Rac1 Activity Is Required for the Activation of Hypoxia-inducible Factor 1*

Kiichi HirotaDagger § and Gregg L. Semenza§||

From the Dagger  Department of Anesthesia, Kyoto University Hospital, Kyoto University, Kyoto 606-8507, Japan and the § Institute of Genetic Medicine, Department of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914

Received for publication, January 24, 2001, and in revised form, March 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses (including erythropoiesis, angiogenesis, and glycolysis) to reduced O2 availability in mammals. Hypoxia induces both the protein expression and transcriptional activity of the HIF-1alpha subunit. However, the molecular mechanisms of sensing and signal transduction by which changes in O2 concentration result in changes in HIF-1 activity are poorly understood. We report here that the small GTPase Rac1 is activated in response to hypoxia and is required for the induction of HIF-1alpha protein expression and transcriptional activity in hypoxic cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian cells exhibit many homeostatic responses to hypoxia, including transcriptional activation of genes encoding proteins that function to increase O2 delivery and that allow metabolic adaptation under hypoxic or ischemic conditions. Although a variety of transcription factors (including AP-1, Egr-1, and nuclear factor kappa B) mediate hypoxia-inducible gene expression in specific contexts, hypoxia-inducible factor 1 (HIF-1)1 is an essential global regulator of oxygen homeostasis (1). HIF-1 is a basic helix-loop-helix/PAS (PER-ARNT-SIM homology domain) protein consisting of HIF-1alpha and HIF-1beta subunits (2). The mechanism by which HIF-1 activity is induced under hypoxic conditions remains to be established. HIF-1alpha and HIF-1beta mRNAs are constitutively expressed in cultured cells, indicating that HIF-1 activity is regulated by post-transcriptional events. HIF-1alpha protein expression and HIF-1 transcriptional activity are precisely regulated by cellular O2 concentration, whereas HIF-1beta protein is constitutively expressed (1). The molecular mechanisms of sensing and signal transduction by which changes in O2 concentration result in changes in HIF-1 activity are complex and involve regulation at multiple levels, including changes in HIF-1alpha protein stability, nuclear localization, and transactivation function in response to hypoxia (1). HIF-1alpha protein expression is negatively regulated in non-hypoxic cells by the ubiquitin-proteasome system (3). Under hypoxic conditions, HIF-1alpha protein levels increase, and the fraction that is ubiquitinated decreases (4). The carboxyl-terminal half of HIF-1alpha contains a domain that negatively regulates protein stability (5, 6) and two transactivation domains that are also negatively regulated under non-hypoxic conditions (7, 8).

Although much has been learned about the role of HIF-1 in controlling the expression of hypoxia-inducible genes, the underlying mechanisms by which cells sense a decrease in O2 concentration and transduce this signal to HIF-1alpha are largely unknown. Presently, four diverse O2-sensing mechanisms have been proposed to mediate the hypoxic transcriptional response (9). Two of these models postulate involvement of an iron-containing unit, in the form of either a heme group or an iron/sulfur cluster, that undergoes a change in activity (10). These models are supported by the observation that exposure of cells to cobaltous ion (CoCl2) or the iron chelator desferrioxamine (DFX) stabilizes HIF-1alpha under non-hypoxic conditions (1). However, no specific proteins with this role have been identified in mammalian cells. Two other models involve the generation of reactive oxygen intermediates by a flavoprotein-containing NAD(P)H oxidase or by mitochondria. In the NAD(P)H model, decreased reactive oxygen intermediate production triggers the transcriptional response to hypoxia (11, 12), whereas in the mitochondrial model, increased reactive oxygen intermediate production by the electron transport chain (ETC) is an initial trigger of the response (13-15). In these latter two models, O2 signals are converted to redox signals.

In addition to changes in cellular redox, hypoxia signal transduction may also require kinase/phosphatase activity because treatment of cells with genistein (a tyrosine kinase inhibitor) or sodium fluoride (a serine/threonine phosphatase inhibitor) blocks hypoxia-induced HIF-1alpha expression (16). In certain cell types, phosphatidylinositol 3-kinase (PI3K) inhibitors such as LY294002 and wortmannin also block hypoxia-induced HIF-1alpha expression (14, 17). Reporter assays involving expression of constitutively activated or dominant-negative forms of PI3K or Akt (protein kinase B) demonstrate that the PI3K/Akt pathway modulates hypoxia-induced HIF-1 activation and induces HIF-1 activity in non-hypoxic cells (17-19). Thus, the signaling pathway from the putative O2 sensor(s) to HIF-1 may contain several intermediate molecules.

In this study, we have focused on the Rho family small GTPase Rac1 as a potential intermediate in the hypoxia signal transduction pathway. Rac1 plays a pivotal role in multiple cellular processes, including cytoskeletal organization, gene transcription, cell proliferation, and membrane trafficking, through direct or indirect interactions with PI3K, p21-activated kinase (PAK), Ras, and p70 S6 kinase (20-23). Rac1 also regulates assembly of the active NAD(P)H oxidase complex (24). Rac1 is expressed in most cells and is recognized as a critical determinant of intracellular redox status. We demonstrate here that Rac1 is activated in response to hypoxia and plays an essential role in the induction of HIF-1alpha protein expression and transcriptional activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- Hep3B cells were maintained in minimal essential medium with Earle's salts and 10% fetal bovine serum (Life Technologies, Inc.). HEK293 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. CoCl2 and DFX were obtained from Sigma. Rotenone, diphenyleneiodonium (DPI), LY294002, wortmannin, PD98059, SB203580, and genistein were obtained from Calbiochem. Rabbit anti-phospho-p38 MAPK (Thr183/Tyr185) polyclonal antibody was obtained from New England Biolabs, Inc. (Beverly, MA).

Plasmid Construction-- Expression vectors pCEP4/HIF-1alpha , pGAL4/HIF-1alpha -(531-826), pGAL4/HIF-1alpha -(531-575), and pGAL4/HIF-1alpha -(786-826) were described previously (7). Expression vector pFA-ATF2, which encodes the GAL4 DNA-binding domain (amino acids 1-129) fused to the transactivation domain (amino acids 1-96) of the transcription factor ATF2 under control of the cytomegalovirus promoter, was obtained from Stratagene (La Jolla, CA). Reporter plasmid p2.1, harboring a 68-base pair hypoxia response element (HRE) from the ENO1 gene inserted upstream of an SV40 promoter and Photinus pyralis (firefly) luciferase coding sequences, and reporter G5E1bLuc, containing five copies of a GAL4-binding site upstream of a minimal E1b gene TATA sequence and firefly luciferase coding sequences, were described previously (7, 25). Reporter plasmid pAP-1-Luc, which contains seven tandem copies of an AP-1-binding site, was described previously (26). HA-tagged expression plasmids pBOS-HA-Rac1 (-WT, -V12, and -N17) and pBOS-HA-Cdc42 (-WT, -V12, and -N17) (27) were kindly provided by Dr. K. Kaibuchi (Nara Institute of Science and Technology). Expression vectors encoding Myc-tagged dominant-negative forms of Rho and Ras (28) were gifts from Dr. K. Irani (The Johns Hopkins University). Plasmid encoding p85Delta , a dominant-negative form of the PI3K p85 regulatory subunit (19), was a gift from Dr. A. J. Giaccia (Stanford University). Expression vector pSRalpha -HA-p38 MAPK, encoding HA-tagged p38 MAPK (29), was a gift from Dr. E. Nishida (Kyoto University).

Hypoxic Treatment-- Tissue culture dishes were transferred to a modular incubator chamber (Billups-Rothenberg, Del Mar, CA), which was flushed with 1% O2, 5% CO2, and 94% N2; sealed; and placed at 37 °C (30).

Reporter Gene Assays-- All reporter assays were performed in Hep3B cells. Cells were transferred to 24-well plates at a density of 5 × 104 cells/well on the day before transfection. Fugene-6 reagent (Roche Molecular Biochemicals) was used for transfection (31). In each transfection, the indicated doses of test plasmids, 200 ng of reporter gene plasmid, and 50 ng of control plasmid pTK-RL (containing a thymidine kinase promoter upstream of Renilla reniformis (sea pansy) luciferase coding sequences; Promega) were premixed with the transfection reagent. In each assay, the total amount of DNA was held constant by addition of empty vector. After treatment, the cells were harvested, and the luciferase activity was determined using the Dual-LuciferaseTM reporter assay system (Promega) (17). The ratio of firefly to sea pansy luciferase activity was determined for each reporter experiment; at least two independent transfections were performed in triplicate.

Immunoblot Assays of HIF-1alpha -- Whole cell lysates were prepared by incubating cells for 30 min in cold radioimmune precipitation assay buffer containing 2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 1 mM NaVO3. Samples were centrifuged at 10,000 × g to pellet cell debris. Aliquots were fractionated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot assay using protein G-purified mouse monoclonal antibody H1alpha 67 at 1:1000 dilution (4). Signal was developed using ECL reagents (Amersham Pharmacia Biotech).

PAK p21-binding Domain (PBD) and Immunoblot Assays-- HEK293 cells were transfected with pBOS-HA-Rac1. After 18 h of serum starvation, cells were exposed to 1% O2, CoCl2, or DFX. Then, cells were lysed in Mg2+ lysis/wash buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, and 2% glycerol) supplemented with EDTA-free Complete protease inhibitor mixture (Roche Molecular Biochemicals) in a controlled atmosphere chamber (Plas-Laboratories, Inc.) maintained at 1% O2. Lysates (200 µg) were incubated with 15 µg of GST-PBD (containing amino acids 69-150 of PAK1), bound to glutathione-agarose beads for 1 h at 4 °C, and washed three times with Mg2+ lysis/wash buffer. The bead pellet was finally suspended in 20 µl of Laemmli sample buffer (32). Bound proteins were fractionated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot assay using anti-HA antibody 12CA5 (Roche Molecular Biochemicals). In each experiment, to confirm that equal amounts of HA-tagged Rac1 protein were expressed, immunoblot assay of the starting lysate with the anti-HA antibody was also performed.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rac1-N17 Suppresses Hypoxia-, CoCl2-, or DFX-induced HIF-1-dependent Gene Transcription-- To examine the role of the small GTPase Rac1 in hypoxia-induced HIF-1 activation, Hep3B cells were cotransfected with reporter p2.1 containing an HIF-1-dependent HRE and an expression vector encoding either a dominant-negative (Rac1-N17) or a constitutively activated (Rac1-V12) form of Rac1. Cells were exposed to 20 or 1% O2 for 16 h and then subjected to luciferase assays. Rac1-N17 expression significantly suppressed hypoxia-induced reporter gene transcription in a dose-dependent manner (Fig. 1A). Rac1-V12 expression had a small but reproducible stimulatory effect.


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Fig. 1.   Effect of small GTPases on HIF-1-mediated gene transcription. A, Hep3B cells were transfected with pTK-RL (50 ng; encoding Renilla luciferase), p2.1 (100 ng; containing an HRE upstream of an SV40 promoter-firefly luciferase reporter gene), and the indicated amounts of expression vectors encoding either no protein (Empty vector) or a dominant-negative (Rac1-N17) or constitutively activated (Rac1-V12) form of Rac1. The total amount of expression vectors was adjusted to 500 ng with empty vector. Cells were exposed to 20 or 1% O2 for 16 h. The ratio of firefly to Renilla luciferase activity was determined and normalized to the value obtained from non-hypoxic cells transfected with empty vector to obtain the relative luciferase activity. Results shown represent means ± S.D. of three independent transfections. Reporter p2.4 contains a 3-base pair mutation that eliminates binding of HIF-1 to the HRE (30). B, Hep3B cells were transfected with pTK-RL; p2.1; and expression vector encoding no protein, Rac1-N17, Cdc42-N17, Rho-DN, or Ras-N17. Transfected cells were exposed to 1% O2, 100 µM CoCl2, or 100 µM DFX. Relative luciferase activities were determined. Results (means ± S.D. of three independent transfections) are expressed as percent of the control (empty vector).

In addition to hypoxia, HIF-1 activity is also induced in cells exposed to CoCl2 or DFX (7). Rac1-N17 expression significantly attenuated reporter gene transcription in response to 100 µM CoCl2 or DFX, although the degree of inhibition was less than the inhibition of the hypoxic response (Fig. 1B). We next tested two other members of the Rho family of small GTPases, Rho and Cdc42. As shown in Fig. 1B, Cdc42-N17 suppressed hypoxia-induced gene transcription, whereas Rho-DN did not. The dominant-negative form of another small GTPase, Ras-N17, also suppressed hypoxia-induced luciferase expression. Transcription of p2.1 in hypoxic HEK293 and NIH3T3 cells was also inhibited by Rac1-N17 (data not shown). Moreover, transcription of a reporter gene containing the HRE from the human VEGF gene in hypoxic Hep3B, HEK293, and NIH3T3 cells was also inhibited by Rac1-N17 (data not shown).

HIF-1alpha Protein Expression in Response to Hypoxia, CoCl2, or DFX Is Differentially Regulated by Rac1-- The biological activity of HIF-1 is mainly determined by the expression and activity of the HIF-1alpha subunit. HEK293 cells overexpressing Rac1-N17 were exposed to hypoxia to examine whether Rac1 is involved in the regulation of HIF-1alpha protein expression. Rac1-N17 completely suppressed the induction of HIF-1alpha expression in response to hypoxia, whereas expression of Rac1-V12 modestly enhanced the induction of HIF-1alpha in hypoxic cells (Fig. 2A). Cdc42-N17 partially suppressed hypoxia-induced expression of HIF-1alpha (Fig. 2A, lane 6), which was consistent with its effects on reporter gene expression (Fig. 1B). Under non-hypoxic conditions, neither Rac1-V12 nor Cdc42-V12 had a detectable effect on HIF-1alpha expression (Fig. 2A, lanes 7 and 8). Compared with Rac1-N17, the dominant-negative form of Rho did not affect HIF-1alpha expression (Fig. 2B). We next examined the effect of Rac1-N17 on chemical-induced HIF-1alpha expression. Neither CoCl2- nor DFX-induced HIF-1alpha expression was significantly affected by Rac1-N17 (Fig. 2C).


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Fig. 2.   Effects of small GTPases on HIF-1alpha protein expression. HEK293 cells were transfected with 4 µg of the indicated expression vector and left untreated (U) or exposed to 1% O2, 100 µM CoCl2, or 100 µM DFX for 4 h before whole cell lysate preparation. 100-µg aliquots were fractionated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot (IB) assay using anti-HIF-1alpha antibody (alpha HIF-1alpha ). The constructs analyzed were as follows: A, empty vector (EV), Rac1-V12, Rac1-N17, Cdc42-V12, and Cdc42-N17; B, empty vector, Rho-DN, and Ras-N17; C, empty vector and Rac1-N17.

HIF-1alpha -mediated Transactivation in Response to Hypoxia, CoCl2, or DFX Is Differentially Regulated by Rac1-- There are two independent transactivation domains (TADs) present in HIF-1alpha designated as the amino-terminal (amino acids 531-575) and carboxyl-terminal (amino acids 786-826) TADs (TAD-N and TAD-C, respectively) (7). Because it has been shown that steady-state levels of fusion proteins consisting of the GAL4 DNA-binding domain fused to HIF-1alpha TADs are similar under hypoxic and non-hypoxic conditions (7), these GAL4-HIF-1alpha fusion constructs can be used to examine the transcriptional activity of HIF-1alpha independent of its protein expression level. Rac1-N17 completely blocked hypoxia-induced transactivation mediated by GAL4-HIF-1alpha -(531-826), which contains both TAD-N and TAD-C (Fig. 3A). Moreover, transactivation by GAL4-HIF-1alpha -(531-826) induced by CoCl2 or DFX was also significantly suppressed by Rac1-N17 expression. It is noteworthy that CoCl2- or DFX-induced transactivation was attenuated by Rac1-N17, although CoCl2- or DFX-induced HIF-1alpha protein expression was not sensitive to Rac1-N17. Rac1-V12 expression stimulated transactivation mediated by GAL4-HIF-1alpha -(531-826), especially in cells not exposed to inducers (20% O2) (Fig. 3A), in contrast to its lack of effect on HIF-1alpha protein expression. Transactivation mediated by GAL4-HIF-1alpha -(531-575), which contains only TAD-N, was also blocked by Rac1-N17 (Fig. 3B). In contrast, GAL4-HIF-1alpha -(786-826), which contains only TAD-C, was constitutively activated and not inhibited by Rac1-N17 (Fig. 3C). These results demonstrate that Rac1 activity is specifically required for HIF-1alpha transcriptional activity that is induced by hypoxia or chemical agents.


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Fig. 3.   Effects of Rac1 on HIF-1alpha transactivation domain function. Constructs encoding the GAL4 DNA-binding domain (amino acids 1-147) fused to the indicated amino acids of HIF-1alpha were analyzed for their ability to transactivate reporter gene G5E1bLuc, containing five GAL4-binding sites. Hep3B cells were cotransfected with pTK-RL (50 ng), G5E1bLuc (100 ng), and expression vectors encoding either no protein (Empty vector) or the indicated small GTPase (250 ng) and GAL4-HIF-1alpha fusion protein (100 ng). Cells were exposed to 20% O2, 1% O2, 100 µM CoCl2, or 100 µM DFX for 16 h and then harvested. The ratio of firefly to Renilla luciferase activity was determined and normalized to the value obtained from cells transfected with plasmid encoding GAL4-(1-147) at 20% O2 to obtain the relative luciferase activity (means ± S.D. of three independent transfections).

Rac1 Is Activated in Response to Hypoxia-- Activated GTP-bound Rac1 regulates distinct downstream signaling pathways by interacting with specific effector molecules, including the serine/threonine protein kinase PAK1 (21). Using recombinant GST-PBD, which contains the Rac1-binding domain of PAK1, we examined whether Rac1 is activated in response to hypoxia. HEK293 cells overexpressing Rac1-WT or Rac1-V12 (GTP-bound) were lysed and used for an affinity precipitation assay (Fig. 4A). GST-PBD bound and precipitated the activated form of Rac1 in lysates from Rac1-V12-expressing cells (Fig. 4A, lane 3) and from Rac1-WT-expressing cells incubated with GTPgamma S (lane 2). GST-PBD did not interact with Rac1-WT loaded with GDP (Fig. 4A, lane 1) or with Rac1-N17 (data not shown). Activated Rac1 was also recovered from Rac1-WT-expressing cells treated with 500 ng/ml epidermal growth factor (Fig. 4A, lane 6) or 100 nM phorbol 12-myristate 13-acetate (lane 7). Exposure to 1% O2 for 2 h (Fig. 4A, lane 5) or to 15 min of reoxygenation after 2 h of hypoxia (lane 8) also activated Rac1. We next investigated the time course of Rac1 activation under hypoxic conditions (Fig. 4B). Rac1 was activated as early as 30 min after exposure to 1% O2 (Fig. 4B, lane 3), and this activation, although diminished after 2 h, lasted for at least 16 h of continuous hypoxia (lanes 4-8). This time course differed from that of epidermal growth factor- or phorbol 12-myristate 13-acetate-induced Rac1 activation, which lasted no more than 15 min (data not shown). Exposure of cells to 75 µM CoCl2 also activated Rac1 (Fig. 4C, lane 3). However, exposure to 130 µM DFX did not activate Rac1 (Fig. 4C, lane 4).


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Fig. 4.   Rac1 activation assay. HEK293 cells overexpressing HA-tagged Rac1-WT or Rac1-V12 were serum-starved for 16 h. A, after the indicated treatment (lane 5, 1% O2 for 2 h; lane 6, 500 ng/ml epidermal growth factor (EGF) for 10 min; lane 7, 100 mM phorbol 12-myristate 13-acetate (PMA) for 10 min; lane 8, 1% O2 for 2 h and then reoxygenation for 15 min (hypoxia-reoxygenation (H/R))), cells were harvested. In lanes 1 and 2, lysates were loaded with GDP or GTPgamma S, respectively, prior to affinity precipitation. B, after exposure to 1% O2 for the indicated times (lanes 2-8) or to 1% O2 for 2 h followed by reoxygenation for 15 min (lane 9), cells were harvested. C, after a 30-min treatment with CoCl2 or DFX, cells were harvested. Cell lysates were incubated with 15 µg of GST-PBD. Activated Rac1 was detected by GST-PBD pull-down and immunoblot (IB) assays with anti-HA antibody (alpha HA). Equal amounts of HA-tagged Rac1 proteins were detected by immunoblot assay in all lysates prior to pull-down assay.

Kinase Inhibitors Inhibit HIF-1 and Rac1 Activation in Response to Hypoxia-- To investigate potential components of the hypoxia signal transduction pathway upstream and downstream of Rac1, we first utilized a PI3K inhibitor, wortmannin. As shown in Fig. 5 (A and B, respectively), treatment with 50 nM wortmannin significantly attenuated HIF-1- and HIF-1alpha TAD-dependent transcriptional activity in response to hypoxia. Hypoxia-induced reporter gene transcription was also inhibited by p85Delta , a dominant-negative form of the PI3K p85 regulatory subunit (Fig. 5C). 10 µM PD98059, a MEK1 inhibitor, and 25 µM SB203580, a p38 MAPK inhibitor, also reduced HIF-1-dependent gene transcription (Fig. 5, A and B).


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Fig. 5.   Effect of kinase inhibitors on HIF-1 transcriptional activity. Hep3B cells were cotransfected with pTK-RL (50 ng) and either p2.1 (100 ng) (A and C) or G5E1bLuc and pGAL4/HIF-1alpha -(531-826) (B). In C, cells were cotransfected with the p85Delta expression vector or empty vector. After transfection, wortmannin (WM), PD98059 (PD), or SB203580 (SB) was added (A and B), and cells were exposed to 20 or 1% O2 (A-C) or 100 µM CoCl2 or DFX (C) for 16 h.

LY294002, wortmannin, and genistein suppressed hypoxia-induced HIF-1alpha expression (Fig. 6A). In contrast, neither PD98059 nor SB203580 affected HIF-1alpha expression (Fig. 6A, lanes 3 and 4). Rotenone and DPI, which inhibit the mitochondrial ETC at complex I, significantly attenuated the expression of HIF-1alpha in hypoxic cells (Fig. 6B), as previously reported (1, 13, 15).


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Fig. 6.   Effect of kinase inhibitors on HIF-1alpha expression. HEK293 cells were pretreated with 50 µM PD98059 (PD), 25 µM SB203580 (SB), 100 µM genistein (GS), 100 µM LY294002 (LY), 100 nM wortmannin (WT), 1 µg/ml rotenone (Rot), or 10 µM DPI and then exposed to 20 or 1% O2 for 4 h prior to whole cell lysate preparation. 100-µg aliquots were fractionated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot assay using anti-HIF-1alpha antibody.

LY294002 and genistein also inhibited Rac1 activation in response to hypoxia (Fig. 7). DPI and rotenone also markedly inhibited hypoxia-induced Rac1 activation. In contrast, neither PD98059 nor SB203580 attenuated Rac1 activation in response to hypoxia. These results, which are consistent with the analysis of HIF-1alpha expression and reporter gene transcription above (Figs. 5 and 6), demonstrate that mitochondrial ETC, tyrosine kinase, and PI3K activities are required for the activation of both Rac1 and HIF-1 in response to hypoxia.


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Fig. 7.   Effect of inhibitors on hypoxia-induced Rac1 activation. HEK293 cells overexpressing HA-tagged Rac1-WT were serum-starved for 16 h and pretreated with 1 or 10 µM DPI, 0.5 or 1 µg/ml rotenone (Rot), 5 or 50 µM LY294002 (LY), 10 or 100 µM genistein (GS), 10 or 50 µM PD98059 (PD), or 5 or 25 µM SB203580 (SB) for 1 h; exposed to 1% O2 for 30 min; and harvested. Lysates were subjected to GST-PBD pull-down assay. IB, immunoblot; alpha HA, anti-HA antibody.

p38 MAPK Is Activated in Response to Hypoxia in a Rac1-dependent Manner-- Because the p38 MAPK inhibitor SB203580 blocked HIF-1-dependent gene transcription and HIF-1alpha TAD function in a hypoxia-specific manner (Fig. 5), we examined whether p38 MAPK activation in response to hypoxia is regulated by Rac1. Hypoxia induced transactivation mediated by GAL4-ATF2-(1-96), which contains the TAD from the transcription factor ATF2 that is known to be phosphorylated by p38 MAPK (33). Rac1-N17 blocked hypoxia-induced transactivation, and Rac1-V12 expression stimulated transactivation mediated by GAL4-ATF2-(1-96) under both non-hypoxic and hypoxic conditions (Fig. 8A). We next analyzed p38 MAPK activation using a rabbit anti-phospho-p38 MAPK (Thr183/Tyr185) polyclonal antibody. Fig. 8B shows that Rac1-N17 completely blocked p38 MAPK phosphorylation in response to hypoxia.


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Fig. 8.   Effect of Rac1 on p38 MAPK activation. A, constructs encoding the GAL4 DNA-binding domain (amino acids 1-147) fused to the transactivation domain (amino acids 1-96) of ATF2 were analyzed for their ability to transactivate reporter gene G5E1bLuc, containing five GAL4-binding sites. Hep3B cells were cotransfected with pTK-RL (50 ng), G5E1bLuc (100 ng), expression vectors encoding Rac1-V12 or Rac1-N17 (250 ng), and GAL4-ATF2 fusion protein (100 ng). Cells were exposed to 20 or 1% O2 for 8 h and then harvested. The ratio of firefly to Renilla luciferase activity was determined and normalized to the value obtained from cells transfected with GAL4-ATF2 at 20% O2 to obtain the relative luciferase activity (means ± S.D. of three independent transfections). B, HEK293 cells were transfected with pSRalpha -HA-p38 MAPK and either Rac1-N17 or empty vector (pEF-BOS). Transfected cells were exposed to 20 or 1% O2 for 6 h or to 100 µM H2O2 for 15 min, and cell lysates were subjected to immunoprecipitation using anti-HA antibody matrix. Precipitates were analyzed by Western immunoblotting (IB) using anti-phospho-p38 MAPK (alpha Pp38) and anti-HA (alpha HA) antibodies.

Rac1-N17 Suppresses Hypoxia-induced AP-1-dependent Gene Transcription-- As the results for the ATF2 TAD indicate, HIF-1 is not the only transcription factor that is activated in response to hypoxia. We therefore explored the possibility that Rac1 regulates the activation of AP-1 in response to hypoxia. Hep3B cells were cotransfected with reporter pAP-1-Luc, containing seven copies of an AP-1-binding site, and expression vector encoding either the dominant-negative (Rac1-N17) or constitutively activated (Rac1-V12) form of Rac1. Cells were exposed to 20 or 1% O2 for 8 h and then subjected to luciferase assays. Rac1-N17 significantly suppressed hypoxia-induced reporter gene transcription (Fig. 9). Furthermore, Rac1-V12 strongly stimulated AP-1-dependent gene transcription in both non-hypoxic and hypoxic cells.


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Fig. 9.   Effect of Rac1 on AP-1-dependent reporter gene expression. Hep3B cells were cotransfected with pTK-RL (50 ng); pAP-1-Luc (100 ng); and expression vectors (250 ng) encoding no protein (Empty vector), Rac1-N17, or Rac1-V12. Cells were exposed to 20 or 1% O2 for 8 h and then harvested. The ratio of firefly to Renilla luciferase activity was determined and normalized to the value obtained from cells transfected with empty vector at 20% O2 to obtain the relative luciferase activity (means ± S.D. of three independent transfections).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The O2-dependent regulation of HIF-1 activity occurs at multiple levels in vivo (1). Among these, the mechanisms regulating HIF-1alpha protein expression and transcriptional activity have been most extensively analyzed. An important recent advance has been the identification of the von Hippel-Lindau tumor suppressor protein (pVHL) as the HIF-1alpha -binding component of the ubiquitin-protein ligase that targets HIF-1alpha for proteasomal degradation in non-hypoxic cells (34-38). Hypoxia may induce changes in the phosphorylation and/or redox status of HIF-1alpha , pVHL, or another component of the ubiquitination machinery. Remarkably, exposure of cells to hypoxia, CoCl2, or DFX induces both HIF-1alpha protein stabilization and transcriptional activation (7, 8), even though these agents are mechanistically distinct. For example, inhibitors of mitochondrial ETC complex I block hypoxia-induced (but not CoCl2- or DFX-induced) HIF-1alpha protein expression (1, 13). For both protein stabilization and transcriptional activation, hypoxia may induce change(s) in the phosphorylation and/or redox status of HIF-1alpha or HIF-1alpha -interacting protein(s).

Rac1 has been shown to modulate both phosphorylation and redox status via its binding to protein kinases (20, 39, 40) and to the NAD(P)H oxidase complex (24), respectively. Our data indicate that Rac1 is required for the induction of HIF-1alpha protein expression, HIF-1alpha TAD function, and HIF-1-dependent gene transcription in response to hypoxia. Although the dramatic inhibitory effects of the dominant-negative form of Rac1 (Rac1-N17) under hypoxic conditions indicate that Rac1 is necessary for these events, the modest stimulatory effects of its constitutively activated form (Rac1-V12) under non-hypoxic conditions indicate that Rac1-independent signals are also required for HIF-1 activation. Below we consider, first, the relationship of Rac1 to other putative components of the hypoxia signal transduction pathway and, second, the mechanisms by which Rac1 may regulate HIF-1alpha expression and activity.

Rac1 and Hypoxia Signal Transduction-- Previous studies have demonstrated that inhibitors of mitochondrial ETC (1, 13, 15), PI3K (13, 14, 17-19), serine/threonine protein phosphatase (14, 16), and protein-tyrosine kinase (16) activities block hypoxia-induced HIF-1alpha expression. The inhibitory effects of DPI, rotenone, LY294002, wortmannin, and genistein on the activation of Rac1 (Fig. 7) indicate that Rac1 is downstream of these putative components of the hypoxia signal transduction pathway (Fig. 10). Hypoxia does not induce PI3K activity (17), and an oxygen-regulated phosphatase or kinase that is required for HIF-1alpha expression has not been identified. Hypoxia-induced hydrogen peroxide generation that is dependent upon mitochondrial ETC activity has been reported (13, 14), but how this signal is transduced to HIF-1alpha is unknown. The present data suggest that activation of Rac1 may represent an intermediate step in this process. In contrast, the p38 MAPK activity that is induced by hypoxia is downstream of Rac1 (Fig. 10). HIF-1alpha protein expression and HIF-1 DNA-binding activity increase exponentially as cellular O2 concentration decreases and rapidly decay upon reoxygenation (2, 41, 42). In contrast, Rac1 has previously been shown to mediate the effects of hypoxia-reoxygenation on the activity of transcription factors such as nuclear factor kappa B and heat shock factor 1 via generation of reactive oxygen intermediates (43, 44). In a recent study, hypoxia-reoxygenation, but not hypoxia, was shown to induce heat shock factor 1 activation as a result of Rac1-mediated H2O2 generation (45). Thus, the involvement of Rac1 in hypoxia-induced HIF-1 activation represents a novel pathway, and delineation of both the upstream signal for Rac1 activation in response to hypoxia as well as the downstream signal leading to HIF-1 activation will require further studies.


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Fig. 10.   Signal transduction pathways leading to induction of HIF-1 activity.

Rac1 and HIF-1alpha Protein Expression-- As in the case of ETC activity, Rac1 activity is specifically required for hypoxia-induced (but not CoCl2- or DFX-induced) HIF-1alpha expression. These results are consistent with data indicating that CoCl2 and DFX directly disrupt the interaction of HIF-1alpha with pVHL (3, 4), i.e. at a step downstream of Rac1.

Rac1 and HIF-1alpha TAD Function-- The carboxyl-terminal half of HIF-1alpha consists of two TADs separated by an inhibitory domain that represses TAD function especially under non-hypoxic conditions (7, 8). TAD-N function (either in the presence or absence of the inhibitory domain) is induced by hypoxia, an effect that is dependent upon Rac1 activity (Fig. 3). In contrast, TAD-C function is independent of both O2 concentration and Rac1, again demonstrating that Rac1 is specifically required to transduce hypoxic signals to HIF-1alpha . Hypoxia also induced p38 MAPK activity in a Rac1-dependent manner, and the p38 inhibitor SB203580 attenuated hypoxia-induced TAD function (Fig. 5). Rac1 is known to interact with the MAPK kinase kinase PAK1 (20), and p38 MAPK has been shown to phosphorylate the HIF-1alpha inhibitory domain in vitro (46). Taken together, these data suggest that in response to hypoxia, activated Rac1 induces p38 MAPK activity, leading to HIF-1alpha phosphorylation and increased TAD function. Rac1-N17 completely blocked hypoxia-induced transactivation, whereas SB203580 had only a partial inhibitory effect, suggesting that in addition to p38 MAPK activation, there may be other pathways by which Rac1 induces HIF-1alpha TAD function in response to hypoxia. The p42/p44 ERK MAPKs phosphorylate HIF-1alpha and stimulate HIF-1 transcriptional activity (46, 47), but this process in not regulated by O2 concentration.

Broader Role for Rac1 in Hypoxia-induced Gene Transcription-- Hypoxia induces the activity of multiple transcription factors in addition to HIF-1. Transcription of an AP-1-dependent reporter gene was induced by hypoxia, and the induction was specifically blocked by Rac1-N17 (Fig. 9), as in the case of the HIF-1-dependent reporter gene (Fig. 1). However, Rac1-V12 markedly induced AP-1-dependent transcription under both hypoxic and non-hypoxic conditions, whereas it had only a minor effect on HIF-1-dependent transcription. These data indicate that Rac1 plays an important role in hypoxia signal transduction in other systems, although the specific mechanisms of transcriptional regulation involved may differ. With these results as a foundation, future studies will be necessary to further delineate the mechanisms and consequences of Rac1 activation in response to hypoxia.

    ACKNOWLEDGEMENTS

We thank Drs. Kozo Kaibuchi, Kaikobad Irani, Amato J. Giaccia, and Eisuke Nishida for providing plasmids and Erik Laughner for excellent technical advice.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants R01-HL55338 and R01-DK39869 (to G. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by the Yamanouchi Foundation for Research on Metabolic Disorders and by a fellowship from the Uehara Memorial Foundation.

|| To whom correspondence should be addressed. Fax: 410-955-0484; E-mail:gsemenza@jhmi.edu.

Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M100677200

    ABBREVIATIONS

The abbreviations used are: HIF-1, hypoxia-inducible factor 1; DFX, desferrioxamine; ETC, electron transport chain; PI3K, phosphatidylinositol 3-kinase; PAK, p21-activated kinase; DPI, diphenyleneiodonium; MAPK, mitogen-activated protein kinase; ATF, activating transcription factor; HRE, hypoxia response element; WT, wild-type; HA, hemagglutinin; PBD, p21-binding domain of PAK; GST, glutathione S-transferase; DN, dominant-negative; TAD, transactivation domain; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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