FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091917736> ?p ?o ?g. }

• W2091917736 endingPage "21172" @default.
• W2091917736 startingPage "21166" @default.
• W2091917736 abstract "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-1α 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-1α protein expression and transcriptional activity in hypoxic cells. 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-1α 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-1α protein expression and transcriptional activity in hypoxic cells. hypoxia-inducible factor 1 desferrioxamine electron transport chain phosphatidylinositol 3-kinase p21-activated kinase diphenyleneiodonium mitogen-activated protein kinase activating transcription factor hypoxia response element wild-type hemagglutinin p21-binding domain of PAK glutathione S-transferase dominant-negative transactivation domain guanosine 5′-O-(3-thiotriphosphate) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase 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 κB) mediate hypoxia-inducible gene expression in specific contexts, hypoxia-inducible factor 1 (HIF-1)1 is an essential global regulator of oxygen homeostasis (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar). HIF-1 is a basic helix-loop-helix/PAS (PER-ARNT-SIM homology domain) protein consisting of HIF-1α and HIF-1β subunits (2Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar). The mechanism by which HIF-1 activity is induced under hypoxic conditions remains to be established. HIF-1α and HIF-1β mRNAs are constitutively expressed in cultured cells, indicating that HIF-1 activity is regulated by post-transcriptional events. HIF-1α protein expression and HIF-1 transcriptional activity are precisely regulated by cellular O2concentration, whereas HIF-1β protein is constitutively expressed (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar). 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-1α protein stability, nuclear localization, and transactivation function in response to hypoxia (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar). HIF-1α protein expression is negatively regulated in non-hypoxic cells by the ubiquitin-proteasome system (3Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar). Under hypoxic conditions, HIF-1α protein levels increase, and the fraction that is ubiquitinated decreases (4Sutter C.H. Laughner E. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4748-4753Crossref PubMed Scopus (263) Google Scholar). The carboxyl-terminal half of HIF-1α contains a domain that negatively regulates protein stability (5Jiang B.-H. Rue E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; 271: 17771-17778Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 6Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar) and two transactivation domains that are also negatively regulated under non-hypoxic conditions (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 8Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliff P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). 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-1α are largely unknown. Presently, four diverse O2-sensing mechanisms have been proposed to mediate the hypoxic transcriptional response (9Semenza G.L. Cell. 1999; 98: 281-284Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). 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 (10Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1998; 242: 1412-1415Crossref Scopus (865) Google Scholar). These models are supported by the observation that exposure of cells to cobaltous ion (CoCl2) or the iron chelator desferrioxamine (DFX) stabilizes HIF-1α under non-hypoxic conditions (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar). 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 (11Fandrey J. Frede S. Jelkmann W. Biochem. J. 1994; 303: 507-510Crossref PubMed Scopus (223) Google Scholar, 12Ehleben W. Bolling B. Merten E. Porwol T. Strohmaier A.R. Acker H. Respir. Physiol. 1998; 114: 25-36Crossref PubMed Scopus (46) Google Scholar), whereas in the mitochondrial model, increased reactive oxygen intermediate production by the electron transport chain (ETC) is an initial trigger of the response (13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar, 14Chandel N.S. McClintock D.S. Feliciano S.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar, 15Agani F.H. Pichiule P. Chavez J.C. LaManna J.C. J. Biol. Chem. 2000; 275: 35863-35867Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). 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-1α expression (16Wang G.L. Jiang B.-H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 216: 669-675Crossref PubMed Scopus (212) Google Scholar). In certain cell types, phosphatidylinositol 3-kinase (PI3K) inhibitors such as LY294002 and wortmannin also block hypoxia-induced HIF-1α expression (14Chandel N.S. McClintock D.S. Feliciano S.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar, 17Zhong H. Chiles K. Felser D. Laughner D. Hanrahan C. Georgescu M. Simon J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar). 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 (17Zhong H. Chiles K. Felser D. Laughner D. Hanrahan C. Georgescu M. Simon J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 18Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 19Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar). 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 (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 21Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (883) Google Scholar, 22Liliental J. Moon S.Y. Lesche R. Mamillapalli R. Li D. Zheng Y. Sun H. Wu H. Curr. Biol. 2000; 10: 401-404Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 23Sun H. King A.J. Diaz H.B. Marshall M.S. Curr. Biol. 2000; 10: 281-284Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Rac1 also regulates assembly of the active NAD(P)H oxidase complex (24Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2578) Google Scholar). 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-1α protein expression and transcriptional activity. 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). Expression vectors pCEP4/HIF-1α, pGAL4/HIF-1α-(531–826), pGAL4/HIF-1α-(531–575), and pGAL4/HIF-1α-(786–826) were described previously (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). 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 theENO1 gene inserted upstream of an SV40 promoter andPhotinus 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 (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 25Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1319) Google Scholar). Reporter plasmid pAP-1-Luc, which contains seven tandem copies of an AP-1-binding site, was described previously (26Hirota K. Matsui M. Murata M. Takashima Y. Chen F.S. Itoh T. Fukuda K. Yodoi J. Biochem. Biophys. Res. Commun. 2000; 274: 177-182Crossref PubMed Scopus (168) Google Scholar). HA-tagged expression plasmids pBOS-HA-Rac1 (-WT, -V12, and -N17) and pBOS-HA-Cdc42 (-WT, -V12, and -N17) (27Kuroda S. Fukata M. Kobayashi K. Nakafuku K. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) 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 (28Irani K. Xia Y. Zweier J.L. Sollot S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1418) Google Scholar) were gifts from Dr. K. Irani (The Johns Hopkins University). Plasmid encoding p85Δ, a dominant-negative form of the PI3K p85 regulatory subunit (19Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar), was a gift from Dr. A. J. Giaccia (Stanford University). Expression vector pSRα-HA-p38 MAPK, encoding HA-tagged p38 MAPK (29Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1989) Google Scholar), was a gift from Dr. E. Nishida (Kyoto University). 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 (30Forsythe J.A. Jiang B.-H. Iyer N.V. Agani F. Leung S.W. Koons R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3135) Google Scholar). 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 (31Hirota K. Murata M. Sachi Y. Nakamura H. Takeuchi J. Mori K. Yodoi J. J. Biol. Chem. 1999; 274: 27891-27897Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). 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 ofRenilla 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) (17Zhong H. Chiles K. Felser D. Laughner D. Hanrahan C. Georgescu M. Simon J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar). The ratio of firefly to sea pansy luciferase activity was determined for each reporter experiment; at least two independent transfections were performed in triplicate. 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 H1α67 at 1:1000 dilution (4Sutter C.H. Laughner E. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4748-4753Crossref PubMed Scopus (263) Google Scholar). Signal was developed using ECL reagents (Amersham Pharmacia Biotech). 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 mmNaCl, 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 (32Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). 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. 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‥ O2for 16 h and then subjected to luciferase assays. Rac1-N17 expression significantly suppressed hypoxia-induced reporter gene transcription in a dose-dependent manner (Fig.1 A). Rac1-V12 expression had a small but reproducible stimulatory effect. In addition to hypoxia, HIF-1 activity is also induced in cells exposed to CoCl2 or DFX (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). 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.1 B). We next tested two other members of the Rho family of small GTPases, Rho and Cdc42. As shown in Fig. 1 B, 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). The biological activity of HIF-1 is mainly determined by the expression and activity of the HIF-1α subunit. HEK293 cells overexpressing Rac1-N17 were exposed to hypoxia to examine whether Rac1 is involved in the regulation of HIF-1α protein expression. Rac1-N17 completely suppressed the induction of HIF-1α expression in response to hypoxia, whereas expression of Rac1-V12 modestly enhanced the induction of HIF-1α in hypoxic cells (Fig.2 A). Cdc42-N17 partially suppressed hypoxia-induced expression of HIF-1α (Fig. 2 A,lane 6), which was consistent with its effects on reporter gene expression (Fig. 1 B). Under non-hypoxic conditions, neither Rac1-V12 nor Cdc42-V12 had a detectable effect on HIF-1α expression (Fig. 2 A, lanes 7 and 8). Compared with Rac1-N17, the dominant-negative form of Rho did not affect HIF-1α expression (Fig. 2 B). We next examined the effect of Rac1-N17 on chemical-induced HIF-1α expression. Neither CoCl2- nor DFX-induced HIF-1α expression was significantly affected by Rac1-N17 (Fig. 2 C). There are two independent transactivation domains (TADs) present in HIF-1α designated as the amino-terminal (amino acids 531–575) and carboxyl-terminal (amino acids 786–826) TADs (TAD-N and TAD-C, respectively) (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). Because it has been shown that steady-state levels of fusion proteins consisting of the GAL4 DNA-binding domain fused to HIF-1α TADs are similar under hypoxic and non-hypoxic conditions (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar), these GAL4-HIF-1α fusion constructs can be used to examine the transcriptional activity of HIF-1α independent of its protein expression level. Rac1-N17 completely blocked hypoxia-induced transactivation mediated by GAL4-HIF-1α-(531–826), which contains both TAD-N and TAD-C (Fig.3 A). Moreover, transactivation by GAL4-HIF-1α-(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-1α protein expression was not sensitive to Rac1-N17. Rac1-V12 expression stimulated transactivation mediated by GAL4-HIF-1α-(531–826), especially in cells not exposed to inducers (20‥ O2) (Fig.3 A), in contrast to its lack of effect on HIF-1α protein expression. Transactivation mediated by GAL4-HIF-1α-(531–575), which contains only TAD-N, was also blocked by Rac1-N17 (Fig. 3 B). In contrast, GAL4-HIF-1α-(786–826), which contains only TAD-C, was constitutively activated and not inhibited by Rac1-N17 (Fig.3 C). These results demonstrate that Rac1 activity is specifically required for HIF-1α transcriptional activity that is induced by hypoxia or chemical agents. Activated GTP-bound Rac1 regulates distinct downstream signaling pathways by interacting with specific effector molecules, including the serine/threonine protein kinase PAK1 (21Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (883) Google Scholar). 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. 4 A). GST-PBD bound and precipitated the activated form of Rac1 in lysates from Rac1-V12-expressing cells (Fig. 4 A, lane 3) and from Rac1-WT-expressing cells incubated with GTPγS (lane 2). GST-PBD did not interact with Rac1-WT loaded with GDP (Fig.4 A, 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. 4 A, lane 6) or 100 nm phorbol 12-myristate 13-acetate (lane 7). Exposure to 1‥ O2 for 2 h (Fig.4 A, 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. 4 B). Rac1 was activated as early as 30 min after exposure to 1‥ O2 (Fig. 4 B, 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.4 C, lane 3). However, exposure to 130 μm DFX did not activate Rac1 (Fig. 4 C,lane 4). 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-1α TAD-dependent transcriptional activity in response to hypoxia. Hypoxia-induced reporter gene transcription was also inhibited by p85Δ, a dominant-negative form of the PI3K p85 regulatory subunit (Fig.5 C). 10 μm PD98059, a MEK1 inhibitor, and 25 μm SB203580, a p38 MAPK inhibitor, also reduced HIF-1-dependent gene transcription (Fig. 5, Aand B). LY294002, wortmannin, and genistein suppressed hypoxia-induced HIF-1α expression (Fig. 6 A). In contrast, neither PD98059 nor SB203580 affected HIF-1α expression (Fig. 6 A, lanes 3 and 4). Rotenone and DPI, which inhibit the mitochondrial ETC at complex I, significantly attenuated the expression of HIF-1α in hypoxic cells (Fig. 6 B), as previously reported (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar, 13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar, 15Agani F.H. Pichiule P. Chavez J.C. LaManna J.C. J. Biol. Chem. 2000; 275: 35863-35867Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). 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-1α 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. Because the p38 MAPK inhibitor SB203580 blocked HIF-1-dependent gene transcription and HIF-1α 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 (33Gupta S. Campbell D. Dérijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar). 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.8 A). We next analyzed p38 MAPK activation using a rabbit anti-phospho-p38 MAPK (Thr183/Tyr185) polyclonal antibody. Fig.8 B shows that Rac1-N17 completely blocked p38 MAPK phosphorylation in response to hypoxia. 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. The O2-dependent regulation of HIF-1 activity occurs at multiple levels in vivo (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar). Among these, the mechanisms regulating HIF-1α 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-1α-binding component of the ubiquitin-protein ligase that targets HIF-1α for proteasomal degradation in non-hypoxic cells (34Maxwell P.H. Wiesener M.S. Chang G.-W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Mahler E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4025) Google Scholar, 35Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.-W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar, 36Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (544) Google Scholar, 37Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1238) Google Scholar, 38Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar). Hypoxia may induce changes in the phosphorylation and/or redox status of HIF-1α, pVHL, or another component of the ubiquitination machinery. Remarkably, exposure of cells to hypoxia, CoCl2, or DFX induces both HIF-1α protein stabilization and transcriptional activation (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 8Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliff P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), 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-1α protein expression (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar,13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar). For both protein stabilization and transcriptional activation, hypoxia may induce change(s) in the phosphorylation and/or redox status of HIF-1α or HIF-1α-interacting protein(s). Rac1 has been shown to modulate both phosphorylation and redox status via its binding to protein kinases (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 39Coso O.A. Chiariello M., Yu, J.-C. Teramoto H. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1555) Google Scholar, 40Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1442) Google Scholar) and to the NAD(P)H oxidase complex (24Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2578) Google Scholar), respectively. Our data indicate that Rac1 is required for the induction of HIF-1α protein expression, HIF-1α 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-1α expression and activity. Previous studies have demonstrated that inhibitors of mitochondrial ETC (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1637) Google Scholar, 13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar, 15Agani F.H. Pichiule P. Chavez J.C. LaManna J.C. J. Biol. Chem. 2000; 275: 35863-35867Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), PI3K (13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar, 14Chandel N.S. McClintock D.S. Feliciano S.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar, 17Zhong H. Chiles K. Felser D. Laughner D. Hanrahan C. Georgescu M. Simon J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 18Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 19Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar), serine/threonine protein phosphatase (14Chandel N.S. McClintock D.S. Feliciano S.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar, 16Wang G.L. Jiang B.-H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 216: 669-675Crossref PubMed Scopus (212) Google Scholar), and protein-tyrosine kinase (16Wang G.L. Jiang B.-H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 216: 669-675Crossref PubMed Scopus (212) Google Scholar) activities block hypoxia-induced HIF-1α 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 (17Zhong H. Chiles K. Felser D. Laughner D. Hanrahan C. Georgescu M. Simon J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar), and an oxygen-regulated phosphatase or kinase that is required for HIF-1α expression has not been identified. Hypoxia-induced hydrogen peroxide generation that is dependent upon mitochondrial ETC activity has been reported (13Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5015-5019Crossref PubMed Scopus (1559) Google Scholar, 14Chandel N.S. McClintock D.S. Feliciano S.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar), but how this signal is transduced to HIF-1α 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-1α protein expression and HIF-1 DNA-binding activity increase exponentially as cellular O2 concentration decreases and rapidly decay upon reoxygenation (2Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar, 41Wang G.L. Semenza G.L. J. Biol. Chem. 1993; 268: 21513-21518Abstract Full Text PDF PubMed Google Scholar, 42Jiang B.-H. Semenza G.L. Bauer C. Marti H.H. Am. J. Physiol. 1996; 271: C1172-C1180Crossref PubMed Google Scholar). In contrast, Rac1 has previously been shown to mediate the effects of hypoxia-reoxygenation on the activity of transcription factors such as nuclear factor κB and heat shock factor 1 via generation of reactive oxygen intermediates (43Kim K.S. Takeda K. Sethi R. Pracyk J.B. Tanaka K. Zhou Y.F., Yu, Z.X. Ferrans V.J. Bruder J.T. Kovesdi I. Irani K. Goldschmidt-Clermont P. Finkel T. J. Clin. Invest. 1998; 101: 1821-1826Crossref PubMed Scopus (136) Google Scholar, 44Ozaki M. Deshpande S.S. Angkeow P. Bellan J. Lowenstein C.J. Dinauer M.C. Goldschmidt-Clermont P.J. Irani K. FASEB J. 2000; 14: 418-429Crossref PubMed Scopus (123) Google Scholar). 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 (45Ozaki M. Deshpande S.S. Angkeow P. Suzuki S. Irani K. J. Biol. Chem. 2000; 275: 35377-35383Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 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. As in the case of ETC activity, Rac1 activity is specifically required for hypoxia-induced (but not CoCl2- or DFX-induced) HIF-1α expression. These results are consistent with data indicating that CoCl2 and DFX directly disrupt the interaction of HIF-1α with pVHL (3Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar, 4Sutter C.H. Laughner E. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4748-4753Crossref PubMed Scopus (263) Google Scholar),i.e. at a step downstream of Rac1. The carboxyl-terminal half of HIF-1α consists of two TADs separated by an inhibitory domain that represses TAD function especially under non-hypoxic conditions (7Jiang B.-H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 8Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliff P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). 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-1α. 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 (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), and p38 MAPK has been shown to phosphorylate the HIF-1α inhibitory domain in vitro (46Sodhi A. Montaner S. Patel V. Zohar M. Bais C. Mesri E.A. Gutkind J.S. Cancer Res. 2000; 60: 4873-4880PubMed Google Scholar). Taken together, these data suggest that in response to hypoxia, activated Rac1 induces p38 MAPK activity, leading to HIF-1α 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-1α TAD function in response to hypoxia. The p42/p44 ERK MAPKs phosphorylate HIF-1α and stimulate HIF-1 transcriptional activity (46Sodhi A. Montaner S. Patel V. Zohar M. Bais C. Mesri E.A. Gutkind J.S. Cancer Res. 2000; 60: 4873-4880PubMed Google Scholar, 47Richard D.E. Berra E. Gothié E. Roux D. Pouysséugur J. J. Biol. Chem. 1999; 274: 32631-32637Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), but this process in not regulated by O2 concentration. 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. We thank Drs. Kozo Kaibuchi, Kaikobad Irani, Amato J. Giaccia, and Eisuke Nishida for providing plasmids and Erik Laughner for excellent technical advice." @default.
• W2091917736 created "2016-06-24" @default.
• W2091917736 creator A5012061869 @default.
• W2091917736 creator A5051276665 @default.
• W2091917736 date "2001-06-01" @default.
• W2091917736 modified "2023-10-10" @default.
• W2091917736 title "Rac1 Activity Is Required for the Activation of Hypoxia-inducible Factor 1" @default.
• W2091917736 cites W1510509865 @default.
• W2091917736 cites W1547863599 @default.
• W2091917736 cites W1721827275 @default.
• W2091917736 cites W1749022239 @default.
• W2091917736 cites W177380973 @default.
• W2091917736 cites W1891283175 @default.
• W2091917736 cites W1937401270 @default.
• W2091917736 cites W1964037605 @default.
• W2091917736 cites W1964918262 @default.
• W2091917736 cites W1972032694 @default.
• W2091917736 cites W1972317299 @default.
• W2091917736 cites W1972681225 @default.
• W2091917736 cites W1973517763 @default.
• W2091917736 cites W1974030744 @default.
• W2091917736 cites W1984665850 @default.
• W2091917736 cites W1988140254 @default.
• W2091917736 cites W1991843895 @default.
• W2091917736 cites W1997031088 @default.
• W2091917736 cites W2000624133 @default.
• W2091917736 cites W2004427637 @default.
• W2091917736 cites W2005613270 @default.
• W2091917736 cites W2010394923 @default.
• W2091917736 cites W2022773473 @default.
• W2091917736 cites W2023172513 @default.
• W2091917736 cites W2034580062 @default.
• W2091917736 cites W2042584344 @default.
• W2091917736 cites W2045055190 @default.
• W2091917736 cites W2046440184 @default.
• W2091917736 cites W2055181868 @default.
• W2091917736 cites W2061352347 @default.
• W2091917736 cites W2072569792 @default.
• W2091917736 cites W2080145523 @default.
• W2091917736 cites W2080374319 @default.
• W2091917736 cites W2085609942 @default.
• W2091917736 cites W2102526923 @default.
• W2091917736 cites W2105286896 @default.
• W2091917736 cites W2123799914 @default.
• W2091917736 cites W2129873928 @default.
• W2091917736 cites W2147718842 @default.
• W2091917736 cites W2149268957 @default.
• W2091917736 cites W2149668250 @default.
• W2091917736 cites W2154975058 @default.
• W2091917736 cites W2161877652 @default.
• W2091917736 cites W2162049443 @default.
• W2091917736 cites W2163069245 @default.
• W2091917736 doi "https://doi.org/10.1074/jbc.m100677200" @default.
• W2091917736 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11283021" @default.
• W2091917736 hasPublicationYear "2001" @default.
• W2091917736 type Work @default.
• W2091917736 sameAs 2091917736 @default.
• W2091917736 citedByCount "155" @default.
• W2091917736 countsByYear W20919177362012 @default.
• W2091917736 countsByYear W20919177362013 @default.
• W2091917736 countsByYear W20919177362014 @default.
• W2091917736 countsByYear W20919177362015 @default.
• W2091917736 countsByYear W20919177362016 @default.
• W2091917736 countsByYear W20919177362017 @default.
• W2091917736 countsByYear W20919177362018 @default.
• W2091917736 countsByYear W20919177362019 @default.
• W2091917736 countsByYear W20919177362020 @default.
• W2091917736 countsByYear W20919177362021 @default.
• W2091917736 countsByYear W20919177362022 @default.
• W2091917736 countsByYear W20919177362023 @default.
• W2091917736 crossrefType "journal-article" @default.
• W2091917736 hasAuthorship W2091917736A5012061869 @default.
• W2091917736 hasAuthorship W2091917736A5051276665 @default.
• W2091917736 hasBestOaLocation W20919177361 @default.
• W2091917736 hasConcept C104317684 @default.
• W2091917736 hasConcept C178790620 @default.
• W2091917736 hasConcept C185592680 @default.
• W2091917736 hasConcept C2779717556 @default.
• W2091917736 hasConcept C2909350873 @default.
• W2091917736 hasConcept C540031477 @default.
• W2091917736 hasConcept C55493867 @default.
• W2091917736 hasConcept C62478195 @default.
• W2091917736 hasConcept C7836513 @default.
• W2091917736 hasConcept C86339819 @default.
• W2091917736 hasConcept C86803240 @default.
• W2091917736 hasConcept C95444343 @default.
• W2091917736 hasConceptScore W2091917736C104317684 @default.
• W2091917736 hasConceptScore W2091917736C178790620 @default.
• W2091917736 hasConceptScore W2091917736C185592680 @default.
• W2091917736 hasConceptScore W2091917736C2779717556 @default.
• W2091917736 hasConceptScore W2091917736C2909350873 @default.
• W2091917736 hasConceptScore W2091917736C540031477 @default.
• W2091917736 hasConceptScore W2091917736C55493867 @default.
• W2091917736 hasConceptScore W2091917736C62478195 @default.
• W2091917736 hasConceptScore W2091917736C7836513 @default.
• W2091917736 hasConceptScore W2091917736C86339819 @default.
• W2091917736 hasConceptScore W2091917736C86803240 @default.
• W2091917736 hasConceptScore W2091917736C95444343 @default.

• next