Introduction

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Angiogenesis, the formation of new vascular tree occurring as a result of vascular sprouting from pre-existing vessels, is essential for developmental growth, the remodeling and regeneration of adult tissues, and solid tumor growth and metastasis (1, 2). This process is tightly controlled by many polypeptide growth factors, which regulate endothelial cell proliferation, migration, and differentiation (3). Among the potential mediators of tumor angiogenesis, vascular endothelial growth factor (VEGF) has several attractive features, such as the fact that it is a potent endothelial cell-specific mitogen that promotes the growth and maintenance of vascular endothelial cells and an angiogenesis inducer in vivo (4).

VEGF has been originally isolated and purified from the medium of cultured pituitary cells (4), and many subsequent studies have shown that high levels of VEGF are produced by various types of tumors. In addition, the importance of VEGF as a mediator of tumor angiogenesis is suggested by studies showing that tumor angiogenesis and subsequent tumor growth are inhibited in vivo by antibodies directed against VEGF (9, 10), by expression of dominant negative VEGF receptors (11), and by antisense VEGF (12). These findings established the role of VEGF in tumor angiogenesis and underscored the importance of the study to identify the regulatory mechanism of VEGF expression in tumors.

Oxygen deprivation is a potent stimulus to induce VEGF expression (13, 14). This process is partly mediated through the post-transcriptional modification of the mRNA, but several studies have shown that hypoxia-inducible factor-1 (HIF-1) plays an important role in the activation of VEGF transcription (14). HIF-1 was shown to consist of two subunits: HIF-1, which belongs to basic helix-loop-helix (bHLH)-PAS proteins, and HIF-1, which is also known as aryl hydrocarbon receptor nuclear translocator (Arnt). HIF-1 is induced by hypoxia and binds to hypoxia response element (HRE), which is present in many hypoxia response genes, such as VEGF and the erythropoietin genes (15). Endothelial PAS domain protein-1 (EPAS1), which is also designated as HIF-1-like factor (HLF), is a recently identified bHLH-PAS protein that shares 48% sequence identity with HIF-1 (16). Analysis of the RNA from a range of human tissues and in situ hybridization revealed that EPAS1 expression is largely restricted to endothelial cells, whereas HIF-1 mRNA expression is ubiquitous and not enriched in endothelial cells (17).

Except for the major difference in abundance and distribution between HIF-1 and EPAS1, regulatory characteristics of these proteins are not much different. After activation by hypoxia, both factors function as a heterodimer with Arnt and can bind to the same DNA element (18). In addition, although both proteins are short-lived under normoxic conditions, exposure of target cells to hypoxia rapidly and dramatically increases the protein levels (19). These studies, however, have not described the regulation of the mRNA expression at the transcriptional levels.

The essential role of HIF-1 in angiogenesis during normal embryonic development is ambiguously shown by the gene-targeting study in which HIF-1^/ homozygous mice are embryonic lethal, at least in part, because of the marked defect in vessel formation (20). In addition, the role of HIF-1/Arnt in tumor angiogenesis has also been demonstrated (21). However, it remains to be determined what role EPAS1 might have in determining the behavior of the tumors in response to hypoxia. Such a consideration may be particularly important for tumors that preferentially express EPAS1 rather than HIF-1.

The present study was undertaken to determine whether hypoxia regulates the HIF-1 and EPAS1 mRNA expression in human lung adenocarcinoma A549 cells. We report here that EPAS1 mRNA levels are considerably higher compared with HIF-1 in these cells, and more importantly, hypoxic stimulation further increased EPAS1 mRNA levels. This induction occurs at the transcriptional levels as determined by the standard decay assays of mRNA. In addition, we found that Src family kinases mediated this process. Transient transfection of EPAS1 expression vector induced the EPAS1 promoter. Furthermore, adenovirus vector expressing EPAS1 increased endogenous EPAS1 mRNA levels, thus suggesting the positive autoregulation of EPAS1 gene transcription by EPAS1 itself. Taken together, the present study provides novel insight into the regulatory mechanisms of EPAS1 gene expression that may contribute to the adaptation of tumor cells under the hypoxic environment.

	Materials and Methods

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Reagents

An oxygen absorbent, Anearopack (22), was obtained from Mitsubishi Gas Chemistry Co., Ltd., Tokyo, Japan. PD98059, SB203580, genistein, wortmannin, herbimycin A, tyrphostin A23, and calphostin C were obtained from Calbiochem (La Jolla, CA). Daidzein was obtained from BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA. EPAS1 polyclonal antibody was obtained from Novus Biologicals, Littleton, CO. Phosphotyrosine monoclonal antibody was purchased from Santa Cruz, Inc., Santa Cruz, CA. EPAS1/pcDNA3, expression vector for EPAS1, was constructed by polymerase chain reaction with the forward primer 5'-GGGCTCGAGATGACAGCTGACAAGAGAAA-3' and reverse primer 5'-CCCCCGCGGGGTGGCCTGGTCCAGAGC TCT-3' into XhoI and SacII site of pcDNA3 (Invitrogen, Groningen, The Netherlands). HLF/pBOS, which was a generous gift from Dr. Yoshiaki Fujii-Kuriyama (Tohoku University, Sendai, Miyagi, Japan) (17), was used as template DNA. The reporter plasmid, human VEGF-Luc, which contains 3.4 kilobases (kb) of the 5'-flanking sequence of the human VEGF, was a generous gift from Dr. Judith Abraham (Scios, Sunnyvale, CA).

Cell Cultures

A549 cells (human lung adenocarcinoma cell line) were obtained from Riken Cell Bank (Tsukuba, Japan). A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 100 units of penicillin, and 50 µg/ml streptomycin. When the cells were confluent, medium was switched to serum-free DMEM for 24 h. The cells were then cultured in a chamber with Anearopack as previously described (23). In the experiment using the protein kinase inhibitors, the cells were treated with various concentrations of reagents for 1 h before exposure to hypoxia.

RNA Extraction and Northern Blot Analysis

Preparation of total cellular RNA and Northern hybridization were performed essentially as previously described (23). Briefly, hybridization was performed in a solution containing 5 × SSPE, 1% sodium dodecyl sulfate (SDS), 5 × Denhardt's solution, and 10 µg/ml salmon sperm DNA at 42°C for 16 h. The filters were washed at room temperature with 2 × saline sodium citrate (SSC), 1% SDS for 10 min twice at 42°C with 0.2 × SSC, 1% SDS for 10 min twice. The signals were detected by autoradiography and exposed to Kodak XAR (Kodak, Rochester, NY). A 642-bp fragment of human VEGF cDNA sequence was used as a probe for Northern blot analyses. The cDNA clone for EPAS1, which hybridizes with mRNA for HIF-1, was obtained by polymerase chain reaction with the forward primer 5'-GGGGAATTCTACCTGAA AGCCT TGGAGGGT-3' and reverse primer 5'-CCCTCTAGAC GAATCCAGGGCATGGTAGAA-3', which are designed to amplify a DNA sequence containing the mouse EPAS1 PAS domain (17). Because of the lack of 3'-untranslated region (UTR) in the exogenous EPAS1 mRNA, we could distinguish endogenous EPAS1 from exogenous EPAS1 in Northern blot analysis.

Western Blotting

Western blotting was performed as previously described (24). EPAS1 protein expression was assayed using an EPAS1 polyclonal antibody at a dilution of 1:1,000. Secondary antibodies were diluted to 1:10,000. Tyrosine phosphorylation was assayed using a phosphotyrosine monoclonal antibody at a dilution of 1:1,000.

Construction of EPAS1 Promoter/Luciferase Gene

Human genomic clone encoding EPAS1 was isolated by screening the human leukocyte genomic library (HL1006d; Clontech, Palo Alto, CA) with the [³²P]-labeled UTR of the mouse EPAS1 cDNA (17). Several positive clones were obtained upon primary screening; one clone that was positive through three rounds of screening was further characterized by restriction mapping, size-fractionation on the gels, and Southern blotting. Selected genomic DNA fragments were subcloned into pBluescript II SK (Stratagene, La Jolla, CA), and DNA sequences were determined by the dideoxy chain termination method on an ABI PRISM model 310 Genetic Analyzer (Applied Biosystems, Foster, CA).

Transient Transfection, Luciferase Assay, and Preparation of Cell Lysates

For transient transfection, cells were seeded at 5 × 10⁵ cells per dish. Cells were transfected with 1 µg of reporter plasmid or, if indicated, 1 µg of expression plasmid by the calcium phosphate precipitation method (25). The following day, cells were switched to fresh medium, and, if indicated, pretreated with genistein or daidzein for 1 h, and were harvested for luciferase assays after 4-h culture in the hypoxic condition. Cells were washed twice with phosphate-buffered saline and lysated in 120 µl of cell cultured lysate reagent (Promega). The cell lysate was scraped and centrifuged to remove cell debris. Luciferase activity was measured immediately with a Berthold Lumat LB9501 luminometer (Berthold Technologies, Bad Wildbag, Germany). Similarly, cells were transfected with 1 µg of reporter plasmid or, if indicated, 1 µg of expression plasmid. The following day, cells were switched to fresh medium, and, if indicated, treated with genistein or daidzein. After 24 h of incubation, cells were harvested for luciferase assay.

Production of Recombinant Adenovirus

The replicate-deficient adenovirus was prepared as described previously (26). Briefly, myc-tagged mouse EPAS1 cDNA was inserted into the cassetted-cosmid vector (pAxCAwt) with chicken -globin poly (A) signal sequences. The recombinant viruses were obtained by in vitro recombination in 293 cells. Adenovirus encoding -galactosidase sequences (pAxCAiLacZ) was used as a control. For 24-48 h after plating, A549 cells were infected with adenovirus and used for the analysis.

Statistical Analysis

Statistical analyses were performed using Student's t test with significant differences determined as P < 0.05. Data are presented as means ± SE for at least three separate experiments.

	Results

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EPAS1 mRNA Is Abundantly Expressed in A549 Cells

To determine the steady-state levels of EPAS1 and HIF-1 mRNAs, total RNA was prepared from A549 cells and analyzed by Northern blot using either EPAS1 or HIF-1 cDNAs as hybridization probes. Approximately 20 µg of cytoplasmic RNA was applied into each well; the integrity of samples was confirmed by methylene blue staining of the 28S rRNA transferred onto the membranes. As shown in Figure 1A, EPAS1 mRNA was abundantly expressed in unstimulated A549 cells, whereas little expression of HIF-1 mRNA was detected.

View larger version (51K): [in this window] [in a new window]   Figure 1.   Time course of hypoxia-induced endothelial PAS domain protein-1 (EPAS1) mRNA and protein expression in A549 cells. (A) Confluent cultures of A549 cells were exposed to hypoxia for 12 h, and total cellular RNA (20 µg) was analyzed by Northern blotting for EPAS1 mRNA and hypoxia-inducible factor-1 (HIF-1) mRNA. 28S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA actually blotted onto a membrane. (B) Confluent cultures of A549 cells were exposed to hypoxia for indicated hours, and total cellular RNA (20 µg) prepared at the indicated times was analyzed by Northern blotting for EPAS1 mRNA. (C) Whole-cell lysates were prepared from A549 cells that had been cultured under hypoxic condition for various hours and were analyzed by Western blotting for EPAS1 and -actinin protein.

Hypoxia Rapidly Induces EPAS1 mRNA and Protein Expression in A549 Cells

The effects of hypoxia on EPAS1 expression in A549 cells was measured by Northern blot analysis and Western blot analysis. The cells were grown under either normoxic or hypoxic conditions for 0-12 h and harvested for the preparation of total RNA. As shown in Figure 1B, exposure of A549 cells to hypoxia increased the levels of EPAS1 mRNA but not those of HIF-1 in a time-dependent manner. An increase in EPAS1 mRNA levels was noticeable as early as 2 h and peaked at 4 h after hypoxic stimulation. Quantification of EPAS1 mRNA levels normalized to 28S ribosomal levels showed 1.9-fold induction at 2 h and 3.2-fold at 4 h after hypoxic stimulation (data not shown). To ascertain that hypoxia increases the EPAS1 protein levels, Western blot analysis was performed on the total cellular lysates prepared from the cells exposed to hypoxia for 0-12 h. Figure 1C shows that hypoxia clearly increased the EPAS1 protein levels. Thus, these results suggest that hypoxic induction of the EPAS1 mRNA expression results in an increase in EPAS1 protein levels in A549 cells.

Hypoxia Increases EPAS1 mRNA at the Transcriptional Level

To determine whether hypoxia increases EPAS1 mRNA levels at the posttranscriptional level, we measured EPAS1 mRNA in the presence of actinomycin D (5 µg/ml), a potent inhibitor of RNA synthesis, in A549 cells (Figure 2). The half-life of EPAS1 mRNA was 10.5 ± 0.4 h under normoxic condition and decreased to 6.6 ± 0.4 h after hypoxic stimulation. This difference was statistically insignificant. In a similar experiment, hypoxia did not alter the -actin mRNA half-life (data not shown). These results suggest that an increase in the EPAS1 mRNA levels by hypoxia was not due to an increase in the stability of the mRNA.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Effects of hypoxia on EPAS1 mRNA stability in A549 cells. Densitometric analysis was performed on Northern blots. The data from three independent experiments are expressed as the means ± SE of the percentage of hybridization signal present at each time following actinomycin D addition, compared with the signal present at 0 h.

A Specific Inhibitor for Src Family Kinases, PP1, Inhibits Hypoxia-induced EPAS1 Expression

Since protein kinases are key components of many signaling pathways in eukaryotic cells, we tested the effects of hypoxia on a set of different protein kinase inhibitors, including PD98059, calphostin C, genistein, SB203580, tyrphostin A23, wortmannin, herbimycin A, PP1, and daidzein. Representative Northern blot analyses performed on total RNA prepared from normoxic cells and cells exposed to hypoxia for 12 h in the presence or absence of various protein kinase inhibitors are illustrated in Figure 3A. The accompanying histograms illustrate the mean EPAS1 mRNA levels (Figure 3B). Among protein kinase inhibitors tested, PP1, herbimycin A, and genistein abolished the increase in EPAS1 mRNA by hypoxia. It is formally possible that genistein inhibits EPAS1 expression independent of an inhibition of tyrosine kinase activity, but it seems unlikely because daidzein, which is considered to be a negative control for the tyrosine kinase effects of genistein, did not inhibit the hypoxic induction of EPAS1. We also excluded the possibility of the nonspecific toxic effect of tyrosine kinase inhibitors tested because mRNA levels for EPAS1 and -actin (data not shown) were not altered in the presence of these protein kinase inhibitors. Given the specific inhibition of Src family kinases by PP1 (27), our data suggest that Src family kinases are involved in the induction of the EPAS1 mRNA levels by hypoxia. As illustrated in Figure 3C, Western blot analysis conducted on the whole-cell lysates prepared from A549 cells indicated that genistein but not daidzein attenuated the increase in EPAS1 protein levels by hypoxia. Consistently, genistein but not daidzein completely inhibited the hypoxia-induced phosphorylation of the protein, whose molecular weight is ~ 125 kD (Figure 3C). The accompanying histograms illustrate the mean phosphorylation levels of the 125-kD protein (Figure 3D). These data support our hypothesis that genistein-sensitive tyrosine kinase mediates an induction of EPAS1 expression in response to hypoxia. We reasoned that if EPAS1 is a critical regulator for hypoxia-induced VEGF expression, then inhibition of EPAS1 by PP1 should blunt the induction of VEGF by hypoxia. Indeed, as shown in Figure 3E, PP1 attenuated the hypoxic induction of VEGF mRNA. These data suggest that induction of EPAS1 by hypoxia is mediated through the Src family kinases and that this pathway plays a role in hypoxia-induced VEGF expression in A549 cells. Because reactive oxygen species are known to be regulators of hypoxia-induced transcription (28), we then examined the effects of the antioxidants on the hypoxia-induced EPAS1 expression. Pretreatment with N-acetyl-L-cysteine (22.5 mM), precursor of glutathione and thiol donor, inhibited hypoxia-induced EPAS1 expression (data not shown), thus suggesting the role of oxidative stress in hypoxia-induced EPAS1 expression.

View larger version (98K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   View larger version (45K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Figure 3.   Effects of protein kinase inhibitors on hypoxia-induced EPAS1 mRNA expression. (A) A549 cells were pretreated with various protein kinase inhibitors, PD98059 (50 µM), calphostin C (200 nM), genistein (100 µM), SB203580 (20 µM), tyrphostin A23 (100 µM), herbimycin A (1 µM), wortmannin (1 µM), PP1 (5 µM), daidzein (100 µM) for 1 h, and then were exposed to hypoxia for 12 h. Total cellular RNA (20 µg) was analyzed by Northern blotting for EPAS1 mRNA. 28S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA actually blotted onto a membrane. (B) Bar graphs show EPAS1 mRNA levels normalized by intensity of 28S RNA. *P < 0.05 relative to control. PD, PD98059; Cal, calphostin C; Gen, genistein; SB, SB203580; Tyr, tyrphostin; Wor, wortmannin; Herb, herbimycin A. (C) A549 cells were pretreated with genistein (100 µM) and daidzein (100 µM) for 1 h, and were exposed to hypoxia for 4 h. Whole-cell lysates were analyzed by Western blotting for EPAS1, phosphotyrosine and -actinin. (D) Bar graphs show phosphorylation levels of 125 kD protein normalized by intensity of -actinin. *P < 0.05 relative to control. (E) A549 cells were pretreated with PP1 (5 µM) for 1 h, and then were exposed to hypoxia for 4 h. Total cellular RNA (20 µg) was analyzed by Northern blotting for vascular endothelial growth factor (VEGF) mRNA. Bar graphs show VEGF mRNA levels normalized by intensity of 28S RNA.

Hypoxia Increases EPAS1 Promoter Activity

To ascertain that hypoxia regulates EPAS1 gene expression at the transcriptional level, transient transfection assays were performed using a reporter gene construct, EPAS-Luc, in which 2.0 kb of the human EPAS1 promoter region is coupled to the luciferase gene. Exposure of the transfected cells to hypoxia increased luciferase activity by 1.5-fold as compared with control (Figure 4). Consistent with the results of Northern blot and Western blot analyses, such an induction in luciferase activity was inhibited in the presence of genistein but not daidzein. These results suggest that hypoxia induces transcription of the EPAS1 gene through activation of protein tyrosine kinase. EPAS1 increased its own promoter activity.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Upregulation of EPAS1 promoter activity by hypoxia. A549 cells were transiently transfected with the endothelial PAS domain protein (EPAS)-Luc reporter gene construct containing 2.0 kilobases of the 5'-flanking region of the human EPAS1 gene. At 20 h posttransfection, transfected cells were exposed to hypoxia for 4 h either in the presence or absence of genistein. The relative luciferase activity values were calculated by dividing the luciferase activity values of samples transfected with EPAS-Luc by the activity value of normoxic samples transfected with promoterless plasmid pGL3BV. The filled bars represent hypoxia; open bars represent normoxia. All results represent means ± SE at least three experiments in duplicate. *P < 0.05 relative to normoxia.

Interestingly, a search of sequence for the putative HRE in the EPAS1 promoter region revealed that a sequence 5'-CACGTG-3', which conforms to the consensus E-box motif, is present at 1.6 kb from the 5' end of the UTR sequence reported previously (16). Because transcription factors that have the bHLH domain are known to bind to the E-box and activate E-box-dependent transcription, we tested whether EPAS1 itself regulates the EPAS1 promoter activity. The result is shown in Figure 5. Transient transfection of the A549 cells with the EPAS promoter-Luc reporter gene along with the EPAS1 expression vector showed that the luciferase activity of VEGF promoter-Luc was markedly enhanced by EPAS1 expression vector as compared with empty expression vector pcDNA3. We then examined whether induced expression of EPAS-Luc expression by EPAS1 expression vector is inhibited by genistein. As shown in Figure 5, EPAS1- induced EPAS-Luc activity was significantly inhibited in the presence of genistein but not daidzein. We obtained essentially the same results using the VEGF-Luc instead of EPAS-Luc. The effects of EPAS1 on the promoter activity appear to be gene-specific because thymidine kinase promoter (TK-Luc) was only minimally affected by EPAS1 expression vector (data not shown). These results suggest that increased expression of EPAS1 by hypoxia leads to the specific activation of its own promoter and VEGF promoter in A549 cells.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Upregulation of EPAS1 promoter activity by EPAS1. A549 cells were transiently transfected with the EPAS1 expression vector and the EPAS-Luc reporter gene either in the presence or absence of various protein kinase inhibitors. At 20 h posttransfection, transfected cells were harvested for luciferase assays. The relative luciferase values were calculated by dividing the luciferase activity values of each by the activity value of control samples cotransfected with pcDNA3. The filled bars represent EPAS1/pcDNA3; open bars represent pcDNA3. All results represent means ± SE of at least three experiments in duplicate.

Adenovirus Expressing EPAS1 Increased Its Own Expression

To test directly whether EPAS1 increases its own mRNA levels, we have constructed an adenoviral vector expressing EPAS1 cDNA under the control of a cytomegalovirus promoter, which we refer to as Ad-EPAS. Infection of the A549 cells with Ad-EPAS but not with the control Ad-LacZ virus, which expresses the -glucosidase gene, modestly but significantly induced endogenous EPAS1 gene expression (Figure 6A). These results suggest that EPAS1 is capable of inducing its own expression via auto-regulatory mechanisms.

View larger version (33K): [in this window] [in a new window]   Figure 6.   Effect of EPAS1 overexpression on EPAS1 mRNA expression. (A) A549 cells were infected with adenoviral vector expressing either EPAS1 or LacZ. At 72 h postinfection, total cellular RNA (20 µg) was analyzed by Northern blotting for EPAS1 mRNA. 28S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA actually blotted onto a membrane. (B) Bar graphs show EPAS1 mRNA levels normalized by intensity of 28S RNA.

	Discussion

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HIF-1 and EPAS1 are two distinct bHLH-PAS proteins, which bind to HRE and induce HRE-dependent transcription in response to hypoxia (13). Each of these proteins functions as a heterodimer with Arnt and binds to the same DNA element. In contrast to the ubiquitous mRNA expression pattern of HIF-1, EPAS1 mRNA expression has been thought to be restricted to endothelial cells. The present study, however, shows that EPAS1 mRNA is by far more abundantly expressed in A549 cells compared with HIF-1. Thus, EPAS1 seems to play a role in a wider range of cell types than previously thought. In this regard, it is worth noting that A549 cells were derived from the adenocarcinoma cells of the lung, where EPAS1 but not HIF-1 mRNA is most abundantly expressed in adult tissues (17). In situ hybridization showed that EPAS1 mRNA is expressed in the epithelial cells of pulmonary alveoli (17). These findings allow us to consider that EPAS1 acts as a potential candidate for mediating hypoxic stimulation in tumor cells derived from lung.

In this study, we demonstrate the hypoxic induction of steady-state levels for EPAS1, mostly due to the activation of transcription rate from the promoter. Previous studies on HIF-1 showed that the HIF-1 and Arnt mRNAs are constitutively expressed in a number of mammalian cell lines under normoxic and hypoxic conditions and that the HIF-1 protein is rapidly and dramatically increased upon exposure of target cells to hypoxia (29, 30). Those studies suggest that functional activity of HIF-1/Arnt complex is regulated by some as yet unknown posttranscriptional mechanism(s) including increased translation or increased stability. In contrast, the present study indicates that exposure of A549 cells to hypoxia induces the transcription from the EPAS1 gene promoter without a concomitant increase in EPAS1 mRNA stability.

The protein levels after exposure to hypoxia appear to have peaked at 2 h, while the mRNA levels peaked at 4-12 h. One possible explanation for such a discrepant result between mRNA and protein levels would be that an inducible expression of EPAS1 protein by hypoxia is regulated at the protein level as well as at the mRNA levels. This assumption is supported by a recent study in which EPAS1 protein levels are described as increased by hypoxia in other cell types (19). Our data suggest that stabilization of EPAS1 protein precedes an increase in EPAS1 transcription by EPAS1 itself.

To define the pathways responsible for the effects of hypoxia on the transcription of the EPAS1 gene, we examined the effects of various protein kinase inhibitors that are known to modulate transcriptional response to extracellular stimuli. Among several protein kinase inhibitors, PP1, herbimycin A, and genistein abolished upregulation of EPAS1 expression by hypoxia. It is of note that neither MEK1 inhibitor PD98059 nor p38MAP kinase inhibitor SB203580 inhibited the induction of EPAS1 mRNA by hypoxia. Moreover, Protein Kinase C (PKC) inhibitor calphostin C was without effect. These results are somewhat surprising because previous studies have demonstrated that activation of the extracellular-regulated signal transduction kinase (ERK) cascade or PKC is implicated in the hypoxic response. Mukhopadhyay and colleagues (31) have demonstrated that a plasmid-overexpressing dominant negative form of the Raf-1 mutant or ERK inhibitor 6-thioguanine clearly inhibit the hypoxia-mediated increase in VEGF mRNA levels. Muller and colleagues (32) have reported the induction of c-fos gene transcription through a MAP kinase-dependent pathway in HeLa cells. In our experiments, neither PD98059 nor calphostin C could inhibit the hypoxic induction of EPAS1 mRNA expression. In this regard, hypoxic stimulation appears to evoke multiple intracellular signaling pathways, which differentially activate the transcription of a certain set of genes.

One of the major findings in this study was that a specific inhibitor for Src family kinases, PP1, blocked hypoxia-induced EPAS1 expression. The importance of tyrosine kinases in transmitting a signal evoked by hypoxia has been reported in other cell systems. In the nitrogen-fixing gene in the bacterium Rhizobium meliloti, hypoxia induces oxygen dissociation from the heme group, which activates tyrosine kinase and results in the phosphorylation of transcription factors (33). In higher organisms, Mukhopadhyay and colleagues have reported that hypoxia activates c-Src, which in turn phosphorylates downstream transcription factors responsible for the hypoxic induction of VEGF (31). In fact, we observed that PP1 inhibited hypoxia- induced VEGF expression. We suggest that Src family kinases, taken together, are involved in the hypoxic induction of VEGF gene expression by EPAS1 in A549 cells.

Recent studies revealed that hypoxia-induced transcription is regulated by reactive oxygen species generated by mitochondria (28). Therefore, we examined whether antioxidants have effects comparable to those of the inhibitors of Src family kinases on the hypoxia-induced EPAS1 expression. N-acetyl-L-cysteine inhibited hypoxia-induced EPAS1 expression. Hydrogen peroxide scavenger catalase and superoxide dismutase inhibitor diethyldithiocarbamic acid did not affect the induction of EPAS1 mRNA by hypoxia. These data suggest that neither hydrogen peroxide nor hydroxyl radical is responsible for hypoxia-induced EPAS1 expression. Although further studies will be required for the identification of the free radical species, recent study of the glutathione-sensitive regulation of the heme oxygenase-1 gene expression (34) led us to speculate that redox reaction involving nitric oxide and S-nitrosothiols may play a role in the hypoxia-induced EPAS1 expression process.

Data obtained by transient transfection assays and adenoviral gene transfer suggest that EPAS1 gene expression is induced by its own product EPAS1. To date only a few genes have been considered to be the target genes of EPAS1. Transcription from the VEGF and Tie-2 gene promoters have been shown to be stimulated by cotransfection of the EPAS1 expression vector, and site-specific mutation analysis of these promoters indicated that HREs in their promoters mediate this effect (16, 17). Although we did not examine the binding of EPAS1 to the promoter sequence, searching of the promoter region revealed E-box motif at -1.6 kb from the 5' end of the UTR sequence reported previously (16). Given that transcription factors containing bHLH motif activate E-box-dependent transcription (35), it is possible that EPAS1 regulates its own gene expression via a binding to E-box. The functional significance of this sequence deserves further study.

In conclusion, hypoxia increases EPAS1 mRNA levels at least partly at the transcriptional level through an activation of the Src family kinases-dependent pathway in A549 cells. In addition, we demonstrate that EPAS1 activates its own promoter as well as VEGF promoter, and this process also seems to be regulated by Src tyrosine kinases. We depicted the model summarized in Figure 7. Identification and purification of Src family kinases involved in hypoxia-induced EPAS1 gene expression may facilitate our understanding of the mechanisms underlying the tumor angiogenesis that is critically dependent upon VEGF.

View larger version (16K): [in this window] [in a new window]   Figure 7.   Model for the mechanism of tumor angiogenesis via activation of EPAS1 by hypoxia. Hypoxia induces EPAS1 mRNA and protein expression via the activation of Src family kinases. EPAS1 protein increases its own promoter activity as well as the transcription of the VEGF gene, a potent inducer of tumor angiogenesis.