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The Journal of Immunology, 2008, 181, 3658 -3664
Copyright © 2008 by The American Association of Immunologists, Inc.
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PP2A Dephosphorylates Elf-1 and Determines the Expression of CD3{zeta} and FcR{gamma} in Human Systemic Lupus Erythematosus T Cells1

Yuang-Taung Juang2,*, Ying Wang{dagger}, Guisen Jiang{ddagger}, Hai-Bin Peng*, Sukran Ergin*, Michelle Finnell*, Abigail Magilavy*, Vasileios C. Kyttaris* and George C. Tsokos2,*

* Division of Rheumatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; {dagger} Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and {ddagger} Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells from patients with systemic lupus erythematosus are characterized by decreased expression of CD3{zeta}-chain and increased expression of FcR{gamma}-chain, which becomes part of the CD3 complex and contributes to aberrant signaling. Elf-1 enhances the expression of CD3{zeta}, whereas it suppresses the expression of FcR{gamma} gene and lupus T cells have decreased amounts of DNA-binding 98 kDa form of Elf-1. We show that the aberrantly increased PP2A in lupus T cells dephosphorylates Elf-1 at Thr-231. Dephosphorylation results in limited expression and binding of the 98 kDa Elf-1 form to the CD3{zeta} and FcR{gamma} promoters. Suppression of the expression of the PP2A leads to increased expression of CD3{zeta} and decreased expression of FcR{gamma} genes and correction of the early signaling response. Therefore, PP2A serves as a central determinant of abnormal T cell function in human lupus and may represent an appropriate treatment target.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells from patients with systemic lupus erythematosus (SLE)3 display various biochemical abnormalities, which are invariably associated with altered cell signaling and gene transcription processes and cell function (1, 2, 3, 4). Prominent among these abnormalities is the decreased expression of CD3{zeta} both at the protein and mRNA levels (5). Various mechanisms appear to contribute to the decreased expression of CD3{zeta} including decreased CD3{zeta} gene transcription linked to decreased formation of the DNA-binding form of the transcription factor Elf-1 (6), decreased stability of the CD3{zeta} mRNA (7, 8), and increased degradation by caspase-3 (9).

Although CD3{zeta} is decreased in SLE T cells, the FcR{gamma}-chain is up-regulated and appears to populate the CD3/TCR complex (10) and lipid rafts (11). FcR{gamma}, unlike CD3{zeta} that associates with ZAP70, associates and signals through pSyk. It appears that the FcR{gamma}-pSyk pathway, along with the aggregated rafts in the surface membrane of SLE T cells (11, 12), contribute to the enhanced early CD3/TCR-mediated signaling events (1). The underlying cause for the "exchange" of two signaling chains in the CD3/TCR complex of SLE T cells is unknown. Because CD3{zeta} and FcR{gamma} protein changes are reflected by similar changes in mRNA levels (5, 10), we considered a link at the transcriptional level, which could explain this observation.

Elf-1 is a 619 aa protein sequence and belongs to the Ets family of transcription factors (13). Elf-1 resides in the cytoplasm as an 80 kDa protein and in the nucleus as a 98 kDa DNA-binding form (14, 15). Elf-1 binds the CD3{zeta} promoter at two GGAA elements and in their absence the promoter activity is reduced significantly (14, 15). Analysis of the promoter of the FcR{gamma} gene also revealed GGAA motifs in the proximal region, suggesting that Elf-1 may be involved in the regulation of the FcR{gamma} gene. Indeed, Elf-1 suppresses the expression of FcR{gamma} gene in basophil and human T cells (16). Elf-1 has been also reported to suppress the transcription of other genes including Fc{epsilon}R{alpha} (17). It appears, therefore, that Elf-1 is a transcription factor that may act as enhancer or repressor. Accordingly, we considered that changes in the levels of expression of the DNA-binding 98 kDa Elf-1 form could account for antithetic changes in the levels of CD3{zeta} and FcR{gamma} expression.

Posttranslational modifications contribute to the difference in the molecular mass between the 80 and 98 kDa forms of Elf-1. Phosphorylation and O-linked glycosylation contribute to the conversion of 80 to 98 kDa form (14), whereas SLE T cells fail to produce the 98 kDa Elf-1 (6). The decreased expression of 98 kDa Elf-1 in SLE T cells could be the result of either decreased phosphorylation or increased dephosphorylation. Recently, we reported that T cells from patients with SLE, but not other rheumatic diseases, express increased levels and activity of the PP2A (18), which contributes to decreased IL-2 production. We report here that PP2A, which is increased in SLE T cells (18), dephosphorylates Elf-1, promotes the expression of the non-DNA binding 80 kDa Elf-1, and leads to decreased expression of CD3{zeta} and increased expression of FcR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and T lymphocyte purification

A total of 24 SLE patients who fulfilled at least 4 of the 11 revised criteria of the American College of Rheumatology for the classification of SLE (19) were enrolled in this study. The patients were 83% African-American, 17% white, with a mean age of 42 ± 12 years and overwhelmingly female (92%). A total of 57% of patients were taking oral prednisone at the time of the study that was temporarily discontinued for least 12 h before the blood was collected. Other immuno-modulatory medications the patients were taking were: hydroxychloroquine (79%), azathioprine (32%), methotrexate (4%), and mycophenalate mofetil (18%). Wherever appropriate, an age-, ethnicity-, and sex-matched healthy volunteer was used as control. Both SLE and healthy volunteers donated 50 ml of blood in heparin-lithium tubes. The purification of T lymphocytes has been described before (20). All T cell cultures, unless stated, were done in RPMI 1640 supplemented with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. COS-7 cells were obtained from ATCC and cultured in RPMI 1640 with 10% FCS.

Plasmids, reagents, and Abs

The expression plasmids of the wild-type as well as the mutant PP2A were gifts from Dr. B. A. Hemmings (Friedrich Miescher Institute, Basel, Switzerland). The Elf-1 expression plasmid was a gift from Dr. J. Leiden (University of Chicago, Chicago, IL). Cycloheximide (CHX) was purchased form Sigma-Aldrich. The protein kinase C (PKC) inhibitor Bis was purchased from Calbiochem. Okadaic acid was purchased from Upstate Cell Signaling Solutions. Both PP1 and PP2A as well as Fas Abs were purchased from Millipore. Abs against CD3{zeta}-chain, Elf-1, Fli-1, Ets-2, and protein kinase N (PKN) are purchased from Santa Cruz Biotechonology; antiactin Ab was purchased from Sigma-Aldrich. The production of Ab against has been described (1). The anti-Fas Ab was purchased from Millipore (clone CH11).

Protein purification and Western blot analysis

To purify nuclear proteins, cell pellet was first re-suspended in protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, and AEBSF) freshly added buffer A (20 mM HEPES, 10 mM KCl, and 0.1 mM EDTA) and incubated on ice for 20 min followed by the addition of 0.6% Nonidet P-40 and subjected to centrifugation. The supernatant was collected and used as cytoplasmic lysate while the pellet was further incubated in protease inhibitors freshly added buffer C (20 mM HEPES, 0.4 M NaCl, and 2 mM EDTA) and vortexed in the cold room for 15 min before they were centrifuged at 13,000 rpm for 15 min. The supernatant was then collected and used as nuclear proteins. For total protein lysates, cells were resuspended in protease inhibitor freshly added RIPA buffer and incubated on ice for 30 min before they were centrifuged at 13,000 rpm for 10 min. The supernatant was then collected and stored in –80°C until use.

Cell transfection

The transfection of primary T cells has been reported before (21). Specifically, freshly purified primary T cells were incubated with plasmids in the nucleofector reagent and subjected to electroporation following the manufacturer’s protocol (Amaxa). To transfect COS-7 cells, cells were plated 1 day before. Plasmids were then incubated with Lipofectamine-2000 reagent (Invitrogen) for 20 min before they were dispensed to the cells.

RT-PCR

The sequence of the primers used for the PCR amplification are as follows: GCGAGGGGGCAGGGCCTGCATGTGAAG-3', 5'-AGCCTCTGCCTCCCAGCCTCTTTCTGAG-3' (CD3{zeta}-chain); 5'-CGGGGATGAAACAATTGAAACTAT-3', 5'-CTCGCTGGGTCCACTNTGATGTA-3'. (Elf-1); and 5'-CATGGGTCAGAAGGATTCCT-3', 5'-AGCTGGTAGCTCTTCTCCA-3' (actin).

EMSA

The performance of the EMSA has been described before (6). The sequences of the oligonucleotides used for EMSA are as follows: 5'-TCGAGAACCTCCAGGGCTTCCTGCCTGTGAACCA-3' (CD3{zeta}); 5'-CCGCCCGGTCTCTTGTGCAGGAAGGGGAAGGGGCCAAA-3' (FcR{gamma}).

Purification of Elf-1 and identification of the potential phosphorylated amino acids by MALDI-TOF mass spectrometry (MS)

Elf-1 was purified by affinity chromatography. In brief, nuclear proteins from Jurkat cells were incubated with concatenated oligonucleotides defining the high-affinity binding sites for Elf-1 (14, 22). The identity of the purified proteins was confirmed by Western blotting. The SDS-PAGE purified Elf-1 was subjected to in-gel trypsin digestion. The resulting peptides were spotted onto the MALDI-TOF MS sample plate with matrix ({alpha}-cyano-4-hydroxycinnamic acid) and internal mass standards. MALDI-TOF MS measurements were then conducted using a delayed extraction time-of-flight mass spectrometer (Voyager-DE STR Biospectrometry Workstation; Applied Biosystems). After spectrum acquisition, the data file was processed using the instrumental Explore program until a peak list was obtained. The phosphorylated peptides were then identified by comparing the measured masses (peak list) with the calculated peptide masses from theoretical trypsin-digested phosphorylated Elf-1 protein.

[Ca2+]i response analysis

Purified T cells were treated with 50 nM okadaic acid or DMSO for 6 h. The cells were then spun down and resuspended in fresh RPMI 1640 medium supplemented with 1% FCS at a concentration of 5 million cells per ml; Indo-1 was added (final concentration 5 µg/ml) in the solution and the cells were incubated at 37°C in the dark for 1 h. The cells were then spun down and resuspended in warm RPMI 1640 plus 1% FCS. Additionally, a CaCl2 solution was prepared by adding CaCl2 in RPMI 1640 plus 1% FCS (final CaCl2 concentration was 1 mM). For each calcium flux experiment, 200 ml of cell solution (1 million cells) were mixed with 800 ml of the CaCl2 solution. The calcium flux was measured using the LSRII flow cytometer (BD Biosciences) as the ratio of violet to blue emission. The cells were initially run through the machine for 1 min to establish a baseline and thereafter anti-CD3 Ab (final concentration 10 ng/ml) was added followed 30 s later by goat anti-mouse cross-linking Ab. The results were analyzed using the FlowJo software package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PP2A increases the conversion of the 98- to the 80-kDa form of Elf-1

Because the formation of the 98-kDa form of Elf-1 involves posttranslational phosphorylation of the 80-kDa form (14) and SLE T cells express increased amounts of PP2A (18), we asked whether overexpression of PP2A can convert the 98-kDa Elf-1 form to the 80 kDa. Overexpression of wild-type PP2A in COS-7 cells converted Elf-1 from the 98- to the 80-kDa form (Fig. 1a); overexpression of two mutated PP2A constructs, one affecting His-118, which is crucial for accelerating phosphatase activity and the other affecting Leu-199, which mediates the metal binding and the contact between PP2A and substrate, failed convert to the 98- to the 80-kDa Elf-1 (Fig. 1a). Wild-type and mutant constructs expressed similar amounts of protein (Fig. 1b), and therefore, the observed effect on Elf-1 can be attributed to phosphatase activity. Subsequently, we transfected human peripheral blood T cells with wild-type PP2A and we noted that the presence of PP2A decreased the levels of 98-kDa whereas it increased the levels of the 80-kDa Elf-1 (Fig. 1, c and d). These results clearly demonstrate that the 98-kDa Elf-1 is subject to dephosphorylation by PP2A.


Figure 1
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  		FIGURE 1. PP2A converts 98- to 80-kDa form of Elf-1. a and b, COS-7 cells were cotransfected with plasmids overexpressing Elf-1 and either control plasmid (pcDNA), wild-type PP2A encoding plasmid, or one of two PP2A mutant constructs (m118 or m199). The transfected cells were lysed and total protein extracts were collected. Thereafter, the expression of both 98- and 80-kDa Elf-1 was analyzed by Western blotting using an anti-Elf-1 (a) and an anti-PP2A Ab. c and d, Primary T cells were transfected with wild-type PP2A expression plasmid or empty vector. The cells were subsequently lysed and total protein extracts were collected and immunoblotted using an Ab against Elf-1. A representative experiment (c) and cumulative data (d) are shown here.

 
The conversion of 98- to the 80-kDa Elf-1 is not a Fas-mediated apoptosis-related event

PP2A has been shown to trigger apoptosis (23), and SLE T cells have been shown to undergo enhanced spontaneous apoptosis (24). The execution of apoptosis involves extensive activation of proteases that digest protein substrates. We asked whether the increased expression of the 80-kDa and the decreased 98-kDa Elf-1 in SLE T cells could be attributed to enhanced apoptosis. PKN protein (120 kDa, a fatty acid- and {rho}-activated Ser/Thr kinase) represents such a protein that during apoptosis is proteolytically digested to generate 105-, 90-, or 55-kDa products, designated apoptotic fragment (AF)1, AF2, and AF3, respectively (25). As shown in Fig. 2, treatment of normal human T cells with anti-Fas Ab (150 ng/ml) resulted in a time-dependent decrease of the 120-kDa PKN and increase of two of its specific proteolytic products, AF1 and AF3. However, this treatment led to increase rather than decrease of the 98-kDa Elf-1, indicating that the conversion of Elf-1 from the 98- to the 80- kD a form is not an apoptosis-related process.


Figure 2
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  		FIGURE 2. The conversion from 98 to 80 kDa Elf-1 is not a result of Fas-initiated apoptosis. Primary T cells were treated with Fas agonist Ab (150 ng/ml) for various time periods. The cells were lysed at the indicated time points and nuclear proteins were collected and analyzed using Western blot and Abs against PKN, AF-1, AF-3, Elf-1, and β-actin. A representative (one of three) experiment is shown here. Longer exposure of the immunoblotted membrane on film was required to demonstrate the presence of AF1.

 
Elf-1 T231 represents a main phosphorylation target responsible for the conversion of 80- to 98-kDa Elf-1 in T cells

We next asked which of the 60 Ser/Thr residues defined by Elf-1 is responsible for the phosphorylation-mediated conversion of the 80- to 98-kDa Elf-1. Affinity chromatography purified Elf-1 from Jurkat cells was subjected to MS analysis, which demonstrated that Elf-1-derived peptides 230–235 displayed molecular masses equal to those predicted for the corresponding peptides modified by phosphorylation (Table I). Since amino acid Thr-231 is the only amino acid within the peptide 230–235 (WTQREK) that can be phosphorylated, we concluded that Thr-231 represents the residue undergoing phosphorylation during the conversion of the 80- to the 98-kDa Elf-1. Interestingly, the sequence stretching between amino acids 228–234 represents a consensus PKC phosphorylation site. We mutagenized Thr-231 to Val and the resulting plasmid, T231V Elf-1 construct, was transfected into primary T cells. As shown in Fig. 3, a and b (cumulative data), T231V Elf-1-transfected T cells display less 98-kDa and increased amounts of 80-kDa Elf-1. To ascertain that the difference in the expression of 98-kDa Elf-1 in the wild-type and T231V Elf-1-transfected T cells was not due to the defects in used plasmids, the quality and quantity of both plasmids were constantly monitored by sequencing (not shown), Elf-1 insertion sites enzyme restriction (Fig. 3c), and DNA concentration measurement. Taken together, the data indicate that the conversion of Elf-1 from 80 to 98 kDa involves Thr-231 phosphorylation.


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  		Table I. Mass spectroscopy analysis revealed Thr-231 as the amino acid that undergoes phosphorylation during the conversion of the 80-kDa form to the 98-kDa form of Elf-1

 

Figure 3
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  		FIGURE 3. Elf-1 mutated at amino acid 231 expresses less 98-kDa form of Elf-1 in primary T cells. Plasmids overexpressing either wild-type plasmid (WT) or plasmid encoding Elf-1 mutated at amino acid 231 (Elf-1 T231V) were transfected into primary T cells. Twenty-four hours later, the transfected cells were lysed and total protein extracts lysates were collected and analyzed by Western blotting. a, A representative immunoblot using the transfected T cell extracts and an Ab against Elf-1 is shown here. b,The immunoblot tracings for 98- and 80-kDa Elf-1 were quantified using QuantityOne (see Materials and Methods). Subsequently, the ratio of 98:80 kDa Efl-1 was calculated. Here, we show cumulative data from four experiments. c, The wild-type (WT) and Elf-1 T231V plasmids used in the aforementioned experiments were digested using EcoRV and XhoI and run in 1% agarose gel.

 
Overexpression of PP2A decreases the binding of Elf-1 to CD3{zeta} and FcR{gamma} gene promoters

Elf-1 regulates the transcription of genes whose promoters define GGAA motifs. CD3{zeta} promoter defines such motifs (15), and we found that the FcR{gamma} gene promoter also contains multiple copies of GGAA motif within its –200 bp proximal region (16). An oligonucleotide defined by the -121/-84 region of the FcR{gamma} promoter bound less (p < 0.05) nuclear protein from SLE T cells than from normal T cells (Fig. 4, a and b, cumulative data, n = 4). The presence of an anti-Elf-1 Ab in a shift assay disrupted the binding of normal nuclear protein to the -121/-84 oligonucleotide (16) indicating the specific binding of Elf-1. Overexpression of PP2A in fresh human T cells resulted in decreased expression of 98 kDa Elf-1, but not Ets-2, in the nucleus (not shown) and nuclear proteins from PP2A-transfected cells displayed limited binding to CD3{zeta} and FcR{gamma} promoter-defined oligonucleotides (Fig. 4c). These data indicate that PP2A can regulate the gene transcription of both CD3{zeta} and FcR{gamma} genes by decreasing the binding of Elf-1 to their promoters.


Figure 4
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  		FIGURE 4. Overexpression of PP2A decreases the binding of Elf-1 to the promoters of both CD3{zeta} and FcR{gamma} genes. Resting T cells from patients with SLE (L) and matched healthy controls (N) were lysed and the nuclear protein was extracted according to the Materials and Methods. The nuclear protein was incubated with a 32P-labeled oligonucleotide and the protein-DNA interaction was analyzed with autoradiography and quantification using QuantityOne. a and b, The nuclear protein from SLE patients and controls was incubated with a 32P-labeled oligonucleotide that contained the two GGAA Ets binding sites that span –121 to –84 on the FcR{gamma} promoter. A representative tracing is shown in a and the cumulative data from four SLE and four normal subjects is shown in b. c, Nuclear proteins as described in a were incubated with 32P-labeled oligonucleotides encoding sequences -147/-119 of the CD3{zeta} promoter and -121/-84 of the FcR{gamma} promoter. Here, we show one (of three) representative experiment.

 
Silencing the expression of PP2A in SLE T cells restores the expression of CD3{zeta} while it suppresses the expression of FcR{gamma} mRNA

Next, we tested whether silencing of the overexpressed PP2A in SLE T cells by small interfering RNA (siRNA) PP2A can restore the expression of CD3{zeta} and suppress the increased expression of FcR{gamma} in SLE T cells. To silence PP2A, we used a mixture of three PP2A-specific siRNA (PP2A siRNA-mix) as well as each of the three PP2A siRNA constructs separately (PP2A siRNA-1, PP2A siRNA-2, and PP2A siRNA-3). Successful silencing of PP2A was confirmed by RT-PCR (Fig. 5a) and real-time RT-PCR (not shown). PP2A mRNA levels were not affected when the cells were treated with scrambled siRNA or siRNA-targeting SRP40 mRNA. As shown in Fig. 5, a and b (cumulative data), CD3{zeta} mRNA was only marginally increased despite of the significantly decreased PP2A mRNA in the PP2A siRNA-mix transfected cells. In contrast, this treatment was sufficient to decrease the expression of FcR{gamma} mRNA (Fig. 5, a and b). Because CD3{zeta}-chain mRNA in SLE T cells is subject to enhanced degradation (7), which could offset the siRNA PP2A-induced increase in CD3{zeta} mRNA, we treated the siRNA PP2A-mix transfected SLE T cells with CHX, an inhibitor of protein synthesis commonly used to block new protein synthesis mediating the degradation of mRNA. As shown in Fig. 5, c and d (cumulative data), the presence of CHX increased significantly the expression of CD3{zeta} mRNA. To address whether the effect of PP2A siRNA-mix on FcR{gamma} mRNA levels is specific, we transfected T cells with each individual siRNA PP2A duplex separately. As shown in Fig. 5e, these constructs decreased the expression of PP2A but not those of GAPDH and CD3{epsilon}. The moderate decrease in PP2A expression was associated with a decrease in FcR{gamma} mRNA levels albeit to a lesser degree than the PP2A siRNA-mix, establishing further the specificity of this approach. Taken together, our findings indicate that increased expression of PP2A is at least in part responsible for the aberrant expression pattern of both CD3{zeta} and FcR{gamma} in SLE T cells, and suppression of the expression of PP2A can restore normal expression pattern for both CD3{zeta}- and FcR{gamma}-chains in SLE T cells.


Figure 5
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  		FIGURE 5. Silencing of PP2A in SLE T cells restores the expression of CD3{zeta} mRNA and suppresses the FcR{gamma} mRNA. a, SLE T cells were transfected with either control siRNA or PP2A siRNA-mix. The cells were lysed and mRNA was isolated as described in the Materials and Methods. The levels of both CD3{zeta} and FcR{gamma} were analyzed using RT-PCR. A representative experiment using T cells derived from two different SLE patients (L1 and L2) is shown here. b, The cumulative data from 10 SLE patients are depicted on this graph. c and d, T cells from patients with SLE cells were transfected as in a and subsequently were treated with CHX for 3.5 h before RNA was purified and analyzed. A representative experiment is shown in c while the cumulative data from four experiments are shown in d. e, Representative experiment of SLE T cells transfected with either control siRNA or one of the three different PP2A siRNA duplexes. The mRNA levels of PP2A, FcR{gamma} were analyzed as in a. GAPDH and CD3{epsilon} mRNA levels are used as controls.

 
Inhibition of PP2A with okadaic acid enhances the expression of CD3{zeta}, suppresses the expression of FcR{gamma}, and limits the CD3-mediated increased intracytoplasmic calcium response in SLE T cells

To determine whether inhibition of PP2A activity in SLE T cells with a pharmacologic agent would correct established aberrant cell function, we treated cells with okadaic acid (50 nM) for 6 h and the cell lysates were blotted with anti-CD3{zeta}, FcR{gamma}, and actin Abs. As seen in Fig. 6a, cells treated with okadaic acid expressed more CD3{zeta}-chain and less FcR{gamma}-chain compared with cells treated with DMSO. Because expression of FcR{gamma} in SLE T cells contributes to increased CD3-mediated free intracytoplasmic calcium response (1), we treated SLE T cells with okadaic acid or DMSO for 6 h and recorded free intracytoplasmic calcium response as the violet/blue emission of Indo-1 loaded cells. The representative tracing (four different patients were studied with similar tracings) of cells after treatment with anti-CD3 Ab, followed by a cross-linking Ab, shows that okadaic acid corrects the increased free intracytoplasmic calcium response (Fig. 6b). Treatment of SLE T cells with okadaic acid for 18 h yielded the same effect on the calcium response. Similarly treated normal cells with the same dose of okadaic acid for 6 or 18 h did not affect the calcium response (not shown).


Figure 6
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  		FIGURE 6. Okadaic acid enhances the expression of CD3{zeta}, suppresses the expression of FcR{gamma}, and limits the CD3-mediated increased intracytoplasmic calcium response in SLE T cells. a, SLE T cells were treated with okadaic acid (50 nM) for 6 h and then were lysed. Protein was isolated from the cellular lysates and was used for immunoblotting experiments with anti-CD3{zeta}, FcR{gamma}, and actin Abs. A representative experiment is shown here. b, Calcium influx in response to anti-CD3 stimulation was measured in SLE T cells treated with 50 nM okadaic acid for 6 h. The treated cells were incubated with Indo-1 for 30 min. The Indo-1 containing cells were treated with anti-CD3 Ab followed by a cross-linking Ab and the ratio of Violet to Blue emission of indo-1 was calculated as a function of time. A representative (n = 4) tracing is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The findings in the current study demonstrate that Elf-1 can function as a molecular valve that regulates the expression of CD3{zeta}- and FcR{gamma}-chains in an opposing manner. This finding echoes previous findings that Elf-1 can act either as a transcription activator or suppressor (26). Although it is not clear at the present time how Elf-1 can serve as a transcriptional enhancer or repressor, it has been shown to be part of a multi-molecule complex that regulates the expression of many genes active in the hemopoietic system (27). Whether Elf-1 is an activator or suppressor probably depends on the molecular partners it interacts with. Comparison of the CD3{zeta} and FcR{gamma} promoters revealed that although they both contain GGAA sites and bind Elf-1, the flanking sequences are not the same, allowing the binding of different transcription factors that may provide proper context for Elf-1 to behave as an enhancer or repressor. In addition, Elf-1 encodes more than 60 amino acids that can either be phosphorylated or O-linked glycosylated. We previously showed that the 98 kDa form exists in three forms that can be distinctly separated by both SDS and isoelectric focusing gels (6). It is possible that each of these three forms has distinct ability to interact with other factors involved in the regulation of transcription.

Increased PP2A expression in SLE T cells was previously shown to dephosphorylate phosphorylated CREB and limit is binding to the c-fos and IL-2 promoters leading to decreased expression of AP1 and IL-2 (18). The current study demonstrates that PP2A can dephosphorylate Elf-1 and alter the transcription CD3{zeta} and FcR{gamma} genes, the products of which are components of the CD3/TCR complex in SLE T cells (1). We show that modulation of the levels of PP2A in T cells either by overexpression or silencing results in changes of the expression of CD3{zeta} and FcR{gamma} mRNA in an opposite manner. We also demonstrate that inhibition of PP2A with okadaic acid suppresses the expression of FcR{gamma} while it increases the expression of CD3{zeta}-chain. Therefore, the PP2A/Elf-1 pathway determines to a point the composition of CD3 complex. Low levels of PP2A result in CD3{zeta} expression, whereas increased expression of PP2A, as is the case in SLE T cells, results in decreased levels of 98 kDa Elf-1, which limits the expression of CD3{zeta} and allows that expression of FcR{gamma}, which becomes a component of the CD3/TCR complex. The effect of PP2A is counterbalanced by PKC but, in SLE T cells, it probably remains unopposed because the expression and activity of PKC in SLE T cells has been reported decreased (18, 28).

We have observed that overexpression in primary T cells of Elf-1 T231V, as compared with the wild-type Elf-1, resulted in the decreased expression of the 98 kDa and simultaneously increased expression of the 80 kDa Elf-1. This data indicates that T231 is crucial for the conversion of Elf-1 from 80 to the 98 kDa. It is not clear though whether phosphorylation of T231 is sufficient for this conversion or phosphorylation at other sites and/or other posttranslational modifications are also involved. Phosphorylation has been shown to be coupled with other posttranslational modifications such as O-linked glycosylation (29), ubiquitination (30). Furthermore, proteins are commonly phosphorylated at multiple sites following a hierarchical order (31). It is possible that the dephosphorylation of T231 prevents other amino acids from being phosphorylated or modified by other posttranslational modalities and altogether these defects account for the decreased conversion of Elf-1 from 80 to 98 kDa.

We have shown that the conversion from the 98 to the 80 kDa form of Elf-1 is not the result of a Fas-related apoptotic event despite the fact that PP2A has been shown to trigger the onset of apoptosis (23) and SLE T cells are known to display enhanced spontaneous apoptosis (24). Instead, our current data and those reported earlier (6, 14) have established that this conversion is a phosphorylation-dependent event.

We found that additional treatment with CHX, an inhibitor of RNA destabilizing factors, is needed for the siPP2A-transfected SLE T cells to stabilize CD3{zeta} mRNA. This finding is in agreement with the well-established observation that CD3 {zeta} mRNA is unstable in SLE T cells (7, 8) and it appears that CHX-sensitive proteins are involved. Previous reports have shown that CHX increases the mRNA stability of genes including IL-6 (32), c-fos (33), and SOX-9 (34) through a mechanism known as superinduction.

Our findings establish that PP2A binds to and dephosphorylates Elf-1 at Thr-231 and that modulation of the levels of PP2A either by forced expression or silencing results in altered CD3{zeta} and FcR{gamma} promoter activity. We propose that the PP2A>Elf-1>CD3{zeta}/FcR{gamma} pathway is of particular significance in SLE T cells because the established increased PP2A activity in SLE T cells (18) results in a structurally variant CD3 complex that contains and handles TCR-initiated signaling not through the normally used {zeta}-chain but rather through the FcR{gamma}-chain. We previously demonstrated that silencing of PP2A in SLE T cells results in corrected production of IL-2 by limiting the dephosphorylation of the IL-2 transcriptional enhancer phosphorylated CREB (18). Our current data prove that enhanced IL-2 production after reduction of PP2A may come through corrected CD3{zeta}-chain expression since replenishment of CD3{zeta} in SLE T cells also results in increased production of IL-2 (35).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grants R01AI42269, R01AI49954, and R01AI068787. Back

2 Address correspondence and reprint requests to Drs. George C. Tsokos and Yuang-Taung Juang, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, E1CLS 957, Boston, MA 02115. E-mail addresses: gtsokos{at}bidmc.harvard.edu and yjuang{at}BIDMC.Harvard.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PKC, protein kinase C; AF, apoptotic fragment; CHX, cycloheximide; MS, mass spectrometry; PKN, protein kinase N; hnRNP, heterogeneous nuclear ribonucleoprotein; siRNA, small interfering RNA. Back

Received for publication November 15, 2007. Accepted for publication June 20, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Tsokos, G. C., M. P. Nambiar, K. Tenbrock, Y. T. Juang. 2003. Rewiring the T-cell: signaling defects and novel prospects for the treatment of SLE. Trends Immunol. 24: 259-263. [Medline]
  2. Fernandez, D., E. Bonilla, P. Phillips, A. Perl. 2006. Signaling abnormalities in systemic lupus erythematosus as potential drug targets. Endocr. Metab. Immune Disord. Drug Targets 6: 305-311. [Medline]
  3. Krishnan, S., B. Chowdhury, G. C. Tsokos. 2006. Autoimmunity in systemic lupus erythematosus: integrating genes and biology. Semin. Immunol. 18: 230-243. [Medline]
  4. Takeuchi, T., K. Tsuzaka, H. Kameda, K. Amano. 2005. Therapeutic targets of misguided T cells in systemic lupus erythematosus. Curr. Drug Targets Inflamm. Allergy 4: 295-298. [Medline]
  5. Liossis, S. N., D. Z. Ding, G. J. Dennis, G. C. Tsokos. 1998. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus: deficient expression of the T-cell receptor {zeta}-chain. J. Clin. Invest. 101: 1448-1457. [Medline]
  6. Juang, Y.-T., K. Tenbrock, M. P. Nambiar, M. F. Gourley, G. C. Tsokos. 2002. Defective production of the 98 kDa form of Elf-1 is responsible for the decreased expression of TCR {zeta}-chain in patients with systemic lupus erythematosus. J. Immunol. 169: 6048-6055. [Abstract/Free Full Text]
  7. Chowdhury, B., C. G. Tsokos, S. Krishnan, J. Robertson, C. U. Fisher, R. G. Warke, V. G. Warke, M. P. Nambiar, G. C. Tsokos. 2005. Decreased stability and translation of T cell receptor {zeta} mRNA with an alternatively spliced 3'-untranslated region contribute to {zeta}-chain down-regulation in patients with systemic lupus erythematosus. J. Biol. Chem. 280: 18959-18966. [Abstract/Free Full Text]
  8. Chowdhury, B., S. Krishnan, C. G. Tsokos, J. W. Robertson, C. U. Fisher, M. P. Nambiar, G. C. Tsokos. 2006. Stability and translation of TCR {zeta} mRNA are regulated by the adenosine-uridine-rich elements in splice-deleted 3' untranslated region of {zeta}-chain. J. Immunol. 177: 8248-8257. [Abstract/Free Full Text]
  9. Krishnan, S., J. G. Kiang, C. U. Fisher, M. P. Nambiar, H. T. Nguyen, V. C. Kyttaris, B. Chowdhury, V. Rus, G. C. Tsokos. 2005. Increased caspase-3 expression and activity contribute to reduced CD3{zeta} expression in systemic lupus erythematosus T cells. J. Immunol. 175: 3417-3423. [Abstract/Free Full Text]
  10. Enyedy, E. J., M. P. Nambiar, S. N. Liossis, G. Dennis, G. M. Kammer, G. C. Tsokos. 2001. Fc {epsilon} receptor type I {gamma} chain replaces the deficient T cell receptor {zeta}-chain in T cells of patients with systemic lupus erythematosus. Arthritis Rheum. 44: 1114-1121. [Medline]
  11. Krishnan, S., M. P. Nambiar, V. G. Warke, C. U. Fisher, J. Mitchell, N. Delaney, G. C. Tsokos. 2004. Alterations in lipid raft composition and dynamics contribute to abnormal T cell responses in systemic lupus erythematosus. J. Immunol. 172: 7821-7831. [Abstract/Free Full Text]
  12. Jury, E. C., P. S. Kabouridis, F. Flores-Borja, R. A. Mageed, D. A. Isenberg. 2004. Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus. J. Clin. Invest. 113: 1176-1187. [Medline]
  13. Leiden, J. M.. 1992. Transcriptional regulation during T cell development: the {alpha} TCR gene as a molecular model. Immunol. Today 13: 22-30. [Medline]
  14. Juang, Y.-T., E. Solomou, B. Rellahan, G. C. Tsokos. 2002. Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the T cell receptor {zeta}-chain. J. Immunol. 168: 2865-2871. [Abstract/Free Full Text]
  15. Rellahan, B. L., J. P. Jensen, A. M. Weissman. 1994. Transcriptional regulation of the T cell antigen receptor {zeta} subunit: identification of a tissue-restricted promoter. J. Exp. Med. 180: 1529-1534. [Abstract/Free Full Text]
  16. Juang, Y. T., L. Sumibcay, M. Tolnay, Y. Wang, V. C. Kyttaris, G. C. Tsokos. 2007. Elf-1 binds to the FcR{gamma} promoter and suppresses its activity. J. Immunol. 179: 4884-4889. [Abstract/Free Full Text]
  17. Nishiyama, C., M. Hasegawa, M. Nishiyama, K. Takahashi, Y. Akizawa, T. Yokota, K. Okumura, H. Ogawa, C. Ra. 2002. Regulation of human Fc{epsilon}RI {alpha}-chain gene expression by multiple transcription factors. J. Immunol. 168: 4546-4552. [Abstract/Free Full Text]
  18. Katsiari, C. G., V. C. Kyttaris, Y. T. Juang, G. C. Tsokos. 2005. Protein phosphatase 2A is a negative regulator of IL-2 production in patients with systemic lupus erythematosus. J. Clin. Invest. 115: 3193-3204. [Medline]
  19. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield, J. G. Schaller, N. Talal, R. J. Winchester. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25: 1271-1277. [Medline]
  20. Juang, Y. T., Y. Wang, E. E. Solomou, Y. Li, C. Mawrin, K. Tenbrock, V. C. Kyttaris, G. C. Tsokos. 2005. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J. Clin. Invest. 115: 996-1005. [Medline]
  21. Kyttaris, V. C., Y. T. Juang, K. Tenbrock, A. Weinstein, G. C. Tsokos. 2004. Cyclic adenosine 5'-monophosphate response element modulator is responsible for the decreased expression of c-fos and activator protein-1 binding in T cells from patients with systemic lupus erythematosus. J. Immunol. 173: 3557-3563. [Abstract/Free Full Text]
  22. John, S., R. Marais, R. Child, Y. Light, W. J. Leonard. 1996. Importance of low affinity Elf-1 sites in the regulation of lymphoid-specific inducible gene expression. J. Exp. Med. 183: 743-750. [Abstract/Free Full Text]
  23. Van, H. C., J. Goris. 2003. Phosphatases in apoptosis: to be or not to be, PP2A is in the heart of the question. Biochim. Biophys. Acta 1640: 97-104. [Medline]
  24. Emlen, W., J. Niebur, R. Kadera. 1994. Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J. Immunol. 152: 3685-3692. [Abstract]
  25. Takahashi, M., H. Mukai, M. Toshimori, M. Miyamoto, Y. Ono. 1998. Proteolytic activation of PKN by caspase-3 or related protease during apoptosis. Proc. Natl. Acad. Sci. USA 95: 11566-11571. [Abstract/Free Full Text]
  26. Honda, S., T. Kobayashi, K. Kajino, S. Urakami, M. Igawa, O. Hino. 2003. Ets protein Elf-1 bidirectionally suppresses transcriptional activities of the tumor suppressor Tsc2 gene and the repair-related Nth1 gene. Mol. Carcinog. 37: 122-129. [Medline]
  27. Lacorazza, H. D., S. D. Nimer. 2003. The emerging role of the myeloid Elf-1 like transcription factorin hematopoiesis. Blood Cells Mol. Dis. 31: 342-350. [Medline]
  28. Tada, Y., K. Nagasawa, Y. Yamauchi, H. Tsukamoto, Y. Niho. 1991. A defect in the protein kinase C system in T cells from patients with systemic lupus erythematosus. Clin. Immunol. Immunopathol. 60: 220-231. [Medline]
  29. Wang, Z., A. Pandey, G. W. Hart. 2007. Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. Mol. Cell. Proteomics 6: 1365-1379. [Abstract/Free Full Text]
  30. Musti, A. M., M. Treier, D. Bohmann. 1997. Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science 275: 400-402. [Abstract/Free Full Text]
  31. Tolnay, M., Y. T. Juang, G. C. Tsokos. 2002. Protein kinase A enhances, whereas glycogen synthase kinase-3 β inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion. Biochem. J. 363: 127-136. [Medline]
  32. Hershko, D. D., B. W. Robb, C. J. Wray, G. J. Luo, P. O. Hasselgren. 2004. Superinduction of IL-6 by cycloheximide is associated with mRNA stabilization and sustained activation of p38 map kinase and NF-{kappa}B in cultured caco-2 cells. J. Cell. Biochem. 91: 951-961. [Medline]
  33. Lee, J. K., J. S. Won, M. R. Choi, Y. H. Kim, H. W. Suh. 2001. Differential effects of forskolin and phobol 12-myristate-13-acetate on the c-fos and c-jun mRNA expression in rat C6 glioma cells. Mol. Cells 12: 11-16. [Medline]
  34. Tew, S. R., T. E. Hardingham. 2006. Regulation of SOX9 mRNA in human articular chondrocytes involving p38 MAPK activation and mRNA stabilization. J. Biol. Chem. 281: 39471-39479. [Abstract/Free Full Text]
  35. Nambiar, M. P., C. U. Fisher, V. G. Warke, S. Krishnan, J. P. Mitchell, N. Delaney, G. C. Tsokos. 2003. Reconstitution of deficient T cell receptor {zeta}-chain restores T cell signaling and augments T cell receptor/CD3-induced interleukin-2 production in patients with systemic lupus erythematosus. Arthritis Rheum. 48: 1948-1955. [Medline]




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