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Departments of Medicine and Microbiology-Immunology, University of California Medical Center, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two distinct types of GPCRs for the lysophospholipid mediators recently have been defined structurally 11, 12, 13, 14, 15, 16 . One subfamily of GPCRs for LPA and S1P consists of at least five homologous seven-transmembrane domain proteins encoded by endothelial differentiation genes (edgs) 1–5. Edg protein GPCRs show amino acid sequence identity of 31–34% as a subfamily, but contain two homology clusters with greater internal similarity of structures and ligand specificity. Human Edg-2 and Edg-4 proteins constitute one cluster, which are 46% identical and 72% similar in amino acid sequence and are both GPCRs for LPA, but not S1P or other lysosphingolipids 11, 12 . Mouse Edg-2 GPCR also is a highly specific LPA receptor 13 . Edg-1, Edg-3, and Edg-5 constitute a second cluster of GPCRs, which are 45–60% identical in their amino acid sequences and specifically bind and transduce signals from S1P and possibly other lysosphingolipids, but not LPA 14, 15 . Xenopus oocytes and murine cells express a second type of GPCR, termed psp24, which is not structurally homologous to Edg protein GPCRs, but specifically transduces LPA-evoked oscillatory Cl- currents by activation of the inositol trisphosphate-Ca2+ system 16 .
The capacities of LPA and S1P to enhance cellular survival recently have been attributed in part to suppression of apoptosis 17, 18 , but the complex mechanisms by which these lipids suppress apoptosis have not been elucidated fully. The present study was designed to delineate mechanisms central to LPA and S1P protection of T cells from apoptosis and to identify associated alterations in cellular levels of proteins of the Bcl superfamily that may mediate suppression of apoptosis by these lipids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sources of chemicals were: S1P, sphingosylphosphorylcholine, sphingosine (S), and C6 ceramide (Biomol, Plymouth Meeting, PA); LPA, phosphatidic acid (PA), 1-ß-D-galactosyl-sphingosine (psychosine, PSSP), hygromycin (Calbiochem, San Diego, CA), and fatty acid-free BSA (faf-BSA) (Sigma, St. Louis, MO). Hybridomas producing mouse mAbs specific for substituent peptides of human Edg-3 (amino acids 1–21), Edg-4 (9–27), and Edg-5 (303–322) were generated from splenocytes of female BALB/c mice, which had been immunized first in multiple s.c. and i.m. sites with 100 µg of keyhole limpet hemocyanin conjugate (Pierce, Rockford, IL) of the respective peptides in CFA, 3 wk later and weekly for five additional weeks with 50 µg of the same conjugate in IFA, and then with 100 µg of unconjugated peptide alone i.v. 2–3 days before removal of the spleen (Ab Solutions, Palo Alto, CA). Each monoclonal IgG was purified by protein A affinity-chromatography (Pierce) and used to develop Western blots at 0.1–0.3 µg/ml. Other Abs were: monoclonal mouse anti-human Bax (clone 2D2, IgG1) and monoclonal mouse anti-Bcl-xL (clone 2H12, IgG2a) (Zymed, South San Francisco, CA), anti-human Bad (clone P3F6), anti-human Bak (G317–2), anti-Fas, anti-CD2, and anti-Bcl-2 (PharMingen, Inc., San Diego, CA), anti-CD3 (Caltag Laboratories, South San Francisco, CA), and anti-CD28 (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA). Rabbit anti-Edg-2 Ab was a gift from Dr. Jerold Chun (University of California, San Diego).
Cell culture and transfection
Human CD4+8+3low T lymphoblasts of the Tsup-1 line 19 were cultured in RPMI 1640 medium (University of California at San Francisco Cell Culture Facility) containing 10% (v:v) FBS, 100 U/ml of penicillin G, 100 µg/ml of streptomycin, and 1 mM ß-mercaptoethanol (hereafter referred to as complete RPMI medium) at 37°C with 5% CO2 in air. Complete RPMI medium was added to cultures every 2–3 days to maintain a density of 0.5–1 x 106 Tsup-1 cells/ml. For all studies of effects of LPA and S1P, batches of 3–5 x 107 Tsup-1 cells were conditioned in 30–50 ml of RPMI/1% FBS for 24 h and RPMI/0.1% FBS for a minimum of 12 h. Transfections of replicate suspensions of 4 x 106 Tsup-1 cells in 2 ml of RPMI/2% FBS were conducted by dropwise addition of a 250-µl preincubated mixture of 5 µg of antisense plasmid DNA, 0.2 µg of DNA of the REP 4 plasmid (Invitrogen, San Diego, CA) encoding hygromycin-resistance, and 15 µl of FuGENE 6 nonliposomal lipofection reagent (Boehringer-Mannheim, Indianapolis, IN), incubation for 16–24 h, and washing once and incubation in 4 ml of RPMI/10% FBS with 800 µg/ml of hygromycin for seven additional days. Then the surviving transfectants were washed and cultured in 4 ml of RPMI/0.1% FBS for 16 h. Antisense plasmids containing full-length cDNA encoding Edg-2, Edg-3, Edg-4, and Edg-5 surface receptors (Rs) in the reverse orientation relative to promoters were constructed in the expression vectors pRc/CMV2 for Edg-2, pcDNA 3.1 for Edg-3 and Edg-4 (Invitrogen, Carlbad, CA), and pSV.SPORT1 for Edg-5 (Life Technologies, Gaithersburg, MD).
RT-PCR analysis of Edg protein receptors
Total cellular RNA was extracted from suspensions of
Tsup-1 cells by the TRIzol method (Life Technologies, Grand Island,
NY), and a Superscript kit (Life Technologies) was used for reverse
transcription (RT) synthesis of cDNAs. PCR began with a
"hot start" at 94°C for 3 min, Taq DNA polymerase was
added, and amplification was conducted with 35 cycles of 30 s at
94°C, 2 min at 55°C, and 1 min at 72°C. A total of 2 µCi of
[
-32P]dCTP was added to some sets of reaction mixtures
to allow quantification of mRNA encoding each Edg receptor relative to
that of the standard glyceraldehyde 3- phosphate dehydrogenase (G3PDH)
20 . Oligonucleotide primer pairs were:
5'-dCCTGGCCAAGGTCATCCATGACAAC and 5'-dTGTCATACCAGGAAATGAGCTTGAC
for G3PDH; 5'-CTACACAAAAAGCTTGGATCACTCA and
5'-CGACCAAGTCTAGAGCGCTTCCGGT for Edg-1 (1100 base pairs (bp));
5'-dGCTCCACACACGGATGAGCAACC and 5'-GTGGTCATTGCTGTGAACTCCAGC for
Edg-2 (621 bp); 5'-dCAAAATGAGGCCTTACGACGCCA and
5'-dTCCCATTCTGAAGTGCTGCGTTC for Edg-3 (701 bp);
5'-dAGCTGCACAGCCGCCTGCCCCGT and 5'-dTGCTGTGCCATGCCAGACCTTGTC for
Edg-4 (775 bp); 5'-CTCTCTACGCCAAGCATTATGTGCT and
5'-ATCTAGACCCTCAGACCACCGTGTTGCCCTC for Edg-5 (500 bp);
5'-dAGTCCTCAAATCATCCCACATCTGC and 5'-dAAGTGGCACTTCCTGTCTCGTAATC for the
type I vasoactive intestinal peptide receptor (VIPR1); and
5'-dTCCCAGCAGGTGTTTCCTGGCCTAC and 5'-dCGAGCCTCTTGTACTGTGACTGGTC for
VIPR2. PCR products were resolved by electrophoresis in a 2 g/100 ml
agarose gel with ethidium bromide staining. G3PDH, VIPR, and Edg
receptor bands were cut from gels and solubilized for ß-scintillation
counting in 0.5 ml of sodium perchlorate solution at 55°C for 1
h (EluQuick; Schleicher and Schuell, Keene, NH). Initially, the G3PDH
cDNA templates in several different-sized portions of each sample were
amplified to determine volumes that would result in G3PDH bands of
equal intensity for each sample. Relative quantities of cDNA encoding
each Edg receptor also were calculated by the ratio of radioactivity to
that in the corresponding G3PDH band 20 .
Induction and assessment of apoptosis
After conditioning at low serum concentrations, replicate suspensions of 5 x 105 Tsup-1 cells in 0.5 ml of RPMI/0.1% FBS were incubated in 24-well plastic plates (Falcon, Oxnard, CA) for 16 h at 37°C in 5% CO2 in air. Some wells were precoated overnight at 4°C with 30 ng of anti-Fas Ab, 0.2 µg of anti-CD2 Ab, or a combination of 0.5 µg each of anti-CD3 and anti-CD28 Abs, and in others 5 µM C6 ceramide was the stimulus for apoptosis. LPA, S1P, and other lipids were dispersed in 0.05 g/100 ml of faf-BSA in medium. The principal assay for quantification of apoptosis was reliable and sensitive endlabeling of free 3'-OH groups of newly-generated nucleosomal DNA 21 . Cells from each well were pelleted at 200 x g for 5 min at 4°C, resuspended in 0.5 ml of phosphate-buffered 4% formaldehyde, kept at room temperature for 10 min, repelleted, resuspended in 150 µl of 80% ethanol, and immobilized and dried on poly-L-lysine precoated glass slides. Each slide was rehydrated in 20 mM Tris-130 mM NaCl, pH 7.6, and endogenous peroxidases were inactivated by treatment with 3% H2O2 in 90% methanol for 5 min at room temperature before endlabeling according to the procedures described in instructions for the Klenow-FragEL kit (Oncogene Research Products–Calbiochem, La Jolla, CA). Percentage apoptosis was calculated from the number of Tsup-1 cells with stained nuclei of a total of 200 counted. Omission of Klenow fragment permitted assessment of those with residual endogenous peroxidase activity, which never exceeded 1%.
For the radioactive assessment of DNA fragmentation, 0.5 x 107 Tsup-1 cells/ml of complete RPMI medium with 0.5% FBS were incubated with 20 µCi/ml of [3H]thymidine (DuPont–New England Nuclear, Boston, MA) for 2 h at 37°C and washed twice in complete RPMI and twice in FBS-free RPMI before addition to 24-well plates as for the Klenow-FragEL assay. The processing of radioactively labeled Tsup-1 cell suspensions was conducted as described 22 , and percentage fragmentation of DNA was calculated by the formula: (S + E)/(S + E + P) x 100%, where radioactivity was determined for S = supernatant in the original well, E = Tris-EDTA-Triton extract of cell pellet, and P = pellet dissolved in SDS. After 16 h of incubation, the release of radioactivity without stimulation was a mean (±SD) of 5.2 ± 2.9% (n = 12). The concomitants of apoptosis also were verified in some studies by fluorescence microscopic detection of characteristic morphological features of Tsup-1 cells stained with bis-benzimide trihydrochloride (Hoechst 33258; 10 µg/ml in glycerol:PBS, 30v:70v).
Western blots
Replicate suspensions of 1 x 107 Tsup-1 cells, which had been incubated with an inducer of apoptosis and/or LPA or S1P for 16 h, were washed three times with 10 ml of cold PBS, resuspended in 200 µl of cold 100 mM NaCl-50 mM Tris-HCl, pH 7.4, containing 5 mM EDTA, 1 mM PMSF, 20 µg/ml of leupeptin, 0.1 mM DL-thiorphan, 5% glycerol (v:v), and 1% Nonidet P-40. After homogenization with a Teflon pestle on ice for 2 min at 250 rpm, each sample was centrifuged at 5000 x g for 10 min at 4°C, and the supernatant was divided into 20 µl aliquots and stored at -70°C. Aliquots of 5000 x g supernatant containing 1–50 µg of protein were mixed with 4x Laemmli’s solution, heated to 100°C for 3 min, and electrophoresed in an SDS-12% polyacrylamide gel for 20 min at 100 v and 1 h at 140 v, along with a rainbow prestained set of molecular mass markers (DuPont–New England Nuclear or Amersham, Arlington Heights, IL). Proteins in each gel were transferred electrophoretically to a nitrocellulose membrane (Hybond; Amersham) for sequential incubation with 5 g/100 ml reconstituted nonfat milk powder for blocking unspecific sites, dilutions of monoclonal mouse anti-Bax, anti-Bad, anti-Bak, anti-Bcl-xL or anti-Bcl-2, and then horseradish peroxidase-labeled goat anti-mouse IgG, before development with a standard ECL kit (Amersham).
ELISA quantification of Bcl-2
Replicate suspensions of 1 x 106 Tsup-1 cells, which had been incubated with an inducer of apoptosis and/or LPA or S1P for 16 h, were transferred to 1.5 ml polypropylene Eppendorf tubes, pelleted at 400 x g for 5 min at 4°C, and resuspended in 200 µl of 50 mM Tris-HCl, pH 7.4, containing 5 mM EDTA, 0.2 mM PMSF, 10 µg/ml of leupeptin, and 1 µg/ml of pepstatin. Then, 40 µl of Ag extraction detergent solution (Calbiochem) was added to each sample followed by 30 min of incubation on ice with vigorous mixing every 5 min and centrifugation at 3000 x g for 10 min at 4°C. Bcl-2 levels in duplicate 50-µl portions of 1:3 dilutions of the 3000 x g supernatant for each sample were measured by an ELISA kit (Oncogene Research Products–Calbiochem) according to the procedures described. ELISA plates were read at 450/540 nm by a Molecular Devices system (Menlo Park, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Tsup-1 line of human
CD4+8+3low T lymphoblastoma cells
is a useful model for studies of the regulation of human T cell
apoptosis induced by different immunologically relevant stimuli 22 .
Tsup-1 cells also bear Rs for many endogenous mediators that influence
thymocyte and T cell apoptosis, including prostaglandins and
neuropeptides 22, 23, 24 . The application of RT-semiquantitative PCR
revealed expression of Edg-2, Edg-3, and Edg-4 Rs (Fig. 1
A). In this and two
additional assays, a radioactive modification of PCR permitted
assessment of the relative quantity of mRNA encoding each Edg receptor
compared with that for G3PDH in unstimulated Tsup-1 cells (Table I). Radioactive PCR confirmed the
known predominant expression of VIPR2 and only marginally detectable
mRNA for VIPR1. The levels of mRNA encoding Edg-2, Edg-3, and Edg-4 Rs
were as high as or higher than that for VIPR2 (n = 3).
In contrast, the amounts of mRNA encoding Edg-1 and Edg-5 Rs were just
at the level of detection and less than half that of Edg-3 receptor,
respectively (Fig. 1 and Table I). Of the stimuli used to induce
apoptosis in Tsup-1 cells, neither anti-Fas Ab nor Abs to other
surface protein Ags altered the levels of mRNA encoding Edg-2 or Edg-4
Rs (Fig. 1, B and D, and Table II). In contrast, a concentration of C6
ceramide that evoked maximal apoptosis reduced the apparent levels of
Edg-2 and Edg-4 receptor mRNA (Fig. 1, B and D,
and Table II). The level of Edg-3 receptor mRNA determined by RT-PCR
and radioactive PCR was increased by each of the apoptosis-inducing
Abs, but was unchanged by C6 ceramide (Fig. 1C and Table II).
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Both LPA and S1P prevented apoptosis induced by anti-Fas Ab
and a combination of anti-CD3 and anti-CD28 Abs with
differences only in lipid concentration dependence (Fig. 2). For anti-Fas Ab-stimulated
apoptosis, LPA and S1P suppression were significant at
10-10 M and reached plateau levels of >80% inhibition by
10-9 M to 10-7 M. At 10-6 M, the
maximal level of protection by S1P was maintained, whereas that
observed with lower concentrations of LPA was lost completely. The net
mean level of apoptosis evoked by anti-CD2 Ab was 20%
(n = 2), and this also was inhibited 94% by
10-8 M LPA, 71% by 10-8 M S1P, and 82% by
10-6 M S1P. As for apoptosis induced by anti-Fas Ab,
that evoked by anti-CD2 Ab was not decreased by 10-6 M
LPA. Similarly, 10-10 M to 10-7 M LPA and S1P
significantly prevented apoptosis induced by the combination of
anti-CD3 and anti-CD28 Abs (Fig. 2). Again the protective
effect of S1P was maintained at 10-6 M, whereas
10-6 M LPA did not prevent apoptosis.
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Loss of protection from apoptosis at a high LPA concentration was in
part attributable to induction of apoptosis by 10-6 M LPA
alone, which attained a mean level (±SD) of 4.7 ± 0.9-fold
higher than the mean level of 3.7% for control Tsup-1 cells in medium,
as contrasted with an increase of only 2.3 ± 0.5-fold for
10-6 M S1P. Although this higher background of apoptosis
for 10-6 M LPA was subtracted from the levels of apoptosis
evoked by defined stimuli in the presence of 10-6 M LPA,
LPA injury and the effects of anti-Fas and other apoptosis-inducing
Abs appear to have been more than additive. The levels of apoptosis
attained means of 2.3-, 2.7-, and 3.0-fold higher than medium-alone
control at 10-9 M, 10-8 M, and
10-7 M LPA, respectively, and 1.3-, 1.5-, and 1.6-fold
higher than medium at 10-9 M, 10-8 M, and
10-7 M S1P, which were significantly higher only for
10-7 M LPA and did not influence the profound protective
effects of these concentrations of the lipids. In contrast to
Ab-induced apoptosis, that evoked by C6 ceramide was suppressed
significantly by S1P, but not by either concentration of LPA (Fig. 2
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The capacities of LPA and S1P to prevent activation-induced apoptosis
of Tsup-1 cells were specific for these lipids and did not extend to
structurally related compounds (Fig. 3).
LPA at 10-7 M again evoked a modest, but significant,
increase in background apoptosis, and 10-7 M S1P in this
study also enhanced apoptosis slightly and with borderline
significance. LPA and S1P, but not PA, S, or PSSP, prevented
anti-Fas Ab induced apoptosis. S1P suppressed apoptosis elicited by
C6 ceramide, but this protection was not mimicked by S or PSSP, and LPA
again lacked protective activity against this stimulus (Fig. 3).
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Possible mechanisms by which LPA and S1P protect Tsup-1
cells from activation-induced apoptosis were examined first by
quantifying any associated changes in cellular content of proteins that
enhance or inhibit apoptosis. Western blot analyses of Bax extracted
from anti-Fas Ab-treated Tsup-1 cells showed that incubation with
10-8 M LPA or 10-8 M S1P decreased the level
of Bax by >80% and >50%, respectively, as assessed by dilutions of
the positive control sample (Fig. 4A). In contrast, neither
10-6 M LPA nor 10-6 M S1P consistently
affected Bax levels in anti-Fas Ab-treated Tsup-1 cells despite the
protective effect of 10-6 M S1P. Densitometric evaluation
of the results of Western blots designed to define the concentration
dependence of LPA and S1P effects on Bax levels in Tsup-1 cells
revealed significant suppression at 10-9 M to
10-7 M and maximal suppression of Bax at 10-8
M for both lysophospholipids (Table III).
The lack of suppression of Bax by 10-6 M S1P, despite
significant protection from apoptosis, implies other functionally
relevant effects of high concentrations of S1P.
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Western blot analyses of equal amounts of protein from extracts of
Tsup-1 cells that had been incubated with 10-7 M LPA or
10-7 M S1P alone or with anti-Fas Ab with and without
10-8 M and 10-6 M LPA and S1P
failed to show any changes in cellular content of Bcl-xL
for any of the samples (Fig. 4B). ELISA assays of extracts
of Tsup-1 cells from similar experimental protocols also did not show
significant changes in cellular levels of Bcl-2 (Table IV). Western blot analyses of Tsup-1
cellular levels of the apoptosis-promoting proteins Bad and Bak did not
reveal any alterations in association with exposures to LPA or S1P
alone nor to LPA or S1P in the presence of anti-Fas.
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The application of newly developed mouse mAbs to Edg-3, Edg-4, and
Edg-5 and a polyclonal Ab to Edg-2 demonstrated expression of the
respective 48- to 55-kDa receptor proteins by Tsup-1 cells (Fig. 5). Transfection of Tsup-1 cells with
ligand-directed pairs of plasmids encoding antisense messages for
either the LPA Rs Edg-2 and Edg-4 or the S1P Rs Edg-3 and Edg-5, as
well as the REP 4 hygromycin-resistance plasmid, followed by 7 days of
selection in hygromycin, selectively reduced respective levels of the
targeted Edg receptor proteins detectable in Western blots, with little
or no suppression of those of the complementary set specific for the
other ligand (Fig. 5). Sham-transfected Tsup-1 cells, which received
only empty vectors, responded to 10-8 M LPA or S1P with
respective reductions of 
85 and 90% in the level of
immunoreactive Bax protein observed after incubation with anti-Fas
Ab alone, as assessed with dilutions of the latter sample (Fig. 6). In contrast, transfection with a
mixture of expression plasmids encoding Edg-2 and Edg-4 antisense
messages prevented the decrease in Bax elicited by LPA, but not by S1P
(Fig. 6). In the reciprocal study, transfection with a mixture of
expression plasmids encoding Edg-3 and Edg-5 antisense messages
prevented the decreases in Bax elicited by S1P, but not by LPA. The
effects of LPA and S1P on apoptosis of Tsup-1 cells transfected with
Edg-2 and Edg-4 antisense plasmids or Edg-3 and Edg-5 antisense
plasmids, followed by hygromycin selection, compared with effects on
apoptosis of sham transfectants, also were evaluated functionally. At
10-9 M and 10-8 M, LPA and S1P
characteristically protected Tsup-1 cell sham transfectants from
anti-Fas Ab-induced apoptosis (Table V). Protection from anti-Fas
Ab-induced apoptosis by LPA was significantly less in Tsup-1 cells
transfected with Edg-2 and Edg-4 antisense plasmids, without a change
in the effectiveness of S1P. Protection from anti-Fas Ab-induced
apoptosis by S1P was significantly less in Tsup-1 cells transfected
with Edg-3 and Edg-5 antisense plasmids, without a change for LPA
(Table V).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which are the most prominent members of
the subfamily found on lymphoid cells and other leukocytes of the
immune system 11, 12, 14 . Radioactive RT-PCR semiquantification of
mRNA specific for each of these Edg Rs (Table I) showed relative levels
similar to or higher than those found for the VIPR2, which has a mean
density of 89,500 receptors/Tsup-1 cell 23 . The level of
mRNA-encoding Edg-5 receptor was much lower and that for Edg-1 receptor
was undetectable (Table I). The conditions and stimuli used to induce
apoptosis of Tsup-1 cells either had no effect or increased the levels
of Edg-2, Edg-3, and Edg-4 R mRNA, with the exception of C6 ceramide
(Fig. 1 and Table II). Ceramide strikingly reduced the levels of mRNA
encoding the Edg-2 and Edg-4 Rs for LPA, without affecting mRNA
encoding the Edg-3 R for S1P. The recent production of mAbs specific
for Edg-3, Edg-4, and Edg-5 Rs and a polyclonal anti-Edg-2
antiserum permitted confirmation but not accurate quantification of
expression of these Edg Rs by Tsup-1 cells (Fig. 5).
The abilities of LPA and S1P to potently suppress activation-induced
apoptosis of Tsup-1 cells were demonstrated by a highly reproducible
primary assay, which quantifies newly-generated 3'-OH groups of
nucleosomal fragments of DNA (Figs. 2 and 3), and were confirmed by
quantification of the release of radioactive prelabeled fragments of
DNA. Both assays revealed elements of stimulus specificity of LPA and
S1P protection from apoptosis. At 10-10 M to
10-7 M, LPA and S1P suppressed with similar
concentration-dependence apoptosis induced by anti-Fas Abs,
anti-CD3 plus anti-CD28 Abs (Fig. 2), and anti-CD2 Abs. LPA
and S1P showed two differences in their patterns of protection from
apoptosis. The first was a loss of protection by LPA but not S1P at the
highest concentration examined of 10-6 M, which was
attributed in part to direct injury of Tsup-1 cells by this level of
LPA but not S1P. The second was lack of protection by LPA from
ceramide-induced apoptosis (Figs. 2 and 3), which appeared to correlate
with the suppression of expression of the Edg-2 and Edg-4 Rs for LPA by
C6 ceramide (Fig. 1 and Table II). In contrast, S1P protected Tsup-1
cells from apoptosis induced by ceramide to the same extent as it
afforded protection from apoptosis evoked by the other stimuli (Figs. 2 and 3), and ceramide did not suppress expression of the Edg-3 receptor
for S1P below the level detected on unstimulated Tsup-1 cells (Fig. 1
and Table II). Although exogenous S is converted to S1P in many types
of cells and PA is similarly metabolized to LPA 1, 2, 3 , neither S nor
PA exhibited the protective effects of LPA and S1P from apoptosis
induced by any of the agonists employed (Fig. 3).
Initial investigations of possible mechanisms by which LPA and
S1P protect Tsup-1 cells from apoptosis employed anti-Fas Abs as
the inducing agent, because its effect was suppressed by both lipids
with similar potency and activity. The level of the proapoptotic
protein Bax 25 extracted from Tsup-1 cells undergoing apoptosis after
anti-Fas Ab stimulation was suppressed by maximally protective
concentrations of 10-9 M to 10-7 M, but not
by 10-6 M LPA or S1P (Fig. 4 and Table III). Thus, effects
of S1P on elements of apoptosis other than Bax must explain its
protective effect at 10-6 M. In contrast, there were no
changes in the levels of other proapoptotic proteins, including Bad and
Bak, or in the antiapoptotic proteins Bcl-xL and Bcl-2 25
in Tsup- 1 cells undergoing apoptosis after exposure to anti-Fas
Abs (Fig. 4 and Table IV).
The dependence of inhibition of activation-induced apoptosis by LPA and
S1P on expression of Edg Rs by Tsup-1 cells was examined next by
transfection of ligand-related combinations of antisense plasmids
directed to the LPA Rs Edg-2 and Edg-4 and the S1P Rs Edg-3 and Edg-5.
The extent and selectivity of suppression of Tsup-1 cell content of
Edg-2 and Edg-4 and Edg-3 and Edg-5 receptor proteins supported the
effectiveness of this antisense approach (Fig. 5). Two of the primary
indicators of the effects of LPA and S1P on Tsup-1 cell apoptosis were
assessed in parallel for cells transfected with antisense plasmids, as
contrasted with those sham transfected with plasmids lacking the
antisense inserts. The pair of antisense plasmids directed to Edg-2 Rs
and Edg-4 Rs for LPA reduced significantly the protective effect of LPA
on anti-Fas Ab-induced apoptosis observed in sham-transfected
controls, but had no effect on protection by S1P (Table V). A pair of
antisense plasmids directed to Edg-3 Rs and Edg-5 Rs for S1P reduced
significantly the protective effect of S1P on anti-Fas Ab-induced
apoptosis observed in sham transfectants, but did not affect the
protection afforded by LPA (Table V). Suppression of the level of Bax
induced by 10-8 M LPA or 10-8 M S1P in sham
transfectants similarly was reduced by combined transfection with
antisense plasmids directed to Edg-2 plus Edg-4 and Edg-3 plus Edg-5,
respectively (Fig. 6). Thus, LPA and S1P effects on activation-induced
apoptosis of Tsup-1 cells depend on expression of a relevant complement
of the Edg Rs specific for each lysophospholipid ligand. That antisense
reduction in Edg receptor expression reduced homologous ligand
protection from apoptosis, and the suppression of Bax in parallel
supported a causal relationship. The discrepancy between protection and
Bax reduction by 10-6 M S1P suggests a dominant role for
non-Bax mechanisms.
The capacity of LPA and S1P to suppress apoptosis often is linked
intimately with their abilities to stimulate cellular proliferation, as
both effects may share biochemical prerequisites and signaling
mechanisms. The present findings implicate alteration in Bax
concentration as one of the mechanisms through which LPA and S1P
protect some types of cells from apoptosis. The concentration-dependent
relationships (Table III) further suggest that LPA- and S1P-induced
changes in Bax contribute to protection from apoptosis predominantly at
lower levels of the lipids. Similar antiapoptotic decreases in cellular
levels of Bax have been observed only rarely as a consequence of the
protective effects of some drugs on neural cells 26 . A greater
understanding of the separate and concerted roles of stimulation of
growth, suppression of Bax, and inhibition of other proapoptotic
activities such as some caspases 17 will require more definitive
dissection of these mechanisms and their interactions. The recently
reported capacity of S1P to inhibit caspases 3, 6, and 7, but not 8, in
the Jurkat line of human T cells, as assessed by cleavage of specific
protein substrates 17 , suggests the potential importance of S1P and
LPA in regulating activities of this family of proteases in T cell
apoptosis. Thus, caspase inhibition by 10-6 M S1P may
explain the Bax-independent protection from apoptosis by this highest
concentration. These earlier studies were restricted to S1P and thus
should first be extended to an analysis of effects of LPA on caspases.
The data available suggest differences between LPA and S1P in their
specificity, potency, and mechanisms of antiapoptotic activities. The
prevention of ceramide-induced apoptosis by S1P, but not LPA, may
reflect principally the demonstrated reduction in expression of Edg-2
and Edg-4 Rs for LPA, without parallel changes in S1P Rs. However,
other contributing factors may include the known antagonism of S1P and
ceramide at sites, such as protein kinase C, that are critical
mediators or regulators of apoptosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Edward J. Goetzl, Immunology and Allergy, UB8B, Box 0711, University of California Medical Center, 533 Parnassus, San Francisco, CA 94143-0711. E-mail address:
3 Abbreviations used in this paper: LPA, 1-oleoyl-lysophosphatidic acid; S1P, sphingosine 1-phosphate; S, sphingosine; PSSP, psychosine; PA, phosphatidic acid; GPCR, G protein-coupled receptor; edg, endothelial differentiation gene; Edg, GPCR encoded by an edg; RT, reverse transcription; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; VIPR1, type I vasoactive intestinal peptide receptor; VIPR2, type II vasoactive intestinal peptide receptor; Rs, surface receptors; bp, base pair.
Received for publication July 24, 1998. Accepted for publication November 9, 1998.
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