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Cell Line Death Pathway in a Human Myelomonocyte -Mediated Inhibition of p38 Kinase Reveals a TNF-α -Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line This information is current as of October 1, 2021. Jishy Varghese, Souvik Chattopadhaya and Apurva Sarin J Immunol 2001; 166:6570-6577; ; doi: 10.4049/jimmunol.166.11.6570 http://www.jimmunol.org/content/166/11/6570 Downloaded from References This article cites 44 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/166/11/6570.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Inhibition of p38 Kinase Reveals a TNF-␣-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line1 Jishy Varghese, Souvik Chattopadhaya, and Apurva Sarin2 TNF-␣ transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 Downloaded from MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-␬B-mediated transactivation, without affecting the nuclear translocation of NF-␬B. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF- triggered signals of activation or apoptosis. The Journal of Immunology, 2001, 166: 6570–6577. umor necrosis factor-␣ is a multifunctional cytokine that gen-activated protein kinase (MAPK) kinase family of protein ki- http://www.jimmunol.org/ transduces signals of survival, differentiation, and death nases (9). T via its receptors TNFRI/p55/p60 and TNFRII/p75/p80 in TNF also activates the p38 MAPK pathway (3, 10, 11). MAPK diverse cell types (1–3). TNF elicits diverse biological conse- family members are required for signal transduction in response to quences by inducing the expression of various genes, and in many many stimuli including cytokines, and p38 MAPK have been im- systems the expression of these is regulated by the transcription plicated in the transcription-activating potential of NF-␬B (12–14). factor, NF-␬B (4, 5). NF-␬B can regulate the expression of genes Transcription factors such as ATF-2 (activating transcription fac- involved in inflammation, cell cycle regulation, immune responses tor-2), Elk-1, etc., are also activated via p38 MAPK, which con- via the expression of cytokines, and antiapoptotic molecules that tributes to TNF-triggered gene expression (11, 15), and existing protect cells from the deleterious effects of TNF (5, 6). The p50/ data support a role for p38 MAPK activation in both death and by guest on October 1, 2021 p65 NF-␬B heterodimer normally exists in the cytosol, with the survival pathways (3, 16–19). DNA-binding dimer held as an inactive complex with the inhibitor In situations in which TNF induces death, the cytoplasmic do- subunit I-␬B3 (inhibitory protein that dissociates from NF-␬B) (7). mains of TNFR, primarily TNFRI, recruit the adapter proteins, Nuclear translocation and activation of NF-␬B occur on the phos- TNFR-associated death domain, and Fas-associated death domain, phorylation and subsequent degradation of I-␬B (8), which re- which in turn activate the initiator caspase, Fas-associated death leases NF-␬B to bind ␬B response elements and initiate transcrip- domain-like IL-1␤-converting enzyme/procaspase 8. Caspase 8 tion. Phosphorylation of I-␬B is achieved by sequential steps triggers apoptotic damage by activating multiple effector caspases, involving multisubunit kinase complexes that belong to the mito- each of which target a limited set of cellular proteins (2). Similarly, procaspase 9 also functions as an initiator/regulator caspase. The N-terminal prodomain of this caspase interacts with Apaf-1 in the presence of cytochrome c and dATP, resulting in the cleavage and National Centre for Biological Sciences, Bangalore, Karnataka, India activation of caspase 9, which activates caspases 3, 6, and 7. Thus, Received for publication October 13, 2000. Accepted for publication March 22, 2001. there are at least two major pathways, initiated by caspases 8 and The costs of publication of this article were defrayed in part by the payment of page 9, which can activate caspase cascades in cells. Effector caspases charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. are required for the execution phase of apoptosis and are respon- 1 This study was funded by a core grant to A.S. from National Centre for Biological sible for many of the morphological changes and cellular damage Sciences, Tata Institute of Fundamental Research, India. that occur during cell death. The essential role of these proteases 2 Address correspondence and reprint requests to Dr. Apurva Sarin, National Centre has been demonstrated in diverse systems (20). TNF-mediated ap- for Biological Sciences, University of Agricultural Sciences, Gandhi Krishi Vigyan optosis is also dependent on caspase activation (1, 2, 21), but par- Kendra Campus, New Bellary Road, Bangalore 560065, Karnataka, India. E-mail address: [email protected] adoxically, a number of studies have shown that inhibition of 3 Abbreviations used in this paper: I-␬B, inhibitory protein that dissociates from NF- caspase activity can lead to necrotic death in many cell types ␬B; ATF-2, activating transcription factor-2; BD-FMK, Boc-Asp(O-methyl)-FMK; (22–25). BID, BH3 interacting domain death agonist; c-IAP, cytoplasmic inhibitor of apoptosis Ј In this study, we identify a novel role for p38 MAPK in the protein; CHX, cycloheximide; DiOC6, 3,3 -diethyloxacarbocyanine; Hsp27, heat- shock protein 27; IETD-FMK, Ile-Glu-Thr-Asp-FMK; JC-1, 5,5Ј,6,6Ј-tetrachloro- TNF-triggered antiapoptotic response in U937 cells. We find that 1,1Ј,3,3Ј-tetraethyl-benzimidazoleyl-carbocyanine iodide; LEHD-FMK, Leu-Glu- p38 MAPK is necessary to TNF-triggered antiapoptotic gene ex- His-Asp-FMK; MAPK, mitogen-activated protein kinase; TRAF-2, TNFR-associated factor-2; VEID-FMK, Val-Glu-Ile-Asp-FMK; ZVAD-FMK, Z-Val-Ala-Asp(O-methyl)- pression, and inhibition of p38 MAPK triggers a TNF-mediated, fluoromethyl ketone. caspase-dependent apoptotic death pathway in U937 cells. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 6571 Materials and Methods volume of PBS, mounted on slides, and photographed using a Pro-Series 3, Cells and reagents CCD camera (Media Cybernetics). U937, a human myelomonocytic cell line, and Jurkat, a T lymphoblastoid Western blot analysis cell line of human origin, were used in all experiments. Murine splenic The 106 cells treated under various conditions were harvested, washed macrophages were isolated by adherence on plastic petri dishes. In each ϫ 6 twice by centrifugation in chilled PBS, lysed by the addition of SDS sam- experiment, unfractionated cells (20 10 cells/ml) from three individual ple buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, spleens were incubated for 45 min in complete medium in tissue culture and 0.1% bromphenol blue), vortexed to reduce sample viscosity, dena- dishes. At the end of incubation, floating cells were removed by gentle tured by boiling, and then cooled on ice. Samples were microcentrifuged swirling and decantation of dish contents. Plates were washed twice with for 5 min before loading on 10% SDS-PAGE gels. Proteins were trans- complete medium using a similar procedure. After the final wash, 2 ml of ferred onto Hybond-ECL nitrocellulose membrane (Amersham, Little cold medium was added to dishes, and adherent cells were removed using Chalfont U.K.). Membranes were blocked for1hinblocking buffer (5% a cell scraper and used in functional assays. rTNF and Abs to TNFR1 and Blotto, 0.1% Tween 20 in PBS) and incubated overnight with primary Ab TNFRII were obtained from R&D Systems (Minneapolis, MN). Cyclohex- in blocking buffer at 4°C, with gentle rocking. Membranes were washed imide (CHX), Hoechst 33342, and propidium iodide were obtained from three times for 5 min each with 10 ml of PBS containing 0.1% Tween-20, Sigma (St. Louis, MO). The p38 MAPK inhibitor PD 169316 was pur- and then incubated for1hat25°C with goat anti-rabbit HRP (Pierce, chased from Calbiochem (San Diego, CA). The peptide inhibitors Z-Val- Rockford, IL), diluted 1/5000 in blocking buffer.
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