Title: Vascular Endothelial Growth Factor-mediated Induction of Manganese Superoxide Dismutase Occurs through Redox-dependent Regulation of Forkhead and IκB/NF-κB
Abstract: The mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) plays a critical cytoprotective role against oxidative stress. Vascular endothelial growth factor (VEGF) was shown previously to induce expression of Mn-SOD in endothelial cells by a NADPH oxidase-dependent mechanism. The goal of the current study was to determine the transcriptional mechanisms underlying this phenomenon. VEGF resulted in protein kinase C-dependent phosphorylation of IκB and subsequent translocation of p65 NF-κB into the nucleus. Overexpression of constitutively active IκB blocked VEGF stimulation of Mn-SOD. In transient transfection assays, VEGF increased Mn-SOD promoter activity, an effect that was dependent on a second intronic NF-κB consensus motif. In contrast, VEGF-mediated induction of Mn-SOD was enhanced by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and by dominant negative Akt and was decreased by constitutively active Akt. Overexpression of a constitutively active (phosphorylation-resistant) form of FKHRL1 (TMFKHRL1) resulted in increased Mn-SOD expression, suggesting that the negative effect of PI3K-Akt involves attenuation of forkhead activity. In co-transfection assays, the Mn-SOD promoter was transactivated by TMFKHRL1. Flavoenzyme inhibitor, diphenyleneiodonium (DPI), and antisense oligonucleotides against p47phox (AS-p47phox) inhibited VEGF stimulation of IκB/NF-κB and forkhead phosphorylation, supporting a role for NADPH oxidase activity in both signaling pathways. Like VEGF, hepatocyte growth factor (HGF) activated the PI3K-Akt-forkhead pathway. However, HGF-PI3K-Aktforkhead signaling was insensitive to diphenyleneiodonium and AS-p47phox. Moreover, HGF failed to induce phosphorylation of IκB/NF-κB or nuclear translocation of NF-κB and had no effect on Mn-SOD expression. Together, these data suggest that VEGF is uniquely coupled to Mn-SOD expression through growth factor-specific reactive oxygen species (ROS)-sensitive positive (protein kinase C-NF-κB) and negative (PI3K-Akt-forkhead) signaling pathways. The mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) plays a critical cytoprotective role against oxidative stress. Vascular endothelial growth factor (VEGF) was shown previously to induce expression of Mn-SOD in endothelial cells by a NADPH oxidase-dependent mechanism. The goal of the current study was to determine the transcriptional mechanisms underlying this phenomenon. VEGF resulted in protein kinase C-dependent phosphorylation of IκB and subsequent translocation of p65 NF-κB into the nucleus. Overexpression of constitutively active IκB blocked VEGF stimulation of Mn-SOD. In transient transfection assays, VEGF increased Mn-SOD promoter activity, an effect that was dependent on a second intronic NF-κB consensus motif. In contrast, VEGF-mediated induction of Mn-SOD was enhanced by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and by dominant negative Akt and was decreased by constitutively active Akt. Overexpression of a constitutively active (phosphorylation-resistant) form of FKHRL1 (TMFKHRL1) resulted in increased Mn-SOD expression, suggesting that the negative effect of PI3K-Akt involves attenuation of forkhead activity. In co-transfection assays, the Mn-SOD promoter was transactivated by TMFKHRL1. Flavoenzyme inhibitor, diphenyleneiodonium (DPI), and antisense oligonucleotides against p47phox (AS-p47phox) inhibited VEGF stimulation of IκB/NF-κB and forkhead phosphorylation, supporting a role for NADPH oxidase activity in both signaling pathways. Like VEGF, hepatocyte growth factor (HGF) activated the PI3K-Akt-forkhead pathway. However, HGF-PI3K-Aktforkhead signaling was insensitive to diphenyleneiodonium and AS-p47phox. Moreover, HGF failed to induce phosphorylation of IκB/NF-κB or nuclear translocation of NF-κB and had no effect on Mn-SOD expression. Together, these data suggest that VEGF is uniquely coupled to Mn-SOD expression through growth factor-specific reactive oxygen species (ROS)-sensitive positive (protein kinase C-NF-κB) and negative (PI3K-Akt-forkhead) signaling pathways. VEGF 1The abbreviations used are: VEGF, vascular endothelial growth factor; KDR, kinase insert domain-containing receptor; VEGFR, VEGF receptor; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; HCAEC, human coronary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; EGM, endothelial growth medium; HGF, hepatocyte growth factor; DPI, diphenyleneiodonium; ERK, extracellular signal-regulated kinase; CREB, cAMP-response element-binding protein; DN, dominant negative; CA, constitutively active; WT, wild type; TM, triple mutant; C/EBP, CAAAT/enhancer-binding protein; mt, mutation; Adv, adenovirus encoding the cDNA of β-galactosidase; AS, antisense; FKHR, forkhead in rhabdomyosarcoma/FOXO1.1The abbreviations used are: VEGF, vascular endothelial growth factor; KDR, kinase insert domain-containing receptor; VEGFR, VEGF receptor; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; HCAEC, human coronary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; EGM, endothelial growth medium; HGF, hepatocyte growth factor; DPI, diphenyleneiodonium; ERK, extracellular signal-regulated kinase; CREB, cAMP-response element-binding protein; DN, dominant negative; CA, constitutively active; WT, wild type; TM, triple mutant; C/EBP, CAAAT/enhancer-binding protein; mt, mutation; Adv, adenovirus encoding the cDNA of β-galactosidase; AS, antisense; FKHR, forkhead in rhabdomyosarcoma/FOXO1. is an endothelial cell-specific mitogen and chemotactic agent that is involved in wound repair, angiogenesis of ischemic tissue, tumor growth, microvascular permeability, vascular protection, and hemostasis (for review, see Refs. 1Tjwa M. 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Subsequent studies have confirmed the importance of ROS in VEGF signal transduction (18Colavitti R. Pani G. Bedogni B. Anzevino R. Borrello S. Waltenberger J. Galeotti T. J. Biol. Chem. 2002; 277: 3101-3108Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 19Ushio-Fukai M. Tang Y. Fukai T. Dikalov S.I. Ma Y. Fujimoto M. Quinn M.T. Pagano P.J. Johnson C. Alexander R.W. Circ. Res. 2002; 91: 1160-1167Crossref PubMed Scopus (421) Google Scholar). The SOD family includes cytosolic Cu,Zn-SOD, mitochondrial manganese SOD, and extracellular Cu,Zn-SOD. By converting superoxide (O2˙¯) to H2O2 and O2, these enzymes inhibit free radical reactions that lead to oxidative damage. Mn-SOD is encoded by the nuclear SOD2 gene and is localized in mitochondria, the major site for oxidative phosphorylation. ROS have been shown to induce mitochondrial damage and dysfunction, leading to an impaired Krebs' cycle and induction of apoptotic pathways (for review, see Refs. 20Taniyama Y. 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St. Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 25Wispe J.R. Warner B.B. Clark J.C. Dey C.R. Neuman J. Glasser S.W. Crapo J.D. Chang L.Y. Whitsett J.A. J. Biol. Chem. 1992; 267: 23937-23941Abstract Full Text PDF PubMed Google Scholar, 26Yen H.C. Oberley T.D. Vichitbandha S. Ho Y.S. St. Clair D.K. J. Clin. Investig. 1996; 98: 1253-1260Crossref PubMed Scopus (394) Google Scholar). In the present study, we wished to extend our previous findings by elucidating the mechanisms underlying VEGF stimulation of Mn-SOD. We show that the effect of VEGF on Mn-SOD expression involves a negative PI3K-Akt-forkhead pathway and a positive PKC-NF-κB pathway, both of which are sensitive to NADPH oxidase activity. The presence of two opposing pathways, one negative and the other positive, is likely to render the VEGF-Mn-SOD axis highly modulatable by the extracellular environment. Cell Culture and Reagents—Human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were grown in endothelial growth medium-2-MV (EGM-2-MV) BulletKit (Clonetics, San Diego, CA) at 37 °C and 5% CO2. Endothelial cells from passage 3-6 were used for all experiments. Cells were serum-starved in 0.5% fetal bovine serum prior to treatment with 50 ng/ml human VEGF165 or 40 units/ml hepatocyte growth factor (HGF) (Pepro Tech Inc., Rocky Hill, NJ). Where indicated, cells were preincubated for 30 min with 50 μm LY294002, 1 μm GF109203X, 50 μm PD98059, or 10 μm diphenyleneiodonium (DPI) (Biomol, Plymouth Meeting, PA). Western and Northern Blot Analyses—Endothelial cells were harvested for total protein, and Western blots were carried out as described previously (15Abid M.R. Guo S. Minami T. Spokes K.C. Ueki K. Skurk C. Walsh K. Aird W.C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 294-300Crossref PubMed Scopus (189) Google Scholar). The following phospho-specific antibodies were used: Ser-256 FKHR, Ser-473 Akt, ERK1/2, c-Jun NH2-terminal kinase (JNK), IκB, NF-κB, and CREB/activating transcription factor (ATF) from Cell Signaling (Beverly, MA); antiphospho-Tyr 4G10 from UBI (Waltham, MA); and anti-β-actin from Sigma. Anti-ERK1/2, anti-Akt, anti-FKHR, anti-IκB, anti-NF-κB (p65), and anti-CREB/activating transcription factor (ATF) antibodies were obtained from Cell Signaling. RNA extraction and Northern blot assays were performed as described previously (17Abid M.R. Tsai J.C. Spokes K.C. Deshpande S.S. Irani K. Aird W.C. FASEB J. 2001; 15: 2548-2550Crossref PubMed Scopus (148) Google Scholar). Immunolocalization Studies—HCAEC were plated onto 4-well chamber slides (Lab-Tek, Christchurch, New Zealand) at a density of 30,000 cells/well. The cells were grown in EGM-2-MV for 48 h and were fixed in ice-cold 3.7% paraformaldehyde for 10 min, washed with phosphate-buffered saline, and subsequently incubated with primary anti-p65 antibody (1:100 dilution) for 2 h. Following extensive washes in phosphate-buffered saline, the cells were incubated with a fluorescein isothiocyanate-labeled secondary antibody (1:200 dilution) for 1 h. Following additional phosphate-buffered saline washes, the slides were mounted in Aquamount (Vector, Burlingame, CA) and viewed under confocal fluorescence microscopy. To-Pro (Molecular Probes, Eugene, OR) was used for identification of nuclear co-localization. Adenoviruses—HCAEC were infected with adenoviruses encoding the cDNAs of β-galactosidase (Adv), dominant negative T308A/S473A-Akt (DN-Akt), constitutively active Gag-Akt (CA-Akt), wild type (WT)FKHRL1, and triple mutant (TM)-FKHRL1 as described previously (15Abid M.R. Guo S. Minami T. Spokes K.C. Ueki K. Skurk C. Walsh K. Aird W.C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 294-300Crossref PubMed Scopus (189) Google Scholar). The Akt viruses were a generous gift from Koji Ueki (Harvard Medical School, Boston, MA). The triple mutant version of FKHRL1 contains T32A, S253A, and S315A and is resistant to agonist-induced phosphorylation. WT- and DN-PKC viruses (a generous gift from George King, Joslin Diabetes Center, Harvard Medical School) were used as described previously (27Minami T. Abid M.R. Zhang J. King G. Kodama T. Aird W.C. J. Biol. Chem. 2003; 278: 6976-6984Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Adenovirus expressing degradation-resistant constitutively active IκBα (IκB S32A/S36A) was a generous gift from Kaikobad Irani (The Johns Hopkins University School of Medicine, Baltimore, MD) (28Deshpande S.S. Angkeow P. Huang J. Ozaki M. Irani K. FASEB J. 2000; 14: 1705-1714Crossref PubMed Scopus (201) Google Scholar). Co-transfection Assays—Control vector (pECE) and vector expressing FKHRL1 (pECE-FKHRL1) were provided by Michael Greenberg, Children's Hospital, Boston, MA. The pGL3-based construct containing a 3340-bp promoter of human Mn-SOD (pGL3-Mn-Luc) was a generous gift from Moon Yim (Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD). A 369-bp fragment (from nucleotide 2410 to nucleotide 2778) of the second intron of Mn-SOD containing the putative NF-κB, C/EBP, and NF-1 consensus sites was generated by PCR using human genomic DNA as template (the sense primer, 5′-TCTTGTCGACCGTTAGTGGTTTGCACAAGGAAGATAATCG-3′, and the reverse primer, 5′-AATAGTCGACTTCACAACTGGGGTATTCCCCAGTCTCTCC-3′, with the SalI site underlined). The resulting fragment was digested with SalI and inserted into the SalI site of the pGL3-Mn-Luc plasmid in forward and reverse orientations, giving rise to pGL3-Mn2-Luc and pGL3-Mn2R-Luc, respectively. To generate the NF-κB mutation (pGL3-Mn2(mt-NFκB)-Luc), a 369-bp fragment was regenerated with the same forward primer as above but with a reverse primer containing the mutations (in lowercase letters) 5′-AATAGTCGACTTCACAACTGGtGTATTttCCAGTCTCTCC-3′. A mutation in the C/EBP site (pGL3-Mn2(mt-CEBP)-Luc) was obtained using a two-step mutagenesis protocol with the primers 5′-GATTAAAAGAGGAGGAAGTTAttACATTCTGGAAGATTTAC-3′ and 5′-GTAAATCTTCCAGAATGTaaTAACTTCCTCCTCTTTTAATC-3′ (mutations in lowercase letters). A total of 0.05 pmol of the Mn-SOD reporter plasmid, 50 ng of a control plasmid containing the Renilla luciferase reporter gene under the control of a cytomegalovirus enhancer/promoter (pRL-CMV), and 0.075 pmol of the forkhead expression vector (or control vector, pECE) were incubated with 2 μl of FuGENE 6 (Roche Applied Science). Twenty-four h later, the cells were washed with phosphate-buffered saline and cultured for 12 h in EBM plus 0.5% fetal bovine serum. The cells were then incubated in the presence or absence of VEGF for 6 h, at which time they were lysed and assayed for luciferase activity using the dual-luciferase reporter assay system (Promega, Madison, WI) and a Lumat LB 9507 luminometer (Berthold, Bad Wildbad, Germany). Experiments were performed in triplicates and repeated at least three times. Transfection with Antisense Oligonucleotide p47phox—HCAEC were grown to 70-80% confluency in 10-cm plates and transfected with 200 nm phosphorothioate antisense p47phox oligonucleotide in Opti-MEM containing Lipofectin (10 μg/ml) for 4 h. The cells were then incubated in EGM-2 for 24 h and serum-starved in 0.5% serum for 12-16 h before VEGF treatment for the times indicated. The antisense sequence (5′-TTTGTCTGGTTGTGTGTGGG-3′) was complementary to nucleotides 394-413 of human p47phox mRNA (29Bey E.A. Cathcart M.K. J. Lipid Res. 2000; 41: 489-495Abstract Full Text Full Text PDF PubMed Google Scholar) and was phosphorothioate-modified and high pressure liquid chromatography-purified (Sequitur, Natick, MA). Assay for NADPH Oxidase Activity in HCAEC and HUVEC—The cells were washed with ice-cold phosphate-buffered saline, collected by a cell scraper, and homogenized with a Dounce homogenizer in a buffer containing 20 mm KH2PO4 (pH 7.0), 1× protease mixture inhibitor (Sigma), 1 mm EGTA, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 0.5 mm phenylmethylsulfonyl fluoride. NADPH oxidase activity of the cell lysate was measured using a modified assay as described previously (30Griendling K.K. Minieri C.A. Ollerenshaw J.D. Alexander R.W. Circ. Res. 1994; 74: 1141-1148Crossref PubMed Scopus (2359) Google Scholar). Briefly, photon emission from the chromogenic substrate lucigenin as a function of acceptance of electron/O2˙¯ generated by the NADPH oxidase complex was measured every 15 s for 20 min in a Berthold luminometer. NADPH oxidase assay buffer containing 250 mm HEPES (pH 7.4), 120 mm NaCl, 5.9 mm KCl, 1.2 mm MgSO4 (7H2O), 1.75 mm CaCl2 (2H2O), 11 mm glucose, 0.5 mm EDTA, 100 μm NADH, and 5 μm lucigenin was used. The data were transformed to relative light units/min/mg of protein using a standard curve generated with xanthine/xanthine oxidase. Measurement of Intracellular ROS Generation—The changes in intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable 2′,7′-dichlorofluorescein diacetate (DCFHDA; Molecular Probes Inc.) to fluorescent dichlorofluorescein (DCF) by fluorescence-activated cell sorter analysis as described previously (17Abid M.R. Tsai J.C. Spokes K.C. Deshpande S.S. Irani K. Aird W.C. FASEB J. 2001; 15: 2548-2550Crossref PubMed Scopus (148) Google Scholar). VEGF-mediated Induction of Mn-SOD Is Attenuated by the PI3K-Akt-Forkhead Signaling Pathway—Previous studies in non-endothelial cells have demonstrated that insulin inhibits Mn-SOD expression via a PI3K-Akt-forkhead-dependent pathway (31Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1237) Google Scholar). In contrast, we have shown that VEGF, although capable of inducing phosphorylation and nuclear exclusion of forkhead in endothelial cells in a PI3K-Akt-dependent manner (15Abid M.R. Guo S. Minami T. Spokes K.C. Ueki K. Skurk C. Walsh K. Aird W.C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 294-300Crossref PubMed Scopus (189) Google Scholar), results in a net increase in Mn-SOD mRNA and protein levels (17Abid M.R. Tsai J.C. Spokes K.C. Deshpande S.S. Irani K. Aird W.C. FASEB J. 2001; 15: 2548-2550Crossref PubMed Scopus (148) Google Scholar). Together, these findings suggest that VEGF-mediated induction of Mn-SOD involves an interplay between positive and negative pathways. To test this hypothesis, HCAEC and HUVEC were preincubated for 30 min in the absence or presence of the PI3K inhibitor LY294002 (50 μm) or wortmannin (100 nm), treated with VEGF (50 ng/ml) for 4 h, and then processed for Northern blot analysis of Mn-SOD. Inhibition of PI3K increased basal and VEGF-induced Mn-SOD expression in both types of endothelial cells (Fig. 1A shows the effect of LY294002 in HCAEC). Lower doses of LY294002 (0.1-10 μm) blocked VEGF-mediated phosphorylation of Akt and enhanced VEGF stimulation of Mn-SOD expression without altering basal Mn-SOD levels (Fig. 1B). Together, these findings strongly suggest that PI3K plays a negative role in the VEGF-Mn-SOD signaling pathway. To determine whether PI3K exerts its negative effect on Mn-SOD expression through Akt, a serine-threonine kinase downstream of PI3K, HCAEC and HUVEC were infected with adenoviruses expressing β-galactosidase, CA-Akt, or DN-Akt and were incubated in the absence or presence of VEGF. Overexpression of DN-Akt accentuated the effect of VEGF on Mn-SOD expression, whereas CA-Akt had the opposite effect (Fig. 1C shows HCAEC). Together, these findings suggest that PI3K-Akt attenuates VEGF-mediated induction of Mn-SOD in endothelial cells. Previous studies of Caenorhabditis elegans and mice have implicated the daf-2/insulin-forkhead signaling pathway in regulating Mn-SOD expression (32Honda Y. Honda S. FASEB J. 1999; 13: 1385-1393Crossref PubMed Scopus (584) Google Scholar, 33Yanase S. Yasuda K. Ishii N. Mech. Ageing Dev. 2002; 123: 1579-1587Crossref PubMed Scopus (129) Google Scholar). The human Mn-SOD gene contains two forkhead consensus sites in the upstream promoter region, one at -1249 (GTAAACAA; inverse of TTGTTTAC) and another at -997 (TTGTTTAA) (31Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1237) Google Scholar). In non-endothelial cells, insulin has been shown to induce PI3K-Akt-dependent phosphorylation and nuclear exclusion of FKHRL1 and to inhibit Mn-SOD expression (31Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1237) Google Scholar). To determine whether Mn-SOD lies downstream of VEGF-PI3K-Akt-forkhead in endothelial cells, HCAEC and HUVEC were infected with adenoviruses overexpressing β-galactosidase, WT-FKHRL1, or a constitutively activated phosphorylation-resistant TM-FKHRL1. The overexpressing cells were serum-starved and then treated in the presence or absence of VEGF for 4 h. VEGF-mediated induction of Mn-SOD was accentuated by overexpression of TM-FKHRL1 but not Adv or WT-FKHRL1 (Fig. 1D shows HCAEC). In the absence of VEGF treatment, TM-FKHRL1 alone had no effect on Mn-SOD expression in serum-starved cells (Fig. 1D). However, in the presence of serum-rich medium, TM-FKHRL1-infected cells demonstrated increased Mn-SOD mRNA levels (Fig. 1E). Together, these data suggest that forkhead-mediated induction of Mn-SOD is dependent on VEGF- or serum-mediated activation of positive pathway(s). In co-transfection assays, the overexpression of TM-FKHRL1 resulted in a 6.6 ± 0.3-fold induction of the 3340-bp human Mn-SOD promoter (Fig. 1F). These findings support the conclusion that the PI3K-Akt signaling pathway negatively regulates expression of Mn-SOD via phosphorylation and nuclear exclusion of forkhead. VEGF-mediated Induction of Mn-SOD Is Positively Regulated through PKC and IκB/NF-κB—Although VEGF triggers a negative PI3K-Akt-forkhead signaling pathway, VEGF signaling results in the net induction of Mn-SOD mRNA and protein. To identify the positive pathway(s) responsible for this effect, endothelial cells were preincubated with other inhibitors of signaling and then treated in the absence or presence of VEGF. As shown in Fig. 2A, preincubation of endothelial cells with the PKC inhibitor GF109203X (1 μm) resulted in marked inhibition of VEGF induction of Mn-SOD, whereas the MEK1/2 inhibitor PD98059 (50 μm) had no such effect. To determine which PKC isoforms are involved in mediating the VEGF response, HCAEC were infected with either WT or DN isoforms of PKC and treated in the absence or presence of VEGF. VEGF induction of Mn-SOD was completely abrogated by DN-PKCδ, significantly inhibited by DN-PKCζ (Fig. 2B), and unchanged by DN-PKCα or DN-PKCϵ (data not shown). These findings suggest that VEGF-mediated induction of Mn-SOD is dependent on novel and atypical PKC isoforms. Previous studies have demonstrated a link between PKCδ/ζ signaling and NF-κB activity in endothelial cells (27Minami T. Abid M.R. Zhang J. King G. Kodama T. Aird W.C. J. Biol. Chem. 2003; 278: 6976-6984Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 34Rahman A. Anwar K.N. Malik A.B. Am. J. Physiol. Cell Physiol. 2000; 279: C906-C914Crossref PubMed Google Scholar, 35Rahman A. Anwar K.N. Uddin S. Xu N. Ye R.D. Platanias L.C. Malik A.B. Mol. Cell. Biol. 2001; 21: 5554-5565Crossref PubMed Scopus (147) Google Scholar). Moreover, NF-κB has been shown to mediate inducible expression of Mn-SOD in a variety of cell types (22Delhalle S. Deregowski V. Benoit V. Merville M.P. Bours V. Oncogene. 2002; 21: 3917-3924Crossref PubMed Scopus (106) Google Scholar, 36Rogers R.J. Chesrown S.E. Kuo S. Monnier J.M. Nick H.S. Biochem. 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In transient transfection assays, VEGF induced Mn-SOD promoter activity in the presence, but not the absence, of the second intronic sequence (Fig. 2D). Mutation of the NF-κB motif (mt-NF-κB) resulted in a loss of induction, whereas mutation of the C/EBP site (at +2648 nucleotides; mt-CEBP) had no such effect (Fig. 2E). The transcription factor NF-κB is normally sequestered in the cytoplasm by IκB. NF-κB activation requires phosphorylation-induced ubiquitination and degradation of cytoplasmic IκB, with subsequent release and nuclear translocation of NF-κB. Incubation of HCAEC with VEGF resulted in increased phosphorylation of IκB in the cytoplasm at 15 min and decreased total cytoplasmic IκB at 30 and 60 min (Fig. 3A). Moreover, VEGF promoted the nuclear translocation of p65 NF-κB at 15 and 30 min (Fig. 3B). Preincubation with the PKC inhibitor GF109203X blocked VEGF-mediated phosphorylation of IκB (Fig. 3C) and nuclear translocation of NF-κB (Fig. 3D). In immunofluorescent assays, VEGF induced the nuclear translocation of p65 NF-κB, an effect that was similarly blocked by GF109203X (Fig. 4). Taken together, these results suggest that VEGF-mediated induction of Mn-SOD is dependent on a PKC-NF-κB signaling pathway.Fig. 4VEGF induces nuclear translocation of p65 NF-κB in
Publication Year: 2004
Publication Date: 2004-08-12
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 100
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