| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Isoform
1 Human Genome Research Institute and Cancer Research Institute and
2 Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea, and
3 Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and HIF-1ß subunits and its activity depends on the amount of HIF-1, which is tightly controlled by cellular oxygen tension. In addition to hypoxia, various nonhypoxic stimuli can stabilize HIF-1 in tumor cells, implying that both hypoxic and nonhypoxic stimuli contribute to the overexpression of HIF-1 in tumors. On the other hand, phorbol esters such as phorbol-12-myristate-13-acetate (PMA) are known to be potent tumor promoters. Here, we identified a novel HIF-1 isoform, which is regulated primarily by PMA. The variant mRNA lacks exon 11 and produces a 785-amino acid isoform (HIF-1785) without altering the reading frame and therefore the COOH-terminal transcriptional activity. HIF-1785 is induced markedly by PMA and heat shock, the latter of which is also known to induce HIF-1. HIF-1785 escapes from lysine acetylation because of the loss of Lys532 and was stabilized under normoxic conditions. Its expression was blocked by reducing agents and by a mitogen-activated protein/extracellular signal-regulated kinase-1 inhibitor and enhanced by hydrogen peroxide. In addition, HIF-1785 overexpression strikingly enhanced tumor growth in vivo. These results suggest that HIF-1785 is induced by PMA under normoxic conditions via a redox-dependent mitogen-activated protein/extracellular signal-regulated kinase-1 pathway and that it plays an important role in tumor promotion. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and HIF-1ß, both of which belong to the per-arnt-sim family of basic helix-loop-helix transcription factors (2)
. HIF-1 activity depends primarily on the amount of HIF-1 protein (3)
. At the protein level, HIF-1 is markedly increased by hypoxia, whereas HIF-1ß is constantly present regardless of oxygen tension.
HIF-1 is composed of 826 amino acids (2)
. Its NH2-terminal contains the basic helix-loop-helix and the per-arnt-sim domains, which are essential for dimerization and DNA binding (4)
, and its COOH-terminal contains two transactivation domains and a nuclear localization signal (5
, 6)
. In the middle section of HIF-1 lies a Pro-Ser-Thr rich oxygen-dependent degradation domain (ODDD, amino acid 401–603), which is responsible for stability of HIF-1 protein (7)
. Under normoxic conditions, HIF-1-prolyl hydroxylases (PHD1–3) hydroxylate two proline residues in the amino acid motif LXXLAP in the NH2-terminal (amino acid 390–417) and the COOH-terminal (amino acid 557–576) of the ODDD (8, 9, 10, 11, 12)
. The von Hippel-Lindau tumor suppressor protein, a part of the E3 ubiquitin ligase protein complex, binds directly to the modified amino acid motifs of HIF-1, resulting in the ubiquitination and proteasomal degradation of HIF-1 (13
, 14)
. Because the enzymatic reaction of prolyl hydroxylation requires molecular oxygen as a substrate, hypoxia limits the hydroxylation, thereby precluding the binding of von Hippel-Lindau tumor suppressor protein and stabilizing HIF-1. In addition, the acetylation of a lysine residue (Lys532) within the ODDD is another mechanism regulating HIF-1 stability. The lysine acetylation enhances the interaction between HIF-1 and von Hippel-Lindau tumor suppressor protein. Because the expression of ARD1, a HIF-1 acetyltransferase, decreased under hypoxic conditions, HIF-1 escapes from the acetylation and is stabilized (15)
.
A growing body of evidence indicates that HIF-1 contributes to tumor progression and metastasis. In human tumors, HIF-1 is overexpressed as a result of intratumoral hypoxia and genetic alterations affecting key oncogenes and tumor suppressor genes (16
, 17)
. The expression level of HIF-1, in biopsies of various solid tumors, is correlated with the tumor aggressiveness, vascularity, treatment failure, and mortality (18)
. In addition, tumor growth and angiogenesis in grafted tumors also depend on HIF-1 activity or the expression level of HIF-1 (19)
. Our recent study (20)
demonstrated that the growth of grafted tumors was halted by the inhibition of HIF-1 and strongly supports the causative role of HIF-1 in tumor promotion.
In this study, we identified a novel splice variant of HIF-1 in mammalian cells. Deprived of exon 11, the variant, designated HIF-1785, produced a HIF-1 of 785 amino acids. The variant was strikingly up-regulated by phorbol 12-myristate 13-acetate (PMA) through a redox-dependent mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1) pathway and was also induced by heat and oxidative stresses under normoxic conditions. In a xenograft experiment, HIF-1785-expressing tumors grew faster than HIF-1-expressing tumors. HIF-1785 seems to play an important role in tumor promotion and in other cellular processes related to HIF-1 activation.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-32P]CTP (500 Ci/mmol) was from NEN Life Science (Boston, MA). Other chemicals were obtained from Sigma-Aldrich Corp (St. Louis, MO) unless otherwise indicated. Culture media and the FCS were purchased from Life Technologies, Inc. (Grand Island, NY).
Cell Culture.
Most cancer cell lines were obtained from American Type Culture Collection and SNU601 cell line, a stomach cancer cell line was obtained from Korean Cell Line Bank (Seoul, Korea). Hep3B and other cell lines were cultured in -modified Eagle’s medium and in DMEM, supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. O2/CO2 levels in the incubator (Vision Sci Co., Seoul, Korea) were either 20%/5% (normoxic) or 1%/5% (hypoxic).
Identification and Cloning of a Human HIF-1 cDNA Variant.
RNAs were isolated using Trizol (Life Technologies, Inc.) and reverse transcribed using the avian myeloblastosis virus reverse transcriptase system (Promega, Madison, WI). Reverse-transcription (RT)-PCR was done in a reaction involving reverse transcription at 48°C for 1 h and 25 PCR cycles at 94°C for 30 s, 53°C for 30 s, and 68°C for 30 s 
2 min. PCR products (5 µl) were electrophoresed on 1 or 2% agarose gel containing ethidium bromide and directly sequenced. On the basis of the nucleotide sequence of the HIF-1 gene (GenBank no. AH006957 for the human gene; GenBank no. AH006789 for the mouse gene; GenBank no. AF057308 for the rat cDNA), eight pairs of PCR primers were designed to amplify HIF-1 and its variant cDNAs, as shown in Fig. 1
.
|
cDNAs of 2.5 kb were amplified by RT-PCR using S3 and A2 primers and cloned using a pCR2.1-TOPO cloning kit (Invitrogen, Carlsbad, CA). To select a colony of Escherichia coli transformed with the HIF-1 cDNA variant, colony PCRs using S1 and A4 primers were performed. The HIF-1 cDNA variants were amplified and sequenced.
Semiquantitative RT-PCR for HIF-1 and Its Variant mRNAs.
To quantitate mRNAs, a highly sensitive, semiquantitative RT-PCR was performed as described previously (21)
. One µg of total RNAs was reverse transcribed at 48°C for 1 h, and the cDNAs were amplified over 20 PCR cycles (94°C for 30 s, 53°C for 30 s, and 68°C for 30 s) in a 50 µl of reaction mixture containing 5 µCi [-32P]dCTP and 250 nM of each primer set. The PCR products (5 µl) were electrophoresed on a 4% polyacrylamide gel, and dried gels were autoradiographed. The nucleotide sequences of the primer pair (5' to 3') for ARD1 were ATGAACATCCGCAATGCG and AGGCTCTAGGAGGCTGAG. Primers for vascular endothelial growth factor, aldolase A, enolase 1, and ß-actin were constructed as described previously (20)
.
Expression Plasmids and Transfection.
Hemagglutinin (HA)-tagged HIF-1 expression plasmid (pcDNA3) was constructed as described previously (3)
. HA-tagged HIF-1 variant (HIF-1785) expression plasmid was made using a PCR-based mutagenesis kit (Stratagene, Cedar Creek, TX). Mutagenesis using specific oligonucleotides (forward, 5-GACACAGATTTAGACTTGGAGATG-3; reverse, 5-CTCAGGTGAACTTTGTCTAGTGCTTC-3) was used to delete nucleotides derived from exon 11. To replace a lysine residue (amino acid 532) in HIF-1 with arginine, the bases AAG in pcDNA3-HA-tagged HIF-1 were replaced with the bases CGA, by using a QuickChange site-directed mutagenesis kit (Stratagene). All constructs were verified by DNA sequencing.
To establish stable transfectant cell lines, 40% of the confluent HEK 293 cells in 60-mm cell culture dishes were transfected with 4 µg of plasmid using the calcium phosphate method. Cells were allowed to stabilizing 36 h and then treated with 0.45 mg/ml G418. Thirty stable transfectants from three different transfections were pooled to avoid bias in gene expression because of variable chromosomal integration.
Reporter Assays.
A luciferase reporter gene, containing the HIF-1-binding erythropoietin enhancer region, was constructed as described previously (3)
. To assay HIF-1 activity, HEK293 cells were cotransfected with 0.6 µg each of reporter gene and plasmid cytomegalovirus-ß-gal and/or plasmids of HIF-1 or its variant, using the calcium phosphate method. pcDNA was added to ensure that the final DNA concentrations in both the control and experimental groups were at the same level. After being allowed to stabilize for 48 h, the cells were incubated under either normoxic or hypoxic conditions, in the absence or in the presence of PMA for 16 h, and lysed to determine luciferase and ß-gal activities.
Immunoblotting of HIF-1 and Its Variant Protein.
Total cell lysate was separated on a 6.5% SDS/polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TTBS) at room temperature for 1 h and then incubated overnight at 4°C with rabbit anti-HIF-1 (22)
diluted 1:1000, rabbit anti-HA (Biodesign International, Saco, ME) diluted 1:4000, or rat anti-ARD1, generated against bacterially expressed full-length peptide of human ARD1, diluted 1:5000 in 5% nonfat milk in TTBS. Horseradish peroxidase-conjugated antirabbit or antirat antiserum was used as a secondary antibody (1:5000 dilution in 5% nonfat milk in TTBS, 2 h of incubation) and the antigen antibody complexes were visualized using an Enhanced Chemiluminescence Plus kit (Amersham Biosciences Corp., Piscataway, NJ). ß-Actin was used as an internal standard and quantified using mouse monoclonal anti-ß-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Xenografts of Human Tumors.
Male nude (BALB/cAnNCrj-nu/nu) mice were purchased from Charles River Japan, Inc. (Shin-Yokohama, Japan). Animals were housed in a specific pathogen-free room under controlled temperature and humidity. All animal procedures were performed according to the procedures in the Seoul National University Laboratory Animal Maintenance Manual. Eighteen mice ages 7–8 weeks were injected s.c. in the flank with 5 x 106 viable cells expressing HIF-1 or variant. Tumor volumes were measured with calipers and calculated using the formula: volume = a x b2/2, where a is the width at the widest point of the tumor and b is the width perpendicular to a.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Splice Variant. cDNAs were amplified in HEK293 cells that had been treated under normoxic, hypoxic, or with CoCl2. In addition to the wild-type cDNA fragment, a smaller product was also reproducibly amplified (Fig. 2A)
. DNA sequencing revealed that the small fragment was a result of the omission of exon 11 and the splicing of exons 10 and 12. Both PCR 2 and the PCR 3, which leave out exon 11, revealed a single band of 847 and 1578 bp, respectively (second lanes in Fig. 2, B and C
), indicating the absence of a splicing site other than that at exon 11 in the variant cDNA. Primer specificity on the variant cDNA was verified because the wild-type HIF-1 DNA was not amplified (third lanes). Because the intensity of the 28-cycle fragment in PCR 5 (variant) was approximately the same as that of the 25 cycle PCR 4 (wild type), the amount of variant mRNA was estimated to be 1/8 [=1/2(28–25)] of that of the wild type (Fig. 2D)
. Among 20 colonies harboring full-length HIF-1 cDNAs, 17 contained wild-type cDNA and 3 contained variant cDNA (Fig. 2E)
. Of note, the 3–20 colony ratio is comparable with the mRNA ratio estimated in Fig. 2D
. The variant mRNA was detected by PCR 7 in both human and murine cell lines, where the wild-type mRNA was also detected by PCR 8 (Fig. 2F)
. The murine cell lines produced a larger PCR 8 product because mouse exon 11 is 42-bases longer than human HIF-1.
|
. The variant cDNA was identical to HIF-1 cDNA, except for the deletion of the 123-bp exon 11. This variant generates a shorter mRNA without altering the reading frame and produces a 785-amino acid polypeptide composed of amino acid residues 1–512 and 554–826, which is designated here as HIF-1785. HIF-1785 preserves most of the functional domains, including both von Hippel-Lindau tumor suppressor protein-interacting subdomains (amino acids 390–417 and 557–576). However, HIF-1785 is deprived of a small part (amino acid 513–553) of the ODDD, which contains the lysine residue (Lys532) responsible for the regulation of HIF-1 stability by acetylation. Because the ODDD is partially missing, it is possible that the protein stability of HIF-1785 is regulated in a different manner.
|
785 by PMA and Hyperthermia. is not regulated by oxygen tension alone; it is also regulated by various other stimuli such as PMA, hyperthermia, and reactive oxygen species (ROS; Refs. 23
, 24
). When cells transfected with pHA-HIF-1 or pHA-HIF-1785 were subjected to hypoxia or treated with PMA, HIF-1785 was markedly induced by PMA in a dose-dependent manner, whereas HIF-1 was induced by hypoxia (Fig. 4A)
. The level of HIF-1785 was much greater in PMA-treated cells than in hypoxia-treated cells. In addition, HIF-1785 was induced by heat in a time-dependent manner, whereas HIF-1 and ß-actin were unaffected (Fig. 4B)
. These results indicate that HIF-1785 is an inducible isoform under normoxic conditions.
|
785 was distinguished from HIF-1. HIF-1785 showed a faster mobility and a sharper band pattern on SDS-PAGE than HIF-1 (Fig. 4C)
. To determine whether PMA induces endogenous HIF-1785, we examined the mobility and band pattern of HIF-1 induced by hypoxia and PMA. Fig. 4D
shows that PMA-induced HIF-1 migrated faster and was sharper than hypoxia-induced HIF-1. This is comparable with the characteristics of expressed HIF-1785, as shown by Fig. 4C
. These results suggest that the different HIF-1 isoforms are induced by hypoxia, whereas PMA specifically induces HIF-1785.
HIF-1785 expression by PMA appears to be regulated at the protein level because its mRNA level was unaltered by PMA (Fig. 4E)
. To further examine whether PMA treatment increases the stability of HIF-1785 protein, we measured the half-life of the protein after blocking de novo protein synthesis with cycloheximide. Fig. 4F
shows that the half-life of HIF-1785 was prolonged to 15.5 min by PMA treatment, whereas the half-life in untreated cells was only 5.4 min.
Transcriptional Activity of PMA-Induced HIF-1785.
Both hypoxia and PMA increased the luciferase activity of erythropoietin enhancer reporter in pcDNA-transfected cells (Fig. 5A)
. In HIF-1-transfected cells, however, only hypoxia and not PMA further augmented reporter activity. In contrast, HIF-1785-transfected cells showed enhanced reporter activity by PMA but not by hypoxia. To confirm the functional role of HIF-1785 in response to PMA, we examined the mRNA levels of hypoxia-inducible genes, namely, vascular endothelial growth factor, aldolase A, and enolase 1 (Fig. 5B)
. In pcDNA-transfected cells, all of the mRNAs of the hypoxia-inducible genes were increased by 10 nM PMA. In HIF-1-transfected cells, the basal levels of these mRNAs were elevated in the control, but their expressions were not additionally increased by PMA. In HIF-1785-transfected cells, however, the levels of these mRNAs were markedly increased by an even lower concentration (2 nM) of PMA. Theses results suggest that HIF-1785 plays an important role in the PMA-induced expression of hypoxia-inducible genes under normoxic conditions.
|
.785 may be accounted by the lack of exon 11. Recently, the lysine residue (Lys532) within exon 11 was identified as the target of ARD1 acetyltransferase, which destabilizes HIF-1 (15)
. To determine whether Lys532 inhibits the nonhypoxic induction of HIF-1 by PMA and hyperthermia, we changed lysine at 532 to arginine (K532R), as shown in Fig. 6
. Surprisingly, the point mutation of Lys532 was sufficient to render HIF-1 responsive to both PMA and hyperthermia (Fig. 6A)
, whereas HIF-1 was not induced (Fig. 6B)
. Thus, Lys532 is likely to play an inhibitory role in stabilization of HIF-1 by PMA and hyperthermia.
|
785, we hypothesize that PMA and hyperthermia provide some signal for the stabilization of HIF-1 isoforms but that wild-type HIF-1 is not stabilized because of acetylation of the exon 11 domain by ARD1. In contrast, HIF-1785 lacking the exon 11 domain is stabilized. This hypothesis is based on the premise that the HIF-1 acetylation process is active in cells treated with PMA and hyperthermia. As reported previously (15)
, the expression of ARD1 mRNA and protein markedly decreased under hypoxic conditions. However, the ARD1 expression was not suppressed by PMA or hyperthermia (Fig. 6C)
, which suggests that HIF-1 is acetylated and degraded under these normoxic conditions. To confirm this hypothesis, we examined whether the stability of HIF-1785 was affected by ARD1. The PMA induction of HIF-1785 was not suppressed in pARD1-transfected cells (Fig. 6E)
, whereas the hypoxic induction of HIF-1 was almost completely blocked (Fig. 6D)
. These results suggest that blockade of acetylation in HIF-1785 provides protein stability under normoxic conditions.
Mechanism of HIF-1785 Regulation.
To determine the nature of the signal transduction pathway mediating the PMA induction of HIF-1785, HEK293 cells and pHIF-1785-transfected cells were incubated with various inhibitors such as N-acetylcysteine, a reducing thiolic compound; wortmannin, a phosphatidylinositol 3'-kinase inhibitor; SB203580, a p38 mitogen-activated protein kinase inhibitor; PD98059, a MEK-1 inhibitor; rapamycin, an FK506-binding protein 12-rapamycin associated protein inhibitor; KT5720, a protein kinase A inhibitor; KT5823, a protein kinase G inhibitor; calphostin C, a protein kinase C inhibitor; herbimycin A, a tyrosine kinase inhibitor. N-Acetylcysteine and PD98059 reduced the induction of endogenous HIF-1785 (Fig. 7A
, left panel), although these inhibitors did not affect the hypoxic induction of HIF-1. In contrast, HIF-1 expression was reduced by SB203580 (Fig. 7A
, right panel). N-Acetylcysteine and PD98059 also inhibited the induction of expressed HIF-1785 (Fig. 7B)
. In addition, another reducing thiolic compound, DTT, inhibited the PMA induction of HIF-1785 (Fig. 7C)
, suggesting that the redox state regulates the stability of HIF-1785. Furthermore, HIF-1785 expression was up-regulated by hydrogen peroxide, and this effect was completely abolished by N-acetylcysteine (Fig. 7D)
. The hyperthermic induction of HIF-1785 was also suppressed by N-acetylcysteine (data not shown). These results suggest that the HIF-1785 stabilization by PMA is regulated by redox state and by a MEK-1 pathway, whereas HIF-1 stabilization by hypoxia is regulated by a p38 mitogen-activated protein kinase pathway.
|
785 in Tumor Growth in Vivo.785, are also well-known tumor promoters (25)
. To investigate the possibility that HIF-1785 promotes tumor growth, we grafted HIF-1- or HIF-1785-expressing cells in nude mice. As expected, HIF-1-expressing cells successfully formed tumors, whereas the cells having empty vector did not. However, tumors expressing HIF-1785 grew faster than tumors expressing HIF-1 (Fig. 8)
. Excised HIF-1785 tumors were larger and more hypervascular than HIF-1 tumors. Thus, HIF-1785 expression seems to be associated with tumor growth.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, HIF-1785, in human and murine cancer cells. This variant lacks exon 11 and produces a 785 amino acid HIF-1 without altering the reading frame and retains normal transactivation activity. Moreover, its expression was found to be strikingly up-regulated by PMA. The expression of HIF-1785 was blocked by reducing agents and enhanced by hydrogen peroxide. Mutational and pharmacological studies revealed that HIF-1785 can escape from lysine acetylation and that this results in the PMA-induced stabilization of HIF-1785 protein. Moreover, HIF-1785-enhanced tumor growth in vivo. Taken together, our results show that HIF-1785 is induced by PMA under normoxic conditions via a redox-dependent MEK-1 pathway and suggest that HIF-1785 plays an important role in the nonhypoxic activation of HIF-1 and in tumor promotion.
To date, four splice variants of human HIF-1 mRNA have been reported. One has an additional 3 bp at the junction of the exons 1 and 2 without a frameshift (26)
. The second loses exon 14 and translates into a 736-amino acid polypeptide (HIF-1a736). This variant is regulated by oxygen tension and transactivates the vascular endothelial growth factor promoter (26)
. The third loses exon 12 and produces a short form of HIF-1557, which was induced specifically by the zinc ion (21)
, and the fourth lacks exons 11 and 12 and produces a shorter form of HIF-1516 (27)
. These truncated variants, which contain the per-arnt-sim domain, inhibit HIF-1 activity by sequestering HIF-1ß and reduce the mRNA expression of hypoxia-inducible genes. Similarly, a splice variant of HIF-3, which also contained the per-arnt-sim domain, inhibited HIF-1 activity in the mouse cornea (28
, 29)
. In this study, we found the fifth splice variant of human HIF-1, HIF-1785. Compared with the protein structures of truncated variants previously found, HIF-1785 retains all of the essential domains of HIF-1 and functions as a transcription activator.
Hypoxia is not the only player in HIF-1 stabilization. Many other stimuli have been found to play important roles in controlling HIF-1 regulation in nonhypoxic environments. Hyperthermia induces the nonhypoxic expression of HIF-1. Katschinski et al. (24)
demonstrated that the expression of HIF-1 was up-regulated in several tissues and in cultured hepatoma cell lines after hyperthermic incubation. They also found a low molecular weight form of HIF-1, which had a sharper band pattern in cells exposed to hyperthermia. Although they suggested that the molecular weight difference was due to reduced phosphorylation of heat-induced HIF-1 protein, the identity of the HIF-1 isoform induced by hyperthermia has not been determined. On the basis of the mobility and bandwidth of heat-induced HIF-1, it is possible that heat-induced HIF-1 is HIF-1785.
In terms of the mechanism underlying the HIF-1 induction by PMA, the activation of the phosphatidylinositol 3'-kinase pathway is possibly involved (30)
. However, PMA stimulates the expression of HIF-1785 in a different way because HIF-1785 expression was not suppressed by a phosphatidylinositol 3'-kinase inhibitor. Instead, an oxidative process seems to be responsible for HIF-1785 stabilization (Fig. 7)
. Similarly, reducing agents also suppressed HIF-1785 induced by hyperthermia or hydrogen peroxide. These results suggest that the stabilization of HIF-1785 is achieved via an oxidative pathway. This is supported by reports that PMA and hyperthermia stimulate the oxidation process. PMA is known to produce oxidative stress via stimulating NADPH oxidase in nonphagocyte cells as well as phagocytes (31)
. Hyperthermia also produces oxidative stress in cancer and muscle cells due to increased energy metabolism (32)
. Therefore, PMA, hyperthermia, and hydrogen peroxide could stimulate an oxidation process to stabilize HIF-1785.
The role of oxidative stress in the regulation of HIF-1 is controversial. Several experiments have demonstrated that strong oxidizing reagents impair the expression of HIF-1 in hypoxic cells (3)
. In contrast, Chaddel et al. (33
, 34)
demonstrated that HIF-1 activation is associated with increased production of ROS from mitochondrial complex III. In addition, inflammatory cytokines producing ROS have been reported to stabilize HIF-1 (35)
. Why is the effect of ROS on HIF-1 expression different? This question may be derived from the misconception that the same isoform of HIF-1 is induced by hypoxia and ROS. On the basis of our results, we speculate that under hypoxic conditions, wild-type HIF-1 is suppressed by oxidative stress but that under normoxic conditions HIF-1785 is induced by oxidative stress.
We also demonstrated the possibility that two mitogen-activated protein kinase pathways, MEK-1/extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, selectively regulate HIF-1785 and HIF-1. Several studies (36
, 37)
have demonstrated that these pathways mediate the stabilization or the transcriptional activation of HIF-1. However, the effects of specific pathway inhibitors were variable and depended on the conditions of HIF-1 induction. In the present study, inhibitors of the MEK-1 and p38 pathways, PD98059 and SB203580, also produced different effects on the expression of HIF-1 isoforms in PMA- and hypoxia-treated cells. These results additionally support our hypothesis that PMA and hypoxia regulate different isoforms of HIF-1 via different mechanisms.
A growing body of evidence indicates that HIF-1 contributes to tumor progression and metastasis (16
, 20)
. Because PMA and ROS, which are well-known tumor promoters, were found to enhance HIF-1 activity by inducing HIF-1785, it is possible that HIF-1785 contributes to tumor promotion by these stimuli. Our results, as shown in Fig. 8
, support this possibility. In addition, it has been reported that HIF-1 also contributes to other cellular processes that occur under normoxic conditions such as the development of normal tissues, the determination of cell death or survival, immune responses, and adaptation to mechanical stresses (23)
. Therefore, HIF-1785 could participate in these normoxic processes.
|
FOOTNOTES |
|---|
Grant support: Korea Science and Engineering Foundation Grant R01-2000-000-00139-0.
Note: Drs. Chun and Lee contributed equally.
Requests for reprints: Jong-Wan Park, Department of Pharmacology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Fax: 82-2-7457996; E-mail: parkjw{at}plaza.snu.ac.kr
Received 6/11/03. Revised 9/29/03. Accepted 10/ 6/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
subunit. J. Biol. Chem., 271: 32253-32259, 1996.. Modulation of transcriptional activity by oxygen tension. J. Biol. Chem., 272: 19253-19260, 1997.. EMBO J., 17: 6573-6586, 1998.[Medline]
is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA, 95: 7987-7992, 1998. targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science (Wash. DC), 292: 464-468, 2001. to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science (Wash. DC), 292: 468-472, 2001. chains activated by prolyl hydroxylation. EMBO J., 20: 5197-5206, 2001.[Medline]
ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc. Natl. Acad. Sci. USA, 97: 10430-10435, 2000. by ARD1-mediated acetylation. Cell, 111: 709-720, 2002.[Medline]
in common human cancers and their metastases. Cancer Res., 59: 5830-5835, 1999. is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res., 60: 4693-4696, 2000. variant induced by zinc ion suppresses HIF-1-mediated hypoxic responses. J. Cell Sci., 114: 4051-4061, 2001.
, but inhibits the nuclear translocation of HIF-1ß, causing HIF-1 inactivation. Biochem. Biophys. Res. Commun., 268: 652-656, 2000.[Medline]
. J. Korean Med. Sci., 17: 581-588, 2002.[Medline]
is dependent on heat shock protein-90 activity. J. Biol. Chem., 277: 9262-9267, 2002.. J. Biol. Chem., 275: 6922-6927, 2000. gene. Biochem. J., 362: 71-79, 2002.[Medline]
locus. J. Biol. Chem., 277: 32405-32408, 2002. 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.. Circ. Res., 90: 143-150, 2002. during hypoxia: a mechanism of O2 sensing. J. Biol. Chem., 275: 25130-25138, 2000.-dependent regulation of HIF-1. FEBS Lett., 505: 269-274, 2001.[Medline]
stabilization and nuclear localization in a human prostate cancer cell line. J. Biol. Chem., 277: 50081-50086, 2002.This article has been cited by other articles:
|
|
 |
|
  J.-H. Lim, Y.-S. Chun, and J.-W. Park Hypoxia-Inducible Factor-1{alpha} Obstructs a Wnt Signaling Pathway by Inhibiting the hARD1-Mediated Activation of {beta}-Catenin Cancer Res., July 1, 2008; 68(13): 5177 - 5184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Kumar, H. Yu, R. Edwards, L. Chen, S. Kazianis, P. Brafford, G. Acs, M. Herlyn, and X. Xu Mutant V600E BRAF Increases Hypoxia Inducible Factor-1{alpha} Expression in Melanoma Cancer Res., April 1, 2007; 67(7): 3177 - 3184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ramanathan, G. Pinhal-Enfield, I. Hao, and S. J. Leibovich Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2A Receptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter Mol. Biol. Cell, January 1, 2007; 18(1): 14 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Lim, J.-W. Park, and Y.-S. Chun Human Arrest Defective 1 Acetylates and Activates {beta}-Catenin, Promoting Lung Cancer Cell Proliferation. Cancer Res., November 15, 2006; 66(22): 10677 - 10682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K S Kimbro and J W Simons Hypoxia-inducible factor-1 in human breast and prostate cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 739 - 749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Oda, K. Hirota, K. Nishi, S. Takabuchi, S. Oda, H. Yamada, T. Arai, K. Fukuda, T. Kita, T. Adachi, et al. Activation of hypoxia-inducible factor 1 during macrophage differentiation Am J Physiol Cell Physiol, July 1, 2006; 291(1): C104 - C113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-J. Yeo, J.-H. Ryu, Y.-S. Cho, Y.-S. Chun, L. E. Huang, M.-S. Kim, and J.-W. Park Amphotericin B blunts erythropoietin response to hypoxia by reinforcing FIH-mediated repression of HIF-1 Blood, February 1, 2006; 107(3): 916 - 923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bilton, N. Mazure, E. Trottier, M. Hattab, M.-A. Dery, D. E. Richard, J. Pouyssegur, and M. C. Brahimi-Horn Arrest-defective-1 Protein, an Acetyltransferase, Does Not Alter Stability of Hypoxia-inducible Factor (HIF)-1{alpha} and Is Not Induced by Hypoxia or HIF J. Biol. Chem., September 2, 2005; 280(35): 31132 - 31140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Gaber, R Dziurla, R Tripmacher, G R Burmester, and F Buttgereit Hypoxia inducible factor (HIF) in rheumatology: low O2! See what HIF can do! Ann Rheum Dis, July 1, 2005; 64(7): 971 - 980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Kaur, F. W. Khwaja, E. A. Severson, S. L. Matheny, D. J. Brat, and E. G. Van Meir Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis Neuro-oncol, April 1, 2005; 7(2): 134 - 153. [Abstract] [PDF]
|
|
|
|
|
|
K. Ukon, K. Tanimoto, T. Shimokuni, T. Noguchi, K. Hiyama, H. Tsujimoto, M. Fukushima, T. Toge, and M. Nishiyama Activator Protein Accelerates Dihydropyrimidine Dehydrogenase Gene Transcription in Cancer Cells Cancer Res., February 1, 2005; 65(3): 1055 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Frantz, K. A. Vincent, O. Feron, and R. A. Kelly Innate Immunity and Angiogenesis Circ. Res., January 7, 2005; 96(1): 15 - 26. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-G. Yoo, M. G. Yeo, D. K. Kim, H. Park, and M.-O. Lee Novel Function of Orphan Nuclear Receptor Nur77 in Stabilizing Hypoxia-inducible Factor-1{alpha} J. Biol. Chem., December 17, 2004; 279(51): 53365 - 53373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-H. Lee, J.-W. Park, and Y.-S. Chun Non-hypoxic transcriptional activation of the aryl hydrocarbon receptor nuclear translocator in concert with a novel hypoxia-inducible factor-1alpha isoform Nucleic Acids Res., October 12, 2004; 32(18): 5499 - 5511. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
