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Endothelial Cell Death Induced by Tumor Necrosis Factor-α Is Inhibited by the Bcl-2 Family Member, A1

Endothelial Cell Death Induced by Tumor Necrosis Factor-α Is Inhibited by the Bcl-2 Family... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 44, Issue of November 1, pp. 27201–27204, 1996 Communication © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. brane including TRADD, FADD/Mort1, and MACH/FLICE Endothelial Cell Death Induced which culminates in the activation of cysteine proteases (4–10). by Tumor Necrosis Factor-a Is TNF stimulation also results in signal transduction pathways mediated by two lipid second messengers, diacylglycerol and Inhibited by the Bcl-2 Family ceramide (3). While ceramide is a transducer of TNF-induced Member, A1* apoptosis (11, 12), diglyceride or phorbol ester pretreatment can protect against ceramide-induced cell death (11, 13). Cer- (Received for publication, July 24, 1996, and in revised form, amides appear to act downstream of FADD/Mort1 since a September 3, 1996) FADD dominant-negative can block TNF-induced apoptosis but not death mediated by ceramide (7). Aly Karsan‡, Esther Yee, and John M. Harlan Although tumor cells and virally infected cells are suscepti- From the Division of Hematology, University of ble to TNF-induced death, many normal cells are not (1, 2). In Washington, Seattle, Washington 98195 this respect, human endothelial cells which play a pivotal role Endothelial cells play a central role in the inflamma- in modulating the inflammatory response are not directly tory process. Tumor necrosis factor-a (TNF) is a multi- killed by TNF (14). However, it has previously been shown that functional cytokine which elicits many of the inflamma- in the presence of RNA or protein synthesis inhibitors, endo- tory responses of endothelial cells. While TNF directly thelial cells as well as other cells can be rendered sensitive to causes apoptosis of tumor cells and virally infected cells, TNF (14–16). Conversely, it has also been demonstrated that normal cells are generally resistant. However, most re- sensitive cells can be made resistant to TNF challenge by prior sistant cells, including human endothelial cells, can be sublethal exposure to TNF (15, 16). These findings have led to rendered susceptible to TNF by inhibiting RNA or pro- the hypothesis and demonstration of TNF-inducible genes tein synthesis. This finding suggests that TNF provides which confer a protective effect on cells. Genes in this category a cell survival signal in addition to a death signal. We include manganous superoxide dismutase (17), plasminogen have previously cloned a human Bcl-2 homologue, A1, activator inhibitor type 2 (18, 19), and the zinc-finger protein, and shown that it is specifically induced by proinflam- A20 (20). Conflicting data have been reported on the effect of matory cytokines but not by endothelial growth factors. the anti-apoptotic protein, Bcl-2 (21). Even in the same cell In this study, we show that retroviral-mediated transfer type, some investigators have reported that Bcl-2 inhibits TNF- of the A1 cDNA to a human microvascular endothelial mediated death while others have claimed that it does not cell line provides protection against cell death initiated (22, 23). by TNF in the presence of actinomycin D. The induction We have recently cloned the human homologue of the Bcl-2 of A1 by TNF in this system is mediated via a protein family member, A1, from phorbol ester-stimulated endothelial kinase C pathway. Since TNF signaling has also been cells and shown that it is induced by the inflammatory cyto- shown to proceed via ceramides, we tested whether ex- kines, TNF and interleukin (IL)-1 (24). This induction is spe- ogenous ceramides could induce A1. Our findings indi- cate that ceramides do not induce A1 but do up-regulate cific in that neither the endothelial growth factors, basic fibro- c-jun and induce endothelial death. Ceramide-activated blast growth factor and vascular endothelial growth factor, nor endothelial death is also inhibited by A1, suggesting interferon-g or transforming growth factor-b induce A1. To that TNF may initiate divergent survival and death determine whether A1 belongs to the family of cytoprotective pathways via separate lipid second messengers. TNF-inducible genes, we generated retroviral constructs of a FLAG-tagged A1 and of Bcl-XL (25) and transduced a micro- vascular endothelial cell line, HMEC-1 (26), with either of these constructs or the empty vector, LNCX (27). In the pres- TNF is an inflammatory cytokine originally defined by its ence of actinomycin D, these cells underwent cell death which tumoricidal activity (1, 2). Subsequently, it has been shown to was inhibited by both A1 and Bcl-XL. TNF induced A1 via a evoke multiple biological responses affecting virtually every PKC-mediated pathway, while ceramide did not induce A1 but cell type (3). Considerable attention has recently been paid to did up-regulate c-jun mRNA. A1 and Bcl-XL also protected the apoptotic pathway elicited by TNF. A series of elegant HMEC-1 cells from ceramide-mediated apoptosis. Hence, A1 is experiments have defined a death pathway emanating from the a Bcl-2 homologue which is induced by TNF and protects TNF receptor 1. Engagement of the TNF receptor results in cell against TNF-mediated cell death. death by recruitment of a complex of proteins to the cell mem- MATERIALS AND METHODS * This work was supported in part by United States Public Health Reagents—Phorbol 12-myristate 13-acetate (PMA), 4-b-phorbol, and Service Grant HL 47151. The costs of publication of this article were mezerein were purchased from Sigma. C -ceramide and dihydroceram- defrayed in part by the payment of page charges. This article must ide were obtained from Biomol and reconstituted in ethanol. TNF was therefore be hereby marked “advertisement” in accordance with 18 purchased from R&D Systems. Calphostin C, H7, and HA1004 (Calbio- U.S.C. Section 1734 solely to indicate this fact. chem) were reconstituted in dimethyl sulfoxide. FLAG M2 monoclonal ‡ Recipient of a Clinician-Scientist Award of the Medical Research antibody was obtained from IBI Scientific and the Bcl-X rabbit poly- Council of Canada. To whom correspondence should be addressed: clonal antibody from Transduction Laboratories. The horseradish per- Division of Hematology, Box 357710, University of Washington, Seattle, oxidase-conjugated secondary antibodies used were purchased from WA 98195. Tel.: 206-685-3054; Fax: 206-685-3062; E-mail: karsan@ Bio-Rad Laboratories. u.washington.edu. Cell Culture—The human dermal microvascular cell line, HMEC-1 The abbreviations used are: TNF, tumor necrosis factor-a; IL, in- (26), was cultured in RPMI 1640 supplemented with 10% bovine calf terleukin; PMA, phorbol 12-myristate 13-acetate; PK, protein kinase; PCR, polymerase chain reaction; MTT, (3-[49,5-dimethylthiazol-2-yl]- serum and endothelial cell growth factor (25 mg/ml) prepared from 2,5 diphenyltetrazolium bromide). bovine hypothalamus (28). Serum-free medium consisted of RPMI 1640 This is an Open Access article under the CC BY license. 27202 TNF-induced Endothelial Cell Death Is Inhibited by A1 supplemented with insulin, transferrin, sodium selenite (Sigma), and 0.1% bovine serum albumin. The PA317 and PE501 packaging lines (27, 29) (provided by A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. All cells were maintained at 37 °C in 5% CO . Gene Transfer—The coding region of Bcl-XL cDNA (25) (provided by L. Boise and C. B. Thompson, University of Chicago) was ligated into the HindIII/HpaI sites of the replication-deficient retroviral vector pLNCX (27) (provided by A. D. Miller). A FLAG octapeptide N-terminal- tagged human A1 was generated by PCR using the following primers: sense, 59-CCAGCTAAGCTTCCACCATGGACTACAAGGACGACGAT- GACAAGCAGACTGTGAATTTGGAT-39, antisense, 59-GGTAAAGAA- TTCTCTGGTCAACAGTATTGCT-39, and ligated into the HindIII/HpaI site of pLNCX. The PCR product was sequenced on both strands to confirm the authenticity of FLAG-A1. The viral long terminal repeat drives expression of Neo while the cytomegalovirus promoter drives transgene expression in pLNCX. Generation of packaging cell lines was performed as described (29). The pLNCBcl-XL, pLNCFLAG-A1 const- ructs, or pLNCX were transiently transfected into the ecotropic packa- ging line, PE501, by calcium-phosphate precipitation. Viral supernata- nts were harvested and used to transduce the amphotropic line PA317 in the presence of 4 mg/ml Polybrene. Polyclonal retrovirus-producing cell lines were obtained by selection in 1 mg/ml G418 (Life Technologies, Inc.). Retroviral supernatants from the PA317 cell lines were used to transduce HMEC-1 cells. Following selection in 200 mg/ml G418 and expansion, HMEC-1 cells were used in survival studies. Polyclonal HMEC-1 lines were used in order to avoid artifacts due to retroviral integration and also because this cell line does not grow in colonies. Western Blotting—Total cellular extracts from the transduced cells were prepared by lysing cells in 1 M Tris, 1 M NaCl, 1% Triton X-100, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mg/ml leupeptin, and FIG.1. A1 inhibits TNF-mediated endothelial cell death. Stable 10 mg/ml aprotinin. Protein from 1 3 10 cells was fractionated on 10% polyclonal cell lines of HMEC-1 cells were generated by transduction SDS-polyacrylamide gel electrophoresis gels and electrotransferred with LNCFLAG-A1, LNCBcl-XL, or LNCX. A, cells were exposed to onto nitrocellulose membranes over1hat4 °C. Filters were blocked TNF and actinomycin D (1 mg/ml) for 18 h, and viability was assessed overnight with TBS containing 5% skim milk. Immunostaining steps by an MTT assay. Viability is expressed as a proportion of cells treated were performed in TBS with 0.05% Tween 20 and 3% bovine serum with actinomycin D only. Results are the mean 6 S.E. of an experiment albumin at room temperature. Filters were incubated with primary and done in triplicate which is representative of four separate experiments. secondary antibodies for 1 h each. Filters were washed in TBS and B, Western blots of the cell lines shown above were probed with the 0.05% Tween 20 four times for 10 min between each step and were FLAG M2 monoclonal antibody or a Bcl-XL polyclonal antibody. C, developed with the ECL reagent (Amersham). Blots were stripped as HMEC-FLAG-A1 or HMEC-Bcl-XL cells were exposed to 50 mg/ml cy- recommended (Amersham) and reprobed with another antibody. cloheximide, and levels of the overexpressed protein were monitored at Viability Assay—For viability assays, transduced or wild type various times by Western blot. HMEC-1 cells were seeded on gelatin-coated 96-well plates at a density of 15,000 cells/well. By the following day, cells had reached confluence coincubated with either cycloheximide or actinomycin D (14). and were washed twice in serum-free medium followed by incubation in To determine whether human A1, which is inducible by TNF, TNF (concentrations as indicated) and actinomycin D (1 mg/ml) or in can abrogate TNF-mediated death, we generated polyclonal C -ceramide or C -dihydroceramide (control for the specificity of ceram- 2 2 FLAG-A1 overexpressing HMEC-1 cell lines. We chose retrovi- ide-mediated effects). At various time points, viable cell numbers were ral transduction to achieve overexpression because human en- estimated by an MTT assay (30). Briefly, medium was removed and replaced with medium containing 1 mg/ml MTT (Sigma) and incubated dothelial cells are extremely difficult to transfect using stand- for 5 h. The medium was then aspirated, and the formazan product was ard methods. As a control, we chose to overexpress Bcl-XL (25), solubilized with dimethyl sulfoxide. Absorbance at 630 nM was sub- another member of the Bcl-2 family, since it is constitutively tracted (to reduce background absorbance) from absorbance at 570 nM expressed in cultured endothelial cells (Fig. 1B) and may be for each well. one of the constitutive TNF cytoprotectants. Also, Bcl-XL has Northern Analysis—Endothelial cells were stimulated for 3 h with been shown to protect the MCF7 breast carcinoma cell line the various factors as indicated. Total cellular RNA (15 mg) was sepa- rated on agarose-formaldehyde gels, blotted onto nitrocellulose filters, from TNF-induced apoptosis (33). Attempts were made to gen- and hybridized overnight with random-primed P-labeled probes as erate clonal cell lines as well, but HMEC-1 cells do not prolif- indicated. The A1 probe was generated by reverse transcriptase-PCR as erate in colonies, so all experiments on transduced cells were described previously (24). The c-jun probe was an approximately 700- done with polyclonal cell populations. base pair HindIII/SacII fragment of the murine cDNA (gift of D. Morris, When a FLAG-A1 construct was overexpressed in HMEC-1 University of Washington, Seattle, WA). The final washing conditions cells, these cells were protected from TNF cytotoxicity. As were 0.1 3 SSC, 0.1% SDS for 15 min at room temperature. Blots were stripped in boiling water prior to reprobing. A b-actin probe (Clontech) shown in Fig. 1A, 18-h incubation of HMEC-1 cells in the was used to confirm equivalent loading of RNA samples. presence of TNF at various concentrations and actinomycin D resulted in dose-dependent cell death. The fact that about half RESULTS AND DISCUSSION of the vector-only transduced cells (HMEC-Neo) were still via- By virtue of their location between blood and tissue, endo- ble suggests the presence of constitutively expressed endoge- thelial cells play a central role in the inflammatory process (31, nous inhibitors of TNF cytotoxicity. This level of endogenous 32). TNF has multiple effects on endothelial cells ranging from protection was also noted in the wild-type HMEC-1 cells (data structural reorganization to the up-regulation of adhesion mol- not shown). One of these cytoprotective molecules may be ecules and the elaboration of chemotactic factors (31, 32). Thus, Bcl-XL since it is constitutively expressed in endothelial cells the integrity of the endothelium during inflammation is of great importance. It has previously been shown that human A. Karsan, E. Yee, G. Poirier, P. Zhou, R. Craig, and J. M. Harlan, endothelial cells are not susceptible to TNF toxicity unless submitted for publication. TNF-induced Endothelial Cell Death Is Inhibited by A1 27203 FIG.2. TNF induction of A1 is mediated by PKC. Total cellular RNA was harvested from wild type HMEC-1 cells following exposure to various treatments for 3 h, and Northern blots were probed with A1 and actin as described under “Materials and Methods.” PMA (160 nM), TNF (10 ng/ml), TNF (10 ng/ml) 1 calphostin C (0.1 mM), TNF (10 ng/ml) 1 H7 (30 mM), TNF (10 ng/ml) 1 HA1004 (30 mM). Cells were pretreated with inhibitors for 30 min prior to TNF addition. FIG.4. A1 inhibits ceramide-mediated endothelial cell death. A, wild type HMEC-1 cells were exposed to C -ceramide or C -dihydro- 2 2 ceramide for 24 h followed by an MTT assay to assess viability. HMEC-1 transductants were exposed to C -ceramide (B) at various concentra- tions for 24 h or to C -ceramide (C) (25 mM) for various time points followed by an MTT assay. Viability is expressed as a proportion of control cells (mean 6 S.E.) treated with ethanol vehicle only. Each panel shows a representative experiment done in triplicate of at least 3 separate experiments. late A1 expression. Several TNF responses have previously been shown to be mediated via PKC (3). Fig. 2 shows that PMA and TNF induced A1 independently. The highly specific PKC inhibitor, calphostin C (0.1 mM), completely blocked TNF-medi- ated A1 induction. Additionally, H7, another PKC inhibitor, also partially blocked A1 induction, but HA1004, which at the FIG.3. Ceramide induces c-jun but not A1. Total cellular RNA was harvested from wild type HMEC-1 cells following exposure to concentrations used (30 mM) blocks PKA and PKG but not PKC various treatments for 3 h, and Northern blots were probed with A1, (35), had no effect. These findings suggest that PKC is involved c-jun, and actin as described under “Materials and Methods.” PMA (160 in the signal to up-regulate A1 expression. nM), phorbol, 4-b-phorbol (160 nM), mezerein (160 nM); Vehicle, ethanol TNF can also signal through another lipid second messenger, vehicle; Ceramide,C -ceramide (mM); Dihydroceramide,C -dihydrocer- 2 2 amide (mM). ceramide, generated by the hydrolysis of sphingolipids (3). Ce- ramides also have pleiotropic effects on cells, dependent on cell (Fig. 1B), but not induced by TNF (data not shown). Indeed, type and the context in which ceramide is generated. In fact, overexpression of Bcl-XL resulted in virtually complete protec- ceramides have been reported to induce both apoptosis and tion against TNF (Fig. 1A). The partial protection conferred by mitogenesis (11, 36, 37). Besides TNF, IL-1, ionizing radiation, A1 may, in part, be due to the shorter half-life of A1 protein. and chemotherapeutic agents have also been reported to gen- When HMEC-FLAG-A1 or HMEC-Bcl-XL cells were exposed to erate ceramides (3, 38–41). Multiple potential targets of cer- cycloheximide (50 mg/ml) to inhibit new protein synthesis, for amide stimulation have been identified including activation of up to 12 h, FLAG-A1 protein was not detectable after 3 to 6 h the atypical PKCz (36, 42). Hence, we investigated whether whereas Bcl-XL levels remained virtually unchanged (Fig. 1C). exogenous membrane-permeable ceramides could result in A1 Thus, after the first few hours of treatment with TNF and mRNA accumulation. Fig. 3 shows that PMA and the non- actinomycin D, following degradation of existing RNA and pro- phorbol PKC activator, mezerein, both induced A1, but the tein, there are probably insufficient levels of A1 to provide inactive phorbol analogue, 4-b-phorbol, did not. Again this protection. Although others have shown that murine A1 inhib- suggests that PKC is involved in A1 induction. The addition of its apoptosis following IL-3 withdrawal in an IL-3-dependent either C -ceramide or the inactive analogue, C -dihydroceram- 2 2 myeloid cell line (34), human A1 is not induced by endothelial ide, did not result in A1 accumulation when tested over a wide growth factors but rather by proinflammatory cytokines (24). range of concentrations. Recent studies have shown that cer- Thus, this model provides a more plausible scenario for the amide-induced apoptosis is associated with induction of c-jun putative function of A1 in endothelial cells. and activation of c-jun N-terminal kinase in human myeloid Because PMA also induces A1 (24), we wondered whether cell lines and bovine endothelial cells (41, 43, 44). 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L., Green, S., Fiore, N., and Williamson, Commun. 168, 1194–1200 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Endothelial Cell Death Induced by Tumor Necrosis Factor-α Is Inhibited by the Bcl-2 Family Member, A1

Karsan, Aly; Yee, Esther; Harlan, John M.
Journal of Biological Chemistry – Nov 1, 1996
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0021-9258
DOI
10.1074/jbc.271.44.27201
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 44, Issue of November 1, pp. 27201–27204, 1996 Communication © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. brane including TRADD, FADD/Mort1, and MACH/FLICE Endothelial Cell Death Induced which culminates in the activation of cysteine proteases (4–10). by Tumor Necrosis Factor-a Is TNF stimulation also results in signal transduction pathways mediated by two lipid second messengers, diacylglycerol and Inhibited by the Bcl-2 Family ceramide (3). While ceramide is a transducer of TNF-induced Member, A1* apoptosis (11, 12), diglyceride or phorbol ester pretreatment can protect against ceramide-induced cell death (11, 13). Cer- (Received for publication, July 24, 1996, and in revised form, amides appear to act downstream of FADD/Mort1 since a September 3, 1996) FADD dominant-negative can block TNF-induced apoptosis but not death mediated by ceramide (7). Aly Karsan‡, Esther Yee, and John M. Harlan Although tumor cells and virally infected cells are suscepti- From the Division of Hematology, University of ble to TNF-induced death, many normal cells are not (1, 2). In Washington, Seattle, Washington 98195 this respect, human endothelial cells which play a pivotal role Endothelial cells play a central role in the inflamma- in modulating the inflammatory response are not directly tory process. Tumor necrosis factor-a (TNF) is a multi- killed by TNF (14). However, it has previously been shown that functional cytokine which elicits many of the inflamma- in the presence of RNA or protein synthesis inhibitors, endo- tory responses of endothelial cells. While TNF directly thelial cells as well as other cells can be rendered sensitive to causes apoptosis of tumor cells and virally infected cells, TNF (14–16). Conversely, it has also been demonstrated that normal cells are generally resistant. However, most re- sensitive cells can be made resistant to TNF challenge by prior sistant cells, including human endothelial cells, can be sublethal exposure to TNF (15, 16). These findings have led to rendered susceptible to TNF by inhibiting RNA or pro- the hypothesis and demonstration of TNF-inducible genes tein synthesis. This finding suggests that TNF provides which confer a protective effect on cells. Genes in this category a cell survival signal in addition to a death signal. We include manganous superoxide dismutase (17), plasminogen have previously cloned a human Bcl-2 homologue, A1, activator inhibitor type 2 (18, 19), and the zinc-finger protein, and shown that it is specifically induced by proinflam- A20 (20). Conflicting data have been reported on the effect of matory cytokines but not by endothelial growth factors. the anti-apoptotic protein, Bcl-2 (21). Even in the same cell In this study, we show that retroviral-mediated transfer type, some investigators have reported that Bcl-2 inhibits TNF- of the A1 cDNA to a human microvascular endothelial mediated death while others have claimed that it does not cell line provides protection against cell death initiated (22, 23). by TNF in the presence of actinomycin D. The induction We have recently cloned the human homologue of the Bcl-2 of A1 by TNF in this system is mediated via a protein family member, A1, from phorbol ester-stimulated endothelial kinase C pathway. Since TNF signaling has also been cells and shown that it is induced by the inflammatory cyto- shown to proceed via ceramides, we tested whether ex- kines, TNF and interleukin (IL)-1 (24). This induction is spe- ogenous ceramides could induce A1. Our findings indi- cate that ceramides do not induce A1 but do up-regulate cific in that neither the endothelial growth factors, basic fibro- c-jun and induce endothelial death. Ceramide-activated blast growth factor and vascular endothelial growth factor, nor endothelial death is also inhibited by A1, suggesting interferon-g or transforming growth factor-b induce A1. To that TNF may initiate divergent survival and death determine whether A1 belongs to the family of cytoprotective pathways via separate lipid second messengers. TNF-inducible genes, we generated retroviral constructs of a FLAG-tagged A1 and of Bcl-XL (25) and transduced a micro- vascular endothelial cell line, HMEC-1 (26), with either of these constructs or the empty vector, LNCX (27). In the pres- TNF is an inflammatory cytokine originally defined by its ence of actinomycin D, these cells underwent cell death which tumoricidal activity (1, 2). Subsequently, it has been shown to was inhibited by both A1 and Bcl-XL. TNF induced A1 via a evoke multiple biological responses affecting virtually every PKC-mediated pathway, while ceramide did not induce A1 but cell type (3). Considerable attention has recently been paid to did up-regulate c-jun mRNA. A1 and Bcl-XL also protected the apoptotic pathway elicited by TNF. A series of elegant HMEC-1 cells from ceramide-mediated apoptosis. Hence, A1 is experiments have defined a death pathway emanating from the a Bcl-2 homologue which is induced by TNF and protects TNF receptor 1. Engagement of the TNF receptor results in cell against TNF-mediated cell death. death by recruitment of a complex of proteins to the cell mem- MATERIALS AND METHODS * This work was supported in part by United States Public Health Reagents—Phorbol 12-myristate 13-acetate (PMA), 4-b-phorbol, and Service Grant HL 47151. The costs of publication of this article were mezerein were purchased from Sigma. C -ceramide and dihydroceram- defrayed in part by the payment of page charges. This article must ide were obtained from Biomol and reconstituted in ethanol. TNF was therefore be hereby marked “advertisement” in accordance with 18 purchased from R&D Systems. Calphostin C, H7, and HA1004 (Calbio- U.S.C. Section 1734 solely to indicate this fact. chem) were reconstituted in dimethyl sulfoxide. FLAG M2 monoclonal ‡ Recipient of a Clinician-Scientist Award of the Medical Research antibody was obtained from IBI Scientific and the Bcl-X rabbit poly- Council of Canada. To whom correspondence should be addressed: clonal antibody from Transduction Laboratories. The horseradish per- Division of Hematology, Box 357710, University of Washington, Seattle, oxidase-conjugated secondary antibodies used were purchased from WA 98195. Tel.: 206-685-3054; Fax: 206-685-3062; E-mail: karsan@ Bio-Rad Laboratories. u.washington.edu. Cell Culture—The human dermal microvascular cell line, HMEC-1 The abbreviations used are: TNF, tumor necrosis factor-a; IL, in- (26), was cultured in RPMI 1640 supplemented with 10% bovine calf terleukin; PMA, phorbol 12-myristate 13-acetate; PK, protein kinase; PCR, polymerase chain reaction; MTT, (3-[49,5-dimethylthiazol-2-yl]- serum and endothelial cell growth factor (25 mg/ml) prepared from 2,5 diphenyltetrazolium bromide). bovine hypothalamus (28). Serum-free medium consisted of RPMI 1640 This is an Open Access article under the CC BY license. 27202 TNF-induced Endothelial Cell Death Is Inhibited by A1 supplemented with insulin, transferrin, sodium selenite (Sigma), and 0.1% bovine serum albumin. The PA317 and PE501 packaging lines (27, 29) (provided by A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. All cells were maintained at 37 °C in 5% CO . Gene Transfer—The coding region of Bcl-XL cDNA (25) (provided by L. Boise and C. B. Thompson, University of Chicago) was ligated into the HindIII/HpaI sites of the replication-deficient retroviral vector pLNCX (27) (provided by A. D. Miller). A FLAG octapeptide N-terminal- tagged human A1 was generated by PCR using the following primers: sense, 59-CCAGCTAAGCTTCCACCATGGACTACAAGGACGACGAT- GACAAGCAGACTGTGAATTTGGAT-39, antisense, 59-GGTAAAGAA- TTCTCTGGTCAACAGTATTGCT-39, and ligated into the HindIII/HpaI site of pLNCX. The PCR product was sequenced on both strands to confirm the authenticity of FLAG-A1. The viral long terminal repeat drives expression of Neo while the cytomegalovirus promoter drives transgene expression in pLNCX. Generation of packaging cell lines was performed as described (29). The pLNCBcl-XL, pLNCFLAG-A1 const- ructs, or pLNCX were transiently transfected into the ecotropic packa- ging line, PE501, by calcium-phosphate precipitation. Viral supernata- nts were harvested and used to transduce the amphotropic line PA317 in the presence of 4 mg/ml Polybrene. Polyclonal retrovirus-producing cell lines were obtained by selection in 1 mg/ml G418 (Life Technologies, Inc.). Retroviral supernatants from the PA317 cell lines were used to transduce HMEC-1 cells. Following selection in 200 mg/ml G418 and expansion, HMEC-1 cells were used in survival studies. Polyclonal HMEC-1 lines were used in order to avoid artifacts due to retroviral integration and also because this cell line does not grow in colonies. Western Blotting—Total cellular extracts from the transduced cells were prepared by lysing cells in 1 M Tris, 1 M NaCl, 1% Triton X-100, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mg/ml leupeptin, and FIG.1. A1 inhibits TNF-mediated endothelial cell death. Stable 10 mg/ml aprotinin. Protein from 1 3 10 cells was fractionated on 10% polyclonal cell lines of HMEC-1 cells were generated by transduction SDS-polyacrylamide gel electrophoresis gels and electrotransferred with LNCFLAG-A1, LNCBcl-XL, or LNCX. A, cells were exposed to onto nitrocellulose membranes over1hat4 °C. Filters were blocked TNF and actinomycin D (1 mg/ml) for 18 h, and viability was assessed overnight with TBS containing 5% skim milk. Immunostaining steps by an MTT assay. Viability is expressed as a proportion of cells treated were performed in TBS with 0.05% Tween 20 and 3% bovine serum with actinomycin D only. Results are the mean 6 S.E. of an experiment albumin at room temperature. Filters were incubated with primary and done in triplicate which is representative of four separate experiments. secondary antibodies for 1 h each. Filters were washed in TBS and B, Western blots of the cell lines shown above were probed with the 0.05% Tween 20 four times for 10 min between each step and were FLAG M2 monoclonal antibody or a Bcl-XL polyclonal antibody. C, developed with the ECL reagent (Amersham). Blots were stripped as HMEC-FLAG-A1 or HMEC-Bcl-XL cells were exposed to 50 mg/ml cy- recommended (Amersham) and reprobed with another antibody. cloheximide, and levels of the overexpressed protein were monitored at Viability Assay—For viability assays, transduced or wild type various times by Western blot. HMEC-1 cells were seeded on gelatin-coated 96-well plates at a density of 15,000 cells/well. By the following day, cells had reached confluence coincubated with either cycloheximide or actinomycin D (14). and were washed twice in serum-free medium followed by incubation in To determine whether human A1, which is inducible by TNF, TNF (concentrations as indicated) and actinomycin D (1 mg/ml) or in can abrogate TNF-mediated death, we generated polyclonal C -ceramide or C -dihydroceramide (control for the specificity of ceram- 2 2 FLAG-A1 overexpressing HMEC-1 cell lines. We chose retrovi- ide-mediated effects). At various time points, viable cell numbers were ral transduction to achieve overexpression because human en- estimated by an MTT assay (30). Briefly, medium was removed and replaced with medium containing 1 mg/ml MTT (Sigma) and incubated dothelial cells are extremely difficult to transfect using stand- for 5 h. The medium was then aspirated, and the formazan product was ard methods. As a control, we chose to overexpress Bcl-XL (25), solubilized with dimethyl sulfoxide. Absorbance at 630 nM was sub- another member of the Bcl-2 family, since it is constitutively tracted (to reduce background absorbance) from absorbance at 570 nM expressed in cultured endothelial cells (Fig. 1B) and may be for each well. one of the constitutive TNF cytoprotectants. Also, Bcl-XL has Northern Analysis—Endothelial cells were stimulated for 3 h with been shown to protect the MCF7 breast carcinoma cell line the various factors as indicated. Total cellular RNA (15 mg) was sepa- rated on agarose-formaldehyde gels, blotted onto nitrocellulose filters, from TNF-induced apoptosis (33). Attempts were made to gen- and hybridized overnight with random-primed P-labeled probes as erate clonal cell lines as well, but HMEC-1 cells do not prolif- indicated. The A1 probe was generated by reverse transcriptase-PCR as erate in colonies, so all experiments on transduced cells were described previously (24). The c-jun probe was an approximately 700- done with polyclonal cell populations. base pair HindIII/SacII fragment of the murine cDNA (gift of D. Morris, When a FLAG-A1 construct was overexpressed in HMEC-1 University of Washington, Seattle, WA). The final washing conditions cells, these cells were protected from TNF cytotoxicity. As were 0.1 3 SSC, 0.1% SDS for 15 min at room temperature. Blots were stripped in boiling water prior to reprobing. A b-actin probe (Clontech) shown in Fig. 1A, 18-h incubation of HMEC-1 cells in the was used to confirm equivalent loading of RNA samples. presence of TNF at various concentrations and actinomycin D resulted in dose-dependent cell death. The fact that about half RESULTS AND DISCUSSION of the vector-only transduced cells (HMEC-Neo) were still via- By virtue of their location between blood and tissue, endo- ble suggests the presence of constitutively expressed endoge- thelial cells play a central role in the inflammatory process (31, nous inhibitors of TNF cytotoxicity. This level of endogenous 32). TNF has multiple effects on endothelial cells ranging from protection was also noted in the wild-type HMEC-1 cells (data structural reorganization to the up-regulation of adhesion mol- not shown). One of these cytoprotective molecules may be ecules and the elaboration of chemotactic factors (31, 32). Thus, Bcl-XL since it is constitutively expressed in endothelial cells the integrity of the endothelium during inflammation is of great importance. It has previously been shown that human A. Karsan, E. Yee, G. Poirier, P. Zhou, R. Craig, and J. M. Harlan, endothelial cells are not susceptible to TNF toxicity unless submitted for publication. TNF-induced Endothelial Cell Death Is Inhibited by A1 27203 FIG.2. TNF induction of A1 is mediated by PKC. Total cellular RNA was harvested from wild type HMEC-1 cells following exposure to various treatments for 3 h, and Northern blots were probed with A1 and actin as described under “Materials and Methods.” PMA (160 nM), TNF (10 ng/ml), TNF (10 ng/ml) 1 calphostin C (0.1 mM), TNF (10 ng/ml) 1 H7 (30 mM), TNF (10 ng/ml) 1 HA1004 (30 mM). Cells were pretreated with inhibitors for 30 min prior to TNF addition. FIG.4. A1 inhibits ceramide-mediated endothelial cell death. A, wild type HMEC-1 cells were exposed to C -ceramide or C -dihydro- 2 2 ceramide for 24 h followed by an MTT assay to assess viability. HMEC-1 transductants were exposed to C -ceramide (B) at various concentra- tions for 24 h or to C -ceramide (C) (25 mM) for various time points followed by an MTT assay. Viability is expressed as a proportion of control cells (mean 6 S.E.) treated with ethanol vehicle only. Each panel shows a representative experiment done in triplicate of at least 3 separate experiments. late A1 expression. Several TNF responses have previously been shown to be mediated via PKC (3). Fig. 2 shows that PMA and TNF induced A1 independently. The highly specific PKC inhibitor, calphostin C (0.1 mM), completely blocked TNF-medi- ated A1 induction. Additionally, H7, another PKC inhibitor, also partially blocked A1 induction, but HA1004, which at the FIG.3. Ceramide induces c-jun but not A1. Total cellular RNA was harvested from wild type HMEC-1 cells following exposure to concentrations used (30 mM) blocks PKA and PKG but not PKC various treatments for 3 h, and Northern blots were probed with A1, (35), had no effect. These findings suggest that PKC is involved c-jun, and actin as described under “Materials and Methods.” PMA (160 in the signal to up-regulate A1 expression. nM), phorbol, 4-b-phorbol (160 nM), mezerein (160 nM); Vehicle, ethanol TNF can also signal through another lipid second messenger, vehicle; Ceramide,C -ceramide (mM); Dihydroceramide,C -dihydrocer- 2 2 amide (mM). ceramide, generated by the hydrolysis of sphingolipids (3). Ce- ramides also have pleiotropic effects on cells, dependent on cell (Fig. 1B), but not induced by TNF (data not shown). Indeed, type and the context in which ceramide is generated. In fact, overexpression of Bcl-XL resulted in virtually complete protec- ceramides have been reported to induce both apoptosis and tion against TNF (Fig. 1A). The partial protection conferred by mitogenesis (11, 36, 37). Besides TNF, IL-1, ionizing radiation, A1 may, in part, be due to the shorter half-life of A1 protein. and chemotherapeutic agents have also been reported to gen- When HMEC-FLAG-A1 or HMEC-Bcl-XL cells were exposed to erate ceramides (3, 38–41). Multiple potential targets of cer- cycloheximide (50 mg/ml) to inhibit new protein synthesis, for amide stimulation have been identified including activation of up to 12 h, FLAG-A1 protein was not detectable after 3 to 6 h the atypical PKCz (36, 42). Hence, we investigated whether whereas Bcl-XL levels remained virtually unchanged (Fig. 1C). exogenous membrane-permeable ceramides could result in A1 Thus, after the first few hours of treatment with TNF and mRNA accumulation. Fig. 3 shows that PMA and the non- actinomycin D, following degradation of existing RNA and pro- phorbol PKC activator, mezerein, both induced A1, but the tein, there are probably insufficient levels of A1 to provide inactive phorbol analogue, 4-b-phorbol, did not. Again this protection. Although others have shown that murine A1 inhib- suggests that PKC is involved in A1 induction. The addition of its apoptosis following IL-3 withdrawal in an IL-3-dependent either C -ceramide or the inactive analogue, C -dihydroceram- 2 2 myeloid cell line (34), human A1 is not induced by endothelial ide, did not result in A1 accumulation when tested over a wide growth factors but rather by proinflammatory cytokines (24). range of concentrations. Recent studies have shown that cer- Thus, this model provides a more plausible scenario for the amide-induced apoptosis is associated with induction of c-jun putative function of A1 in endothelial cells. and activation of c-jun N-terminal kinase in human myeloid Because PMA also induces A1 (24), we wondered whether cell lines and bovine endothelial cells (41, 43, 44). 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Journal of Biological Chemistry – Unpaywall

Published: Nov 1, 1996

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