Lysophosphatidic Acid and Sphingosine 1-Phosphate Protection of T Cells from Apoptosis in Association with Suppression of Bax This information is current as of September 26, 2021. Edward J. Goetzl, Yvonne Kong and Baisong Mei J Immunol 1999; 162:2049-2056; ; http://www.jimmunol.org/content/162/4/2049 Downloaded from References This article cites 25 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/162/4/2049.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 *average by guest on September 26, 2021 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lysophosphatidic Acid and Sphingosine 1-Phosphate Protection of T Cells from Apoptosis in Association with Suppression of Bax1 Edward J. Goetzl,2 Yvonne Kong, and Baisong Mei Members of a subfamily of G protein-coupled receptors (GPCRs), encoded by five different endothelial differentiation genes (edgs), specifically mediate effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on cellular proliferation and dif- ferentiation. Mechanisms of suppression of apoptosis by LPA and S1P were studied in the Tsup-1 cultured line of human T lymphoblastoma cells, which express Edg-2 and Edg-4 GPCRs for LPA and Edg-3 and Edg-5 GPCRs for S1P. At 10210 Mto1027 M, both LPA and S1P protected Tsup-1 cells from apoptosis induced by Abs to Fas, CD2, and CD3 plus CD28 in combination. Apoptosis elicited by C6 ceramide was inhibited by S1P, but not by LPA, in part because ceramide suppressed expression of Edg-2 Downloaded from and Edg-4 surface receptors for LPA without affecting Edg-3 surface receptors for S1P. At 1029 Mto1027 M, LPA and S1P significantly suppressed cellular levels of the apoptosis-promoting protein Bax, without altering the levels of Bcl-xL or Bcl-2 assessed by Western blots and immunoassays. Transfections of pairs of antisense plasmids for Edg-2 plus Edg-4 and Edg-3 plus Edg-5, and hygromycin selection of transfectants with reduced expression of the respective Edg R proteins in Western blots, inhibited both protection from apoptosis and reduction in cellular levels of Bax by LPA and S1P. Thus, LPA and S1P protection from apoptosis is mediated by distinct Edg GPCRs and may involve novel effects on Bax regulatory protein. The Journal of http://www.jimmunol.org/ Immunology, 1999, 162: 2049–2056. he lysophospholipid mediators lysophosphatidic acid ferentiation genes (edgs) 1–5. Edg protein GPCRs show amino (LPA)3 and sphingosine 1- phosphate (S1P) are generated acid sequence identity of 31–34% as a subfamily, but contain two T by complex enzymatic pathways from membranes of homology clusters with greater internal similarity of structures and many different types of stimulated cells (1–3). LPA and S1P are ligand specificity. Human Edg-2 and Edg-4 proteins constitute one both characterized by wide-spread cellular production, micromolar cluster, which are 46% identical and 72% similar in amino acid maximal concentrations in serum and some tissue fluids, high lev- sequence and are both GPCRs for LPA, but not S1P or other ly- els of binding to serum albumin, and biodegradation by multiple sosphingolipids (11, 12). Mouse Edg-2 GPCR also is a highly by guest on September 26, 2021 enzymatic mechanisms (4, 5). In extracellular fluids, these lipids specific LPA receptor (13). Edg-1, Edg-3, and Edg-5 constitute a are potent stimuli of cellular proliferation, differentiation, survival, second cluster of GPCRs, which are 45–60% identical in their adhesion, aggregation, and other specific functions (6–8). The ex- amino acid sequences and specifically bind and transduce signals istence of G protein-coupled receptors (GPCRs) for LPA and S1P from S1P and possibly other lysosphingolipids, but not LPA (14, was suggested initially by specific ligand structural dependence of 15). Xenopus oocytes and murine cells express a second type of their effects, ligand-induced desensitization of some cellular re- GPCR, termed psp24, which is not structurally homologous to Edg sponses, and pertussis toxin inhibition of their cellular Ca21 mo- protein GPCRs, but specifically transduces LPA-evoked oscilla- bilizing and proliferative activities (9, 10). tory Cl2 currents by activation of the inositol trisphosphate-Ca21 Two distinct types of GPCRs for the lysophospholipid media- system (16). tors recently have been defined structurally (11–16). One subfam- The capacities of LPA and S1P to enhance cellular survival ily of GPCRs for LPA and S1P consists of at least five homologous recently have been attributed in part to suppression of apoptosis seven-transmembrane domain proteins encoded by endothelial dif- (17, 18), but the complex mechanisms by which these lipids sup- press apoptosis have not been elucidated fully. The present study was designed to delineate mechanisms central to LPA and S1P Departments of Medicine and Microbiology-Immunology, University of California protection of T cells from apoptosis and to identify associated Medical Center, San Francisco, CA 94143 alterations in cellular levels of proteins of the Bcl superfamily that Received for publication July 24, 1998. Accepted for publication November 9, 1998. may mediate suppression of apoptosis by these lipids. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 The research described was supported by Grant HL31809 from the National Insti- Chemical reagents and Abs tutes of Health (E.J.G.). The sources of chemicals were: S1P, sphingosylphosphorylcholine, sphin- 2 Address correspondence and reprint requests to Dr. Edward J. Goetzl, Immunology gosine (S), and C6 ceramide (Biomol, Plymouth Meeting, PA); LPA, phos- and Allergy, UB8B, Box 0711, University of California Medical Center, 533 Parnas- b sus, San Francisco, CA 94143-0711. E-mail address: [email protected] phatidic acid (PA), 1- -D-galactosyl-sphingosine (psychosine, PSSP), 3 hygromycin (Calbiochem, San Diego, CA), and fatty acid-free BSA (faf- Abbreviations used in this paper: LPA, 1-oleoyl-lysophosphatidic acid; S1P, sphin- BSA) (Sigma, St. Louis, MO). Hybridomas producing mouse mAbs spe- gosine 1-phosphate; S, sphingosine; PSSP, psychosine; PA, phosphatidic acid; GPCR, G protein-coupled receptor; edg, endothelial differentiation gene; Edg, GPCR en- cific for substituent peptides of human Edg-3 (amino acids 1–21), Edg-4 coded by an edg; RT, reverse transcription; G3PDH, glyceraldehyde 3-phosphate (9–27), and Edg-5 (303–322) were generated from splenocytes of female dehydrogenase; VIPR1, type I vasoactive intestinal peptide receptor; VIPR2, type II BALB/c mice, which had been immunized first in multiple s.c. and i.m. vasoactive intestinal peptide receptor; Rs, surface receptors; bp, base pair. sites with 100 mg of keyhole limpet hemocyanin conjugate (Pierce, Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2050 LYSOPHOSPHOLIPID SUPPRESSION OF T CELL APOPTOSIS Rockford, IL) of the respective peptides in CFA, 3 wk later and weekly for 0.2 mg of anti-CD2 Ab, or a combination of 0.5 mg each of anti-CD3 and five additional weeks with 50 mg of the same conjugate in IFA, and then anti-CD28 Abs, and in others 5 mM C6 ceramide was the stimulus for with 100 mg of unconjugated peptide alone i.v. 2–3 days before removal of apoptosis. LPA, S1P, and other lipids were dispersed in 0.05 g/100 ml of the spleen (Ab Solutions, Palo Alto, CA). Each monoclonal IgG was pu- faf-BSA in medium. The principal assay for quantification of apoptosis was rified by protein A affinity-chromatography (Pierce) and used to develop reliable and sensitive endlabeling of free 39-OH groups of newly-generated Western blots at 0.1–0.3 mg/ml. Other Abs were: monoclonal mouse anti- nucleosomal DNA (21). Cells from each well were pelleted at 200 3 g for human Bax (clone 2D2, IgG1) and monoclonal mouse anti-Bcl-xL (clone 5 min at 4°C, resuspended in 0.5 ml of phosphate-buffered 4% formalde- 2H12, IgG2a) (Zymed, South San Francisco, CA), anti-human Bad (clone hyde, kept at room temperature for 10 min, repelleted, resuspended in 150 P3F6), anti-human Bak (G317–2), anti-Fas, anti-CD2, and anti-Bcl-2 ml of 80% ethanol, and immobilized and dried on poly-L-lysine precoated (PharMingen, Inc., San Diego, CA), anti-CD3 (Caltag Laboratories, South glass slides. Each slide was rehydrated in 20 mM Tris-130 mM NaCl, pH San Francisco, CA), and anti-CD28 (Bristol-Myers Squibb Pharmaceutical 7.6, and endogenous peroxidases were inactivated by treatment with 3% Research Institute, Seattle, WA). Rabbit anti-Edg-2 Ab was a gift from H2O2 in 90% methanol for 5 min at room temperature before endlabeling Dr. Jerold Chun (University of California, San Diego). according to the procedures described in instructions for the Klenow- FragEL kit (Oncogene Research Products–Calbiochem, La Jolla, CA). Per- Cell culture and transfection centage apoptosis was calculated from the number of Tsup-1 cells with Human CD41813low T lymphoblasts of the Tsup-1 line (19) were cultured stained nuclei of a total of 200 counted.