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Deciphering Vascular Endothelial Cell Growth Factor/Vascular Permeability Factor Signaling to Vascular Permeability

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m202391200

ISSN

1083-351X

Autores

Ali Pedram, Mahnaz Razandi, Ellis R. Levin,

Tópico(s)

Angiogenesis and VEGF in Cancer

Resumo

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology. Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology. The vascular endothelial cell growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial cell growth factor; ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase: NPRC, natriuretic peptide receptor, clearance; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; TJ, tight junction; VPF, vascular permeability factor; ZO, zona occludens; DMEM, Dulbecco's modified Eagle's medium; BAEC, bovine aortic endothelial cells; l-NAME, monomethyll-arginine; EC, endothelial cell; BNP, brain natriuretic peptide; NP, natriuretic peptides; PKG, protein kinase G; MAP, mitogen-activated protein; ANOVA, analysis of variance 1The abbreviations used are: VEGF, vascular endothelial cell growth factor; ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase: NPRC, natriuretic peptide receptor, clearance; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; TJ, tight junction; VPF, vascular permeability factor; ZO, zona occludens; DMEM, Dulbecco's modified Eagle's medium; BAEC, bovine aortic endothelial cells; l-NAME, monomethyll-arginine; EC, endothelial cell; BNP, brain natriuretic peptide; NP, natriuretic peptides; PKG, protein kinase G; MAP, mitogen-activated protein; ANOVA, analysis of varianceglycoprotein is an important angiogenesis factor that was originally isolated as a vascular permeability factor (VPF) (1Senger D.R. Galli S.J. Dvorak A.M. Peruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3380) Google Scholar, 2Ferrara N. Trends Cardiovasc. Med. 1993; 3: 224-225Crossref Scopus (169) Google Scholar). VEGF/VPF (henceforth designated VPF) potently stimulates fluid transgression through endothelial cell (EC) tight junctions (TJ) (3Milton S.G. Knutson K.P. J. Cell. Physiol. 1990; 144: 498-504Crossref PubMed Scopus (63) Google Scholar, 4Larson D.M. Ryan U.S. Endothethelial Function. 3. CRC Press, Inc., Boca Raton, FL1988: 75-84Google Scholar). This permeability factor also modulates the formation and function of vesiculovascular organelles in venules (5Feng D. Nagy J.A. Hipp J. Pyne K. Dvorak H.F. Dvorak A.M. J. Physiol. (Lond.). 1997; 504: 747-761Crossref Scopus (91) Google Scholar) and the development of EC fenestrations (6Roberts W.G. Palade G.E. J. Cell Sci. 1995; 108: 2369-2379Crossref PubMed Google Scholar). These mechanisms underlie the enhanced vascular permeability seen in response to VPF, which is implicated in the ascites associated with ovarian and other carcinomas (1Senger D.R. Galli S.J. Dvorak A.M. Peruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3380) Google Scholar, 7Gossmann A. Helbich T.H. Mesiano S. Shames D.M. Wendland M.F. Roberts T.P. Ferrara N. Jaffe R.B. Brasch R.C. Am. J. Obstet. Gynecol. 2000; 183: 956-963Abstract Full Text PDF PubMed Scopus (51) Google Scholar), the pathogenesis of diabetic retinopathy (8Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3361) Google Scholar), and the ovarian hyperstimulation syndrome (9Levin E.R. Rosen G.F. Yee W. Cassidenti D. Meldrum D. Pedram A. J. Clin. Invest. 1998; 102: 1978-1985Crossref PubMed Scopus (146) Google Scholar).The angiogenesis-promoting actions of VPF result after binding and signaling through the transmembrane receptors Flk-1 (10Millauer B.S. Wizigmann-Voos H. Schnurch R. Martinez N.P.H. Moeller W. Risau A. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar), Flt-1 (11Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar), and neuropilin (12Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar). The Flk-1 tyrosine kinase receptor (VEGF-R2) has been proposed to participate in VPF permeability (13Murohara T. Horowitz B.S. Silver M. Tsurumi Y. Chen D. Sullivan A. Isner J.M. Circulation. 1998; 97: 99-107Crossref PubMed Scopus (469) Google Scholar), but signaling through other related receptors also appears to be important (14Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 49: 34884-34892Abstract Full Text Full Text PDF Scopus (98) Google Scholar). Flk-1 receptors activate membrane-associated kinases, such as Src and phosphatidylinositol 3-kinase (PI3K) (11Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 15Guo D. Jia Q. Song H.-Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), and Src is critical to the role of the participation of VPF in the development of local edema following brain insult (16Van Bruggen N. Thbodeaux H. Plamer J.T. Lee W.P., Fu, L. Cairns B. Tumas D. Gerlai R. Williams S.-P. van Lookeren Campagne M. Ferrara N. J. Clin. Invest. 1999; 104: 1613-1620Crossref PubMed Scopus (390) Google Scholar, 17Elicieri B.P. Paul R. Schwartzberg P.L. Hood J.D. Leng J. Cheresh D.A. Mol. Cell. 1999; 4: 915-924Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). PI3K contributes to the ability of VPF to promote EC migration (18Radisavljevic Z. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 20770-20774Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), but its precise role in permeability is unclear. Signaling to the generation of nitric oxide (NO) (13Murohara T. Horowitz B.S. Silver M. Tsurumi Y. Chen D. Sullivan A. Isner J.M. Circulation. 1998; 97: 99-107Crossref PubMed Scopus (469) Google Scholar, 18Radisavljevic Z. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 20770-20774Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) and subsequent activation of the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (19Kroll J. Waltenberger J. J. Biol. Chem. 1997; 272: 32521-32527Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar), prostacyclin generation (13Murohara T. Horowitz B.S. Silver M. Tsurumi Y. Chen D. Sullivan A. Isner J.M. Circulation. 1998; 97: 99-107Crossref PubMed Scopus (469) Google Scholar), or VPF-induced protein kinase C activity (20Wu H.M. Yuan Y. Zawieja D.C. Tinsley J. Granger H.J. Am. J. Physiol. 1999; 276: H535-H542PubMed Google Scholar) have all been proposed to contribute to increased vascular permeability. How activation of various signals is integrated into a functional pathway responsible for permeability is undetermined.Regarding NO, this gas disrupts both cytoskeletal protein complexing in epithelial cells and the arrangement of the actin cytoskeleton (21Clancy R.M. Rediske J. Tang X. Nijher N. Frenkel S. Philips M. Abramson S.B. J. Clin. Invest. 1997; 100: 1789-1796Crossref PubMed Scopus (108) Google Scholar, 22Salzman A.L. Menconi M.J. Unno N. Ezzell R.M. Casey D.M. Gonzalez P.K. Fink M.P. Am. J. Physiol. 1995; 268: G361-G373PubMed Google Scholar, 23Bacallao R. Garfinkel A. Monke S. Zampighi G. Mandel L.J. J. Cell Sci. 1994; 107: 3301-3313Crossref PubMed Google Scholar, 24Antonetti D.A. Barber A.J. Hollinger L.A. Wolpert E.B. Gardner T.W. J. Biol. Chem. 1999; 274: 23463-23467Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). This results in the dilation of cell tight junctions due to ATP depletion (23Bacallao R. Garfinkel A. Monke S. Zampighi G. Mandel L.J. J. Cell Sci. 1994; 107: 3301-3313Crossref PubMed Google Scholar). We previously implicated NO as participating in VPF-induced permeability in the pathogenesis of the human ovarian hyperstimulation syndrome (9Levin E.R. Rosen G.F. Yee W. Cassidenti D. Meldrum D. Pedram A. J. Clin. Invest. 1998; 102: 1978-1985Crossref PubMed Scopus (146) Google Scholar). In parallel, poorly defined VPF signaling pathways can result in the phosphorylation of TJ proteins that exist in complex, such as zona occludens-1 (ZO-1) or occludin (24Antonetti D.A. Barber A.J. Hollinger L.A. Wolpert E.B. Gardner T.W. J. Biol. Chem. 1999; 274: 23463-23467Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). Phosphorylation of TJ proteins results in a lower transcellular resistance of EC (25Staddon J.M. Herrenknecht K. Smales C. Rubin L.L. J. Cell Sci. 1995; 108: 609-619Crossref PubMed Google Scholar), serving as an index of barrier function. Upon phosphorylation, TJ proteins assume abnormal relationships with other members of this complex, thereby creating "leaky" endothelial cell-cell contacts (26Hirase T. Kawashima S. Wong E.Y.M. Ueyama T. Rikitake Y. Tsukita S. Yokoyama M. Staddon J.M. J. Biol. Chem. 2001; 276: 10423-10431Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 27Kevil C.G. Oshima T. Alexander B. Coe L.L. Alexander J.S. Am. J. Physiol. 2000; 279: C21-C30Crossref PubMed Google Scholar). Thus, VPF-induced signaling to the cytoskeleton and to associated TJ proteins is potentially important and could present therapeutic targets to prevent vascular pathobiology (7Gossmann A. Helbich T.H. Mesiano S. Shames D.M. Wendland M.F. Roberts T.P. Ferrara N. Jaffe R.B. Brasch R.C. Am. J. Obstet. Gynecol. 2000; 183: 956-963Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 8Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3361) Google Scholar, 9Levin E.R. Rosen G.F. Yee W. Cassidenti D. Meldrum D. Pedram A. J. Clin. Invest. 1998; 102: 1978-1985Crossref PubMed Scopus (146) Google Scholar).Relevant to vascular biology, the natriuretic peptides (NP) are a family of small proteins that modulate salt and water balance, as well as vascular tone (reviewed in Ref. 28Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2028) Google Scholar). Atrial and brain natriuretic peptides (ANP and BNP) are produced predominantly in the heart, whereas C-type natriuretic peptide (CNP) is synthesized by the endothelial cell. These peptides inhibit vascular cell growth mainly after binding the guanylate cyclase A (reactive to ANP or BNP) or B (CNP specific) receptors (29Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 334: 68-71Crossref Scopus (499) Google Scholar, 30Chinkers M. Garbers D.L. Chang M.S. Lowe D.G. Chin H. Goeddel D.V. Schultz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (882) Google Scholar). This stimulates the production of cGMP and subsequent activation of PKG, inducing target genes or the modulation of K+ channels (28Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2028) Google Scholar). A second class of NP receptors is the clearance receptor (NPRC) (31Maack T. Suzuki M. Almeida F.A. Nussenzveig D. Scarborough R.M. McEnroe G.A. Lewicki J.A. Science. 1987; 238: 675-678Crossref PubMed Scopus (847) Google Scholar), and this protein may contribute to the inhibition of cardiomyocyte, vascular endothelial cell, or astrocyte proliferation (28Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2028) Google Scholar, 32Itoh H. Pratt R.E. Dzau V.J. Hypertension. 1992; 19: 758-761Crossref PubMed Scopus (129) Google Scholar, 33Levin E.R. Frank H.J.L. Am. J. Physiol. 1991; 261: E183-E189PubMed Google Scholar).Because VPF-induced permeability underlies the pathobiology of several disorders (7Gossmann A. Helbich T.H. Mesiano S. Shames D.M. Wendland M.F. Roberts T.P. Ferrara N. Jaffe R.B. Brasch R.C. Am. J. Obstet. Gynecol. 2000; 183: 956-963Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 8Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3361) Google Scholar, 9Levin E.R. Rosen G.F. Yee W. Cassidenti D. Meldrum D. Pedram A. J. Clin. Invest. 1998; 102: 1978-1985Crossref PubMed Scopus (146) Google Scholar, 16Van Bruggen N. Thbodeaux H. Plamer J.T. Lee W.P., Fu, L. Cairns B. Tumas D. Gerlai R. Williams S.-P. van Lookeren Campagne M. Ferrara N. J. Clin. Invest. 1999; 104: 1613-1620Crossref PubMed Scopus (390) Google Scholar), we determined a relevant signaling pathway and the resulting morphological consequences in EC. Furthermore, we identify the NP as inhibitors of these processes, in vitroand in vivo.DISCUSSIONVPF-induced vascular permeability is a critical contributor to the pathophysiology of diabetic retinopathy, ovarian, and other cancer-related ascites and the cerebral edema and injury following vascular insufficiency (1Senger D.R. Galli S.J. Dvorak A.M. Peruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3380) Google Scholar, 7Gossmann A. Helbich T.H. Mesiano S. Shames D.M. Wendland M.F. Roberts T.P. Ferrara N. Jaffe R.B. Brasch R.C. Am. J. Obstet. Gynecol. 2000; 183: 956-963Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 8Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3361) Google Scholar, 16Van Bruggen N. Thbodeaux H. Plamer J.T. Lee W.P., Fu, L. Cairns B. Tumas D. Gerlai R. Williams S.-P. van Lookeren Campagne M. Ferrara N. J. Clin. Invest. 1999; 104: 1613-1620Crossref PubMed Scopus (390) Google Scholar, 17Elicieri B.P. Paul R. Schwartzberg P.L. Hood J.D. Leng J. Cheresh D.A. Mol. Cell. 1999; 4: 915-924Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). Permeability of EC also contributes to angiogenesis. We report that the ability of VPF to modify 1) the actin cytoskeletal architecture, 2) TJ protein phosphorylation and localization, and 3) the permeability barrier function of vascular endothelial cells results from signaling. The tyrosine kinase activity of the Flk-1 receptor mediates the ability of VPF to signal to ERK and EC proliferation (59Parenti A. Morbidelli L. Cui X.L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Stimulation of the p38 MAP kinase or PI3K/AKT/S6 kinases by VPF underlies EC migration (54Wang W. Dentler W.L. Borchardt R.T. Am. J. Physiol. 2001; 280: H434-H440Crossref PubMed Google Scholar,59Parenti A. Morbidelli L. Cui X.L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 60Rousseau S. Houle F. Landry J. Huot J. Oncogene. 1997; 15: 2169-2177Crossref PubMed Scopus (718) Google Scholar) and also proliferation (66Dayanir V. Meyer R.D. Lashkar K. Rahimi N. J. Biol. Chem. 2001; 276: 17686-17692Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Interestingly, AKT can inhibit p38 kinase activation in some settings, thus promoting EC survival, a function of VPF (67Gratton J.-P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). We showed previously that JNK (and molecules downstream from this kinase) affects VPF-induced cyclin D1 synthesis, Cdk4 activity, and G1/S cell cyclin progression in EC (37Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 1998; 273: 26722-26728Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). This occurs through a signaling cross-talk, where ERK stimulation leads to the upstream activation of SEK-1 and JNK. Thus, multiple signaling proteins contribute to the various function of this important vascular factor.Here we define the signaling events that are responsible for VPF-induced permeability and establish that the natriuretic peptides inhibit these functions (Fig. 7). VPF-induced Src is essential to the vascular permeability and cerebral edema that follows ischemic stroke (17Elicieri B.P. Paul R. Schwartzberg P.L. Hood J.D. Leng J. Cheresh D.A. Mol. Cell. 1999; 4: 915-924Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 47Paul R. Zhang Z.G. Eliceiri B.P. Jiang Q. Boccia A.D. Zhang R.I. Chopp M. Cheresh D.A. Nat. Med. 2001; 7: 222-227Crossref PubMed Scopus (304) Google Scholar), but what occurs downstream of Src is not clear. We find that Src activation is necessary for VPF stimulation of ERK and JNK MAP kinases and the activation of PI3K/AKT (Fig.8). By inhibiting each of these downstream proteins, we identify and link important signaling effectors of VPF and Src. These effectors result in the egress of molecules through the EC barrier. We also find that p38 MAP kinase and NO contribute to this effect of VPF. As shown previously, Flk-1 activation of Src results in enhanced NO production (57He H. Venema V.J., Gu, X. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. Chem. 1999; 274: 25130-25135Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), and activation of endothelial nitric-oxide synthase contributes to VPF-induced angiogenesis and vascular permeability (68Fukumura D. Gohongi T. Kadambi A. Izumi Y. Ang J. Yun C.-O. Buerk D.G. Huang P.L. Jain R.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2604-2609Crossref PubMed Scopus (795) Google Scholar). Here we show that inhibition of NO generation partially prevents VEGF-induced ERK. It must be appreciated, however, that AKT can directly activate NO (69Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar) upon activation by PI3K in response to VEGF (58Papapetropoulos A. Guillermo-Cardena G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1013) Google Scholar).We show that Src also activates JNK in the setting of VPF, and this occurs in part from ERK activation and ERK-JNK cross-talk (37Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 1998; 273: 26722-26728Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We report here a novel auto-activation loop for VPF signaling, where JNK and PI3K/AKT bi-directionally and positively regulate each other's activity. There is precedent that platelet-derived growth factor or epidermal growth factor activates a uni-directional cross-talk from PI3K to JNK (70Logan S.K. Falasca M., Hu, P. Schlessinger J. Mol. Cell. Biol. 1997; 17: 5784-5790Crossref PubMed Scopus (123) Google Scholar). Analogous to our model, the small GTP-binding protein Rac has been found to be both upstream (71Coniglio S.J. Jou T.-S. Symons M. J. Biol. Chem. 2001; 276: 28113-28120Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and downstream of PI3K (72Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar), leading to the proposal of a positive feedback loop between these two signaling proteins (71Coniglio S.J. Jou T.-S. Symons M. J. Biol. Chem. 2001; 276: 28113-28120Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These molecules are relevant to VPF action in that VPF-induced PI3K/AKT contributes to cell migration (18Radisavljevic Z. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 20770-20774Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The importance of JNK and PI3K/AKT cross-activation by VPF is demonstrated here in that signaling from these Ser/Thr kinases leads to alteration of 1) the morphological actin structure and TJ protein localization, and 2) enhanced permeability of the EC (see below). In diabetic proliferative retinopathy or the hypervascularity of prematurity, the newly formed blood vessels are "leaky," and this has been attributed in part to the actions of VPF, stimulated by hypoxic conditions in the retina (8Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3361) Google Scholar, 73Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). Increased vascular permeability can lead to retinal hemorrhage and edema. Therefore, inhibition of the VPF-induced signaling that we define here (prevented in this case by natriuretic peptides) may be therapeutically useful.How does VPF-induced signaling result in leaky EC? This permeability factor promotes fenestrations in EC and stimulates vesiculovascular organelle formation/function in venules (5Feng D. Nagy J.A. Hipp J. Pyne K. Dvorak H.F. Dvorak A.M. J. Physiol. (Lond.). 1997; 504: 747-761Crossref Scopus (91) Google Scholar, 6Roberts W.G. Palade G.E. J. Cell Sci. 1995; 108: 2369-2379Crossref PubMed Google Scholar). VPF also promotes the paracellular transgression of fluid through EC tight junctions (3Milton S.G. Knutson K.P. J. Cell. Physiol. 1990; 144: 498-504Crossref PubMed Scopus (63) Google Scholar, 4Larson D.M. Ryan U.S. Endothethelial Function. 3. CRC Press, Inc., Boca Raton, FL1988: 75-84Google Scholar). The important barrier function of EC is highly dependent upon the architecture of the TJ. TJ proteins such as the ZO family (ZO-1, -2, and -3) physically complex with occludin (74Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar), claudin (75Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (973) Google Scholar), junctional adhesion molecule (76Martin-Padura I. Lostaglio S. Schneeman M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1132) Google Scholar), and cingulin (77Bazzoni G. Martinez-Estrada M. Orsenigo F. Cordenonsi M. Citi S. Dejana E. J. Biol. Chem. 2000; 27: 20520-20526Abstract Full Text Full Text PDF Scopus (369) Google Scholar), and several of these proteins associate with actin at cell junctions to form a seal (78Wittchen E.S. Haskins J. Stevenson B.R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Rearrangement of the actin cytoskeleton disrupts the TJ protein(s) complex formation, lowering transcellular resistance and barrier function (23Bacallao R. Garfinkel A. Monke S. Zampighi G. Mandel L.J. J. Cell Sci. 1994; 107: 3301-3313Crossref PubMed Google Scholar, 79Blum M.S Toninelli E. Anderson J.M. Balda M.S. Zhou J. O'Donnell L. Pardi R. Bender J.R. Am. J. Physiol. 1997; 273: H286-H294PubMed Google Scholar). This also leads to the development of endothelial cell fenestrations that are known to be induced by VPF (6Roberts W.G. Palade G.E. J. Cell Sci. 1995; 108: 2369-2379Crossref PubMed Google Scholar).We found that after VPF treatment, a redistribution of ZO-1 and occludin proteins at the EC contact sites occurred, and this was blocked by ANP. It has been reported recently that VPF can 1) alter ZO-1 and occludin concentrations at the EC tight junction, and 2) lower transendothelial cell electrical resistance by undetermined mechanisms (80Wang W. Dentler W.L. Borchardt R.T. Am. J. Physiol. 2001; 280: H434-H440Crossref PubMed Google Scholar). Here we implicate the phosphorylation of these TJ proteins by JNK and PI3K/AKT, as the basis for the morphological alteration of the TJ. Preventing this phosphorylation by expressing dominant negative signaling molecules or by exposing the cells to ANP significantly led to the inhibition of VPF-enhanced permeability. Previous work (81Kevil C.G. Payne D.K. Mire E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) has implicated ERK in the increased EC permeability induced by VPF, which correlated with the loss of VE-cadherin and occludin localization at EC junctions. Also, oxidant stress induced by H2O2causes ERK-dependent phosphorylation and redistribution of occludin in EC (27Kevil C.G. Oshima T. Alexander B. Coe L.L. Alexander J.S. Am. J. Physiol. 2000; 279: C21-C30Crossref PubMed Google Scholar). In contrast, PKC induces serine phosphorylation of occludin, leading to the incorporation of occludin into the TJ of epithelial cells (82Andreeva A.Y. Krause E. Muller E.-C. Blasig I.E. Utepbergenov D.I. J. Biol. Chem. 2001; 276: 38480-38486Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Additionally, tyrosine phosphorylation of ZO-1 and occludin in EC occurs after exposure to VPF, induced by unknown kinases (24Antonetti D.A. Barber A.J. Hollinger L.A. Wolpert E.B. Gardner T.W. J. Biol. Chem. 1999; 274: 23463-23467Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). There are numerous potential sites for JNK and AKT-induced Ser/Thr phosphorylation (as well as tyrosine) within ZO-1 and occludin, and a detailed mutagenesis study of the required residues for TJ assembly and VPF function is underway.We also showed that VPF signaling through these pathways redistributes actin fibers in the EC. In part, this resulted from the generation of NO, which has been linked to ATP depletion, and the subsequent dilation of tight junctions (9Levin E.R. Rosen G.F. Yee W. Cassidenti D. Meldrum D. Pedram A. J. Clin. Invest. 1998; 102: 1978-1985Crossref PubMed Scopus (146) Google Scholar, 21Clancy R.M. Rediske J. Tang X. Nijher N. Frenkel S. Philips M. Abramson S.B. J. Clin. Invest. 1997; 100: 1789-1796Crossref PubMed Scopus (108) Google Scholar, 23Bacallao R. Garfinkel A. Monke S. Zampighi G. Mandel L.J. J. Cell Sci. 1994; 107: 3301-3313Crossref PubMed Google Scholar). 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