Notch4 Inhibits Endothelial Apoptosis via RBP-Jκ-dependent and -independent Pathways

Notch4, a member of the Notch family of transmembrane receptors, is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions, partly by regulating the propensity of cells to live or die. Various studies have demonstrated a role for Notch1 in modulating apoptosis, either in a positive or negative manner. In this study, we determined that constitutively active Notch4 (Notch4 intracellular domain) inhibited endothelial apoptosis triggered by lipopolysaccharide. Notch signals are transmitted by derepression and coactivation of the transcriptional repressor, RBP-Jκ, as well as by less well defined mechanisms that are independent of RBP-Jκ. A Notch mutant lacking the N-terminal RAM domain showed only partial antiapoptotic activity relative to Notch4 intracellular domain but stimulated equivalent RBP-Jκ-dependent transcriptional activity. Similarly, constitutively active RBP-Jκ activated a full transcriptional response but only demonstrated partial antiapoptotic activity. Additional studies suggest that Notch4 provides endothelial protection in two ways: inhibition of the JNK-dependent proapoptotic pathway in an RBP-Jκ-dependent manner and induction of an antiapoptotic pathway through an RBP-Jκ-independent up-regulation of Bcl-2. Our findings demonstrate that Notch4 activation inhibits apoptosis through multiple pathways and provides one mechanism to explain the remarkable capacity of endothelial cells to withstand apoptosis. Notch4, a member of the Notch family of transmembrane receptors, is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions, partly by regulating the propensity of cells to live or die. Various studies have demonstrated a role for Notch1 in modulating apoptosis, either in a positive or negative manner. In this study, we determined that constitutively active Notch4 (Notch4 intracellular domain) inhibited endothelial apoptosis triggered by lipopolysaccharide. Notch signals are transmitted by derepression and coactivation of the transcriptional repressor, RBP-Jκ, as well as by less well defined mechanisms that are independent of RBP-Jκ. A Notch mutant lacking the N-terminal RAM domain showed only partial antiapoptotic activity relative to Notch4 intracellular domain but stimulated equivalent RBP-Jκ-dependent transcriptional activity. Similarly, constitutively active RBP-Jκ activated a full transcriptional response but only demonstrated partial antiapoptotic activity. Additional studies suggest that Notch4 provides endothelial protection in two ways: inhibition of the JNK-dependent proapoptotic pathway in an RBP-Jκ-dependent manner and induction of an antiapoptotic pathway through an RBP-Jκ-independent up-regulation of Bcl-2. Our findings demonstrate that Notch4 activation inhibits apoptosis through multiple pathways and provides one mechanism to explain the remarkable capacity of endothelial cells to withstand apoptosis. The Notch proteins comprise a family of transmembrane receptors that have been highly conserved through evolution as mediators of cell fate and are comprised of four members in mammals (Notch1 to -4) (1Fleming R.J. Semin. Cell Dev. Biol. 1998; 9: 599-607Crossref PubMed Scopus (159) Google Scholar). Following intracellular processing of the full-length protein by a furin-like convertase, Notch is expressed at the cell surface as a heterodimeric receptor (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 3Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4874) Google Scholar). Engagement by ligand results in a two-step cleavage of the Notch heterodimer. These cleavage events release the intracellular domain of Notch (NotchIC) 1The abbreviations used are: NotchIC, Notch intracellular domain; LPS, lipopolysaccharide; JNK, c-Jun NH2-terminal kinase; HMEC, human microvascular endothelial cell(s); HUVEC, human umbilical vein endothelial cell(s); HA, hemagglutinin epitope tag; MTT, 3-[4′,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ΔΨm, mitochondrial transmembrane potential; TMRE, tetramethyl rhodamine ethyl ester; pNA, p-nitroaniline; NLS, nuclear localization signal; CMV, cytomegalovirus; YFP, yellow fluorescent protein. 1The abbreviations used are: NotchIC, Notch intracellular domain; LPS, lipopolysaccharide; JNK, c-Jun NH2-terminal kinase; HMEC, human microvascular endothelial cell(s); HUVEC, human umbilical vein endothelial cell(s); HA, hemagglutinin epitope tag; MTT, 3-[4′,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ΔΨm, mitochondrial transmembrane potential; TMRE, tetramethyl rhodamine ethyl ester; pNA, p-nitroaniline; NLS, nuclear localization signal; CMV, cytomegalovirus; YFP, yellow fluorescent protein. from its membrane tether, whereupon NotchIC translocates to the nucleus and interacts with the DNA-binding factor, RBP-Jκ (CBF1). RBP-Jκ is a DNA binding protein that has dual function: it represses transcription in the absence of NotchIC and activates transcription in its presence (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In the nucleus, NotchIC and RBP-Jκ associate with other factors to form a multimeric complex that results in transcriptional activation of various basic helix-loop-helix factors of the HES (Hairy and enhancer of Split) and HRT (Hairy-related transcription factor, also called HEY, HESR, Gridlock, and HERP) families, through the release of a corepressor complex from RBP-Jκ, and recruitment of a co-activator complex (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). There is cell type-dependent activation of the HRTs, but all three HRTs have been shown to be expressed in vascular cells (4Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1012) Google Scholar, 5Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.M. Lee M.E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 6Nakagawa O. Nakagawa M. Richardson J.A. Olson E.N. Srivastava D. Dev. Biol. 1999; 216: 72-84Crossref PubMed Scopus (245) Google Scholar, 7Henderson A.M. Wang S.J. Taylor A.C. Aitkenhead M. Hughes C.C. J. Biol. Chem. 2001; 276: 6169-6176Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). RBP-Jκ-independent Notch activity has also been described, but Notch signaling through this mechanism is less well elucidated. Because Notch function requires ligand-dependent cleavage of the intracellular domain, enforced expression of NotchIC results in a constitutively active, signaling form of the receptor, which results in altered cell fate decisions in several models (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 3Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4874) Google Scholar). The intracellular domain can be divided into two major subdomains; C-terminal to the transmembrane domain, there is a RAM domain, which is followed by an ankyrin domain composed of six Cdc10/ankyrin repeats. The region C-terminal to the ankyrin repeats acts as a putative transactivation domain in Notch1 but not Notch4 (8Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar). Notch1, -2, and -4 have been reported to be expressed in endothelial cells in vivo, and similar results have been reported in cultured endothelial cells (9Liu Z.J. Shirakawa T. Li Y. Soma A. Oka M. Dotto G.P. Fairman R.M. Velazquez O.C. Herlyn M. Mol. Cell Biol. 2003; 23: 14-25Crossref PubMed Scopus (407) Google Scholar). Several studies point to a role for Notch and its ligands in influencing vascular development (10Gridley T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5377-5378Crossref PubMed Scopus (66) Google Scholar). Notch signaling is required for arterial-venous differentiation in zebrafish (11Lawson N.D. Scheer N. Pham V.N. Kim C.H. Chitnis A.B. Campos-Ortega J.A. Weinstein B.M. Development. 2001; 128: 3675-3683Crossref PubMed Google Scholar). Mutant mice that are null for Notch1 show defects in the vasculature, and the severity of these vascular defects is enhanced in mice that are null for both Notch4 and Notch1 (12Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar). A homozygous Notch2 hypomorphic allele disrupts development of vasculature of the glomerulus, heart, and eye (13McCright B. Gao X. Shen L. Lozier J. Lan Y. Maguire M. Herzlinger D. Weinmaster G. Jiang R. Gridley T. Development. 2001; 128: 491-502Crossref PubMed Google Scholar). Interestingly, constitutive activation of Notch4 also causes defects in vascular remodeling (14Leong K.G. Hu X. Li L. Noseda M. Larrivee B. Hull C. Hood L. Wong F. Karsan A. Mol. Cell Biol. 2002; 22: 2830-2841Crossref PubMed Scopus (152) Google Scholar, 15Uyttendaele H. Ho J. Rossant J. Kitajewski J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5643-5648Crossref PubMed Scopus (242) Google Scholar). The regulation of vascular cell survival and death is critical during vascular development and homeostasis as well as in diverse pathological processes including inflammation (16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (381) Google Scholar, 17Chavakis E. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 887-893Crossref PubMed Scopus (232) Google Scholar, 18Guevara N.V. Chen K.H. Chan L. Pharmacol. Res. 2001; 44: 59-71Crossref PubMed Scopus (28) Google Scholar). Despite continual exposure to various inflammatory cytokines and exogenous toxins, endothelial cells have a remarkable capacity to resist apoptosis, and this may be a mechanism to preserve vascular integrity in pathological situations (19Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 20Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). However, we have shown that in certain circumstances inflammatory mediators, such as tumor necrosis factor and bacterial lipopolysaccharide (LPS), are to endothelial apoptosis. In we have shown that endothelial apoptosis by the protein family c-Jun NH2-terminal C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). Notch1 activation to endothelial cells are of is the mechanism (9Liu Z.J. Shirakawa T. Li Y. Soma A. Oka M. Dotto G.P. Fairman R.M. Velazquez O.C. Herlyn M. Mol. Cell Biol. 2003; 23: 14-25Crossref PubMed Scopus (407) Google Scholar). Notch and its ligands are expressed at in vascular we that Notch activation may a role in endothelial survival in inflammatory situations N. L. J. Weinmaster G. Dev. 2001; PubMed Scopus Google Scholar, J.R. S. Kitajewski J. C.R. Stark K.L. Genes Dev. 2000; 14: Google Scholar, V. C. D. T. L. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we the activity of Notch4, a Notch member that is expressed in the demonstrate that activated Notch4 is to endothelial apoptosis in response to the inflammatory LPS, in at two Notch activation is to through an RBP-Jκ-dependent Notch also provides antiapoptotic activity by an RBP-Jκ-independent dual antiapoptotic mechanism the activation of Notch a of the human microvascular endothelial cells to as by the for and and cultured as (14Leong K.G. Hu X. Li L. Noseda M. Larrivee B. Hull C. Hood L. Wong F. Karsan A. Mol. Cell Biol. 2002; 22: 2830-2841Crossref PubMed Scopus (152) Google Scholar, S. J. Full Text PDF PubMed Google Scholar). umbilical vein endothelial cells and cultured as A. Yee E. R. Harlan J.M. J. 1997; Google Scholar). at in The Notch4 intracellular region (14Leong K.G. Hu X. Li L. Noseda M. Larrivee B. Hull C. Hood L. Wong F. Karsan A. Mol. Cell Biol. 2002; 22: 2830-2841Crossref PubMed Scopus (152) Google a C-terminal hemagglutinin epitope and of the full-length The by as a and into the The lacking the RAM domain lacking the RAM and with an the for the and lacking all six ankyrin repeats and also into the as a constitutively active RBP-Jκ. by of the region of the of E. the region for the transactivation domain L. A. E. Nucleic Acids Res. 23: PubMed Scopus Google Scholar). The with and to the of the for from the of human RBP-Jκ of R. F. S. G. R.M. Mol. Cell Biol. 1998; PubMed Google Scholar). The of S. D. four of an RBP-Jκ binding into the T. E. Mol. Cell Biol. 1996; PubMed Scopus Google Scholar). and with the various as (19Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). by in of for the and by for yellow fluorescent protein expression a for the used in to to the on at a of the cells in as with or cell by 3-[4′,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium as (19Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). expressed as a of cells of mitochondrial transmembrane cells with tetramethyl rhodamine ethyl and for on a The mitochondrial used as a positive for the of of cleavage activity with a to the of cell from cells to with in a and at The release of the by active at and to cell cultured on with for and with for binding by with with the for and for with an with for and with a and with a of by as T. A. J. M.D. G. J. Immunol. 2001; PubMed Scopus Google Scholar). and and in at for with as and in of The to a for at and at a of and a of and of The the expressed of the and as a for the cells for at in The and cells for and dual to the with on a of from to the and The divided by the determined the relative from cell of and of Following and on in the negative used for as and at for and at for Notch4 Cell of the Notch family have been shown to have either or proapoptotic on the Notch cell or F. R. S. H. B. 2002; PubMed Scopus Google Scholar, K. B. D. C. D. 2000; PubMed Google Scholar). Because Notch4 is from the other Notch members and we Notch4 to regulate endothelial apoptosis by inflammatory have shown that can endothelial an activated form of Notch4 to to cell death C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). in inhibited death in response to LPS, as by a death pathway to apoptosis C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). that endothelial cells to mitochondrial integrity by the of cells to transmembrane The TMRE, to the of results in a mitochondrial which can be by of by that activated Notch4 is to in response to interacts with receptors to of mitochondrial C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). of activity the showed that inhibited activation of in to Because are a microvascular endothelial cell we also endothelial cells from apoptosis. either with activated Notch4 by an to or with the as a The of cells as determined by used to apoptosis by The of cells determined by only on the and the results of three such that activated Notch4 inhibits apoptosis of endothelial cells in response to The for the Notch4 Notch intracellular domain two major that have been in binding to the RBP-Jκ. These are the RAM domain at the and the ankyrin which of these are in the antiapoptotic activity of lacking of these and of these by and the Notch4 intracellular domain and the mutant to the nucleus, the mutant expressed in the a of nuclear expression also The localization of the mutant by studies not there is nuclear expression of this the localization of the mutant is with the presence of an nuclear localization in the region NH2-terminal to the ankyrin repeats as has been T. A. Honjo T. J. 1999; PubMed Google Scholar). In to nuclear localization of the an nuclear localization to the of the and this showed nuclear expression The various Notch4 for to apoptosis. in of the ankyrin repeats the of In the mutant only activity. Because the mutant a in nuclear we the mutant to the nucleus similar antiapoptotic activity to Interestingly, the mutant not to endothelial cells to the as but this mutant showed a similar of protection as the mutant nuclear expression is not for the mutant to full activity. These findings that the ankyrin repeats are for antiapoptotic and the partial protection by the mutant that the RAM may one of multiple pathways by The RAM domain may be required for derepression and activation of its RBP-Jκ. the for RBP-Jκ-dependent is the pathways required for the antiapoptotic activity of and is this function with to of signaling the critical pathways activated by Notch members in endothelial cells to be A major signaling pathway by Notch in other cell of the transcriptional RBP-Jκ (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). the RAM domain as a region required for RBP-Jκ-dependent Notch have not this to be K. Y. S. T. T. T. Honjo T. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, L. V. Mol. Cell Biol. 2000; PubMed Scopus Google Scholar). the of the various to RBP-Jκ, an RBP-Jκ-dependent to a used to for Notch4 activity. of the various cell with an RBP-Jκ-dependent demonstrate that the RBP-Jκ-dependent activated in response to the Notch4 the or the that the Notch4 RAM is not required for RBP-Jκ in endothelial cells. However, the mutant lacking the ankyrin repeats not to the RBP-Jκ at the of this domain for Notch4 function RBP-Jκ has been shown to transcription of basic helix-loop-helix factors of the HES and HRT (4Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1012) Google Scholar). In has been shown to be expressed in endothelial cells and to an role in endothelial function (4Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1012) Google Scholar). to the of the mutant Notch4 to an RBP-Jκ-dependent of from of the mutant Notch4 cell These results that the mutant not to the both the and the the expression of to a similar as findings suggest that only the ankyrin repeats are for Notch4 to through RBP-Jκ, the RAM domain is for this activity. the that the and the showed similar activity is in with the that nuclear localization is for RBP-Jκ activation by Notch4 through RBP-Jκ-dependent and mutant activates RBP-Jκ-dependent to a similar as the this mutant only provides partial protection apoptosis. these we that Notch4 is to endothelial cells apoptosis through RBP-Jκ-dependent and RBP-Jκ activation only antiapoptotic we a negative RBP-Jκ into endothelial cells. this mutant only the Notch4 antiapoptotic we also not to activity this not this not to inhibition of RBP-Jκ a RBP-Jκ-independent activity. an a constitutively active RBP-Jκ a constitutively active RBP-Jκ the activation domain to the of RBP-Jκ, and this into to the RBP-Jκ-dependent to the as but only apoptosis with the function of the findings demonstrate that activated Notch4 is to endothelial apoptosis by and that RBP-Jκ-dependent and signals are both required for full activity. Notch4 Activation and RBP-Jκ-dependent and has been shown to and we have shown that inhibition or of activation endothelial cells apoptosis C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar, R. J.P. Noseda M. Karsan A. J. 2003; PubMed Scopus Google Scholar). activated Notch4 to activation in endothelial as one mechanism of its antiapoptotic activity. that Notch4 activation by and that of the RAM domain not this In of the ankyrin repeats not the mutant is to RBP-Jκ-dependent the that the constitutively active mutant also that activation by LPS, one mechanism for the RBP-Jκ-dependent antiapoptotic activity. have demonstrated that Notch1 the antiapoptotic protein in but not all cell 1998; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). have that various family members are to endothelial cells apoptosis by or A. Yee E. R. Harlan J.M. J. 1997; Google Scholar, X. Yee E. Harlan J.M. Wong F. Karsan A. 1998; PubMed Google Scholar). Notch4 and the to Interestingly, only to mutant on and by Notch4 activation that of the RAM domain not RBP-Jκ we that Notch4 expression an RBP-Jκ-independent in constitutive activation of RBP-Jκ not to in endothelial that up-regulation by activated Notch4 through an RBP-Jκ-independent Our findings show that activated Notch4 is to endothelial apoptosis through multiple mechanisms and suggest that Notch activation a role in vascular studies by Taylor K.L. A.M. Hughes C.C. Res. 2002; PubMed Scopus Google the that Notch activation may be required for the of a endothelial by vascular endothelial factor the vascular defects in and Notch mice suggest that apoptosis may a role in the Interestingly, the of Notch1 Notch4 not endothelial differentiation in mice (10Gridley T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5377-5378Crossref PubMed Scopus (66) Google Scholar). The vascular is but remodeling of this endothelial not (10Gridley T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5377-5378Crossref PubMed Scopus (66) Google Scholar). it is that Notch activation is required to endothelial only in or Notch a critical role in the of cell and in this is to the regulation of apoptosis. Notch1 activation has been both in and in cell In and tumor Notch1 antiapoptotic activity F. R. S. H. B. 2002; PubMed Scopus Google Scholar, B.M. J. Immunol. 1999; Google Scholar). has that activated Notch1 with and inhibits apoptosis through the activation of A. R. S. O. A. S. 2001; PubMed Scopus Google Scholar). In in and human Notch1 activation has been reported to apoptosis K. B. D. C. D. 2000; PubMed Google Scholar, T. R. Y. M. D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In studies suggest multiple of apoptosis inhibition by the one B.M. J. Immunol. 1999; Google Notch1 in a as inhibited transcription and cell but not apoptosis in cells B.M. J. Immunol. 1999; Google Scholar). In 1998; 9: Full Text Full Text PDF PubMed Scopus Google have demonstrated that in the and cell apoptosis. Interestingly, these demonstrated up-regulation of in but not both cell death 1998; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). The findings demonstrate the cell of Notch signaling activity and the multiple pathways of used a mutant lacking the RAM domain to to pathways of protection of endothelial cells. The mutant showed partial activity the to RBP-Jκ-dependent transcriptional activity to the as the the RAM domain is not to of the ankyrin repeats all of the antiapoptotic the by the RAM domain the of the ankyrin repeats. Interestingly, a constitutively active RBP-Jκ mutant also transcriptional activity but only partial antiapoptotic the findings suggest that signals antiapoptotic activity that is both and independent of RBP-Jκ. the mutant the constitutively active RBP-Jκ to both inhibited RBP-Jκ-independent is transmitted K. D. T. A.M. T. S. 1998; PubMed Scopus Google Scholar, N. S. F. M. A. N. K. K. Weinmaster G. H. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Interestingly, in the by 1998; 9: Full Text Full Text PDF PubMed Scopus Google cells that in response to also it not reported the cell that induction also it is that the RBP-Jκ-independent induction of is In that inhibition of activation by Notch4 activation in endothelial cells is Our Notch in inhibition of the have that enforced expression of family members is to endothelial cell death in response to as well as other A. Yee E. R. Harlan J.M. J. 1997; Google Scholar, X. Yee E. Harlan J.M. Wong F. Karsan A. 1998; PubMed Google Scholar). Similarly, inhibition of activation a negative mutant inhibited endothelial apoptosis in response to or C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar, R. J.P. Noseda M. Karsan A. J. 2003; PubMed Scopus Google Scholar). mechanisms of in this are is to to the mitochondrial membrane and of mitochondrial transmembrane and release of Y. J. Cell Physiol. 2003; PubMed Scopus Google Scholar). apoptosis by proapoptotic members of the family and cell death K. R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, K. A. D. R.J. Mol. Cell Biol. 2002; 22: PubMed Scopus Google Scholar, S. S. K. B. S. R.J. A. A.C. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). by and Notch signals a antiapoptotic pathway that the death we not the of in these a showed that endothelial death by (9Liu Z.J. Shirakawa T. Li Y. Soma A. Oka M. Dotto G.P. Fairman R.M. Velazquez O.C. Herlyn M. Mol. Cell Biol. 2003; 23: 14-25Crossref PubMed Scopus (407) Google Scholar). that endothelial death is also on activation and is inhibited by findings suggest that the mechanisms explain the protection in the A. Yee E. R. Harlan J.M. J. 1997; Google Scholar, R. J.P. Noseda M. Karsan A. J. 2003; PubMed Scopus Google Scholar). Our studies with tumor necrosis endothelial apoptosis demonstrate that there is only a of F. M. A. The of Notch activity on apoptosis is with the pathway critical for not tumor necrosis endothelial cell which is through the death receptor pathway C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar, X. Yee E. Harlan J.M. Wong F. Karsan A. 1998; PubMed Google Scholar, P.J. Wong F. K. R. Karsan A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Our that activated Notch4 is to multiple pathways on the The Notch4 activity is into a pathway that is by RBP-Jκ and one that is independent of RBP-Jκ. cells are to various in vivo, continual exposure to various and proapoptotic cytokines A. Harlan J.M. R. S.J. B. and Scholar). The findings reported in this Notch activation in one mechanism to explain this capacity for

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