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CD40-dependent Activation of Phosphatidylinositol 3-Kinase/Akt Pathway Mediates Endothelial Cell Survival and in Vitro Angiogenesis*

  • Maria Chiara Deregibus
    Affiliations
    Cattedra di Nefrologia, Dipartimento di Medicina Interna, Università di Torino, and Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126, Italy
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  • Stefano Buttiglieri
    Affiliations
    Cattedra di Nefrologia, Dipartimento di Medicina Interna, Università di Torino, and Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126, Italy
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  • Simona Russo
    Affiliations
    Cattedra di Nefrologia, Dipartimento di Medicina Interna, Università di Torino, and Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126, Italy
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  • Benedetta Bussolati
    Affiliations
    Cattedra di Nefrologia, Dipartimento di Medicina Interna, Università di Torino, and Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126, Italy
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  • Giovanni Camussi
    Correspondence
    To whom correspondence should be addressed: Cattedra di Nefrologia, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126, Torino, Italy. Tel.: 39-011-6336708; Fax: 39-011-6631184
    Affiliations
    Cattedra di Nefrologia, Dipartimento di Medicina Interna, Università di Torino, and Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126, Italy
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  • Author Footnotes
    * This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), by Istituto Superiore di Sanità(Targeted Project AIDS), by Italian Ministry of University and Research (MIUR) FIRB project (RBNE01HRS5–001) and COFIN 01, by Italian Ministry of Health (Ricerca Finalizzata 02), and by the special project Oncology, Compagnia San Paolo/FIRMS.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 12, 2003DOI:https://doi.org/10.1074/jbc.M300711200
      CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis andin vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.
      TNF
      tumor necrosis factor
      TRAF
      TNF receptor-associated factor
      DMEM
      Dulbecco's modified Eagle's medium
      FCS
      fetal calf serum
      WT
      wild type
      HMEC
      human microvascular endothelial cells
      TUNEL
      terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling
      Ab
      antibody
      PI3K
      phosphatidylinositol 3-kinase
      ND
      negative-dominant
      ELISA
      enzyme-linked immunosorbent assay
      FACS
      fluorescence-activated cell sorter
      HRP
      horseradish peroxidase
      CD40 is a member of the tumor necrosis factor (TNF)1 receptor superfamily, which provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells (
      • Clark E.A.
      • Ledbetter J.A.
      ,
      • Grewal I.S.
      • Flavell R.A.
      ). Among the molecular mechanisms that link immunity to inflammation, the interaction between CD40 and its counterreceptor CD154 has rapidly emerged as a key system in the regulation of vascular pathophysiological processes such as atherogenesis (
      • Schonbeck U.
      • Libby P.
      ,
      • Lutgens E.
      • Daemen M.J.
      ), tumor neoangiogenesis (
      • Kluth B.
      • Hess S.
      • Engelmann H.
      • Schafnitzel S.
      • Riethmuller G.
      • Feucht H.E.
      ,
      • Biancone L.
      • Cantaluppi V.
      • Boccellino M.
      • Del Sorbo L.
      • Russo S.
      • Albini A.
      • Stamenkovic I.
      • Camussi G.
      ), and inflammation (
      • Grewal I.S.
      • Flavell R.A.
      ,
      • Reul R.M.
      • Fang J.C.
      • Denton M.D.
      • Geehan C.
      • Long C.
      • Mitchell R.N.
      • Ganz P.
      • Briscoe D.M.
      ). Under physiological conditions, CD40 is expressed at low levels on endothelial cells but is up-regulated in areas of inflammation (
      • Karmann K.
      • Hughes C.C.
      • Schechner J.
      • Fanslow W.C.
      • Pober J.S.
      ). Ligation of endothelial CD40 by CD154, either expressed on activated monocytes or T cells (
      • Grewal I.S.
      • Flavell R.A.
      ) or disgorged by platelets upon activation (
      • Henn V.
      • Slupsky J.R.
      • Grafe M.
      • Anagnostopoulos I.
      • Forster R.
      • Muller-Berghaus G.
      • Kroczek R.A.
      ), induces production of various inflammatory cytokines and chemokines, pro-coagulant activity, adhesion molecules, metalloproteinases, and inflammatory mediators (
      • Thienel U.
      • Loike J.
      • Yellin M.J.
      ,
      • Slupsky J.R.
      • Kalbas M.
      • Willuweit A.
      • Henn V.
      • Kroczek R.A.
      • Muller-Berghaus G.
      ,
      • Dechanet J.
      • Grosset C.
      • Taupin J.L.
      • Merville P.
      • Banchereau J.
      • Ripoche J.
      • Moreau J.F.
      ,
      • Schonbeck U.
      • Mach F
      • Bonnefoy J.Y.
      • Loppnow H.
      • Flad H.D.
      • Libby P.
      ). These mediators have been implicated in the development and progression of atherosclerosis. In situ analysis of human atherosclerotic lesions revealed the co-expression of CD154 and CD40 on vascular endothelium and smooth muscle cells (
      • Mach F.
      • Schonbeck U.
      • Sukhova G.K.
      • Bourcier T.
      • Bonnefoy J.Y.
      • Pober J.S.
      • Libby P.
      ). Blockade of CD40-CD154 interaction in atherosclerosis was found not only to diminish the formation and progression of mouse atheroma but also to foster changes in lesions associated with plaque destabilization (
      • Schonbeck U.
      • Sukhova G.K.
      • Shimizu K.
      • Libby P.
      ,
      • Mach F.
      • Schonbeck U.
      • Sukhova G.K.
      • Atkinson E.
      • Libby P.
      ). Recently, it has also been shown that the engagement of CD40 on endothelial cells by CD154 induces in vitro vessel-like tubule formation and expression of matrix metalloproteinases, two events involved in neovascularization (
      • Mach F.
      • Schonbeck U.
      • Fabunmi R.P.
      • Murphy C.
      • Atkinson E.
      • Bonnefoy J.Y.
      • Graber P.
      • Libby P.
      ). In vivo, the stimulation of CD40 triggered neoangiogenesis in mice (
      • Biancone L.
      • Cantaluppi V.
      • Boccellino M.
      • Del Sorbo L.
      • Russo S.
      • Albini A.
      • Stamenkovic I.
      • Camussi G.
      ,
      • Melter M.
      • Reinders M.E.J.
      • Sho M.
      • Pal S.
      • Geehan C.
      • Denton M.D.
      • Mukhopadhyay D.
      • Briscoe D.
      ). Moreover, blockade of CD40-CD154 interaction prevented vascularization and tumor growth in an experimental model of Kaposi's sarcoma (
      • Biancone L.
      • Cantaluppi V.
      • Boccellino M.
      • Del Sorbo L.
      • Russo S.
      • Albini A.
      • Stamenkovic I.
      • Camussi G.
      ).
      CD40 signaling is initiated by receptor oligomerization upon binding the trimeric ligand CD154 (
      • Foy T.M.
      • Aruffo A.
      • Bajorath J.
      • Buhlmann J.E.
      • Noelle R.J.
      ). CD40 signaling elicits different outcomes in distinct cell types, ranging from proliferation, survival, and differentiation to growth suppression and apoptosis (
      • Biancone L.
      • Cantaluppi V.
      • Camussi G.
      ,
      • Young L.S.
      • Eliopoulos A.G.
      • Gallagher N.J.
      • Dawson C.W.
      ), and implies a complex regulation of CD40 signal transduction (
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      ). Indeed, the CD40 cytoplasmic C terminus lacks intrinsic kinase activity and adaptor proteins of the TNF receptor-associated factor (TRAF) family appear to mediate the activation of the CD40 signaling cascade (
      • Pullen S.S.
      • Miller H.G.
      • Everdeen D.S.
      • Dang T.T.A.
      • Crute J.J.
      • Kehry M.R.
      ,
      • Hu H.M.
      • O'Rourke K.
      • Boguski M.S.
      • Dixit V.M.
      ,
      • Cheng G.
      • Cleary A.M.
      • Ye Z.S.
      • Hong D.I.
      • Lederman S.
      • Baltimore D.
      ,
      • Ishida T.
      • Mizushima S.
      • Azuma S.
      • Kobayashi N.
      • Tojo T.
      • Suzuki K.
      • Aizawa S.
      • Watanabe T.
      • Mosialos G.
      • Kieff E.
      • Yamamoto T.
      • Inoue J.
      ,
      • Werneburg B.G.
      • Zoog S.J.
      • Dang T.T.
      • Kehry M.R.
      • Crute J.J.
      ). It has been recently found that CD40 mediated Akt activation in dendritic and B cells (
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N.
      • Gu H.
      • Choi Y.
      ). Several studies indicate that Akt-dependent signaling plays a critical role in the regulation of vascular homeostasis and angiogenesis (for review, see Ref.
      • Shiojima I.
      • Walsh K.
      ). Various growth factors, including vascular endothelial growth factor (VEGF) and angiopoietin-1 as well as mechanical stimuli, activate Akt in endothelial cells. However, the effect of stimulation of endothelial CD40 on the activation of Akt and the role of Akt in CD40-induced angiogenesis are unknown.
      The aim of the present study was to investigate whether Akt is directly activated by the engagement of endothelial CD40 by its ligand CD154 and whether Akt mediates biological effects relevant for CD40-induced angiogenesis such as cell survival, proliferation, migration, and vessel-like structure formation.

      RESULTS

      HMEC expressed CD40, as evaluated by cytofluorimetric analysis (Fig. 1A). TUNEL assay showed a marked increase in HMEC apoptosis after 24 h of starving, without FCS or treatment with 0.25 μg/ml of vincristine (Fig.1B). Stimulation of CD40 with sCD154 prevented apoptosis induced both by serum deprivation or treatment with vincristine. Soluble CD40-muIg fusion protein inhibited the anti-apoptotic effect elicited by sCD154, preventing the interaction between sCD154 and the CD40 expressed by HMEC (Fig. 1B). This result suggests that the anti-apoptotic effect of sCD154 was mediated by the CD40-dependent signaling. Treatment of HMEC with two unrelated PI3K pharmacological inhibitors, wortmannin (0.1 μm) and LY294002 (10 μm) abrogated the anti-apoptotic effect of sCD154, suggesting that this effect was dependent on the activation of PI3K. Wortmannin and LY294002 also inhibited the sCD154-induced in vitro cell motility (Fig.2). The tube formation in Matrigel was absent in unstimulated HMEC cells (Fig.3A). As shown in Fig.3B, sCD154 induced a rapid formation of vessel-like tubular structures of endothelial cells. Soluble CD40-muIg fusion protein inhibited vessel-like formation elicited by sCD154 (not shown). The sCD154-induced in vitro vessel-like formation was significantly reduced by treatment with wortmannin and LY294002 (Fig.3, C and D).
      Figure thumbnail gr1
      Figure 1Expression of CD40 by HMEC and effect of CD40 stimulation on HMEC apoptosis.Panel A, cytofluorimetric analysis of CD40 expression by HMEC. The figure is representative of three individual experiments. In each experiment the Kolmogorov-Smirnov statistical analysis between anti-CD40 IgG2a mAb (solid line) and the isotypic control (dotted line) was significant (p < 0.05). Panel B, apoptosis was evaluated by TUNEL assay as percentage of apoptotic cells after 24-hour serum withdrawal (noFCS) or treatment with 0.25 μg/ml vincristine. Where indicated, cells were stimulated with 100 ng/ml of sCD154 (plus 1 μg/ml enhancer) alone or in the presence of 20 ng/ml CD40-muIg fusion protein (CD40FP) or of 0.1 μm wortmannin or 10 μm LY294002. As control, cells were incubated in the presence of 10% FCS or with FCS plus 0.1 μm wortmannin or plus 10 μm LY294002. Data are expressed as mean ± 1 S.D. from three different experiments.
      Figure thumbnail gr2
      Figure 2Effect of PI3K pharmacological inhibitors on sCD154-induced motility of HMEC. Time course of HMEC motility (1 × 105 cells) induced by sCD154 (100 ng/ml, plus 1 μg/ml enhancer) in the presence or absence of 0.1 μmwortmannin or 10 μm LY294002. Motility was measured by time-lapse cinematography and digital image analysis as described under “Experimental Procedures.” As control, HMEC were incubated with vehicle alone. Results are expressed as means ± 1 S.D. from three individual experiments. Analysis of variance with Newmann-Keul's multicomparison test was performed for sCD154 versus control or for sCD154 versus sCD154 + wortmannin or sCD154 + LY294002. *, p < 0.05.
      Figure thumbnail gr3
      Figure 3Micrographs representative of in vitro formation of vessel-like structures by HMEC after CD40 stimulation. Tube formation by HMEC (5 × 104cells) plated on growth factor reduced Matrigel (see “Experimental Procedures”) was evaluated after stimulation of HMEC for 5 h at 37 °C with vehicle alone (A), sCD154 (100 ng/ml, plus 1 μg/ml enhancer) (B), or sCD154 (100 ng/ml, plus 1 μg/ml enhancer) in the presence of 0.1 μm wortmannin (C) or 10 μm LY294002 (D). ND-Akt HMEC were stimulated with vehicle alone (E) or sCD154 (F). Magnification: ×120.
      Apoptosis of HMEC induced by serum deprivation and treatment with vincristine was associated with an impairment of Akt phosphorylation (P-Akt) that was restored by treatment with sCD154 (Fig.4). This effect was dependent on CD40 stimulation since it was inhibited by the soluble CD40-muIg fusion protein. When PI3K was inhibited by wortmannin and LY294002, the CD40-induced phosphorylation of Akt was significantly prevented.
      Figure thumbnail gr4
      Figure 4Effect of treatment with sCD154 and PI3Kpharmacological inhibitors on Akt phosphorylation in HMEC. Cell lysates (30 μg of protein) were immunoblotted with anti-P-Akt, -Akt, or -β-actin antibodies. Panel A, densitometric analysis and panel B, representative immunoblot of P-Akt expression (panel A, black column) and Akt expression (panel A, open column). Row 1, control HMEC cultured in DMEM containing 10% FCS; row 2, HMEC serum starved for 24 h; row 3, HMEC serum-starved treated with sCD154 (100 ng/ml, plus 1 μg/ml enhancer); row 4, HMEC serum-starved treated with sCD154 and 20 ng/ml CD40-muIg fusion protein; row 5, HMEC treated with 0.25 μg/ml vincristine;row 6, HMEC treated with 0.25 μg/ml vincristine plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer); row 7, HMEC treated with 0.25 μg/ml vincristine plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer) and 20 ng/ml CD40-muIg fusion protein; row 8, HMEC treated with 0.25 μg/ml vincristine plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer) and 0.1 μm wortmannin;row 9, HMEC treated with 0.25 μg/ml vincristine plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer) and 10 μmLY294002. Panel A, data are expressed as mean ± 1 S.D. from three different experiments.
      To evaluate whether Akt enzymatic activity was directly induced by CD40 stimulation, Akt activity was measured in HMEC following incubation with sCD154 for different times. As shown in Fig.5, A and B, sCD154 induced a rapid and sustained enhancement of Akt enzymatic activity. This Akt activation was not inhibited by cycloheximide, an inhibitor of protein synthesis, suggesting that a CD40-induced synthesis of secondary mediators is not critical for Akt activation (not shown). It is known that TNF receptor family members, including CD40, transduce signals through TRAF family proteins. In particular an interaction between TRAF6, c-Cbl protein, and PI3K in dendritic cells (
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N.
      • Gu H.
      • Choi Y.
      ) has been shown. In the present study we evaluated whether endothelial CD40 after stimulation with sCD154 associates TRAF family proteins, c-Cbl, and the p85 subunit of PI3K. Immunoprecipitation of CD40 followed by Western blotting showed that stimulation of endothelial CD40 induced rapid association of TRAF2 and 6, c-Cbl, and the p85 PI3K subunit (Fig.5C). Such association was temporarily correlated with enhanced Akt activity (Fig. 5A). The association of TRAF3 to CD40 was minimal (Fig. 5C).
      Figure thumbnail gr5
      Figure 5Time course of Akt kinase activity and association of PI3K, c-Cbl, and TRAFs with CD40 upon sCD154 stimulation of HMEC. HMEC were treated for the indicated times with sCD154 (100 ng/ml, plus 1 μg/ml enhancer) and lysed. As control (Ctrl), cells were treated with vehicle alone. Kinase reactions and Western blot analysis were performed in anti-Akt immunoprecipitates from the corresponding lysates, as described under “Experimental Procedures.” Akt activity was assessed using GSK-3α/β as substrate for phosphorylation (P-GSK-3α/β).A, densitometric analysis of Akt kinase activity expressed as fold increase with respect to unstimulated cells (mean ± 1 S.D. from three different experiments). B, representative Western blot analysis showing P-GSK-3α/β generation and specific bands detected by the anti-Akt antibody. C, after treatment of HMEC with sCD154 (100 ng/ml, plus 1 μg/ml enhancer) for the indicated times, CD40 was immunoprecipitated from cell lysates. The immunoprecipitates were probed with antibodies to PI3K, c-Cbl, TRAF2, TRAF6, and TRAF3. Data are representative from three independent experiments.
      In order to evaluate the relevance of Akt in the biological activities elicited by CD40 stimulation in HMEC, we developed negative-dominant HMEC for Akt by transfecting the cells with Akt1 cDNA containing the mutation of lysine 179 to methionine. Expression of ND-Akt has been shown to interfere with the activation of endogenous Akt1 (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). Fig. 6 (A and B) compares Akt activity after CD40 stimulation with sCD154 in ND-Akt and in WT-Akt HMEC, transfected with an empty vector as control. At variance with the WT-Akt HMEC, ND-Akt HMEC did not display enhancement of Akt activity, despite the observation that the pathway of CD40-dependent PI3K activation was intact. Indeed, no differences in the association of CD40 with TRAF6, c-Cbl, and PI3K was observed after stimulation with sCD154 (Fig. 6C). As shown in Fig. 7, the anti-apoptotic effect of CD40 stimulation was abrogated in ND-Akt but not in WT-Akt HMEC. ND-Akt showed an enhanced apoptosis also in basal conditions. In addition, the proliferative response of HMEC to CD40 stimulation observed in WT-Akt, was impaired in ND-Akt cells (Fig.8A). Moreover, the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC plated on Matrigel was reduced in ND-Akt cells (Fig. 3, E and F), suggesting a role of Akt activation in the coordinate migration of endothelial cells. Indeed, as shown by the time-lapse analysis of HMEC motility, WT-Akt exhibited an enhanced motility after stimulation with sCD154 compared with control whereas ND-Akt did not (Fig. 8B and Fig.9).
      Figure thumbnail gr6
      Figure 6Comparison of Akt kinase activity and association of PI3K, c-Cbl, and TRAF-6 with CD40 upon sCD154 stimulation in ND-Akt and WT-Akt HMEC. Akt-negative-dominant HMEC were generated by transfecting the cells with Akt1 cDNA containing the mutation of lysine 179 to methionine. Control WT-Akt were transfected with the empty vector. ND-Akt and WT-Akt HMEC were treated for the indicated times with sCD154 (100 ng/ml, plus 1 μg/ml enhancer) and lysed. Kinase reactions and Western blot analysis were performed in anti-Akt immunoprecipitates from the corresponding lysates, as described under “Experimental Procedures.”A, densitometric analysis of Akt kinase activity was expressed as fold increase with respect to unstimulated cells (mean ± 1 S.D. from three different experiments). ND-Akt,dark column; WT-Akt, open column. B, representative Western blot showing P-GSK-3α/β generation and specific bands detected by the anti-Akt antibody in ND-Akt and WT-Akt HMEC. C, after treatment of ND-HMEC with sCD154 (100 ng/ml, plus 1 μg/ml enhancer) for the indicated times, CD40 was immunoprecipitated from cell lysates, and the immunoprecipitates were probed with antibodies to PI3K, c-Cbl, and TRAF6. Data are representative of three independent experiments.
      Figure thumbnail gr7
      Figure 7Abrogation of anti-apoptotic effect of CD40 stimulation in ND-Akt HMEC. A, DNA fragmentation was detected after DNA extraction and electrophoresis on 2% agarose gel.Row 1, control WT-Akt HMEC cultured in DMEM containing 10% FCS; row 2, WT-Akt HMEC serum-starved for 24 h;row 3, WT-Akt HMEC serum-starved treated with sCD154 (100 ng/ml, plus 1 μg/ml enhancer); row 4, control ND-Akt HMEC cultured in DMEM containing 10% FCS; row 5, ND-Akt HMEC serum-starved for 24 h; row 6, ND-Akt HMEC serum-starved treated with sCD154 (100 ng/ml, plus 1 μg/ml enhancer).B, apoptosis of HMEC evaluated by TUNEL assay after 24 h incubation with different stimuli (see “Experimental Procedures”). ND-Akt (dark column) and WT-Akt (open column) HMEC were incubated with 10% FCS, without FCS (noFCS), without FCS plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer) or with 0.25 μg/ml vincristine or 0.25 μg/ml vincristine plus sCD154 (100 ng/ml, plus 1 μg/ml enhancer). Data are expressed as mean ± 1 S.D. from three different experiments.
      Figure thumbnail gr8
      Figure 8Reduction of proliferation and motility induced by CD40 stimulation in ND-Akt HMEC. A, proliferation was evaluated by BrdUrd incorporation in ND-Akt and WT-Akt HMEC after 18-hour stimulation with vehicle alone or sCD154 (100 ng/ml, plus 1 μg/ml enhancer). OD, optical density.Panel B, cell motility, measured by time-lapse cinematography and digital image analysis, was evaluated after 4 h of stimulation with vehicle alone or sCD154 (100 ng/ml, plus 1 μg/ml enhancer) in ND-Akt and WT-Akt HMEC. Data are expressed as mean ± 1 S.D. from three different experiments.
      Figure thumbnail gr9
      Figure 9Micrographs representative of time-lapse analysis of WT-Akt and NT-Akt HMEC motility after stimulation with sCD154. Motility was performed by digital saving at 15-min intervals. Migration tracks (magnification: ×120) were generated by marking the position of the nucleus of individual cells in each image (see “Experimental Procedures”). WT-Akt (A andB) and ND-Akt (C and D) HMEC were stimulated for 4 h at 37 °C with vehicle alone (Aand C) or with sCD154 (100 ng/ml, plus 1 μg/ml enhancer) (B and D).

      DISCUSSION

      In the present study we demonstrated that resistance to apoptosis and the in vitro vessel-like formation elicited in endothelial cells after CD40 stimulation were dependent on the activation of Akt. This activation was triggered by PI3K that associated with CD40 after its ligation by sCD154.
      The stimulation of endothelial CD40 plays an important role in the phenotypic modulation of the endothelium to an activated state (
      • Henn V.
      • Slupsky J.R.
      • Grafe M.
      • Anagnostopoulos I.
      • Forster R.
      • Muller-Berghaus G.
      • Kroczek R.A.
      ). CD40 was shown to induce expression of collagenase and stromelysin on human monocytes/macrophages, and of collagenase, stromelysin, gelatinase B, and activated gelatinase A on vascular smooth muscle and endothelial cells (
      • Thienel U.
      • Loike J.
      • Yellin M.J.
      ,
      • Slupsky J.R.
      • Kalbas M.
      • Willuweit A.
      • Henn V.
      • Kroczek R.A.
      • Muller-Berghaus G.
      ,
      • Dechanet J.
      • Grosset C.
      • Taupin J.L.
      • Merville P.
      • Banchereau J.
      • Ripoche J.
      • Moreau J.F.
      ,
      • Schonbeck U.
      • Mach F
      • Bonnefoy J.Y.
      • Loppnow H.
      • Flad H.D.
      • Libby P.
      ). Ligation of endothelial CD40 by CD154, either expressed on activated monocytes or T cells or disgorged from platelet granules after activation, stimulated the production of various inflammatory cytokines by endothelial cells (
      • Thienel U.
      • Loike J.
      • Yellin M.J.
      ,
      • Dechanet J.
      • Grosset C.
      • Taupin J.L.
      • Merville P.
      • Banchereau J.
      • Ripoche J.
      • Moreau J.F.
      ). Moreover, it has been reported that surface-expressed CD154 is rapidly cleaved with generation of a circulating soluble CD154, which remains trimeric and biologically active (
      • Henn V.
      • Slupsky J.R.
      • Grafe M.
      • Anagnostopoulos I.
      • Forster R.
      • Muller-Berghaus G.
      • Kroczek R.A.
      ). A soluble form of CD154 released from the surface of tumor cells (
      • Bussolati B.
      • Russo S.
      • Deambrosis I.
      • Cantaluppi V.
      • Volpe A.
      • Ferrando U.
      • Camussi G.
      ) may contribute to endothelial activation in tumor angiogenesis. In vivo stimulation of endothelial CD40 was shown to trigger neoangiogenesis and its inhibition limited neoangiogenesis and allowed apoptotic regression in an experimental model of tumor neoangiogenesis (
      • Biancone L.
      • Cantaluppi V.
      • Boccellino M.
      • Del Sorbo L.
      • Russo S.
      • Albini A.
      • Stamenkovic I.
      • Camussi G.
      ).
      In the present study we found that stimulation of endothelial CD40 with sCD154 prevented apoptosis induced both by serum deprivation or treatment with vincristine and induced motility and vessel-like formation by HMEC. These effects were mediated by CD40-dependent signaling because it was abrogated by preventing the interaction between sCD154 and CD40 by a soluble CD40-muIg fusion protein. In addition, we found that apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by sCD154. The anti-apoptotic effect of sCD154 as well as cell motility, vessel-like formation, and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of PI3K, wortmannin and LY294002, suggesting that the effect was dependent on the activation of this kinase.
      PI3K/Akt is one of the central pathways involved in survival signaling (
      • Talapatra S.
      • Thompson C.B.
      ). Several receptors, including those for VEGF (
      • Gerber H.P.
      • Mc Murtrey A.
      • Kowalskj J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ), IGF-1 (
      • Weihua W.
      • Lee W.L.
      • Wu Y.Y.
      • Chen D.
      • Liu T.J.
      • Jang A.
      • Sharma P.M.
      • Wang P.H.
      ), and IL-3 (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ), transmit survival signals through these pathways. PI3K activation catalyzes the transfer of a phosphate group from ATP to the D3 position of phosphatidylinositol (PI), thus generating 3′-phosphatidylinositol phosphates (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). 3′-Phosphatidylinositol phosphates serve as binding sites for proteins that possess a pleckstrin homology domain such as Akt. The binding of Akt to 3′-phosphatidylinositol phosphates results in its translocation from cytosol to plasma membrane and phosphorylation of threonine 308 and serine 473 residues. Phosphorylation of threonine 308 and membrane localization depend on the activation of a phosphatidylinositol-dependent kinase-1 that also contains a pleckstrin homology domain (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ). Several studies have shown that Akt is the major effector of PI3K survival signaling (
      • Talapatra S.
      • Thompson C.B.
      ,
      • Zhang L.
      • Zhou W.
      • Velculescu V.E.
      • Kern S.E.
      • Hruban R.H.
      • Hamilton S.R.
      • Vogelstein B.
      • Kinzler K.W.
      ,
      • Zhou H.
      • Xin-Ming L.
      • Meinkoth J.
      • Pittman R.N.
      ).
      In the present study we found that a rapid increase in Akt enzymatic activity was induced by CD40 stimulation. Although, CD40 is known to stimulate the synthesis of cytokines that may activate Akt (
      • Melter M.
      • Reinders M.E.J.
      • Sho M.
      • Pal S.
      • Geehan C.
      • Denton M.D.
      • Mukhopadhyay D.
      • Briscoe D.
      ), we found that Akt activation was rapid and independent from protein synthesis, suggesting a direct effect of CD40 stimulation.
      CD40 is a member of the TNF receptor family, which lacks intrinsic enzymatic activity but is linked to intracellular signaling cascades through TRAF proteins (
      • Pullen S.S.
      • Miller H.G.
      • Everdeen D.S.
      • Dang T.T.A.
      • Crute J.J.
      • Kehry M.R.
      ,
      • Hu H.M.
      • O'Rourke K.
      • Boguski M.S.
      • Dixit V.M.
      ,
      • Cheng G.
      • Cleary A.M.
      • Ye Z.S.
      • Hong D.I.
      • Lederman S.
      • Baltimore D.
      ,
      • Ishida T.
      • Mizushima S.
      • Azuma S.
      • Kobayashi N.
      • Tojo T.
      • Suzuki K.
      • Aizawa S.
      • Watanabe T.
      • Mosialos G.
      • Kieff E.
      • Yamamoto T.
      • Inoue J.
      ,
      • Werneburg B.G.
      • Zoog S.J.
      • Dang T.T.
      • Kehry M.R.
      • Crute J.J.
      ). Activation of CD40-dependent signaling pathways is thought to be mediated primarily by recruitment of several members of the TRAF protein family to the multimerized CD40 cytoplasmic domain (
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      ). The CD40 cytoplasmic domain contains a membrane-proximal site that binds TRAF6 and a membrane-distal site that binds TRAF1, TRAF2, and TRAF3 (
      • Pullen S.S.
      • Miller H.G.
      • Everdeen D.S.
      • Dang T.T.A.
      • Crute J.J.
      • Kehry M.R.
      ,
      • Hu H.M.
      • O'Rourke K.
      • Boguski M.S.
      • Dixit V.M.
      ). We found that TRAF6 and TRAF2 co-precipitated with CD40 after stimulation of HMEC with sCD154. No significant enhancement of TRAF3 binding to CD40 was observed after sCD154 stimulation of endothelial cells. It has been described that TRAF3 up-regulation by shear stress abrogates CD40 signaling in endothelial cells (
      • Urbich C.
      • Mallat Z.
      • Tedgui A.
      • Clauss M.
      • Zeiher A.M.
      • Dimmeler S.
      ). In contrast, the binding of CD40-TRAF2 domain has been associated with the activation of transcription factors and of apoptosis-regulating proteins (
      • Shu H.B.
      • Halpin D.R.
      • Goeddel D.V.
      ,
      • Malinin N.L.
      • Boldin M.P.
      • Kovalenko A.V.
      • Wallach D.
      ,
      • Takeuchi M.
      • Rothe M.
      • Goeddel D.V.
      ,
      • McCarthy J.V.
      • Ni J.
      • Dixit V.M.
      ,
      • Yuasa T.
      • Ohno S.
      • Kehrl J.H.
      • Kyriakis J.M.
      ,
      • Nishitoh H.
      • Saitoh M.
      • Mochida Y.
      • Takeda K.
      • Nakano H.
      • Rothe M.
      • Miyazono K.
      • Ichijo H.
      ). Recently it has been found that in dendritic cells TRAF6 in concert with c-Cbl mediates the binding and the activation of PI3K by TNF receptor family members (
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N.
      • Gu H.
      • Choi Y.
      ). In the present study we found that the enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. c-Cbl, a cytoplasmic adapter molecule implicated in the negative regulation of signaling from a variety of tyrosine kinase receptors, has been recently identified as a positive modulator of the TNF receptor superfamily (
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N.
      • Gu H.
      • Choi Y.
      ). Information on the critical role of c-Cbl in the interaction of PI3K and in the subsequent activation of Akt was obtained in c-Cbl negative-dominant B cells (
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N.
      • Gu H.
      • Choi Y.
      ). In this context, it has been proposed that c-Cbl recruited PI3K by favoring association with TRAF6.
      In order to evaluate the relevance of Akt activation to biological activity dependent on the stimulation of endothelial CD40, we developed Akt negative-dominant cells by transfecting HMEC with Akt1 cDNA containing the mutation of lysine 179 to methionine (
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). Expression of ND-Akt has been shown to interfere with activation of the endogenous Akt1, suggesting that it displaced endogenous Akt from critical protein-protein interactions (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). CD40 stimulation in ND-Akt HMEC failed to enhance Akt activity, despite the observation that the association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, thein vitro formation of vessel-like tubular structures, and the enhanced motility of HMEC.
      In conclusion, these results suggest that the PI3K/Akt signaling axis was activated by endothelial CD40 stimulation and regulated multiple critical steps in angiogenesis, including endothelial cell survival, proliferation, migration, and vessel-like structure formation.

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