Protein Kinase C {epsilon} Dependence of the Recovery from Down-regulation of S1P1 G Protein-coupled Receptors of T Lymphocytes*

Markus H. Graeler, Yvonne Kong, Joel S. Karliner {ddagger} and Edward J. Goetzl §

From the Departments of Medicine and Microbiology-Immunology, University of California, San Francisco, California 94143-0711 and {ddagger}The Cardiology Section of the Veterans Affairs Medical Center, San Francisco, California 94121

Received for publication, April 7, 2003 , and in revised form, May 13, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nM S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 µM S1P were suppressed after 1 h of preincubation with 100 nM S1P, but recovered fully after 12–24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKC{epsilon}-selective inhibitors and was absent in T cells from PKC{epsilon}-null mice. The same PKC{epsilon} inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P1 Rs was suppressed by a selective inhibitor of PKC{epsilon} after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P1 Rs thus differ from those for S1P induction of down-regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The lysophospholipids sphingosine 1-phosphate (S1P)1 and lysophosphatidic acid (LPA) are generated and secreted by many types of stimulated cells (1, 2). At concentrations of 0.1–1 micromolar found normally in plasma and other extracellular fluids, S1P and LPA evoke cellular proliferation and diverse other functional responses through at least eight members of a family of homologous G protein-coupled receptors (GPCRs) (3, 4). Originally termed endothelial differentiation gene-encoded or Edg receptors (Rs), those specific for S1P now are officially re-named S1P1 (Edg-1), S1P2 (Edg-5), S1P3 (Edg-3), S1P4 (Edg-6), and S1P5 (Edg-8), and those for LPA are LPA1 (Edg-2), LPA2 (Edg-4), and LPA3 (Edg-7). Stimulated mononuclear phagocytes and platelets are the predominant sources of S1P and LPA in the immune system. Blood and lymphoid tissue CD4 and CD8 T cells, B cells, and mononuclear phagocytes all express S1P and LPA GPCRs, in cell type-specific patterns, which are regulated distinctively by their respective ligands and by cellular immune activation (58).

T cells express predominantly S1P1 and S1P4, of which S1P1 transduces two distinct effects of S1P on T cell migration and also regulates other T cell functional responses (7, 8). S1P is chemotactic for T cells at 0.001–0.1 µM, enhances chemotactic responses to chemokines at 0.01–0.1 µM, and suppresses T cell chemotaxis to numerous stimuli at 0.3–3 µM. As T cell antigen receptor-dependent activation of T cells suppresses expression of S1P GPCRs and functional responses to S1P in parallel, the S1P-S1P1 R axis is considered most important in controlling recruitment and stimulation of naïve and memory T cells by setting their response threshold to other stimuli.

One unanswered critical question about S1P1 Rs of T cells and other types of cells is how they are maintained at a level of full functional expression in tissues and fluids where there are completely saturating micromolar concentrations of S1P. Epitope-tagged or fluorescent protein-containing recombinant S1P1 Rs introduced by transfection into several different cell lines were down-regulated by S1P through phosphorylation, internalization, and translocation to a caveolar compartment by a G protein-coupled receptor kinase 2-dependent process (9, 10). Rapid down-regulation and internalization, but not S1P1 R binding and signaling functions, were facilitated by N-linked glycans of S1P1 Rs (11). However, recovery and stabilization of cell-surface expression of S1P1 Rs after down-regulation have not been examined previously. We now report that T cell S1P1 R recovery from S1P-induced down-regulation requires protein kinase C {epsilon} (PKC{epsilon}) activity and AP-1 transcriptional complex, and involves PKC{epsilon}-dependent late phosphorylation of the S1P1 Rs.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Isolation, Transfection, Culture, and S1P Treatment of Cells—Mouse CD4 T cells were isolated from splenocytes of 6 to 8 week-old C57BL/6 female mice at a minimum purity of 97% using metallic beads bearing anti-CD4 monoclonal antibodies (MoAbs) and two cycles of magnetic retention chromatography (Miltenyi-Biotec, Auburn, CA), as described (8). HTC4 rat hepatoma cells, which lack any endogenous S1P or LPA Rs, were LipofectAMINE-transfected with a pcDNA3.1 (+) construct (Invitrogen) encoding influenza hemagglutinin peptide (HA)-NH2-terminally tagged human S1P1 Rs and selected by culture with 400 µg/ml of geneticin. The level of expression of S1P1 Rs was established by TaqMan real-time PCR (8). Suspensions of 0.5 x 106 purified CD4 T cells per ml of RPMI 1640 with 5% charcoal-adsorbed FBS were transfected with 50 µg/ml each of the c-Jun plus c-Fos antisense or corresponding sense phosphorothioated oligonucleotides (Biomol, Plymouth Meeting, PA), which had been preincubated with FuGene reagent (Roche Diagnostics), and further incubated for 16 h prior to exposure to S1P. Two-ml aliquots of some suspensions of purified CD4 T cells in RPMI 1640 with 50 µg/ml fatty acid-free BSA (FAF-BSA) (Calbiochem) and of HTC4-S1P1(HA) cells in Dulbecco's modified Eagle's medium with 50 µg/ml FAF-BSA were preincubated for up to 48 h in 6-well plates to which were added one or more biochemical inhibitors and/or 107 M S1P at 12 h intervals or 1 h prior to assessment of S1P1 R expression, phosphorylation, or functional signaling.

Quantification of Migration of Mouse CD4 T Cells—Migration of mouse purified CD4 T cells was analyzed in Transwell chambers (Costar, Cambridge, MA) with human type IV collagen (Sigma)-coated 5-µm pore width polycarbonate filters and incubation for 4 h, as described previously (6). T cell suspensions were 1 x 107/ml in RPMI 1640 with 5% heated and charcoal-extracted fetal bovine serum, from which 0.1 ml portions of each were loaded into top compartments of chemotactic chambers. S1P, CCL21 (Exodus-2) and CCL5 (RANTES) (Peprotech, Inc., Rocky Hill, NJ) in 0.6 ml of the same buffer were the positive chemotactic stimuli. T cells that had migrated through the filter and into the lower compartment were counted and the chemotactic response (CT) expressed as a percentage of the total T cells initially added to the upper compartment. CT of T cells pretreated with 100 nM S1P and/or an oligonucleotide or pharmacological agent was expressed as a percentage of the concurrent CT of untreated T cells (100%). The inhibition of chemokine-evoked CT by 1 and 3 µM S1P, termed CI, was expressed as percentage inhibition and compared with percentage inhibition of control T cells not preincubated with inhibitors and/or S1P or preincubated with control compounds. The significance of differences between migration altered with S1P and/or inhibitors and control CT or CI was calculated with a paired or two-sample Student's t test.

PKC{epsilon}-Null Mice—Mice with a selective absence of PKC{epsilon}, but normal tissue levels of other subtypes of PKC, were obtained from Dr. Robert Messing (Gallo Research Center, Emeryville, CA) (12).

Western Blot Analyses of S1P1 GPCRs—Replicate suspensions of HTC4-S1P1(HA)-stable transfectants were incubated with various protein kinase inhibitors and phosphatase inhibitors before preincubation with S1P for 1 h or 12 and 24 h. Membranes of the transfectants then were prepared by homogenization in 50 mM Tris-HCl, 100 mM NaCl with 1 mM EDTA, 1 mM dithiothreitol, and 1% glycerol (pH 7.4) with HALT Protease Inhibitor Mixture (Pierce), recovered by centrifugation at 15,000 x g for 20 min at 4 °C, and solubilized in 0.1% Nonidet P-40. One hundred µl of anti-HA rat MoAb-agarose matrix (Roche Molecular Biochemicals) was added to each preparation of solubilized membranes in 400 µl of 50 mM Tris-HCl with 0.1% Nonidet P-40, incubated for 1 h at 4 °C, washed twice with the same solubilizing buffer, resuspended in 40 µl of electrophoresis loading solution, and heated for 3 min at 100 °C. Immunoprecipitated proteins then were resolved by SDS-PAG electrophoresis, transferred, and stained as described previously (5) sequentially with 1 µg/ml mouse anti-Thr(P) MoAb(H-2, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-HA rat MoAb.

Nuclear Extraction and ELISA Quantification of c-Fos and Phosphorylated c-Jun/JunD—Replicate suspensions of 4 x 106 mouse spleen CD4 T cells were preincubated either with various protein kinase inhibitors for 30 min before incubation with S1P for 1 h or with S1P for 24 h, which included re-additions at 12 and 1 h and where the protein kinase inhibitors were introduced at 23, 12, and 1 h. c-Fos and phosphorylated c-Jun and JunD ("c-Jun") were measured in nuclear extracts of the T cells by an ELISA, according to protocol instructions, where AP-1 complexes bind to fixed oligonucleotides containing the 12-O-tetradecanoylphorbol-13-acetate-responsive element 5'-TGA(C/G)TCA-3' and AP-1 constituents are quantified by binding of antibodies to accessible epitopes (Active Motif, Carlsbad, CA).

Lysophospholipids and Biochemical Inhibitors—LPA and S1P (Sigma); LipofectAMINE (Invitrogen); the broadly specific PKC inhibitor calphostin C, myristoylated peptide subtype-selective PKC inhibitors PKC{epsilon} V1–2 (N-myristoyl-D-A-V-S-L-K-P-T) and PKC{beta} C2–4 (N-myristoyl-S-L-N-P-D-W-N-D-T), the protein phosphatase inhibitor okadaic acid, and phosphorothioated antisense and sense c-Fos and c-Jun oligonucleotides for the first 18 bases following the AUG sequence (Biomol); and the PKA-selective inhibitor KT5720 and broadly specific PKC inhibitor Ro318220 (Calbiochem) were obtained from the suppliers noted.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The addition of S1P to S1P-deprived cells of many types results in down-regulation and internalization of S1P1 Rs, but T cells freshly isolated from fully saturating ambient S1P levels of 100–300 nM express a full complement of S1P1 Rs (9, 10). To determine the course of recovery and persistence of T cell functional S1P1 Rs, mouse CD4 T cells were isolated, preincubated in medium without S1P for 16 h, and then incubated for 48 h with 100 nM S1P present for the entire period or for shorter times prior to assessment of chemotaxis. In the absence of re-exposure to S1P, mean control chemotactic responses ± S.D. to 108 M and 107 M S1P, respectively, were 17 ± 4% and 24 ± 6% for all intervals of preincubation as a group and mean control inhibition of chemotaxis to CCL21 by 106 M and 3 x 106 M S1P as a group were 47 ± 14% and 53 ± 6%. A 1-h exposure, which down-regulates S1P1 Rs, significantly decreased the chemotactic response of CD4 T cells to 108 M and 107 M S1P and concurrently reduced suppression of CD4 T cell chemotactic responses to CCL21 by 106 M and 3 x 106 M S1P (Fig. 1). Similar decreases were observed for suppression of chemotactic responses to CCL5 by S1P. At 12 h and for up to 48 h of exposure to S1P, the S1P1R-mediated direct chemotactic effects and chemotactic inhibitory effects of S1P both returned to control levels similar to those for CD4 T cells not exposed to down-regulating levels of S1P.



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FIG. 1.
Time course of S1P-induced down-regulation and recovery of S1P1 (Edg-1) GPCR transduction of migration effects in mouse CD4 T cells. The CD4 T cells were deprived of S1P for 16 h, after which 100 nM S1P was re-introduced at 48, 36, 24, and 12 h (48 on the abscissa), 12 and 24 h (24), 12 h (12), or 1 h (1). Each column and bar depicts the mean ± S.D. of the results of four separate studies conducted in duplicate. Control values (100%) in the left frame are migration of CD4 T cells preincubated for the same times without S1P and ranged from 15 to 22% of the total number of CD4 T cells initially added to the top compartment of the chemotactic chambers with 108 M S1P, 19 to 30% with 107 M S1P, and 21 to 32% with 200 nM CCL21. The range of control values for inhibition of chemotaxis to 200 nM CCL21 when preincubation was for 1, 12, 24, and 48 h without S1P was 43–58% for 106 M S1P and 47–62% for 3 x 106 M S1P in the T cell compartment (right frame). The statistical significance of each difference between the values for an S1P-preincubated sample and the respective control was calculated by a paired Student's t test and shown by + = p < 0.05 and * = p < 0.01.

 

The capacity of inhibitors of potentially relevant signal transducers to prevent recovery of function of down-regulated S1P1 Rs also was assessed in chemotactic assays. After 24 h of exposure of S1P-deprived CD4 T cells to S1P, the direct chemotactic effects and inhibitory actions on chemokine-evoked chemotaxis of 108 and 106 M S1P, respectively, had returned to control levels (Fig. 2A, right frame). The PKC type-specific inhibitors calphostin C and Ro318220, and the PKC{epsilon} subtype-selective inhibitor m-PKC{epsilon}V1–2 significantly suppressed the recovery of both functions of the S1P1 Rs. In contrast, the PKA inhibitor KT5720 and the PKC{alpha}/{beta}/{gamma} inhibitory peptide m-PKC{beta}C2–4 had no effect on recovery of down-regulated S1P1 Rs (Fig. 2A). In addition, neither calphostin C nor m-PKC{epsilon}V1–2 altered the S1P-elicited down-regulation of functional S1P1 Rs after 1 h of exposure of CD4 T cells to S1P (Fig. 2A, left frame). To confirm involvement of PKC{epsilon} in the recovery of down-regulated S1P1 Rs, similar studies were conducted with CD4 T cells from selective PKC{epsilon}-null mice. The patterns of down-regulation of functional S1P1 Rs by a 1-h exposure to 100 nM S1P, reflected in reduced direct chemotactic responses and suppressed inhibition of chemotaxis to CCL21 relative to controls not preincubated with S1P, were identical in wild-type and PKC{epsilon}-null mice (Fig. 2B, left two sets). In contrast, only the wild-type CD4 T cells had recovered S1P1 R-mediated chemotactic and chemotactic inhibitory responses after 24 h of exposure to S1P (Fig. 2B, first of right two sets). PKC{epsilon}-null mouse-derived CD4 T cells continued to show impaired direct chemotactic responses to S1P without suppression by S1P of CCL21-evoked chemotaxis after 24 h (Fig. 2B, second of right two sets), which was a pattern indistinguishable from that observed after 1 h (Fig. 2B, second of left two sets).



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FIG. 2.
PKC{epsilon} dependence of recovery of effects of S1P1 (Edg-1) GPCRs on CD4 T cell migration. A, prevention of recovery by pharmacological inhibitors of signal transduction. Each column and bar depicts the mean ± S.D. of the results of three or four separate studies conducted in duplicate. Inhibitor concentrations were: KT5720, 180 nM; calphostin C, 0.5 µM; RO318220, 100 nM; and the myristoylated peptides, 50 µg/ml. Control chemotactic values (100% on the left ordinate, left one or two bars) without prior exposure to S1P ranged from 14 to 20% of the total number of CD4 T cells initially added to the top compartment of the chemotactic chambers at 1 and 24 h with 108 M S1P and 18 to 26% with 107 M S1P. The range for chemotaxis to 200 nM CCL21 was 34–41% for 1 and 24 h preincubation without S1P, which represents 0% inhibition on the right ordinate for the one or two right-hand bars in each set. Control values for inhibition of chemotaxis to 200 nM CCL21 (right-hand ordinate) were 43–58% at 1 and 24 h for 106 M S1P and 47–62% for 3 x 106 M S1P in the T cell compartment. A negative value for inhibition indicates enhancement. The statistical significance of effects of all inhibitors were calculated relative to the respective responses with no inhibitor (0) at 1 and 24 h, and the symbols are the same as described in the legend to Fig. 1. B, lack of recovery in CD4 T cells of PKC{epsilon}-null mice. Each column and bar depicts the mean ± S.D. of the results of two separate studies conducted in triplicate. Control chemotactic values (100% on the left ordinate) without prior exposure to S1P ranged from 16 to 25% and 11 to 18% of the total number of CD4 T cells initially added to the top compartment of the chemotactic chambers for wild-type and PKC{epsilon}-null mice, respectively, with 108 M S1P and 20 to 24% and 14 to 19% with 107 M S1P. Chemotaxis to 200 nM CCL21 was 36–40% and 34–37% for wild-type and PKC{epsilon}-null mice, respectively (0% for inhibition of chemotaxis to CCL21 on the right-hand ordinate). Control values for inhibition of chemotaxis to 200 nM CCL21 without S1P preincubation (right-hand ordinate) were 38–42% at 1 and 24 h for wild-type mice and 49–54% for PKC{epsilon}-null mice with 106 M S1P and 47–53% for wild-type mice and 50–66% with 3 x 106 M S1P in the T cell compartment. Statistical significance was calculated for differences in corresponding values at the same time between wild-type and PKC{epsilon}-null mice and symbols are the same as in Fig. 1.

 

PKC{epsilon} in T cells has been linked to recruitment and functional activation of the AP-1 and N-FAT-1 transcription factors (13). Two approaches were used to examine independently the course and PKC{epsilon} dependence of activation of AP-1 by S1P and the involvement of components of AP-1 in recovery of down-regulated functional S1P1 Rs of CD4 T cells. First, the characteristics of S1P activation of c-Fos and c-Jun/JunD in CD4 T cells were investigated, as some growth effects of S1P involve engagement of the c-Fos promoter. S1P increased the nuclear contents of c-Fos and phosphorylated c-Jun/JunD by 1 h, and the levels were sustained for up to 24 h in the continued presence of a plasma concentration of 100 nM S1P (Fig. 3A). These increases were blocked significantly by the type-specific PKC inhibitor calphostin C and the PKC{epsilon}-selective inhibitor m-PKC{epsilon}V1–2 at 24 h, but not at 1 h, and not at either time point by the PKC{alpha}/{beta}/{gamma} inhibitory peptide m-PKC{beta}C2–4 (Fig. 3A) (14). Second, the involvement of AP-1 components in recovery of chemotactic signaling by down-regulated S1P1 Rs of CD4 T cells was demonstrated by introducing c-Fos plus c-Jun antisense oligonucleotides into CD4 T cells, in amounts proven to decrease AP-1 sufficiently for functional alterations (15), prior to assessing S1P effects on chemotaxis. Suppression of both direct chemotactic effects of 107 M S1P and of the inhibition by 106 M S1P of CCL21-induced chemotaxis by 1 h of prior reexposure to S1P was not affected by the mixture of AP-1 antisense oligonucleotides nor the corresponding sense oligonucleotides (Fig. 3B, left-hand set). In contrast, recovery of both chemotactic stimulation by S1P and S1P inhibition of chemokine-elicited chemotaxis after 24 h was prevented by c-Fos plus c-Jun antisense oligonucleotides (Fig. 3B, set furthest to the right), whereas timely recovery was complete for CD4 T cells pretreated only with corresponding sense oligonucleotides. The migration response patterns were no different after 24 h than 1 h for the antisense oligonucleotide-pretreated CD4 T cells.



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FIG. 3.
PKC{epsilon} dependence of the role of AP-1 in S1P effects on CD4 T cell migration. A, quantification by ELISAs of the involvement of PKC{epsilon} in S1P effects on expression of c-Fos and c-Jun/JunD in nuclear extracts of CD4 T cells. Each column and bar depicts the mean ± S.D. of the results of two separate studies conducted in duplicate. All samples were incubated for 24 h with inhibitors and S1P added at 24, 12, and 1 h for the four "24-h" sets and at 1 h prior to termination for the four "1-h" sets. The c-Jun values include phosphorylated c-Jun and JunD. Mean net base-line values without S1P were 0.11 and 0.14 for c-Fos and c-Jun/JunD, respectively, at 1 h and 0.16 and 0.12 at 24 h. B, prevention of recovery of S1P1 GPCR effects on migration of CD4 T cells by c-Fos + c-Jun antisense oligonucleotides. Each column and bar depicts the mean ± S.D. of the results of two separate studies conducted in triplicate. Control values (100% on the left ordinate) without S1P pretreatment ranged from 23 to 28% and 31 to 35% of the total number of CD4 T cells initially added to the top compartment of the chemotactic chambers for sense (S)-and antisense (AS)-treated sets, respectively, with 107 M S1P (left-hand bar) and 23 to 29% and 24 to 27% for 200 nM CCL21 (middle bar). The range of control values without S1P pretreatment for inhibition of chemotaxis to 200 nM CCL21 was 37–45% and 39–44% for sense- and antisense-treated sets, respectively, with 106 M S1P in the T cell compartment (right-hand bar and right ordinate). Statistical significance was calculated for differences in corresponding values at the same time between sense- and antisense-treated sets, and symbols are the same as described in the legend to Fig. 1.

 

S1P1(HA) Rs of S1P-deprived HTC4-transfectants re-exposed to 100 nM S1P were threonine-phosphorylated after 1 and 24 h (Fig. 4). Selective inhibition of PKC{epsilon} suppressed S1P-evoked threonine phosphorylation after 24 h, but not after 1 h. Protein phosphatase inhibition enhanced threonine phosphorylation of S1P1 Rs slightly after 24 h but not after 1 h. Thus only late threonine phosphorylation of S1P1(HA) Rs appears to be PKC{epsilon}-dependent and occurs in the same time period as reappearance of functional S1P1 Rs. The PKC{epsilon} dependence of recovery and persistence of functional S1P1 Rs in the presence of micromolar concentrations of S1P, which fully saturate the S1P1 Rs, thus also may require S1P1 R specific phosphorylation.



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FIG. 4.
Western blot analyses of threonine phosphorylation of S1P1 (Edg-1) GPCRs in HTC4-S1P1(HA) transfectants. The upper row shows immunolabeling of phosphothreonine (p-Thr) in the ~55-kDa immunoprecipitated HA epitope-tagged S1P1 GPCRs, and the lower row shows the total amount of S1P1(HA) receptor. All samples were incubated for 24 h, but with inhibitors and S1P added at 24, 12, and 1 h for the three 24-h sets and at 1 h prior to termination for the three 1-h sets. N-Myristoyl-PKC{epsilon} V1–2 was used at 100 µM and okadaic acid at 100 nM. The left-hand values are for molecular mass standards.

 

Elucidation of the different requisites for S1P and phorbol ester induction of rapid phosphorylation and internalization of epitope-tagged recombinant S1P1 receptors in a line of hamster fibroblast transfectants revealed independent mechanisms (9). Immediate down-regulation evoked by S1P depended on 12 amino acids of the carboxyl terminus, was resistant to suppression by PKC inhibitors, and was mediated in part by G protein-coupled receptor kinase-2. In contrast, elicitation of immediate down-regulation by a phorbol ester was not dependent on the carboxyl terminus and was completely suppressed by PKC inhibitors. Dissociation of early S1P-evoked down-regulation of S1P1 Rs from PKC activity was confirmed for T cells by the lack of effect of PKC inhibitors or of PKC{epsilon} genetic deletion on down-regulation of functional S1P1 Rs (Fig. 2, A and B). Late recovery and persistence of S1P1 Rs down-regulated by S1P were shown to depend on PKC{epsilon} by the suppressive effects of selective and broadly specific PKC inhibitors, PKC{epsilon} gene deletion, and antisense inhibition of portions of the S1P1-coupled signaling pathways (Figs. 2, 3, 4).

Coupling of expression of S1P1 Rs to the activation of PKC{epsilon} was considered because of the known capacity of PKC{epsilon} to recruit components of the AP-1 transcription complex, which are implicated in S1P signaling (13). These observations have been extended to the demonstration of involvement of PKC{epsilon}-evoked AP-1 in late-phase recovery of down-regulated S1P1 Rs (Fig. 3). A sustained rise in expression of the c-Fos and phosphorylated c-Jun/JunD components of AP-1 resulted in peak levels for all at 24 h after S1P stimulation (Fig. 3). Antisense suppression of these same components of AP-1 blocked PKC{epsilon}-dependent late recovery of S1P1 Rs from down-regulation by S1P, without altering PKC{epsilon}-independent S1P induction of early down-regulation. Although the site(s) of S1P-evoked and PKC{epsilon}-dependent phosphorylation of S1P1 Rs have not been established, threonine (Fig. 4), and presumably serine, are preferred phosphorylation targets consistent with the specificity of PKC. That PKC{epsilon}-dependent phosphorylation of Edg-1, recruitment of AP-1 components, and recovery of down-regulated Edg-1 receptors follow a similar late time course suggests mechanistic relationships. However, the precise roles and interrelationships of late phosphorylation of S1P1 Rs and AP-1 regulation of specific transcriptional events remain to be elucidated. Ongoing studies are examining effects of AP-1 antisense oligonucleotides on late phosphorylation of S1P1 receptors, distinctive characteristics of S1P1 R phosphorylation in PKC{epsilon}-null mice, and a range of mutant Edg-1 Rs to determine which will not be PKC{epsilon}-phosphorylated.

That distinct mechanisms govern S1P1 R down-regulation and recovery has numerous implications for cell biology and immunology. S1P effects on S1P1 Rs of T cells differ substantially from those of T cell antigen receptor activation, which persistently suppresses expression of all S1P GPCRs with a distinctive rank order of efficacy. It explains how S1P1 Rs may be internalized by acute exposure to S1P, but avoid persistent down-regulation in the presence of plasma and lymph levels of 100–300 nM S1P. It also suggests the possibility that agents may be developed which will distinguish and separately regulate down-regulation and recovery of S1P1 Rs pharmacologically.


    FOOTNOTES
 
* This work was supported by Grants HL-31809 (to E. J. G.) and HL-68738 (to J. S. K.) from the National Institutes of Health. 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. Back

§ To whom correspondence and reprint requests should be addressed: University of California-UB8B, UC Box 0711, 53 Parnassus at 4th, San Francisco, CA 94143-0711. Tel.: 415-476-5339; Fax: 415-476-6915; E-mail: egoetzl{at}itsa.ucsf.edu.

1 The abbreviations used are: S1P, sphingosine 1-phosphate; LPA, lysophosphatidic acid; Edg, endothelial differentiation gene-encoded receptor; R, receptor; GPCR, G protein-coupled receptor; FAF, fatty acid-free; BSA, bovine serum albumin; MoAb, monoclonal antibody; HA, influenza hemagglutinin peptide epitope; PKC, protein kinase C; CT, T cell chemotactic response; CI, inhibition of T cell chemotaxis to chemokine; ELISA, enzyme-linked immunosorbent assay. Back


    ACKNOWLEDGMENTS
 
We are grateful to Robert Chan for expert graphics and electronic submission of this manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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