; Viloria-Petit, A.; Filmus, J.; Mansour, S. J.; Ahn, N. G.; Kerbel, R. S. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res. 60:490 – 498; 2000. Chen, C.; Pore, N.; Behrooz, A.; Ismail-Beigi, F.; Maity, A. Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J. Biol. Chem. 276:9519 – 9525; 2001. An, W. G.; Kanekal, M.; Simon, M. C.; Maltepe, E.; Blagosklonny, M. V.; Neckers, L. M. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature 392:405– 408; 1998. Carmeliet, P.; Dor, Y.; Hebert, J.-M.; Fukumuras, D.; Brusselmans, K.; Dewerchin, M.; Neeman, M.; Bono, F.; Abramovitch, R.; Maxwell, P.; Koch, C. J.; Ratcliffe, P.; Moons, L.; Jain, R. K.; Collen, D.; Keshet, E. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature 394: 485– 490; 1998. Halterman, M. W.; Miller, C. C.; Federoff, H. J. Hypoxia-Inducible Factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J. Neurosci. 19:6818 – 6824; 1999. Halterman, M. W.; Federoff, H. J. HIF-1alpha and p53 promote hypoxia-induced delayed neuronal death in models of CNS ischemia. Exp. Neurol. 159:65–72; 1999. Ravi, R.; Mookerjee, B.; Bhujwalla, Z. M.; Sutter, C. H.; Artemov, D.; Zeng, Q.; Dillehay, L. E.; Madan, A.; Semenza, G. L.; Bedi, A. Regulation of tumor angiogenesis by p53-induced deg-
[65] [66]
[67]
[68]
[69]
855
radation of hypoxia-inducible factor-1alpha. Genes Dev. 14:34 – 44; 2000. Jin, K. L.; Mao, X. O.; Greenberg, D. A. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl. Acad. Sci. USA 97:10242–10247; 2000. Katoh, O.; Takahashi, T.; Oguri, T.; Kuramoto, K.; Mihara, K.; Kobayashi, M.; Hirata, S.; Watanabe, H. Vascular endothelial growth factor inhibits apoptotic death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL1 acting as an antiapoptotic factor. Cancer Res. 58:5565–5569; 1998. Baek, J. H.; Jang, J. E.; Kang, C. M.; Chung, H. Y.; Kim, N. D.; Kim, K. W. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene 19:4621– 4631; 2000. Lin, Z.; Weinberg, J. M.; Malhotra, R.; Merritt, S. E.; Holzman, L. B.; Brosius, F. C. GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation. Am. J. Physiol. 278:E958 –E966; 2000. Zaman, K.; Ryu, H.; Hall, D.; O’Donovan, K.; Lin, K. I.; Miller, M. P.; Marquis, J. C.; Baraban, J. M.; Semenza, G. L.; Ratan, R. R. Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/ CREB and increased expression of glycolytic enzymes, p21(waf1/ cip1), and erythropoietin. J. Neurosci. 19:9821–9830; 1999. ABBREVIATIONS
Ang II—angiotensin II ARNT-1—Aryl/hydrocarbon Receptor Nuclear Translocator-1 bFGF— basic fibroblast growth factor EPO—Erythropoietin ERK—Extracellular Regulated Kinase (p42/p44 MAPK) HIF-1—Hypoxia-Inducible Factor-1 HIF-1␣—Hypoxia-Inducible Factor-1alpha HMEC-1—Human Microvascular Endothelial Cells-1 MAPK—Mitogen Activated Protein Kinase PC12—Pheocytochroma Cells 12 PI-3K—Phosphatidylinositol-3 Kinase ROS—Reactive Oxygen Species SAPK1—Stress Activated Protein Kinases 1 (JNK, c-Jun N-terminal Kinases) SAPK2—Stress Activated Protein Kinase 2 (p38/Hog) TAD—transactivation domain TH—tyrosine hydroxylase VEGF—Vascular Endothelial Growth Factor VSMC—Vascular Endothelial Smooth Muscle Cells
PII S0891-5849(01)00657-8
Review Article TRANSDUCTION PATHWAYS INVOLVED IN HYPOXIA-INDUCIBLE FACTOR-1 PHOSPHORYLATION AND ACTIVATION E. MINET, G. MICHEL, D. MOTTET, M. RAES,
and
C. MICHIELS
Laboratoire de Biochimie et Biologie Cellulaire, Faculte´s Universitaires Notre-Dame de la Paix, Namur, Belgium (Received 13 February 2001; Accepted 3 July 2001)
Abstract—Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor which is activated by hypoxia and involved in the adaptative response of the cell to oxygen deprivation. During hypoxic stress, HIF-1 triggers the overexpression of genes coding for glycolytic enzymes and angiogenic factors. To be active HIF-1 must be phosphorylated. HIF-1 is a substrate for various kinase pathways including PI-3K and the MAP kinases ERK and p38. Several transduction pathways have been proposed which act downstream of putative oxygen sensors and lead to the activation of these kinases. In this review, we summarize some of the latest advances describing the possible signaling pathways leading to HIF-1 phosphorylation and subsequent activation. The physiological relevance of these regulations is also discussed. © 2001 Elsevier Science Inc. Keywords— Hypoxia, Hypoxia-inducible factor-1, Mitogen-activated protein kinases, Phosphoinositide-3 kinase, Signaling, Tumorigenesis, Free radicals
INTRODUCTION
cerebral strokes, as well as thrombosis [1]. Mammalian cells have developed adaptative systems allowing them to survive to moderate or even severe hypoxia. This process involves an increase in the expression of genes coding for proteins responsible for the anaerobic production of ATP [2–5], namely aldolase A, enolase-␣, lactate dehydrogenase, pyruvate kinase, and glucose transporter-1. Hypoxic cells also secrete vascular endothelial growth factor (VEGF) [4,6], an angiogenic growth factor allowing neovascularization of the hypoxic tissue. Several transcription factors have been reported to be involved in the response to hypoxic stress (AP-1, NF-B, HIF-1) [7]. Among these transcription factors HypoxiaInducible Factor-1 (HIF-1) is the most potent inducer of the expression of genes such as those coding for glycolytic enzymes, VEGF and erythropoietin (EPO) [2–5]. HIF-1 is a heterodimer composed of the HIF-1␣ and ARNT-1 subunits [8]. Both subunits belong to the basic Helix-Loop-Helix-Per/ARNT/AhR/Sim (bHLH-PAS) transcription factor family [8]. HIF-1␣ as well as ARNT-1 are constitutively expressed [9]. However, HIF-1␣ appears to be the HIF-1 subunit regulated by hypoxia as under this condition, HIF-1␣ degradation is inhibited [10 –12], whereas in contrast, it is rapidly degraded by the ubiquitin-proteasome system under nor-
Vascular injuries can lead to an inadequate flow of the blood, which results in a decrease in the delivery of oxygen and nutrients to the tissues [1]. These ischemic conditions can lead to tissue necrosis, cardiovascular and Emmanuel Minet has obtained his Ph.D. thesis at the University of Namur. This work was focused on the transcriptional and post-translational regulation of HIF-1. He is now a post-doctoral fellow at the Dana Farber Cancer Institute in Boston. Gaetan Michel is a Ph.D. student in chemistry. He has developed a 3D model of the DNA-binding domain of HIF-1 when bound to HRE. He is now studying the effect of mutations within the DNA-binding domain of HIF-1␣. Denis Mottet is a Ph.D. student in Biology. His research is aimed at identifying new proteins interacting with HIF-1. Martine Raes obtained her doctorate in biological sciences in 1983 at the University of Namur, where she is now in charge of the Laboratory of Biochemistry and Cellular Biology. Her research is focused on signal transduction in inflammatory conditions and during the development of atherosclerosis. Carine Michiels was awarded her doctorate on the protective effect of antioxidant enzymes against oxidative stress in 1989. Since then, her research interests include understanding the effects of hypoxia on cells at the transcriptional as well as on biochemical levels. She is also currently engaged in the development of new anti-ischemic molecules. Address correspondence to: Dr. Carine Michiels, Laboratoire de Biochimie et Biologie Cellulaite, Faculte´s Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, 5000 Namur; Tel: ⫹32 (81) 72 4321; Fax: ⫹32 (81) 72 41 35; E-Mail: [email protected]. 847
848
E. MINET et al. Table 1. Examples of Kinases Activated Under Hypoxia and/or Involved in the Activation of HIF-1 in Different Cell Types
Cell type
Kinase and stimulus for kinase activation
Transcription factor phosphorylated
Target gene studied
References
PC12
p38␣, p38␥ (hypoxia) ERK (hypoxia) ERK (hypoxia) ERK (hypoxia) p38 ␣, p38 ␥ (GPCR transformation) ERK (GPCR transformation) PI-3K (hypoxia ⫹ Ha-Ras transformation) JNK1 (hypoxia)
AP-1 HIF-2 elk-1 HIF-1 HIF-1 HIF-1 HIF-1 c-jun
TH HRE-luc reporter c-fos HRE-luc reporter VEGF VEGF VEGF bFGF
[22] [21] [26] [28] [32] [32] [30] [31]
HeLa HMEC-1 NIH3T3 NIH3T3 MCF-7
moxic conditions [10 –12]. Under hypoxia, HIF-1␣ is seen to dissociate from the chaperone protein heat shock protein 90 (Hsp90) [13] and then translocates into the nucleus [14], where it dimerizes with the nuclear protein ARNT-1 to form the HIF-1 complex. To be fully activated, HIF-1 requires suitable redox conditions [15] as well as interaction with coactivators, namely CRB binding protein/p300 (CBP/p300) and steroid receptor coactivator-1 (SRC-1) [14]. The first evidence indicating that HIF-1 is a phosphoprotein was produced by Wang and Semenza [16]. Using electrophoretic mobility shift assay (EMSA) they showed that when nuclear extracts of hypoxic Hep3B cells were treated with phosphatase, the HIF-1/DNA complex is disrupted [16]. They went on to demonstrate that the serine/threonine kinase inhibitor 2-aminopurine, the tyrosine kinase inhibitor genistein, as well as the serine threonine phosphatase inhibitor NaF, are able to inhibit HIF-1-DNA-binding activity and HIF-1␣ stabilization in under hypoxic conditions [17]. Moreover, the MAP kinase inhibitor, PD98059, is able to inhibit HIF-1 transcriptional activity [18]. These data suggest that phosphorylation as well as dephosphorylation could both be involved in HIF-1 activation. Similar results have already been described for c-jun, which needs to be dephosphorylated in its DNA-binding domain and phosphorylated in its transactivation domain to be active [19]. Several kinases are known to be activated under hypoxia and are therefore possible candidates for HIF-1 activation. This is the case for some of the MAP kinase family members and for PI-3 kinase.
KINASES ACTIVATED DURING HYPOXIA
Under hypoxic conditions, the activation of several MAP kinases has been demonstrated in different cell types and is associated with the activation of transcription factors such as AP-1 (activated protein-1, c-Jun, c-Jos, Fra-1, ATF-2), elk-1, and nuclear factor-B (NFB) [7]. However, all the kinases and the transcription factors that are involved, as well as the target genes that
are over-expressed, could differ according to the cell type investigated (Table 1). One of the most comprehensive studies of MAP kinase activation in hypoxic cells has been performed by Conrad et al. in PC12 cells [20,21]. PC12 cells are rat pheocytochroma cells that can be differentiated into neurones following NGF stimulation. Using Western blots and in vitro kinase assays, it was demonstrated that a moderate hypoxia (6% O2) strongly activates both the p38␣ and p38␥ isoforms of the p38/SAP kinase 2 pathway in these cells [20]. Under the same conditions, ERK are also activated but to a lesser extent; however, JNK activity does not increase [20]. When PC12 cells are incubated for 6 h under more severe hypoxic conditions (1% O2), ERK1 activity is strongly increased [21]. In addition, exposure of PC12 cells to hypoxia causes membrane depolarization and extracellular calcium influx as assessed by Fura-2 fluorescence imaging [22]. The calcium influx observed in the early stage of hypoxia is mediated by L-type Ca⫹⫹ channels and is involved in the activation of downstream effectors such as several calmodulin kinases and cAMP responsive element binding protein (CREB) [23]. However, whether hypoxia activates voltage-gated channels is controversial. We should consider that in other cell types such as carotid body glomus cells, hypoxia inhibits the calcium current [23]. However, it could be possible that different L-type Ca⫹⫹ channel subunits are expressed as a function of cell type, and trigger a cell-typespecific response stimulating different downstream effectors. When pheocytochroma PC12 cells are incubated in a calcium-free medium and placed under hypoxic conditions, p38␥ is not activated [22,24]. p38␥ activation is still observed in PKA-deficient PC12 cells, which implies that p38␥ activation is not dependent on PKA activity [24]. The hypoxic activation of ERK also seems to result from the calcium influx. Interestingly, W13 and calmidazolium chloride, two calcium-calmodulin pathway inhibitors, inhibit ERK activation in hypoxic PC12 cells, as demonstrated by Western blot detection of the phosphorylated form of ERK [21]. However, the exact
Transduction pathways involved in HIF-1
mechanism linking calcium-calmodulin and HIF-1 activation is not known. Under hypoxia, the tyrosine hydroxylase (TH) gene is over-expressed [22,25]. TH participates in the synthesis of catecholamines in the carotid body, hence triggering an adaptative respiratory response. This gene is a target gene for AP-1 and HIF-1, two transcription factors activated by hypoxia [7,22]. However, when hypoxic PC12 cells are incubated in calcium-free medium, TH gene induction is not observed [22]. This indicates that in PC12 cells, calcium influx contributes to the transcriptional response to hypoxia, probably through pathways involving p38 and ERK activation. However, the link between the increase in calcium and the activation of MAP kinases is still unknown. In HeLa cells, the activation of the ERK pathway is involved in the induction of the c-fos gene transcription in hypoxic conditions [26]. Indeed, it has been demonstrated that in hypoxic HeLa cells, PD98059 inhibits the c-fos gene induction and that ERK2 is hyperphosphorylated [26]. Furthermore, in vitro kinase assays showed that immunoprecipitated ERK from hypoxic HeLa cells are able to phosphorylate the transcription factor elk-1, which triggers the hypoxic induction of the c-fos gene transcription [26]. The hypoxic activation of the ERK pathway has also been observed in pulmonary artery fibroblasts and in human microcirculation endothelial cells-1 (HMEC-1) [27,28]. The Raf-1/ERK pathway has also been associated with the hypoxic induction of the VEGF gene [29]. In hypoxic NIH3T3 cells expressing a dominant negative form of c-src or of Raf-1, VEGF hypoxic induction is observed to be inhibited. VEGF gene induction is also inhibited in the presence of 6-thioguanine, an ERK pathway inhibitor [29]. In contrast, in NIH3T3R, a Ha-Ras transformed NIH3T3 cell line, the use of ERK dominant negative mutants does not inhibit the VEGF overexpression under hypoxia [30]. In this particular cell line, wortmannin, a PI-3K/Akt pathway inhibitor, is able to inhibit the hypoxic induction of VEGF gene transcription. This was confirmed by the use of dominant negative mutants for the p85 subunit (⌬p85) of PI-3K or for Akt [HA-Akt(K119M)] [30]. Both ⌬p85 and HAAkt(K119M) mutants were able to inhibit the hypoxic over-expression of the VEGF gene [30]. Taken together, these results indicate that hypoxia triggers ERK and PI-3K activation in several cell lines (Table 1). However, it is not clear whether the ERK pathway is also involved in the increase in the transcription of the VEGF gene during hypoxia in the NIH3T3R cells. The kinases of the JNK pathway are not activated by hypoxia in PC12 cells [20]. However, they are involved in the activation of AP-1 and in the phosphorylation of c-Jun in hypoxic MCF-7 cells [31]. An increase in JNK1
849
phosphorylation and activity was detected by Western blot and kinase assays in hypoxic MCF-7 cells incubated under severe hypoxia (0.1% O2). In these cells, JNK1 activation is concomitant with the phosphorylation of c-Jun [31]. Additional evidence recently indicated that p38 can also regulate HIF-1 [32]. HIF-1␣ phosphorylation by p38 could be involved in the inhibition of the inhibitory domain located within the carboxy-terminal region of HIF-1␣ [32]. However, it is currently unclear whether this pathway is involved in hypoxia-induced apoptosis or cell survival. MAP and many other kinases are potentially activated by hypoxia but the responses vary according to the cell type. Thus, depending on the particular set of kinases activated in a given cell line, it is possible that the cellular response to hypoxia could be either adaptative or apoptotic. Accordingly, kinase activation under hypoxic conditions could activate different sets of transcription factors orienting the transcriptional response in different directions, depending on the tissues and on the severity of the hypoxic stress. HIF-1 ACTIVATION DEPENDS ON SEVERAL KINASE PATHWAYS
As previously described, HIF-1 is a phosphorylated protein and its phosphorylation is involved in HIF-1␣ subunit stabilization as well as in the regulation of HIF-1 transcriptional activity [16 –18]. Here, we describe the role played by PI-3K/Akt and ERK in HIF-1 stabilization and activation under hypoxic conditions.
HIF-1 stabilization is dependent on the PI-3 kinase/Akt pathway The hypoxic expression of VEGF is regulated by HIF-1. During hypoxia, HIF-1 is activated and binds several HIF-1 Responsive Elements (HRE) located within the VEGF gene promoter and 5⬘UTR [4,6]. The use of dominant negative mutants for the PI-3 kinase or for the Akt kinase inhibits the hypoxia-induced overexpression of VEGF, suggesting that the PI-3K/Akt pathway is involved in HIF-1 activation [30]. The co-transfection of a HIF-1 reporter plasmid either with the p85 or with the Akt dominant negative vector demonstrated that the disruption of the PI-3K/Akt pathway impaired the activation of HIF-1 as well as VEGF gene transcription in hypoxic NIH3T3R cells [30,33] (Table 1). This result indicates that HIF-1 is a protein regulated by the PI-3K/ Akt pathway (Fig. 1). Moreover, this pathway is downregulated by the tumor suppressor factor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [33,34], which is a phosphatidylinositol 3,4,5-triphos-
850
E. MINET et al.
Fig. 1. Putative role of the PI-3K/Akt pathway in hypoxia-induced stabilization of HIF-1␣ (1). Under normoxia, the HIF-1␣ sub-unit is degraded by the ubiquitin-proteasome system. Under hypoxia, HIF-1␣ degradation is inhibited through a PI-3K/Akt pathway. PI-3K could be activated by mitochondrial ROS (2) or by an NADPH-oxidase-like oxygen sensor associated to Rac1, a small G protein (3). PIP3 activates Akt, which is then involved in the HIF-1␣ stabilization process (4). When PIP3 degradation by PTEN is inhibited either due to PTEN mutation(s) or possibly under hypoxia, the stabilization of HIF-1␣ is enhanced (5).
phate phosphatase [35] (Fig. 2). Mutations within the PTEN catalytic domain increase the phosphorylation and activation of Akt [35]. In glioblastoma-derived cell lines lacking functional PTEN, VEGF expression is high even under normoxia [33]. Restoration of wild-type PTEN expression suppresses VEGF expression in normoxic cells [33,34]. Moreover, expression of a constitutively active Akt as well as the absence of functional PTEN leads to constitutive stabilization of the HIF-1␣ subunit [33]. Finally, inhibitors of PI-3K such as wortmannin and LY294002 are able to inhibit the stabilization of the HIF-1␣ subunit in hypoxic Hep3B cells as demonstrated by Western blotting [33,34]. These results indicate that HIF-1␣ stabilization under hypoxia is dependent on a PI-3K/Akt pathway [30,33,34] (Fig. 2). However, the mechanisms of PI-3K activation by hypoxia remain unknown. One possible hypothesis is that ROS generation in hypoxic cells is increased [36] and that ROS activate the PI-3K/Akt pathway [37] (Fig. 2). Similarly, the activation of Akt following angiotensin II stimulation in vascular smooth muscle cells (VSMC) has been shown to be dependent on reactive oxygen species (ROS) production [38]. Indeed, in angiotensin II-stimulated VSMC, catalase overexpression or DPI incubation inhibits Akt activation [38]. Chandel et al. have
shown that hypoxia triggers higher mitochondrial ROS production in Hep3B cells when compared to Hep3B-° cells, which are deplete of mitochondrial DNA and incapable of mitochondrial respiration [36,37]. It is therefore possible that mitochondrial ROS are involved in Akt activation and HIF-1␣ stabilization (Fig. 2). However, even if HIF-1␣ stabilization was observed in VSMC stimulated with angiotensin II [39], the use of a PI-3K/ Akt inhibitor (LY294002) in these cells failed to inhibit HIF-1␣ stabilization [39] indicating that other pathways are involved in HIF-1␣ stabilization, depending on the stimulus. HIF-1 transcriptional activity is partially dependent on the ERK pathway Because the hypoxic induction of the VEGF gene was inhibited in NIH3T3 cells expressing inactive c-src or Raf-1 proteins [29], it has been hypothesized that the Raf-1/MEK1/ERK pathway and the modulation of Raf-1 activity by c-src could be involved in HIF-1 activation. However, kinase assay experiments performed in embryonic fibroblasts failed to detect c-src activation in hypoxic conditions (1% O2) [40], while after 1 h of anoxic incubation, a significant activation of c-src was observed.
Transduction pathways involved in HIF-1
851
Fig. 2. Schematic representation of putative upstream activators of the ERK pathway that could be involved in the regulation of HIF-1␣ phosphorylation and HIF-1 transcriptionnal activity. Under hypoxia, HIF-1␣ is stabilized and translocated into the nucleus. In hypoxic cells (HeLa, HMEC-1, PC12), or in growth factor or Ang II-stimulated cells, the ERK pathway is activated and ERK phosphorylate HIF-1␣. This phosphorylation enhances HIF-1 transcriptional activity. The hypoxic activation of the ERK pathway may be due to calcium influx triggered by a glucose/sodium symport or by membrane depolarization. Mitochondrial ROS or NADPH-oxidase associated to Rac1 are putative activators of the ERK pathway. Finally, the hypoxia-induced VEGF production as well as other growth factors and oncogenes are ERK activators that can participate in enhancing HIF-1 activity.
Similarly, no increase in c-src activity was detected in hypoxic Hep3B cells [40]. In c-SRC⫺/⫺ fibroblasts as well as in Hep3B cells expressing a dominant negative form of c-src, no inhibition of HIF-1 DNA-binding activity as well as of the overexpression of HIF-1-regulated genes (GLUT-1, VEGF) was observed [40]. This indicates that c-src is unlikely to be part of the HIF-1 activation pathway under hypoxia. Even if the c-src kinase is not the upstream kinase in the process of HIF-1 activation, this does not exclude that the ERK pathway plays a role in HIF-1 activity. Figure 2 summarizes possible ways to activate ERK under hypoxia, resulting in HIF-1 activation. Salceda et al. showed by reporter gene assays and EMSA experiments that in hypoxic Hep3B cells the MEK1 inhibitor PD98059 was able to block the transcriptional activity of HIF-1 without affecting its DNA-binding activity [18]. This suggested that the ERK pathway plays a role in regulating the transcriptional activity of HIF-1 and is not involved in HIF-1 stabilization. More recently, Richard et al. demonstrated in an in vitro kinase assay that the
HIF-1␣ subunit was phosphorylated by either active ERK1 or ERK2, but not by active JNK or p38 SAP kinases [41]. However, when measuring ERK1 or ERK2 activity in serum-deprived CCL39 cells incubated under hypoxia, no activation of these kinases was detected [41]. The same authors also used the Raf-1:ER cell line, a clonal cell line derived from CCL39 cells, where the ERK pathway can be selectively activated by estradiol. When these cells are stimulated with estradiol under normoxia, the expression of a HIF-1 reporter gene is strongly induced indicating that ERK can trigger HIF-1 transcriptional activity. It must be noted that in this case, there is no increase in HIF-1␣ protein level [41]. Consistent with the results obtained by Salceda et al. in Hep3B cells [18], Muller et al. in HeLa cells [26], and Conrad et al. in PC12 cells [21], we demonstrated that in HMEC-1 cells, an endothelial cell line, hypoxia moderately activates ERK [28]. Using PD98059 or ERK dominant negative mutants together with a HIF-1 reporter gene, we showed that in hypoxic HMEC-1, the transcriptional activity of HIF-1 is dependent on the MEK1/ERK
852
E. MINET et al.
transduction pathway. Using an in vitro kinase assay, the carboxy-terminal domain of HIF-1␣, which encompasses the transactivation domains of the protein, was demonstrated to be directly phosphorylated by ERK1 [28]. Taken together, these results indicate that the activation of the ERK pathway could directly participate in the activation of HIF-1 transcriptional activity. Several hypotheses could explain how ERK are activated under hypoxia. Firstly, hypoxia-induced calcium influx has been proposed to trigger the activation of ERK in hypoxic PC12 cells [21]. Calcium influx has also been observed in endothelial cells incubated under hypoxia [42]. In endothelial cells, under hypoxia, more glucose is used for the anaerobic production of ATP through glycolysis and the activity of the glucose/sodium symport is increased [42]. As in the case of PC12 cells, calcium could thus be the messenger leading to ERK activation in hypoxic endothelial cells. Secondly, hypoxia is known to induce an increase in mitochondrial ROS production in Hep3 cells in comparison to Hep3B-° cells depleted of mitochondrial DNA and incapable of mitochondrial respiration [36,37]. ERK1 and ERK2 are activated by ROS in different cell types including JB6 (mouse epidermal cells) [43], MCT (mouse proximal tubular cells) [44], and rat endothelial cells [45]. Therefore, ROS production could be another way to activate the ERK pathway (Fig. 2). However, other studies have demonstrated that the ROS production decreases in hypoxic cells and consequently other mechanisms were proposed for the activation of HIF-1 [46]. For example, an NADPH-oxidaselike oxygen sensor could be the upstream activator of the transduction pathways leading to HIF-1 phosphorylation and activation [47]. Moreover, it was recently reported that in endothelial cells and in hepatocytes, the overexpression of a Rac1 dominant negative mutant was able to inhibit hypoxia-induced VEGF overexpression [48]. Rac1 is an NADPH-oxidase-associated small G protein belonging to the Rho family. The Rho GTP-binding proteins are mainly involved in the SAP kinase transduction pathway, but it was demonstrated in NIH3T3 and in HEK 293 cells that Rac was able to activate elk-1 and the c-fos gene transcription through a Raf-1/MEK-1/ERKdependent pathway; indicating that Rac can be a ERK activator [49] (Fig. 2). Finally, VEGF and other growth factors are activators of the ERK pathway [50]. Thus, the secreted VEGF present in hypoxic tissues could be involved in an autoregulatory feedback loop leading to ERK activation and HIF-1 phosphorylation (Fig. 2). As described here, several possible pathways leading to ERK activation have been proposed on the basis of results obtained using different cell types. It still remains to be determined whether all or only some of these
pathways are required working cooperatively or whether HIF-1 activation relies on specific mechanisms depending on the cell type. PHYSIOLOGICAL RELEVANCE OF HIF-1 PHOSPHORYLATION
Although other transcription factors such as AP-1, Egr-1, or NF-B mediate the hypoxia-inducible expression of specific genes in specific cell types [9], HIF-1 appears to be unique with respect to its function as a global regulator of oxygen homeostasis. The increase in the expression of glycolytic enzymes, the glucose transporter GLUT-1, or VEGF all contribute towards the survival of cells and tissues undergoing oxygen deficiency [51]. In the process of HIF-1 activation by hypoxia, phosphorylation of the HIF-1␣ subunit is thus crucial for this adaptative response. Despites its utility, HIF-1 has a downside because it plays an important role in tumorigenesis. It is known that the loss of HIF-1␣ in embryonic stem cells dramatically retards solid tumor growth [4,52]. In addition, HIF-1␣ upregulation is observed in most human cancers [53] and has been proposed as a marker for unfavorable prognosis in early stage invasive cervical cancer [54]. Tumor cells respond to hypoxia developing in growing tumors by HIF-1-mediated VEGF expression. Increased VEGF expression triggers neoangiogenesis that will sustain tumor growth. Some oncoproteins such as src and abl form complexes with PI-3K leading to cell transformation [55]. It has been demonstrated that the loss of src or abl ability to form complexes with PI-3K results in a significant decrease in their transforming activity [55]. Constitutive activation of the PI-3K/Akt pathway by interference with oncoproteins or following inactivation of antioncogenes such as PTEN lead to the stabilization of HIF-1 [33] and hence may be involved in tumor vascularization. PTEN loss of function has been correlated with angiogenesis and advanced tumor stage in human prostate cancer [56,57]. It has recently been demonstrated that even under normoxic conditions, NIH3T3 cells transformation by viral oncogenes such as the G protein-coupled receptor (GPCR) from the Kaposi’s sarcoma-associated herpes virus, can lead to the activation of HIF-1, VEGF overexpression, and possibly, to Kaposi’s sarcoma angiogenesis. In these cells, the GPCR protein is able to induce the activation of both p38 and ERK pathways that trigger HIF-1 activation [32]. Oncogenic ras mutations are also very common in human cancer and can lead to VEGF expression via either the PI-3K or MAPK pathway depending upon the cell type [58]. The induction of VEGF in Ha-Ras-transformed NIH3T3 cells [30] and of Glut-1 in ras-overex-
Transduction pathways involved in HIF-1
pressing rat fibroblasts [59] is dependent on PI-3K and HIF-1. One needs to emphasize that tumor cells subjected to hypoxia must not only overexpress VEGF to sustain angiogenesis but also undergo metabolic adaptation to survive. Upregulating GLUT-1 and glycolytic enzyme expression through HIF-1 is part of this adaptation as exemplified by the work of Chen et al. [59]. Tumor growth is the result of the imbalance between cell proliferation and cell death. The role of HIF-1 in apoptosis is highly controversial. HIF-1␣ is known to stabilize the proapoptotic protein p53 under hypoxia [60]. HIF-1␣ and p53 then conspire to promote hypoxiainduced apoptosis as shown in ES cells [61] and in neurones [62,63]. p53 inhibits HIF-1 activity and induces mdm-2-mediated degradation of HIF-1␣ [61,64]. Amplification of normal HIF-1-dependent responses to hypoxia via p53 loss of function contributes to the angiogenic switch during tumorigenesis and this, in addition to the inhibition of apoptosis, will favor tumor growth [64]. However, other recent data indicate that HIF-1 may be protective against apoptosis. VEGF has been shown to protect against apoptosis in neurons [65] and in hematopoietic cells [66], this effect depends on the PI-3K/Akt signaling pathway initiated at the VEGF receptor. A positive protective feedback loop has been suggested by the work of Baek et al. [67] because hypoxia-induced VEGF enhances viability via suppression of serum deprivation-induced apoptosis. The ERK pathway plays an important role in this regulation. Overexpression of Glut-1, another HIF-1 target gene, also protects cells against apoptosis [68]. Moreover, activation of HIF-1 and of its target genes has been shown to be involved in the anti-apoptotic effect of iron chelator in cultured neurons [69]. However, the role of kinases in this protective effect has yet to be studied. CONCLUSION
The activation of HIF-1 is a multi-step process requiring HIF-1␣ subunit stabilization [10 –12], redox-regulation [15], phosphorylation, and interaction with Hsp90 [13] and coactivator(s) [14]. Here, we present some of the recent data highlighting the activation of several kinases in hypoxic cells and their possible role in the activation of the HIF-1 transcription factor. Firstly, the PI-3 kinase/Akt pathway is involved in HIF-1␣ stabilization under hypoxia [30,33,34,37], but it must be noted that HIF-1␣ stabilization induced by growth factors does not seem to be dependent on the Akt pathway [39]. Secondly, the ERK pathway is involved in the activation of the transcriptional activity of HIF-1, under normoxic and/or hypoxic conditions, depending on the cell type and on the stimuli [18,28,41]. Thirdly, p38-mediated
853
regulation of HIF-1 has recently been suggested by Sodhi et al. [32]. The upstream activators of these kinases have yet to be identified, but mitochondrial ROS generated during hypoxia [36,37,45] or an NADPH-like oxidase associated with signal transducers are good candidates [47]. Finally, the precise role of calcium in several transduction pathways involved in the hypoxic response [21,22,24,42] remains to be elucidated. Acknowledgements — C. Michiels is Research Associate and E. Minet Research Assistant of the National Funds for Scientific Research (FNRS, Belgium). G. Michel and D. Mottet are fellows of FRIA (Fonds pour la Recherche dans I’Industrie et I’Agriculture, Belgium). This text presents results of the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsability is assumed by its authors. This work was partly supported by FRRC and SSTC.
REFERENCES [1] Semenza, G. L. HIF-1 and human disease: one highly involved factor. Genes Dev. 14:1983–1991; 2000. [2] Semenza, G. L.; Jiang, B. H.; Passantino, S. W.; Condorcet, J. P.; Maire, P.; Giallongo, A. Hypoxia response element in the aldolase A, enolase 1, and lactate deshydrogenase A promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 271:32529 –32537; 1996. [3] Semenza, G. L. HIF-1: mediator of physiological and pathological responses to hypoxia. J. Appl. Physiol. 88:1474 –1480; 2000. [4] Ryan, H. E.; Lo, J.; Johnson, R. S. HIF-1a is required for solid tumor formation and embryonic vascularization. EMBO J. 17: 3005–3015; 1998. [5] Iyer, N. V.; Kotch, L. E.; Agani, F.; Leung, S. W.; Laughner, E.; Wenger, R. H.; Gassmann, M.; Gearhart, J. D.; Lawler, A. M.; Yu, A. Y.; Semenza, G. L. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 12:149 –162; 1998. [6] Levy, A. P.; Levy, N. S.; Wegner, S.; Goldberg, M. A. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J. Biol. Chem. 270:13333–13340; 1995. [7] Faller, D. Endothelial cell responses to hypoxic stress. Clin. Exp. Pharmacol. Physiol. 26:74 – 84; 1999. [8] Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. HypoxiaInducible Factor-1 is a basic-helix-loop-helix-PAS hetrodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92:5510 –5514; 1995. [9] Semenza, G. L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell. Dev. Biol. 15:551– 578; 1999. [10] Salceda, S.; Caro, J. Hypoxia-inducible factor 1␣ (HIF-1␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. J. Biol. Chem. 272:22642–22647; 1997. [11] Huang, L. E.; Gu, J.; Schau, M.; Bunn, H. F. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 95:7987–7992; 1998. [12] Kallio, P. J.; Wilson, W. J.; O’Brien, S.; Makino, Y.; Poellinger, L. Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J. Biol. Chem. 274:6519 – 6525; 1999. [13] Minet, E.; Motte, D.; Michel, G.; Roland, I.; Raes, M.; Remacle, J.; Michiels, C. Hypoxia-induced activation of HIF-1 : role of HIF-1alpha-Hsp90 interaction. FEBS Lett. 460:251–256; 1999. [14] Kallio, P. J.; Okamoto, K.; O’Brien, S.; Carrero, P.; Makino, Y.; Tanaka, H.; Poellinger, L. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17:6573– 6586; 1998.
854
E. MINET et al.
[15] Lando, D.; Pongratz, I.; Poellinger, L.; Whitelaw, M. L. A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor. J. Biol. Chem. 275:4618 – 4627; 2000. [16] Wang, G. L.; Semenza, G. L. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 268:21513–21518; 1993. [17] Wang, G. L.; Jiang, B. J.; Semenza, G. L. Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1. Biochem. Biophys. Res. Commun. 212:550 –556; 1995. [18] Salceda, S.; Beck, I.; Srinivas, V.; Caro, J. Complex role of protein phosphorylation in gene activation by hypoxia. Kidney Int. 51:556 –559; 1997. [19] Boyle, W. J.; Smeal, T.; Defize, L. H.; Angel, P.; Woodgett, J. R.; Karin, M.; Hunter, T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64:573–584; 1991. [20] Conrad, P. W.; Rust, R. T.; Han, J.; Millhorn, D. E.; BeitnerJohnson, D. Selective activation of p38alpha and p38gamma by hypoxia. J. Biol. Chem. 274:23570 –23576; 1999. [21] Conrad, P. W.; Freeman, T. L.; Beitner-Johnson, D.; Millhorn, D. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK. J. Biol. Chem. 274:33709 –33713; 1999. [22] Raymond, R.; Millhorn, D. Regulation of tyrosine hydroxylase gene expression during hypoxia: role of Ca2⫹ and PKC. Kidney Int. 51:536 –541; 1997. [23] Premkumar, D. R.; Mishra, R. R.; Overholt, J. L.; Simonson, M. S.; Cherniack, N. S.; Prabhakar, N. R. L-type Ca(2⫹) channel activation regulates induction of c-fos transcription by hypoxia. J. Appl. Physiol. 88:1898 –1906; 2000. [24] Conrad, P. W.; Millhorn, D. E.; Beitner-Johnson, D. Hypoxia differentially regulates the mitogen- and stress-activated protein kinases. Role of Ca2⫹/CaM in the activation of MAPK and p38 gamma. Adv. Exp. Med. Biol. 475:293–302; 2000. [25] Norris, M. L.; Millhorn, D. E. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. J. Biol. Chem. 270:23774 –23779; 1995. [26] Muller, J.; Krauss, B.; Kaltschmidt, C.; Baeuerle, P.; Rupec, R. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway. J. Biol. Chem. 272:23435–23439; 1997. [27] Scott, P. H.; Paul, A.; Belham, C. M.; Peacock, A. J.; Wadsworth, R. M.; Gould, G. W.; Welsh, D.; Plevin, R. Hypoxic stimulation of the stress-activated protein kinases in pulmonary fibroblasts. Am. J. Respir. Crit. Care. Med. 158:958 –962; 1998. [28] Minet, E.; Arnould, T.; Michel, G.; Roland, I.; Mottet, D.; Raes, M.; Remacle, J.; Michiels, C. ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett. 468:53–58; 2000. [29] Mukhopadhyay, D.; Tsiokas, L.; Zhou, X. M.; Foster, D.; Brugge, J. S.; Sukhatme, V. P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 375:577–591; 1995. [30] Mazure, N.; Chen, Y.; Laderoute, K.; Giaccia, A. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-rastransformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 90:3322–3331; 1997. [31] Le, Y. J.; Corry, P. M. Hypoxia-induced bFGF gene expression is mediated through the JNK signal transduction pathway. Mol. Cell. Biochem. 202:1– 8; 1999. [32] Sodhi, A.; Montaner, S.; Patel, V.; Zohar, M.; Bais, C.; Mesri, E. A.; Gutkind, J. S. The Kaposi’s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogenactivated protein kinase and p38 pathways acting on hypoxiainducible factor 1 alpha. Cancer Res. 60:4873– 4880; 2000. [33] Zundel, W.; Schindler, C.; Haas-Kogan, D.; Koong, A.; Kaper, F.; Chen, E.; Gottschalk, A. R.; Ryan, H. E.; Johnson, R. S.; Jefferson, A. B.; Stokoe, D.; Giaccia, A. J. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev. 14:391–396; 2000.
[34] Zhong, H.; Chiles, K.; Feldser, D.; Laughner, E.; Hanrahan, C.; Georgescu, M. M.; Simons, J. W.; Semenza, G. L. Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60:1541–1545; 2000. [35] Sun, H.; Lesche, R.; Li, D. M.; Liliental, J.; Zhang, H.; Gao, J.; Gavrilova, N.; Mueller, B.; Liu, X.; Wu, H. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 96:6199 – 6204; 1999. [36] Chandel, N.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA 95:11715–11720; 1998. [37] Chandel, N. S.; McClintock, D. S.; Feliciano, C. E.; Wood, T. M.; Melendez, A.; Rodriguez, A. M.; Schumacker, P. T. Reactive oxygen species generated at mitochondrial complex III stabilize HIF-1 alpha during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275:25130 –25138; 2000. [38] Ushio-Fukai, M.; Alexander, R. W.; Akers, M.; Yin, Q.; Fujio, Y.; Walsh, K.; Griendling, K. K. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J. Biol. Chem. 274:22699 –22704; 1999. [39] Richard, D. E.; Edurne, B.; Pouysse´ gur, J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor-1 alpha in vascular smooth muscle cells. J. Biol. Chem. 275:26765–26771; 2000. [40] Gleadle, J.; Ratckliffe, P. Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase. Blood 89:503–509; 1997. [41] Richard, D. E.; Berra, E.; Gothie, E.; Roux, D.; Pouyssegur, J. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1 alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. J. Biol. Chem. 274:32631–32637; 1999. [42] Michiels, C.; Arnould, T.; Remacle, J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions. Biochim. Biophys. Acta 1497:1–10; 2000. [43] Bae, G. U.; Seo, D. W.; Kwon, H. K.; Lee, H. Y.; Hong, S.; Lee, Z. W.; Ha, K. S.; Lee, H. W.; Han, J. W. Hydrogen peroxide activates p70(S6k) signaling pathway. J. Biol. Chem. 274:32596 – 32602; 1999. [44] Hannken, T.; Schroeder, R.; Zahner, G.; Stahl, R. A.; Wolf, G. Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells. J. Am. Soc. Nephrol. 11: 1387–1397; 2000. [45] Natarajan, V.; Scribner, W. M.; al-Hassani, M.; Vepa, S. Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells. Environ. Health Perspect. 106(Suppl. 5):1205–1212; 1998. [46] Zulueta, J. J.; Yu, F. S.; Hertig, I. A.; Thannickal, V. J.; Hassoun, P. M. Release of hydrogen peroxide in response to hypoxiareoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. Am. J. Respir. Cell. Mol. Biol. 12:41– 49; 1995. [47] Bunn, H. F.; Poyton, R. O. Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76:839 – 885; 1996. [48] Gorlach, A.; Acker, H.; Cool, R. H.; Frandrey, J.; Schini-Kerth, V.; Busse, R.; Kietzmann, T. Rac1 modulates activation of hypoxia-inducible factor-1. Hypoxia and its role in angiogenesis. Ascona, Switzerland 2000. [49] Frost, J. A.; Steen, H.; Shapiro, P.; Lewis, T.; Ahn, N.; Shaw, P. E.; Cobb, M. H. Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. EMBO J. 16:6426 – 6438; 1997. [50] Gupta, K.; Kshirsagar, S.; Li, W.; Gui, L.; Ramakrishnan, S.; Gupta, P.; Law, P. Y.; Hebbel, R. P. VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on
Transduction pathways involved in HIF-1
[51] [52]
[53]
[54]
[55] [56] [57]
[58]
[59]
[60] [61]
[62] [63] [64]
MAPK/ERK and SAPK/JNK signaling. Exp. Cell Res. 247:495– 504; 1999. Semenza, G. L. Surviving ischemia: adaptive responses mediated by hypoxia-inducible factor 1. J. Clin. Invest. 106:809 – 812; 2000. Ryan, H. E.; Poloni, M.; McNulty, W.; Elson, D.; Gassmann, M.; Arbeit, J. M.; Johnson, R. S. Hypoxia-inducible factor-1 alpha is a positive factor in solid tumor growth. Cancer Res. 60:4010 – 4015; 2000. Zhong, H.; De Marzo, A. M.; Laughner, E.; Lim, M.; Hilton, D. A.; Zagzag, D.; Buechler, P.; Isaacs, W. B.; Semenza, G. L.; Simons, J. W. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res. 59:5830 –5835; 1999. Birner, P.; Schindl, M.; Obermair, A.; Plank, C.; Breitenecker, G.; Oberhuber, G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res. 60:4693– 4696; 2000. Krasilnikov, M. A. Phosphatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry 65:59 – 67; 2000. Giri, D.; Ittmann, M. Inactivation of the PTEN tumor suppressor gene is associated with increased angiogenesis in clinically localized prostate carcinoma. Hum. Pathol. 30:419 – 424; 1999. McMenamin, M. E.; Soung, P.; Perera, S.; Kaplan, I.; Loda, M.; Sellers, W. R. Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res. 59:4291– 4296; 1999. Rak, J.; Mitsuhashi, Y.; Sheehan, C.; Tamir, A.; Viloria-Petit, A.; Filmus, J.; Mansour, S. J.; Ahn, N. G.; Kerbel, R. S. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res. 60:490 – 498; 2000. Chen, C.; Pore, N.; Behrooz, A.; Ismail-Beigi, F.; Maity, A. Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J. Biol. Chem. 276:9519 – 9525; 2001. An, W. G.; Kanekal, M.; Simon, M. C.; Maltepe, E.; Blagosklonny, M. V.; Neckers, L. M. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature 392:405– 408; 1998. Carmeliet, P.; Dor, Y.; Hebert, J.-M.; Fukumuras, D.; Brusselmans, K.; Dewerchin, M.; Neeman, M.; Bono, F.; Abramovitch, R.; Maxwell, P.; Koch, C. J.; Ratcliffe, P.; Moons, L.; Jain, R. K.; Collen, D.; Keshet, E. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature 394: 485– 490; 1998. Halterman, M. W.; Miller, C. C.; Federoff, H. J. Hypoxia-Inducible Factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J. Neurosci. 19:6818 – 6824; 1999. Halterman, M. W.; Federoff, H. J. HIF-1alpha and p53 promote hypoxia-induced delayed neuronal death in models of CNS ischemia. Exp. Neurol. 159:65–72; 1999. Ravi, R.; Mookerjee, B.; Bhujwalla, Z. M.; Sutter, C. H.; Artemov, D.; Zeng, Q.; Dillehay, L. E.; Madan, A.; Semenza, G. L.; Bedi, A. Regulation of tumor angiogenesis by p53-induced deg-
[65] [66]
[67]
[68]
[69]
855
radation of hypoxia-inducible factor-1alpha. Genes Dev. 14:34 – 44; 2000. Jin, K. L.; Mao, X. O.; Greenberg, D. A. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl. Acad. Sci. USA 97:10242–10247; 2000. Katoh, O.; Takahashi, T.; Oguri, T.; Kuramoto, K.; Mihara, K.; Kobayashi, M.; Hirata, S.; Watanabe, H. Vascular endothelial growth factor inhibits apoptotic death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL1 acting as an antiapoptotic factor. Cancer Res. 58:5565–5569; 1998. Baek, J. H.; Jang, J. E.; Kang, C. M.; Chung, H. Y.; Kim, N. D.; Kim, K. W. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene 19:4621– 4631; 2000. Lin, Z.; Weinberg, J. M.; Malhotra, R.; Merritt, S. E.; Holzman, L. B.; Brosius, F. C. GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation. Am. J. Physiol. 278:E958 –E966; 2000. Zaman, K.; Ryu, H.; Hall, D.; O’Donovan, K.; Lin, K. I.; Miller, M. P.; Marquis, J. C.; Baraban, J. M.; Semenza, G. L.; Ratan, R. R. Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/ CREB and increased expression of glycolytic enzymes, p21(waf1/ cip1), and erythropoietin. J. Neurosci. 19:9821–9830; 1999. ABBREVIATIONS
Ang II—angiotensin II ARNT-1—Aryl/hydrocarbon Receptor Nuclear Translocator-1 bFGF— basic fibroblast growth factor EPO—Erythropoietin ERK—Extracellular Regulated Kinase (p42/p44 MAPK) HIF-1—Hypoxia-Inducible Factor-1 HIF-1␣—Hypoxia-Inducible Factor-1alpha HMEC-1—Human Microvascular Endothelial Cells-1 MAPK—Mitogen Activated Protein Kinase PC12—Pheocytochroma Cells 12 PI-3K—Phosphatidylinositol-3 Kinase ROS—Reactive Oxygen Species SAPK1—Stress Activated Protein Kinases 1 (JNK, c-Jun N-terminal Kinases) SAPK2—Stress Activated Protein Kinase 2 (p38/Hog) TAD—transactivation domain TH—tyrosine hydroxylase VEGF—Vascular Endothelial Growth Factor VSMC—Vascular Endothelial Smooth Muscle Cells