From the
Departments of Medicine and Microbiology-Immunology, University of
California, San Francisco, California 94143-0711 and
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.
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ABSTRACT |
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INTRODUCTION |
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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 0.0010.1 µM, enhances chemotactic responses to chemokines at 0.010.1 µM, and suppresses T cell chemotaxis to numerous stimuli at 0.33 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 (PKC
) activity and AP-1 transcriptional complex, and
involves PKC
-dependent late phosphorylation of the S1P1
Rs.
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EXPERIMENTAL PROCEDURES |
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Quantification of Migration of Mouse CD4 T CellsMigration 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-Null MiceMice with a selective absence
of PKC
, 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 GPCRsReplicate 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/JunDReplicate 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 InhibitorsLPA and S1P
(Sigma); LipofectAMINE (Invitrogen); the broadly specific PKC inhibitor
calphostin C, myristoylated peptide subtype-selective PKC inhibitors PKC
V12 (N-myristoyl-D-A-V-S-L-K-P-T) and PKC
C24
(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.
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RESULTS AND DISCUSSION |
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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 subtype-selective
inhibitor m-PKC
V12 significantly suppressed the recovery of both
functions of the S1P1 Rs. In contrast, the PKA inhibitor KT5720 and
the PKC
/
/
inhibitory peptide m-PKC
C24 had no
effect on recovery of down-regulated S1P1 Rs
(Fig. 2A). In
addition, neither calphostin C nor m-PKC
V12 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
in the recovery of down-regulated S1P1 Rs, similar
studies were conducted with CD4 T cells from selective PKC
-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
-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
-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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PKC 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
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
-selective inhibitor m-PKC
V12 at 24
h, but not at 1 h, and not at either time point by the
PKC
/
/
inhibitory peptide m-PKC
C24
(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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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 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
-dependent and occurs in the same time period as reappearance of
functional S1P1 Rs. The PKC
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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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
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
by the suppressive
effects of selective and broadly specific PKC inhibitors, PKC
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
was considered because of the known capacity of PKC
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
-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
-dependent late recovery of
S1P1 Rs from down-regulation by S1P, without altering
PKC
-independent S1P induction of early down-regulation. Although the
site(s) of S1P-evoked and PKC
-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
-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
-null mice, and a range of
mutant Edg-1 Rs to determine which will not be PKC
-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 100300 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.
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FOOTNOTES |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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