Notch4 Inhibits Endothelial Apoptosis via RBP-Jκ-dependent and -independent Pathways
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m312102200
ISSN1083-351X
AutoresFarrell MacKenzie, Patrick J. Duriez, Fred Wong, Michela Noseda, Aly Karsan,
Tópico(s)Cerebrovascular and genetic disorders
ResumoNotch4, 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 able to induce endothelial apoptosis. In particular, we have shown that LPS induces endothelial apoptosis by activating the mitogen-activated protein kinase family member, c-Jun NH2-terminal kinase (JNK) (20Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). Although Notch1 activation appears to promote endothelial viability when cells are starved of serum, little is known about 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). Since Notch and its ligands are expressed at high levels in vascular endothelium, we postulated that Notch activation may play a protective role in maintaining endothelial survival in inflammatory situations (21Villa N. Walker L. Lindsell C.E. Gasson J. Iruela-Arispe M.L. Weinmaster G. Mech Dev. 2001; 108: 161-164Crossref PubMed Scopus (336) Google Scholar, 22Shutter J.R. Scully S. Fan W. Richards W.G. Kitajewski J. Deblandre G.A. Kintner C.R. Stark K.L. Genes Dev. 2000; 14: 1313-1318PubMed Google Scholar, 23Lindner V. Booth C. Prudovsky I. Small D. Maciag T. Liaw L. Am. J. Pathol. 2001; 159: 875-883Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In this paper, we examined the functional activity of Notch4, a Notch member that is expressed selectively in the endothelium. We demonstrate that activated Notch4 is able to inhibit endothelial apoptosis in response to the inflammatory mediator, LPS, in at least two different ways. First, Notch activation is able to inhibit LPS-mediated JNK activation, through an RBP-Jκ-dependent pathway. Notch also provides antiapoptotic activity by up-regulating Bcl-2 via an RBP-Jκ-independent mechanism. This dual antiapoptotic mechanism makes the activation of Notch a particularly potent inhibitor of the intrinsic apoptotic pathway. Transformed human microvascular endothelial cells (HMEC-1, here-after referred to as HMEC) were provided by the Centers for Disease Control and Prevention (Atlanta, GA) and cultured as previously described (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, 24Ades E.W. Candal F.J. Swerlick R.A. George V.G. Summers S. Bosse D.C. Lawley T.J. J. Invest. Dermatol. 1992; 99: 683-690Abstract Full Text PDF PubMed Google Scholar). Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as previously described (25Karsan A. Yee E. Poirier G.G. Zhou P. Craig R. Harlan J.M. Am. J. Pathol. 1997; 151: 1775-1784PubMed Google Scholar). Cells were maintained at 37 °C in 5% CO2. The Notch4 intracellular region (Notch4IC) construct, described previously (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), contains a C-terminal hemagglutinin epitope tag (HA) and includes amino acids 1476–2003 of the 2003 residue full-length Notch4. The Notch4IC deletion mutants were constructed by PCR, using Notch4IC as a template, and inserted into the LNCX retroviral vector. The Notch4IC mutants (see Fig. 3A) include constructs (i) lacking the entire RAM domain (ΔRAM; encodes amino acids 1518–2003); (ii) lacking the RAM and N-terminally fused with an SV40-derived NLS (NLS-ΔRAM; the NLS tag codes for the amino acid sequence DPKKKRKV); and (iii) lacking all six ankyrin repeats (ΔAnk encodes amino acids 1476–1578 and 1801–2003). Notch4IC was also cloned into the MSCV-IRES-YFP (MIY) retroviral vector, as was RBP-VP16, a constitutively active RBP-Jκ. RBP-VP16 was constructed by PCR amplification of the 3′ region of the murine RBP-VP16 cDNA (gift of E. Manet) containing the coding region for the VP16 transactivation domain (26Waltzer L. Bourillot P.Y. Sergeant A. Manet E. Nucleic Acids Res. 1995; 23: 4939-4945Crossref PubMed Scopus (70) Google Scholar). The product was digested with AflII and ligated to the corresponding AflII site of the cDNA for FLAG-RBP-Jκ derived from the RBP-2N isoform of human RBP-Jκ (gift of R. Schmid) (27Oswald F. Liptay S. Adler G. Schmid R.M. Mol. Cell Biol. 1998; 18: 2077-2088Crossref PubMed Google Scholar). The 4×RBP-Jκ luciferase plasmid (gift of S. D. Hayward) includes four copies of an RBP-Jκ binding element cloned into the pGL2pro (Promega) firefly luciferase plasmid (28Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell Biol. 1996; 16: 952-959Crossref PubMed Scopus (395) Google Scholar). HMEC and HUVEC were transduced with the various constructs as described previously (19Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Polyclonal HMEC lines were isolated by selection in 300 μg/ml of G418 (Invitrogen) for the LNCX constructs and by sorting for yellow fluorescent protein (YFP) expression using a FACS 440 (BD Biosciences) for the MIY constructs. Polyclonal HMEC lines were used in order to avoid artifacts due to the retroviral integration site. Viability Assay—For viability assays, HMEC were seeded on 96-well plates at a density of 15,000 cells/well. On the following day, cells were incubated in LPS (concentrations as indicated) with ALLN (25 μm) or cycloheximide (50 μg/ml). After 16 h, viable cell numbers were estimated by 3-[4′,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described (19Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Viability was expressed as a proportion of cells incubated without LPS. Loss of ΔΨm—To measure mitochondrial transmembrane potential (ΔΨm), 5 × 105 cells were incubated with tetramethyl rhodamine ethyl ester (TMRE) (Molecular Probes, Inc., Eugene, OR) and analyzed for fluorescence on a flow cytometer. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (Sigma) was used as a positive control for the detection of loss of ΔΨm. Caspase Activity Assay—DEVD-p-nitroaniline (pNA) (caspase 3/7) cleavage activity was quantitated with a colorimetric assay kit according to the manufacturer's instructions (R & D Systems). Briefly, 200 μg of whole cell lysates from HMEC cells exposed to LPS (100 ng/ml) was combined with DEVD-pNA (200 μm) in a 96-well plate and incubated at 37 °C. The release of the chromophore by active caspases was quantitated at 405 nm and normalized to untreated cell lysates. Transduced HMEC lines were cultured overnight on chamber slides, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with cold methanol for 3 min. Nonspecific binding was blocked by incubation with 5% goat serum. Cells were stained with the mouse anti-HA monoclonal primary antibody (1:100 dilution) for 1 h and then for 30 min with an AlexaFluor 488-conjugated goat anti-mouse IgG secondary antibody (1:500 dilution). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole for 5 min, and coverslips were mounted with 50% glycerol. Slides were viewed using a Zeiss Axioplan II Imaging inverted microscope (Carl Zeiss Canada), and images were captured with a 1350EX cooled CCD digital camera (QImaging). Transient transfection of luciferase reporter plasmids was carried out by electroporation as described (29Ear T. Giguere P. Fleury A. Stankova J. Payet M.D. Dupuis G. J. Immunol. Methods. 2001; 257: 41-49Crossref PubMed Scopus (25) Google Scholar). Transduced HMEC lines were grown until ∼80% confluence and then trypsinized and resuspended in HMEC medium. Cells (1.5 × 106/transfection) were pelleted at 1000 rpm for 5 min, washed with PBS, pelleted as previous, and then resuspended in 0.4 ml of electroporation buffer (20 mm HEPES, 137 mm sodium chloride, 5 mm potassium chloride, 0.7 mm sodium phosphate, 6 mm d-glucose, pH 7.0) containing luciferase reporter plasmid DNA. The cell/DNA mixture was transferred to a 4-mm gap electroporation cuvette (Bio-Rad), left for 10 min at room temperature, and then electroporated at a fixed capacitance of 900 microfarads and 200 V using a Bio-Rad Gene Pulser II instrument. For each transfection, 2.5 μg of 4×RBP-Jκ-binding promoter luciferase and 1 μg of RL-CMV was used. The RL-CMV reporter contains the Renilla luciferase cDNA expressed under control of the CMV immediate early enhancer/promoter and serves as a normalization control for transfection efficiency. After electroporation, the cells were left for 10 min at room temperature before plating in prewarmed HMEC medium. The medium was changed 24 h later, and cells were harvested for assay 48 h after transfection. Lysis and dual luciferase reporter assays were performed according to the manufacturer's recommendations (Promega) with luminescence measured on a Tropix tube luminometer (BIO/CAN Scientific). Luminescence values of mock transfections were subtracted from sample luminescence readings to give the net firefly and net Renilla luciferase units. The net firefly units divided by the net Renilla units determined the relative luciferase units. Total RNA was isolated from confluent cell monolayers using TRIzol reagent (Invitrogen). First strand cDNA was synthesized using 50-μl reactions containing 2.5 μg of RNA and 200 units of SuperScript II reverse transcriptase (Invitrogen). Following RNase H (2 units/reaction) (Invitrogen) treatment, PCRs were performed, and amplicons were resolved on TAE-agarose gels. No PCR products were detected in the negative control reactions performed without reverse transcriptase. Conditions used for amplification were as follows: HRT2, sense primer (5′-tgagcataggattccgagagtgc-3′) and antisense primer (5′-gaaggacagagggaagctgtgtg-3′) amplified at 57 °C annealing temperature for 28 cycles; glyceraldehyde-3-phosphate dehydrogenase, sense primer (5′-cccatcaccatcttccag-3′) and antisense primer (5′-atgaccttgcccacagcc-3′) amplified at 55 °C annealing temperature for 22 cycles. Activated Notch4 Inhibits Endothelial Cell Apoptosis—Members of the Notch family have previously been shown to have either anti- or proapoptotic effects depending on the Notch member, cell type, or apoptotic stimulus (30Jundt F. Anagnostopoulos I. Forster R. Mathas S. Stein H. Dorken B. Blood. 2002; 99: 3398-3403Crossref PubMed Scopus (343) Google Scholar, 31Ohishi K. Varnum-Finney B. Flowers D. Anasetti C. Myerson D. Bernstein I.D. Blood. 2000; 95: 2847-2854Crossref PubMed Google Scholar). Because Notch4 is structurally distinct from the other Notch members and exhibits endothelium-selective expression, we tested whether Notch4 was able to regulate endothelial apoptosis elicited by inflammatory mediators. We have previously shown that LPS can induce endothelial apoptosis; thus, HMEC expressing an activated form of Notch4 were exposed to LPS to induce cell death (20Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). As seen in Fig. 1A, Notch4IC inhibited HMEC death in response to LPS, as measured by MTT assays. LPS utilizes a mitochondria-dependent death pathway to induce apoptosis (20Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). We thus confirmed that endothelial cells were able to maintain mitochondrial integrity by confirming the ability of Notch4IC-expressing cells to retain their transmembrane potential. The cationic fluorophore, TMRE, partitions preferentially to the mitochondria. Loss of ΔΨm results in a mitochondrial permeability transition, which can be detected by loss of TMRE fluorescence by flow cytometry. Fig. 1B demonstrates that activated Notch4 is able to maintain ΔΨm over time in response to LPS stimulation. LPS interacts with Toll-like receptors to activate caspase 3/7 downstream of mitochondrial disruption (20Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (98) Google Scholar). Determination of caspase 3/7 activity using the caspase 3/7-specific substrate, DEVD-pNA, showed that Notch4IC inhibited activation of caspase 3/7 in HMEC exposed to LPS (Fig. 1C). Because HMEC are a transformed microvascular endothelial cell line, we tested whether Notch4IC also protected primary endothelial cells from apoptosis. HUVEC were transduced either with activated Notch4 linked by an internal ribosome entry site to YFP (MIY-Notch4IC), or with the empty vector as a control. The percentage of cells demonstrating phosphatidylserine exposure, as determined by annexin V binding, was used to quantitate apoptosis by flow cytometry (Fig. 2, right upper quadrant). The proportion of apoptotic cells was determined by gating only on the YFP-positive HUVEC (Fig. 2, right upper and lower quadrants). Fig. 2 shows the results of three such experiments, demonstrating that activated Notch4 inhibits apoptosis of primary endothelial cells in response to LPS stimulation. The Ankyrin Repeats Are Required for the Notch4 Antiapoptotic Function—The Notch intracellular domain contains two major subdomains that have been implicated in binding to the downstream effector, RBP-Jκ. These are the RAM domain at the NH2 terminus and the ankyrin repeat region. To determine which of these domains are important in the antiapoptotic activity of Notch, deletion mutants were generated lacking each of these subdomains (Fig. 3A, ΔRAM and ΔAnk). Expression of these constructs was confirmed by immunoblotting (Fig. 3B) and immunofluorescent staining (Fig. 3C). Whereas the intact Notch4 intracellular domain and the ΔAnk mutant localized mainly to the nucleus, the ΔRAM mutant was expressed mainly in the cytoplasm, although a small amount of nuclear expression was also seen. The localization pattern of the ΔRAM mutant was confirmed by subcellular fractionation studies (data not shown). Although there is some nuclear expression of this construct, the mainly cytoplasmic localization of the ΔRAM mutant is consistent with the presence of an important nuclear localization signal in the region NH2-terminal to the ankyrin repeats as has previously been suggested (32Lee J.S. Haruna T. Ishimoto A. Honjo T. Yanagawa S.I. J. Virol. 1999; 73: 5166-5171Crossref PubMed Google Scholar). In order to ensure nuclear localization of the ΔRAM mutant, an SV40 nuclear localization signal (NLS) was fused to the NH2 terminus of the ΔRAM construct (NLS-ΔRAM), and this construct showed mainly nuclear expression (Fig. 3C). The various Notch4 mutants were tested for their ability to protect HMEC against LPS-initiated apoptosis. As seen in Fig. 3D, deletion of the ankyrin repeats abrogated the cytoprotective effect of Notch4. In contrast, the ΔRAM mutant only partially lost cytoprotective activity. Because the ΔRAM mutant exhibited a defect in nuclear entry, we tested whether the ΔRAM mutant targeted to the nucleus would provide similar antiapoptotic activity to Notch4IC. Interestingly, the NLS-ΔRAM mutant was not able to protect endothelial cells to the same extent as Notch4IC, but rather this mutant showed a similar degree of protection as the nontargeted ΔRAM mutant (Fig. 3E). Thus, increased nuclear expression is not sufficient for the ΔRAM mutant to provide full cytoprotective activity. These findings indicate that the ankyrin repeats are essential for antiapoptotic function, and the partial protection conferred by the ΔRAM mutant suggests that the RAM motif may signal one of multiple cytoprotective pathways induced by Notch4. Alternatively, The RAM domain may be required for "full" derepression and activation of its downstream effector RBP-Jκ. Only the Ankyrin Repeats Are Required for Notch4-activated RBP-Jκ-dependent Signaling—Little is known about the downstream pathways required for the antiapoptotic activity of Notch, and nothing is known regarding this function with respect to Notch4. Further, because of cell-specific signaling events, the critical downstream pathways activated by Notch members in endothelial cells remain to be defined. A major signaling pathway utilized by Notch homologues in other cell types involves derepression/activation of the transcriptional repressor RBP-Jκ (2Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Although the RAM domain was initially identified as a crucial region required for RBP-Jκ-dependent Notch activity, others have not found this to be true (33Tamura K. Taniguchi Y. Minoguchi S. Sakai T. Tun T. Furukawa T. Honjo T. Curr. Biol. 1995; 5: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 34Aster J.C. Xu L. Karnell F.G. Patriub V. Pui J.C. Pear W.S. Mol. Cell Biol. 2000; 20: 7505-7515Crossref PubMed Scopus (240) Google Scholar). To test the ability of the various constructs to derepress/activate RBP-Jκ, an RBP-Jκ-dependent promoter construct fused to a luciferase reporter was used to assay for Notch4 activity. Transient transfections of the various cell lines with an RBP-Jκ-dependent promoter-luciferase construct (Fig. 4A) demonstrate that the RBP-Jκ-dependent promoter was activated equally in response to the wild-type Notch4 construct, the ΔRAM mutant, or the NLS-ΔRAM mutant. This finding suggests that the Notch4 RAM motif is not required for RBP-Jκ derepression/activation in endothelial cells. However, the mutant lacking the ankyrin repeats was not able to activate the RBP-Jκ promoter at all, confirming the essential requirement of this domain for Notch4 function (Fig. 4A). RBP-Jκ has been shown to promote transcription of downstream basic helix-loop-helix factors of the HES and HRT families (4Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1012) Google Scholar). In particular, HRT2 has been shown to be expressed in endothelial cells and appears to play an important role in endothelial function (4Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1012) Google Scholar). Thus, to confirm the ability of the ΔRAM mutant Notch4 construct to activate an endogenous RBP-Jκ-dependent promoter, RT-PCR of HRT2 from RNA of the mutant Notch4 cell lines was performed. These results confirmed that the ΔAnk mutant was not able to activate the HRT2 promoter, whereas both the ΔRAM and the NLS-ΔRAM mutants increased the expression of HRT2 mRNA to a similar extent as wild-type Notch4IC (Fig. 4B). Thus, our findings suggest that only the ankyrin repeats are necessary for Notch4 to signal through RBP-Jκ, whereas the RAM domain is dispensable for this activity. Further, the finding that the ΔRAM and the NLS-ΔRAM mutants showed similar protective activity is in keeping with the idea that minimal nuclear localization is sufficient for functional RBP-Jκ activation by Notch. Notch4 Inhibits Apoptosis through RBP-Jκ-dependent and -independent Pathways—The NLS-ΔRAM mutant activates RBP-Jκ-dependent promoters to a similar extent as the wild-type Notch4IC construct, yet this mutant only provides partial protection against apoptosis. Given these findings, we posited that Notch4 is able to protect endotheli
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