Mitogen-Activated Protein 3 Kinase 6 Mediates Angiogenic and Tumorigenic Effects via Vascular Endothelial Growth Factor Expression
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080190
ISSN1525-2191
AutoresNobuaki Eto, Makoto Miyagishi, Reiko Inagi, Toshiro Fujita, Masaomi Nangaku,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoGenome-wide screening using a small interfering RNA (siRNA) library has revealed novel molecules that are involved in a wide range of physiological responses. The expression of vascular endothelial growth factor (VEGF) is increased under hypoxic conditions, and plays a crucial role in tumor angiogenesis and tissue responses to ischemia. Here, we used a siRNA expression vector library to elucidate molecules that modify VEGF expression. Screening using an siRNA library revealed that MAPKKK6 (MEKK6/MAP3K6) regulates VEGF expression under both normoxic and hypoxic conditions in vitro, although the biological function of MAP3K6 remains unknown. Attenuation of VEGF expression by MAP3K6 inhibition was demonstrated by transient transfection of double-stranded RNA as well as by stable transfection of short hairpin RNA-expressing vectors against MAP3K6. Conditioned medium of MAP3K6-knocked down cells attenuated both endothelial proliferation and capillary network formation in a VEGF-dependent manner in vitro. In addition, tumor cells with down-regulation of MAP3K6 expression showed significant suppression of tumor growth in vivo, which was accompanied by significant repression of vessel formation and VEGF expression in these tumors. The results of this study suggest that MAP3K6 regulates VEGF expression in both normoxia and hypoxia, and that regulation of VEGF by MAP3K6 may play a crucial role in both angiogenesis and tumorigenesis. Genome-wide screening using a small interfering RNA (siRNA) library has revealed novel molecules that are involved in a wide range of physiological responses. The expression of vascular endothelial growth factor (VEGF) is increased under hypoxic conditions, and plays a crucial role in tumor angiogenesis and tissue responses to ischemia. Here, we used a siRNA expression vector library to elucidate molecules that modify VEGF expression. Screening using an siRNA library revealed that MAPKKK6 (MEKK6/MAP3K6) regulates VEGF expression under both normoxic and hypoxic conditions in vitro, although the biological function of MAP3K6 remains unknown. Attenuation of VEGF expression by MAP3K6 inhibition was demonstrated by transient transfection of double-stranded RNA as well as by stable transfection of short hairpin RNA-expressing vectors against MAP3K6. Conditioned medium of MAP3K6-knocked down cells attenuated both endothelial proliferation and capillary network formation in a VEGF-dependent manner in vitro. In addition, tumor cells with down-regulation of MAP3K6 expression showed significant suppression of tumor growth in vivo, which was accompanied by significant repression of vessel formation and VEGF expression in these tumors. The results of this study suggest that MAP3K6 regulates VEGF expression in both normoxia and hypoxia, and that regulation of VEGF by MAP3K6 may play a crucial role in both angiogenesis and tumorigenesis. 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The use of genome-wide RNAi screening in C. elegans using libraries of in vitro-transcribed long double-stranded (ds) RNAs has proven extremely useful in gene discovery and functional annotation in various processes, including early embryonic development, lethality, sterility, genome instability, and longevity.20Fraser AG Kamath RS Zipperlen P Martinez-Campos M Sohrmann M Ahringer J Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.Nature. 2000; 408: 325-330Crossref PubMed Scopus (1401) Google Scholar, 21Lee SS Lee RY Fraser AG Kamath RS Ahringer J Ruvkun G A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.Nat Genet. 2003; 33: 40-48Crossref PubMed Scopus (785) Google Scholar, 22Kamath RS Fraser AG Dong Y Poulin G Durbin R Gotta M Kanapin A Le Bot N Moreno S Sohrmann M Welchman DP Zipperlen P Ahringer J Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.Nature. 2003; 421: 231-237Crossref PubMed Scopus (2807) Google Scholar Until recently, however, loss-of-function genetic screening in mammalian cells was hampered because dsRNAs also have nonspecific inhibitory effects, which are known as the interferon response. Although the use of synthetic siRNAs allows cells to escape the interferon response,23Elbashir SM Harborth J Lendeckel W Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8266) Google Scholar vector-based RNAi is also useful in maintaining RNAi activity for much longer periods in stable transfection studies.24Yu JY DeRuiter SL Turner DL RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci USA. 2002; 99: 6047-6052Crossref PubMed Scopus (966) Google Scholar, 25Matsumoto S Miyagishi M Akashi H Nagai R Taira K Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.J Biol Chem. 2005; 280: 25687-25696Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 26Paul CP Good PD Winer I Engelke DR Effective expression of small interfering RNA in human cells.Nat Biotechnol. 2002; 20: 505-508Crossref PubMed Scopus (758) Google Scholar, 27Lee NS Dohjima T Bauer G Li H Li MJ Ehsani A Salvaterra P Rossi J Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.Nat Biotechnol. 2002; 20: 500-505Crossref PubMed Scopus (145) Google Scholar, 28Miyagishi M Taira K U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol. 2002; 20: 497-500Crossref PubMed Scopus (681) Google Scholar In this study, we attempted to identify novel regulatory components of the VEGF expression pathway by screening 320 short hairpin RNA expression vectors that target genes for kinases and transcription factors. We identified MAP3K6 as a novel regulator of VEGF expression. Although MAP3K6 was found to weakly activate JNK,29Wang XS Diener K Tan TH Yao Z MAPKKK6, a novel mitogen-activated protein kinase kinase kinase, that associates with MAPKKK5.Biochem Biophys Res Commun. 1998; 253: 33-37Crossref PubMed Scopus (53) Google Scholar its biological validity has not been well documented. Our data demonstrate that regulation of VEGF expression by MAP3K6 plays a crucial role in angiogenesis. HEK293T and HeLa S3 cells were cultured in Dulbecco's modified Eagle's medium (Nissui, Tokyo, Japan) buffered with 25 mmol/L HEPES (Sigma, St. Louis, MO) at pH 7.4, supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C under a humidified atmosphere of 5% CO2/95% air. Hypoxic conditions were established at 0.2% O2 with an Anaerocult A mini pack (Merck KGaA, Darmstadt, Germany), and at 1.0% by exposure to 1.0% O2/5% CO2, with the balance as nitrogen, in an APM-30D multigas incubator (Astec, Fukuoka, Japan). Rat kidney vascular endothelial cells30Adler S Eng B Integrin receptors and function on cultured glomerular endothelial cells.Kidney Int. 1993; 44: 278-284Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 31Tanaka T Kojima I Ohse T Ingelfinger JR Adler S Fujita T Nangaku M Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model.Lab Invest. 2005; 85: 1292-1307Crossref PubMed Scopus (200) Google Scholar were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 10% NuSerum (BD Biosciences, San Jose, CA). To construct an optimal vector that reflects VEGF expression in a hypoxic milieu, we constructed a luciferase reporter vector by incorporating the promoter region of human VEGF into the pGL4-basic plasmid (Promega, Madison, WI), termed pGL4-VEGFp. To obtain a 2.7-kbp human VEGF promoter region, human genomic DNA was amplified in a two-step polymerase chain reaction (PCR). In the first step, PCR amplification was done using (forward, 5′-ACCTCCACCAAACCACAGCAACAT G-3′ and reverse, 5′-TCGCACGCACGTCCCCAGCCG-3′) synthetic oligonucleotides, with the annealing/extension step set at 68°C for 360 seconds. In the second step, the first PCR product was amplified using forward, 5′-GCGCTCGAGTCCCATTCTCAGTCCATGCCTCCAC-3′ and reverse, 5′-AAGTCCATGGTTTCGGAGGCCCGACCG-3′ synthetic oligonucleotides, with the annealing/extension step set at 68°C for 360 seconds. The obtained fragment was digested with XhoI and NcoI, and then cloned into pGL4-basic at XhoI through NcoI sites. The insert was confirmed to be identical to the human VEGF promoter by sequence analysis. Further, to construct an optimal vector that responds maximally in a hypoxic milieu, we constructed a second luciferase reporter vector by incorporating seven tandem repeats of HIF-responsive elements (HREs) and a human minimal CMV promoter (hmCMVp) into the pGL3-basic plasmid (Promega), to give pHRE-Luc.32Tanaka T Kojima I Ohse T Inagi R Miyata T Ingelfinger JR Fujita T Nangaku M Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity.Am J Physiol. 2005; 289: F1123-F1133Crossref PubMed Scopus (92) Google Scholar In brief, complementary oligonucleotides containing a HRE and NheI recognition site at both ends (5′-CTAGCCCACAGTGCATACGTGGGCTTCCACAGGTCGTCTG-3′ and 5′-CTAGCAGACGACCTGTGGAAGCCCACGTATGCACTGTGGG-3′) were synthesized, annealed, and cloned into pGL3-basic at NheI through BglII sites in tandem repeats, directly or via blunt-ended ligation. A synthetic fragment composed of hmCMVp was inserted at the BglII-HindIII site. The short hairpin type of a siRNA (shRNA) expression plasmid library was purchased from iGENE Therapeutics Inc. (Tsukuba, Ibaraki, Japan).25Matsumoto S Miyagishi M Akashi H Nagai R Taira K Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.J Biol Chem. 2005; 280: 25687-25696Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar In brief, the vector includes a human U6 promoter, a puromycin resistance gene, and BspMI cloning sites and is termed pcPUR hU6. Synthetic oligonucleotides in which sense and antisense nucleotides were connected by an 11-base hairpin loop were formed as a single chain. After annealing, DNA fragments were ligated into the BspMI sites of pcPUR hU6. Target sites of oligonucleotides for each gene were predicted by an algorithm developed by the manufacturer. HEK293T cells (1 × 105) were cultured in a 96-well plate in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 1% antibiotics. Mixtures of three plasmids, ie, 400 ng of siRNA library, 400 ng of pGL4-VEGFp, and 3 ng of pCMV-Renilla were co-transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described in the manufacturer's protocol. Forty-eight hours after transfection, fresh medium was added to the cells followed by exposure to 0.2% O2. After 24 hours of incubation, the cells were assayed by a dual luciferase assay system (Promega), with luciferase activity normalized by Renilla gene activity. The pcPUR hU6 vector containing a targeted sequence against the HIF-1α gene and seven tandem thymidine repeats (T7) served as positive and negative control, respectively. Screening was performed in duplicate. Hit clones were considered to be those which suppressed VEGF reporter activity in comparison with the negative control in three independent experiments. VEGF concentration in the culture medium was determined by an ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The data were adjusted by the total protein amount of the cells. Total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) and reverse-transcribed with an Im-Prom II reverse transcription kit (Promega). A measure of one-twentieth (v/v) cDNA was used as a template for subsequent quantification. PCR was run on an iCycler (Bio-Rad, Hercules, CA) using iQt SYBR Green PCR supermix (Bio-Rad). The relative amount of VEGF and MAP3K6 gene expression was calculated and corrected for that of ribosomal protein LS28. The sets of primers for VEGF, MAP3K6, and LS28 were: forward, 5′-TCTGAGCAAGGCCCACAGGGA-3′ and reverse, 5′-CCCTGATGAGATCGAGTACATCTT-3′; forward, 5′-CTGCTGCTTCCTCTATGCACT-3′ and reverse, 5′-CCCGTCTCCGTGTACTCATAA-3′; and forward, 5′-ATGGTCGTGCGGAACTGC-3′ and reverse, 5′-TTGTAGCGGAAGGAATTGCG-3′, respectively. Two sets of double-strand RNA (siPerfect) for MAP3K6 and the negative control dsRNA were generated by the manufacturer (RNAi Co., Ltd., Tokyo, Japan) according to an original algorithm developed by it. Two hundred and fifty pmol of dsRNA were transfected into 5.0 × 105 HeLa S3 cells using Lipofectamine 2000 (Invitrogen) in a six-well plate, as described in the manufacturer's protocol. Twenty-four hours after transfection, cells were placed under normoxic or hypoxic conditions (1% O2) for 24 hours. The pcPUR hU6 vector targeted against the MAP3K6 gene was transfected into HeLa S3 cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were selected by exposure to 1 μg/ml of puromycin for 120 hours. After selection, surviving colonies of cells were isolated and resuspended in fresh medium. Transfected cells having the pcPUR hU6 vector containing seven tandem repeats of thymidine (T7) served as control. To obtain a full length of mouse MAP3K6 cDNA, IMAGE clone (BC120565) was purchased from Open Biosystems (Huntsville, AL). Mouse MAP3K6 cDNA was isolated by EcoRI and XhoI digestion from pCR-XL-TOPO, and was subcloned in pCDNA3.1-TOPO. Transfection of this expression plasmid into HeLa S3 cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Expression of MAP3K6, VEGF, and ASK-1 in HeLa S3 cells as well as that of MAP3K6 and VEGF in the tumors was monitored by Western blot analysis. The tumors were collected in lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L dithiothreitol, 2 μg/ml leupeptin, 2 μg/ml pepstatin A) and homogenates were prepared manually using a tissue homogenator (Niti-on, Chiba, Japan). Sixty μg of whole cell extracts or 40 μg of tumor homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and electrophoretically transferred to a nylon membrane. The membranes were blocked with 5% nonfat dry milk in TBS Tween-20 (0.1%, v/v) at room temperature for 1 hour. For MAP3K6 detection, the membranes were incubated with 1:100 anti-human MAP3K6 primary antibody (Abnova, Taipei, Taiwan) at 4°C overnight followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG. For the detection of VEGF and ASK-1, the membranes were incubated with 1:300 anti-human VEGF(147) primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 1:250 anti-human ASK-1 (H-300) primary antibody (Santa Cruz Biotechnology) at 4°C overnight, respectively. Hybridizations with horseradish peroxidase-conjugated anti-rabbit IgG were performed the following day. Immunoreactive protein was visualized by a chemiluminescence protocol (ECL; Amersham, Arlington Heights, IL). Electric densities were measured using an image processing and analysis program, Image J software (National Institutes of Health, Bethesda, MD). The angiogenic effect of VEGF on the endothelial cells was assessed using CM. Control HeLa and MAP3K6 stably knocked-down HeLa S3 cells were grown to confluence in 100-mm culture dishes. After changing the medium to Dulbecco's modified Eagle's medium with 10% fetal bovine serum, the cells were cultured under hypoxic conditions (1% O2) for an additional 24 hours. Supernatants were then collected, centrifuged to remove cellular debris, and passed through a 0.22-μm pore filter (Millipore, Tokyo, Japan). Endothelial cell proliferation in the CM was examined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction to formazan (MTS assay, Promega). Cells were seeded at 2.0 × 104 on 96-well culture plates and incubated overnight. The medium was then aspirated and replaced by the CM, and the cells were incubated for an additional 24 hours. After this incubation, the cells were loaded with MTS reagent, and cell proliferation was measured using a microtiter plate reader at 492 nm. Capillary network formation was examined by Matrigel assays (BD Biosciences). ECs (2.0 × 104) were seeded on four-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) coated with Matrigel, and cultured in the CM for 8 hours under isolation from control and MAP3K6 knock-down cells. After incubation, capillary network formation was observed with a phase-contrast microscope and quantitatively evaluated by measuring capillary tube length. A total of 20 ng/ml of recombinant VEGF purchased from R&D Systems was added to the CM isolated from MAP3K6 knock-down cells. BALB/cAJc-nu/nu male mice, 7 to 8 weeks of age, were purchased from CLEA Japan (Tokyo, Japan) and inoculated subcutaneously on the flank with 5 × 106 of control or MAP3K6 stably knock-down HeLa S3 cells. Tumor volumes were measured at the indicated days after transplantation with electronic calipers (Mitutoyo, Kanagawa, Japan) and calculated using the formula length (mm) × width (mm) × height (mm)/2 and expressed in mm3. Tumor weight was measured at 28 days after initiation of transplantation. All experiments were performed in accordance with the guidelines of the Committee on Ethical Animal Care and Use at the University of Tokyo. For measurement of microvessel density in the tumors, frozen sections from xenograft tissues were fixed in acetone for 1 minute and allowed to air-dry. After 3 hours of incubation at room temperature with anti-mouse CD31 monoclonal antibody (BD Biosciences) at 1:20 dilution, the sections were incubated with biotinylated secondary antibodies at a 1:1000 dilution for 1 hour, followed by a 30-minute incubation with horseradish peroxidase-conjugated avidin D (Vector Laboratories, Burlingame, CA). Color was developed by incubation with diaminobenzidine (Wako Pure Chemical Industries, Tokyo, Japan) and the sections were counterstained with Mayer's hematoxylin (Wako Pure Chemical Industries). Quantification of tumor vessel counts was performed in a blinded manner with 10 randomly selected fields at a ×100 magnification per section. VEGF expression in the tumors was also monitored by immunohistochemical analysis. The tumors at 14 days were fixed in 10% neutralized formalin after removal, and then processed, embedded in paraffin, and cut into 3-μm sections. The sections were incubated with primary antibody against human VEGF(A20) (Santa Cruz Biotechnology). After overnight incubation at 4°C with the primary antibody at 1:100 dilution, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies at 1:500 dilution for 1 hour. Color was developed by incubation with diaminobenzidine (Wako Pure Chemical Industries) and hydrogen peroxide and the sections were counterstained with Mayer's hematoxylin (Wako Pure Chemical Industries). A Proteome Profilter antibody array of human phosphono-mitogen-activated protein kinases (MAPKs) and other serine/threonine kinases was purchased from R&D Systems. The array was a nitrocellulose membrane on which capture antibodies against a variety of MAPKs and other serine/threonine kinases had been spotted in duplicate, including ERK1/2/3, JNK1/2/3, p38α/β/δ/γ, RSK1/2, GSK-3α/β, Akt1/2, MSK2, HSP27, and p70 S6 kinase. Control and MAP3K6 stably knocked-down HeLa S3 cells were cultured in hypoxic conditions for 45 minutes or 24 hours. Three hundred μg of protein from whole lysates of the cells were hybridized with the antibody array according to the manufacturer's protocol. Relative changes in phosphorylation state between two samples were measured with Image J software. Statistical analyses were performed using Stat-View software (ver. 5.0; SAS Institute, Cary, NC). Comparisons among groups were done using analysis of variance, with Bonferroni/Dunn correction. Nonparametric data were analyzed with the Mann-Whitney test. P values of <0.05 were considered statistically significant. A VEGF-reporter vector was obtained using a 2.7-kbp fragment of the human VEGF gene located in its 5′-flanking promoter region. To validate the assay, HEK293T cells were transiently transfected with VEGF reporter vector and were subjected to a graded oxygen content series for 24 hours. A decrease in oxygen concentration affected reporter activity, with a fivefold increase seen at concentrations of 0.2% (Figure 1A). Co-expression with the VEGF reporter and shRNA expression vector of HIF-1α, which mediates various hypoxic responses, significantly ameliorated the hypoxic response of the reporter compared with control, ie, VEGF reporter-transfected cells without the shRNA expression vector. Moreover, co-expression with the VEGF reporter and shRNA expression vector against nonrelated genes, such as GFP or the tandem repeated sequence of T (T7), did not affect reporter activity (Figure 1B). These results indicated that the 2.7-kbp fragment of the human VEGF promoter can be used to monitor the hypoxic response to VEGF expression. For our screening analysis, we selected 320 vectors designed against genes for kinases and transcription factors. We selected shRNA against HIF-1α and T7 to serve as a positive and negative control, respectively. HEK239T cells were co-transfected with the VEGF reporter vector and a shRNA expression vector. Screening was performed in duplicate and quantitatively scored. We identified several shRNA expression vectors that modified expression of the VEGF reporter vector after 24 hours of incubation under hypoxic conditions. Figure 1C shows clones that suppressed VEGF reporter activity compared with the negative control in three independent experiments. To confirm the inhibitory effects of these shRNA expression vectors, we measured VEGF content in the CM by ELISA (Figure 1D). Results showed that knock-down of ENT7A, ENT11D, SRN5A, or SNR7A significantly suppressed both VEGF reporter activity and VEGF expression (Supplemental Table S1 available at http://ajp.amjpathol.org). Of these four molecules, ENT11D and SRN5A were more effective than the others. We selected SRN5A (mitogen-activated kinase kinase kinase 6, also called MAP3K6/MEKK6) for further biological analysis because MAP3K6 weakly activates JNK, although the biological function of this molecule remains primarily unknown. To confirm the effect of this kinase, we performed transient transfection of dsRNA into HeLa S3 cells instead of the shRNA expression vector using two distinct synthesized dsRNAs that target different sites of the MAP3K6 gene. Quantitative PCR analysis showed that both dsRNA significantly reduced MAP3K6 (70 to 73% reduction, Figure 2A) and VEGF mRNA expression (28 to 34% reduction, Figure 2B) after 24 hours of incubation under hypoxic conditions. In addition, a decrease in MAP3K6 expression in dsRNA-transfected cells at the protein level was confirmed by Western blot analysis (Figure 2C). O
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