A Splice Variant of the TCR mRNA Lacking Exon 7 Leads to the Down-Regulation of TCR , the TCR/CD3 Complex, and IL-2 Production in Systemic
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免疫学杂志 2005年第6期
Abstract
The reduction or absence of TCR -chain () expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of mRNA that lacks the 36-bp exon 7 ( mRNA/exon 7(–)) and is accompanied by the down-regulation of protein in T cells from SLE patients. In this study, we show that EX7– mutants (MA5.8 cells deficient in protein that have been transfected with mRNA/exon 7(–)) exhibit a reduction in the expression of TCR/CD3 complex and protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab, compared with that in wild-type (WT) mutants (MA5.8 cells transfected with the WT mRNA). Furthermore, real-time PCR analyses demonstrated that mRNA/exon 7(–) in EX7– mutants was easily degraded compared with mRNA by the WT mutants. Pulse-chase experiment showed protein produced by this EX7– mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of mRNA/exon 7(–) might also be responsible for the reduced expression of the TCR/CD3 complex, including protein, in SLE T cells.
lntroduction
Systemic lupus erythematosus (SLE)3 is an autoimmune disease of unknown etiology (1, 2, 3). The disease is characterized by a large number of immunological abnormalities that appear to result from defects in T cells, B cells, and monocytes (4, 5). T cells are considered to be central to the pathogenesis of SLE because a dysfunction in their regulatory action may be responsible for the altered immune responses and overproduction of pathogenic autoantibodies (6). Abnormalities in peripheral blood T cells (PBTs) from SLE patients include T lymphocytopenia, low proliferative responses to lectin, anti-CD3 and anti-CD2 stimulation (7, 8), and a lower production of Th-1 type cytokines, such as IL-2 (9, 10, 11). Although the costimulatory pathway is up-regulated, the TCR/CD3 pathway appears to be down-regulated (12, 13). We and other groups have reported that a reduction in tyrosine phosphorylation and the diminished expression of TCR protein () play crucial roles in the pathogenesis of SLE (14, 15, 16). Clinically, a reduction in expression is not correlated with either the disease activity of SLE or the dose of prednisolone (17). In contrast, we and other groups have reported alterations in the mRNA open reading frame (ORF) or the 3'-untranslated region of mRNA in T cells from SLE patients (18, 19, 20, 21, 22). has crucial roles in signal transduction through the TCR/CD3 complex (23, 24, 25) and in the efficient transport of the assembled TCR complexes to the cell surface (26). is composed of three ITAM domains that are sufficient to couple chimeric receptors to early and late signaling events (24, 25, 27, 28, 29, 30, 31, 32). The mutation of tyrosines within the ITAM or the nonphosphorylated and monophosphorylated motif abrogates the signal transduction ability (32, 33), suggesting that these tyrosines and their phosphorylation have crucial roles in protein function. Furthermore, contains the GTP/GDP binding site, which is located immediately before the third ITAM (34). We have previously reported that 14 of 21 patients with SLE had decreased expression of in PBTs. And 2 of the 14 SLE patients were lacking of the exon 7 portion of mRNA (14). Exon 7 of mRNA spans the GTP/GDP binding site proximal to the third ITAM. Thus, the aberrant protein lacking exon 7 may result in skewed signal transduction, without efficient interaction with Shc and/or GTP/GDP. However, the involvement of mRNA with an altered ORF, specifically in exon 7, in the decreased expression of in SLE T cells has not been previously reported. To investigate the effect of mRNA lacking exon 7 on the intracellular and cell surface expression of the protein and TCR/CD3 complex, cDNA lacking exon 7 ( cDNA/exon 7(–)) or wild-type (WT) cDNA were transfected using a recombinant retrovirus system into murine T cell hybridomas (MA5.8) (35) deficient for expression. In this study, we report that not only the expression of protein, but also the expression of the TCR/CD3 complex, was down-regulated on the cell surface of the MA5.8 mutant cells expressing mRNA/exon 7(–) because of a reduction in mRNA stability.
Materials and Methods
Cell lines and inhibition of RNA synthesis
The MA5.8 cells (lacking endogenous expression) were kindly provided by Prof. Takashi Saito (Chiba University, Chiba, Japan), and the RetroPackPT67 (BD Clontech) was used as the dualtropic packaging cell line.
For experiments involving the inhibition of RNA synthesis, cell cultures were incubated with 4 μg/ml actinomycin D in the culture medium. Samples were collected for up to 48 h after drug exposure.
DNA transfection and infection
The DNA transfection and infection protocols have been previously described (36). Full-length WT human cDNA ( cDNA; +136+1627 (1492 bp)) and human cDNA lacking exon 7 ( cDNA/exon 7(–); +136+564, +600+1627 (1456 bp)) were amplified from the PBTs of a normal healthy control and an SLE patient (KS), respectively, using RT-PCR (Fig. 1). Each cDNA was ligated into a SalI-cut pDON-AI (Takara Bio), and each of the pDON-AI construct was sequenced in both directions to verify the nucleotide sequences. Purified pDON-AI containing the WT cDNA insert, the cDNA/exon 7(–) insert, or without any DNA insert were then transfected into 5.0 x 106 RetroPackPT67 cells using a cationic liposome kit (TransFast Transfection Reagent; Promega). Forty-eight hours after transfection, 10 ml of DMEM was added to the cells, and supernatant containing the same amount of the vector retrovirus was subsequently used to infect 1.0 x 107 MA5.8 cells in the presence of 8 μg/ml polybrene. After 24 h of incubation, G418 was added to select the infected cells, and 30 random colonies were selected and cultured together to construct MA5.8 mutants (WT, EX7–, and NEG, respectively).
FIGURE 1. RT-PCR of human cDNA lacking exon 7. A, WT human full-length cDNA ( cDNA) (1492 bp) and human cDNA lacking exon 7 ( cDNA/exon 7(–)) (1456 bp) were amplified by RT-PCR from PBTs obtained from a healthy normal control and an SLE patient (KS), respectively, and electrophoresed on an agarose gel (1.0%). B, mRNA/exon 7(–) is lacking the 36-bp region corresponding to exon 7. The arrows indicate the specific primers used to amplify the full-length cDNA.
RT-PCR
Whole mRNA was isolated from the cell samples and the mRNA was converted to whole cDNA by reverse transcriptase, according to a previously described method (18). Using 5 μl of the whole cDNA as the template, specific cDNA was amplified by PCR using specific primers and TaqDNA polymerase (Applied Biosystems). The PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles. The primers for amplifying the human full-length cDNA were arranged upstream of the ORF 5'-TCAGCCTCTGCCTCCCAGCCTCTTTCT-3' (+136 to +162) and 3' end of exon 8 5'-GCAGAGCAGAGAGCGTTTTCCATCCAT-3' (+1627 to +1601) of human mRNA (37). To amplify the murine CD3 cDNA (25), specific primers were arranged as follows: 5'-ATCCTGTGCCTCAGCCTCCTAGCTGT-3' (+25 to +50) and 5'-ATGGGCTCATAGTCTGGGTTGGGAA-3' (+494 to +488). As a positive control, murine -actin cDNA was amplified by PCR using the following primers: 5'-GGCCAACCGTGAAAAGATGA-3' (+419 to +438) and 5'-CACGCTCGGTCAGGATCTTC-3' (+669 to +650). PBTs were isolated from whole blood according to a previously described method (18).
Real-time PCR
The primers for human were located in two different exons of each gene to avoid the amplification of any contaminating genomic DNA (37): the forward primer was 5'-TGCTGGATCCCAAACTCTGC-3' (+254 to +272) (exon 3) and the reverse primer was 5'-CCCGGCCACGTCTCTTG-3' (+434 to +449) (exon 5). The TaqMan probe was 5'-ATGGAATCCTCTTCATCTATGGTGTCATTCTCAC-3' (+284 to +317) and had a fluorescent reporter dye (FAM) covalently linked to its 5'-end and a downstream quencher dye (TAMRA) linked to its 3'-end. The primers for murine CD3 cDNA were located in two different exons of each gene (38): the forward primer was 5'-GGACAGTGGCTACTACGTCTGCTA-3' (+307 to +330) (exon 4) and the reverse primer was 5'-TGATGATTATGGCTACTGCTGTCA-3' (+423 to +400) (exon 7). The TaqMan probe was 5'-CACCTCCACACAGTACTCACACACTCGA-3' (+400 to +373). In addition, the forward primer of 5'-GGCCAACCGTGAAAAGATGA-3' (+419 to +438) and the reverse primer of 5'-CACGCTCGGTCAGGATCTTC-3' (+669 to +650) for the murine -actin cDNA were designed in exon 3 and exon 4, respectively. The TaqMan probe for the murine -actin cDNA was 5'-TTTGAGACCTTCAACACCCCAGCCA-3' (+450 to +474).
Amplification and detection of specific products were performed using an ABI PRISM 7700 sequence detection system (Applied Biosystems) according to a previously described amplification protocol (36). To prepare a template DNA standard, a target DNA fragment was amplified by PCR and was fused into pCRII vector by using TA cloning kit (Invitrogen Life Technologies), amplified, and refined. The amount of standard DNA construct per well was adjusted to 10 pg and then serially diluted, yielding samples containing 10, 1, 10–1, 10–2, and 10–3 pg, which were then used to construct standard plots.
Cell surface biotinylation, immunoprecipitation (IP), and SDS-PAGE
Cells (1.0 x 107 cells/ml) were biotinylated in bicarbonate buffer to label the cell surface proteins using a previously described method (36). Cells were then lysed, and the cleared lysates were immunoprecipitated for 2 h at 4°C with 2 μg of mouse anti-human mAb (TIA-2) (Coulter Immunology), rabbit anti-mouse TCR mAb (Santa Cruz Biotechnology), rabbit anti-mouse TCR mAb (Santa Cruz Biotechnology), goat anti-mouse CD3 mAb (Santa Cruz Biotechnology), goat anti-mouse CD3 mAb (Santa Cruz Biotechnology), or goat anti-mouse CD3 mAb (Santa Cruz Biotechnology) bound to 15 μl of equilibrated protein G-Sepharose (Amersham Biosciences). The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE.
Western blot analysis
Cells were lysed with the lysis buffer and disrupted by sonication according to a previously described method (17). After centrifuging at 10,000 x g for 5 min, the supernatant was loaded on a 15% SDS-PAGE gel using a reducing method. The proteins were electrophoretically blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore), and the membranes were soaked at 37°C for 1 h in blocking agents (Blockace; Dainippon Pharmaceuticals). The blots were then probed with a mouse anti-human mAb (TIA-2) at 16°C for 1 h. TIA-2 was visualized using a peroxidase-conjugated anti-mouse IgG (Amersham Biosciences). Biotinylated proteins were detected using streptavidin-peroxidase (Southern Biotechnology Associates). After washing three times, the signals were detected by chemiluminescence-enhancing reagents (Amersham Biosciences). The treated membranes were visualized on ECL x-ray film (Amersham Biosciences). The density of the specific bands was quantified as index by scanning with a Scan Jet II (Hewlett Packard) and National Institutes of Health Image Software (version 1.56).
Flow cytometric analysis
The flow cytometric analysis procedure has been previously described (17). Briefly, MA5.8 or the transfectants were stained with a FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (Coulter Immunology) or an FITC-conjugated mouse anti-human mAb (TIA-2) (Coulter Immunology). The analysis was performed using a FACScan flow cytometer and consort-30 software. An FITC-conjugated Armenian hamster anti-mouse IgG (Coulter Immunology) and an FITC-conjugated mouse anti-human IgG (Coulter Immunology) were used as negative controls.
Pulse-chase experiment
Cells (3.0 x 107) were collected and washed twice with PBS. Cells were labeled in methionine-free RPMI 1640 medium (Sigma-Aldrich) containing 0.21 mCi of ProMix [35S]methionine in vitro cell labeling mix (Amersham Biosciences). Five hours later, the medium was removed, cells were washed three times with PBS and were chased with RPMI 1640 medium containing methionine for 0, 2, and 4 h. Cells were then washed three times with PBS and incubated for 15 min with 200 μl of lysis buffer. The cell lysates were centrifuged at 10,000 x g for 5 min. The supernatant was retained for protein assay using the BCA protein kit (Pierce). Equal amounts of proteins for each condition were then immunoprecipitated. Protein bands were detected by autoradiography using BAS5000 system (Fuji Photo Film).
Intracellular staining for , CD3, and endoplasmic reticulum (ER)
Cells were resuspended in PBS, laid on poly-L-lysine-coated slides for 15 min at 37°C, and fixed for 10 min with 4% paraformaldehyde, and permeabilized for 10 min at room temperature with washing buffer (HEPES-buffered PBS containing 0.1% Triton X-100). Cells were then stained with FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (BD Pharmingen) in PBS containing 1% BSA, a mouse anti-human mAb (6B10.2) (Santa Cruz Biotechnology), followed by Alexa Fluor 568 goat anti-mouse IgG (H+L) (Molecular Probes), and a rabbit polyclonal Ab to calreticulin (Novus Biologicals) followed by Alexa Fluor 647 F(ab')2 of goat anti-rabbit IgG (H+L) (Molecular Probes). The samples were mounted in DakoCytomation Fluorescent Mounting Medium (DakoCytomation) and were examined using a Leica TCS SP2 confocal microscope (Leica Camera).
Ab stimulation and IL-2 quantification
Anti-mouse CD3 mAb (KT3) (Coulter Immunology) was bound for 16 h to a 24-well, flat-bottom plate in PBS. The wells were rinsed with fresh PBS three times before the addition of the cells. Fifty microliters of transfected cells (1.0 x 106 cells/ml) were added to each well and incubated at 37°C in 7.0% CO2. Culture supernatants were harvested, and their aliquots were collected and frozen at 1, 2, and 3 days after stimulation. The harvested supernatants were assayed using a standard IL-2 assay. Recombinant murine IL-2 (BD Pharmingen) was used as a standard.
Statistical analysis
Statistical significance was calculated using the Student’s t test for unpaired data using Statview software (version 4.5; Abacus). A value of p < 0.05 was considered statistically significant.
Results
Western blot analysis of protein in MA5.8 mutants expressing mRNA/exon 7(–)
In a Western blot analysis using an anti-human mAb (TIA-2), the production of the exon 7-deleted (16 kDa) by the EX7– mutants was only 9.3% (index ratio, 1.8:19.3) of the WT (18 kDa) produced by the WT mutants (Fig. 2). Therefore, we concluded hat the expression of protein was reduced in mutants containing mRNA/exon 7(–).
FIGURE 2. Western blot analysis of human expressed by MA5.8 mutants. Cell lysates from MA5.8 and its mutants (NEG, WT, and EX7–) were electrophoresed on 15% SDS-polyacrylamide gels using a reducing method and blotted onto a PVDF membrane. The membranes were then incubated with a mouse anti-human mAb (TIA-2) followed by a peroxidase-conjugated anti-mouse IgG. After treatment with chemiluminescence-enhancing reagents, the membranes were visualized on ECL x-ray films, and the densities of the 18-kDa WT protein ([wild-type]) and the 17-kDa short-form exon 7-deleted protein ([exon 7(–)]) bands (indicated by the arrows) were quantified as index. *, Western blot of the MA5.8 mutants using a hamster anti-mouse -actin mAb.
Analysis of protein and TCR/CD3 complex on the cell surface of MA5.8 mutants expressing mRNA/exon 7(–) by FACS and IP
To investigate the expression of protein and the TCR/CD3 complex on the cell surface, MA5.8 and its mutants were stained with an FITC-conjugated anti-mouse CD3 mAb (145-2C11) or an FITC-conjugated anti-human mAb (TIA-2) and analyzed by flow cytometry (Fig. 3). This experiment was performed in triplicate. Although the expression of protein on the cell surface of EX7– mutants (mean channel fluorescence value, 34.48 ± 5.07 (mean ± SD)) was significantly (p < 0.05) up-regulated, compared with the MA5.8 cells (12.52 ± 1.50) and the NEG (13.78 ± 1.58) mutants, it was significantly (p < 0.05) lower than that of the WT mutants (112.19 ± 18.27). The CD3 expression level on the EX7– mutants (18.44 ± 2.50) was significantly (p < 0.05) higher than those on the MA5.8 cells (7.00 ± 1.00) and the NEG mutants (7.49 ± 1.25). However, it was significantly (p < 0.05) lower than that on the WT mutants (41.74 ± 6.51).
FIGURE 3. Flow cytometric analysis of the MA5.8 mutants. The surface expression of the TCR/CD3 complex and the protein on MA5.8 and its mutants (NEG, WT, and EX7–) was quantified as the mean channel fluorescence value using FITC-conjugated anti-mouse CD3 mAb (145-2C11) and FITC-conjugated anti-human mAb (TIA-2), respectively. Each experiment was performed in triplicate. The mean channel fluorescence value of the EX7– mutants was compared with that of MA5.8 and the other MA5.8 mutants (MA5.8, NEG, and WT), respectively. Statistical significance was calculated using Student’s t test. Bars show the mean ± SD.
To confirm the cell surface expression of protein and the TCR/CD3 complex, we subjected MA5.8 and its mutants to surface biotinylation, IP, SDS-PAGE analysis under nonreducing conditions, and Western blot analysis (Fig. 4). IP of the WT mutants with both hamster anti-mouse CD3 mAb (145-2C11) and mouse anti-human mAb (TIA-2) yielded the following surface labeled proteins: mature forms of the TCR heterodimers (m) (67–95 kDa), a homodimer (34 kDa) (indicated as the open arrowheads), CD3 (26 kDa), CD3 (28 kDa), and CD3 (23 kDa), indicating that TCR/CD3 complex is producing on the WT mutants. The protein bands of the TCR/CD3 components were confirmed by the Western blot and IP of a whole cell lysate of the WT mutants with Abs against each TCR/CD3 component. IP of the WT mutants with nonspecific hamster and mouse IgG did not yield any of these proteins (data not shown). Interestingly, IP of the EX7– mutants with anti-CD3 mAb or anti-human mAb demonstrated a reduced expression of the cell surface short homodimer (32 kDa) (indicated as the closed arrowheads) as well as a decreased expression of the cell surface CD3 and CD3 accompanied with the absence of CD3, indicating the down-regulation of TCR/CD3 complex on the cell surface of EX7– mutants.
FIGURE 4. A, Western blot and IP of TCR/CD3 components in WT mutant. The cell lysates from WT mutants were electrophoresed on 12% SDS-polyacrylamide gels using a nonreducing method and were blotted onto a PVDF membrane. The membranes were then incubated with a mouse anti-human mAb (TIA-2) followed by a peroxidase-conjugated anti-mouse IgG (lane W). The cell lysates from WT were immunoprecipitated using a goat anti-mouse CD3 mAb (145-2C11), rabbit anti-mouse TCR and TCR mAbs, and goat anti-mouse CD3 and CD3 mAbs bound to protein G-Sepharose. The pellets were electrophoresed on 12% SDS-polyacrylamide gels using a nonreducing method and were blotted onto a PVDF membrane. The membranes were then incubated with the Ab against each TCR/CD3 component, followed by a peroxidase-conjugated anti-goat or anti-rabbit IgG (lane IP). m, im, and im indicate the mature forms of the TCR -chains, the immature forms of TCR, and the immature forms of the TCR -chains, respectively. B, IP of cell surface TCR/CD3 complexes and protein in MA5.8 and its mutants. MA5.8 and its mutants (NEG, EX7–, and WT) were biotinylated and lysed in a cell lysis buffer. The cell lysates were immunoprecipitated using goat anti-mouse CD3 mAb (145-2C11) or mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE. Biotinylated proteins were blotted onto PVDF membranes and detected using streptavidin-peroxidase. After treatment with chemiluminescence-enhancing reagents, the membranes were visualized on ECL x-ray films. M, The protein molecular markers. The open and closed arrowheads indicate the protein bands of the WT (34-kDa) and the exon 7-deleted short-form (32-kDa) homodimer, respectively.
Confocal microscopic analysis of MA5.8 cells
To explore the intracellular localization of TCR/CD3 complex including in the MA5.8 mutants, WT and EX7– mutants were fixed, permeabilized, and stained with anti-, anti-CD3, and anti-calreticulin Abs, respectively (Fig. 5). The staining of the WT mutant with 6B10.2 (anti-, red in Fig. 5) and 145-2C11 (anti-CD3, green in Fig. 5) observed in a confocal microscopy showed the ring-shaped pattern (indicated as the white arrows), indicating the cell surface expression of TCR/CD3 complex including in the WT mutants. In contrast, the staining pattern of the EX7– mutants with 6B10.2 (anti-) or 145-2C11 (anti-CD3) was similar to that with anti-calreticulin Ab (indicated as the yellow arrows), indicating the staining of the ER detected by anti-calreticulin (blue in Fig. 5) as well as some weak dots on the cell surface (indicated as the yellow arrow), indicating low expression of the original TCR/CD3 complex of MA5.8 cells. From these observations, most of the TCR/CD3 complex in the EX7– mutants could be retained in the ER.
FIGURE 5. Intracellular staining for , CD3, and ER. The EX7– and WT mutants were laid on poly-L-lysine-coated slides, fixed with 4% paraformaldehyde, and permeabilized with washing buffer. Cells were then stained with FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (green color), a mouse anti-human mAb (6B10.2), followed by Alexa Fluor 568 goat anti-mouse IgG (H+L) (red color), and a rabbit polyclonal Ab to calreticulin followed by Alexa Fluor 647 F(ab')2 of goat anti-rabbit IgG (H+L) (blue color). The samples were mounted and were examined using a confocal microscope. White arrows show the ring-shaped pattern of the WT mutants with 6B10.2 and 145-2C11, whereas yellow arrows indicate the cytoplasmic pattern of the EX7– mutants with 6B10.2, 145-2C11, and anti-calreticulin Ab.
Decrease in IL-2 production in MA5.8 mutants expressing mRNA/exon 7(–)
To evaluate the effect of exon 7 deletion in mRNA, MA5.8 mutants were stimulated with anti-mouse CD3 mAb (145-2C11) (Fig. 6). IL-2 production in the WT, NEG, or MA5.8 mutants on day 1, 2, or 3 after stimulation was compared statistically with that in the EX7– mutants. IL-2 production in the EX7– mutants on day 1 (1.50 ± 2.12 ng/ml), day 2 (3.00 ± 4.24 ng/ml), and day 3 (4.50 ± 0.71 ng/ml) was significantly (p < 0.01) lower than that in the WT mutants on day 1 (54.00 ± 0.00 ng/ml), day 2 (81.50 ± 3.54 ng/ml), and day 3 (89.50 ± 2.12 ng/ml), respectively. Consequently, IL-2 production in the MA5.8 mutants expressing mRNA/exon 7(–) was lower than that in the MA5.8 mutants expressing WT mRNA.
FIGURE 6. IL-2 production in MA5.8 mutants after stimulation with anti-CD3 Ab. Anti-mouse CD3 mAb (145-2C11) was bound to a 96-well, flat-bottom plate. MA5.8 and its mutants (NEG, WT, and EX7–) were then added to the wells and incubated. The culture supernatants were collected 1, 2, and 3 days after stimulation and assayed using a standard IL-2 assay. Each experiment was performed in triplicate. IL-2 production in WT, NEG, or MA5.8 on day 1, 2, or 3 after stimulation was compared statistically with that in EX7– mutants by using Student’s t test. Bars show the mean ± SD.
mRNA stability assay
To evaluate the relationship between the reduction in protein expression and exon 7 deletion, we examined the stability of mRNA. WT and EX7– mutants were exposed to actinomycin D to inhibit transcription. The cell cultures were incubated with 4 μg/ml actinomycin D, and the cells were collected at 0, 6, 12, 24, and 48 h after drug exposure. One microgram of whole mRNA was isolated from the cell samples and converted to whole cDNA by reverse transcriptase. Using 5 μl of the whole cDNA as the template, , CD3, and -actin cDNA in the WT or the EX7– mutants were quantified by real-time PCR. To validate the real-time PCR, the standard curves for human , murine CD3, and murine -actin gene were constructed from the pCRII fused with cDNA (1492 bp), CD3 cDNA (470 bp), and -actin cDNA (250 bp), respectively. The critical threshold cycle (Ct) for , CD3, and -actin cDNA was inversely proportional (correlation coefficient of all three genes was 0.999) to the logarithm of the initial amount of the standard template DNA
(Fig. 7). Then the Ct for these cDNA were measured by the real-time PCR. These cDNA were measured by three separate experiments, and the statistical significance was calculated using the Student’s t test. As a result, demonstrated in Table I and Fig. 8A, the transcript of CD3 and -actin in the two MA5.8 mutants was gradually degraded over time after the treatment of actinomycin D. Decrease in the expression of these mRNA itself was not affected following the transfection of mRNA because there was no observed difference in the protein expression of and CD3 of the WT or EX7– mutants at 0, 24, and 48 h after the transfection by Western blot (data not shown). However, there seemed to be a difference in the kinetics of mRNA stability between the WT and EX7– mutants because the transcripts of -actin in the WT mutants were easily degraded compared with the EX7– mutants at 6 h after the actinomycin D treatment. The amount of or CD3 transcript was evaluated as the relative quantity against -actin cDNA (Table I and Fig. 8B). As a result, the relative amount of mRNA in the EX7– mutants (0.008 ± 0.001) was already significantly (p < 0.01) lower than that in the WT mutants (0.014 ± 0.002) before the actinomycin D treatment. And the relative amount of the WT mRNA in the WT mutants increased constantly, while that of the mRNA/exon 7(–) in the EX7– mutants did not change and was significantly (p < 0.01) lower than that of the WT transcript over time (Fig. 8Bi). In contrast, relative amount of CD3 mRNA in both MA5.8 mutants was almost the same at 0, 12, and 48 h after the actinomycin D treatment. From these observations, we can conclude that the mRNA/exon 7(–) in the EX7– mutants was less stable than the WT mRNA in the WT mutants. In contrast, the stability of the CD3 mRNA was similar in the EX7– mutants and the WT mutants (Fig. 8Bii).
FIGURE 7. Standard curves for quantifying the amount for , CD3, and -actin cDNA. Human ORF cDNA (1492 bp), murine CD3 cDNA (470 bp), and murine -actin cDNA (250 bp) were fused with pCRII vector, respectively. Real-time PCR was performed with the serial dilution (10, 1, 10–1, 10–2, and 10–3 pg) of the plasmid DNA as the template to estimate the critical Ct.
Table I. mRNA stability assay for the EX7– or WT mutants
FIGURE 8. Reduction in mRNA stability in the absence of the 36-bp portion of exon 7. MA5.8 mutants (WT and EX7–) were cultured and incubated with 4 μg/ml actinomycin D in the culture medium. Samples were collected at various time points, and the mRNA was subsequently extracted and converted to whole cDNA. A, Using 5 μl of the whole cDNA as the template, , CD3, and -actin cDNA in the WT (i) or the EX7– mutants (ii) were quantified by real-time PCR. Each experiment was performed in triplicate. B, The amount of (i) or CD3 (ii) mRNA expression was evaluated as the relative quantity against -actin cDNA in WT (?) or EX7– mutants (). Bars show the mean ± SD. *, p < 0.01; **, p < 0.001 of EX7– vs WT.
Pulse-chase experiment using MA5.8 mutants
To confirm whether decreased stability of the mRNA/exon 7(–) in the EX7– mutants could be related to the reduced amount of protein, we compared the production of in the WT and EX7– mutants. After labeling with [35S]methionine in methionine-free medium for 5 h, WT and EX7– mutants were chased with the complete medium for 0, 2, and 4 h. The cell lysate was then incubated with mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE followed by the autoradiography. As shown in Fig. 9, short homodimer (32 kDa) produced by the EX7– mutants (indicated as the closed arrowheads) was gradually decreased over time, whereas the expression level of the WT homodimer (34 kDa) produced by the WT mutants (indicated as the open arrowheads) did not change even after 4 h from chasing. From these observations, reduced mRNA/exon 7(–) due to altered transcription and/or mRNA stability could lead to decreased protein formation; as degradation of mRNA occurs, less protein is made.
FIGURE 9. Pulse-chase experiment of MA5.8 mutants. A total of 3.0 x 107 of MA5.8 mutants (WT and EX7–) was collected and washed twice with PBS. Cells were labeled in methionine-free RPMI 1640 medium containing ProMix [35S]methionine in vitro cell labeling mix. Five hours later, the medium was removed, and cells were chased with RPMI 1640 medium containing methionine for 0, 2, and 4 h. Cells were then washed with PBS and incubated for 15 min with lysis buffer. The cell lysate was then incubated with mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE. Protein bands were detected by autoradiography using BAS5000 system. M, The protein molecular markers of 14C-methylated proteins.
Discussion
We previously reported that mRNA/exon 7(–), a splice variant of mRNA, was detected in SLE T cells (14). The cytoplasmic domain of is sufficient for coupling to receptor-associated signal transduction (25). This cytoplasmic domain contains three ITAM domains that, when phosphorylated, serve as docking sites for signaling proteins like ZAP70, actin, PI3K, and Shc. In particular, PI3K preferentially binds to ITAM1 (39), whereas Shc and the actin cytoskeleton interact predominantly with ITAM3 (40, 41). The ITAM sequence alone is sufficient to couple chimeric receptors to early and late signaling events (27). Mutations at tyrosines within the ITAM or nonphosphorylated and monophosphorylated motifs abrogate the signal transduction ability (32), suggesting a crucial role for phosphorylation of tyrosines. The GTP/GDP binding site, a glycine-rich sequence of GxxxxGKGxxGxxxG, is a unique portion that has the capacity to bind GTP/GDP, but not GMP or ATP, and is responsible for the G protein signaling pathway (38).
The mRNA mutation of the exon 7 deletion found in SLE patients influenced the ITAM3 domain and the GTP/GDP binding site, two regions that are critical for signal transduction involving the protein. In this study, we attempted to confirm that a reduction in protein expression occurs in cells containing mRNA/exon 7(–) using a recombinant retrovirus system described by Bolliger et al. (42) and Weissman et al. (43).
The down-regulation and smaller size of the protein in EX7–, as confirmed by the Western blot analysis, suggests that the production of the smaller protein is down-regulated when it is translated from mRNA/exon 7(–) because of the exon 7 deletion. These observations were also confirmed by IP and FACS analyses. Reportedly, TCR/CD3 complexes cannot be expressed on the cell surface without binding to the homodimer in the cytoplasm (44, 45, 46). Therefore, in the MA5.8 mutants expressing mRNA/exon 7(–), the TCR/CD3 complex might be down-regulated on the cell surface because of the reduction in the expression of homodimer in IP using biotinylated cell surface proteins. Confocal microscopic analysis also revealed reduced cell surface expression of the protein and the retention of TCR/CD3 complex in the cytoplasm of the EX7– mutant. Other groups have shown that the expression of the detergent-insoluble membrane-associated form of was reduced in SLE T cells (47), supporting our results. The reduction in IL-2 production in the EX7– mutants revealed that the signal from the TCR was not transduced into the cytoplasm by anti-CD3 Ab stimulation in this MA5.8 mutant. The results obtained using the MA5.8 mutants in this study may explain the mechanism behind the reduction in protein expression in SLE T cells.
We examined the stability of mRNA to investigate the reduction in protein expression in the MA5.8 mutants expressing mRNA/exon 7(–). From our observations, mRNA/exon 7(–) in the EX7– mutants appeared to be less stable and more easily degraded than the WT mRNA in the WT mutants. In pulse-chase experiment, protein produced by the EX7– mutants was gradually decreased while the expression level of the protein by the WT mutants did not change over time. From these observations, unstable mRNA/exon 7(–) in the EX7– mutants could be related to the reduced amount of protein. Therefore, it is conceivable that a reduction in mRNA/exon 7(–) stability may lead to a reduction in the expression of homodimer, leading to the absence of TCR/CD3 complex expression on the cell surface. The lower basal levels of mRNA/exon 7(–) that were observed in the EX7– mutants before the treatment of actinomycin D, compared with that of the WT mRNA in WT, may also be caused by mRNA instability in the EX7– mutants. Moreover, other reasons for reduced protein including increased degradation by a ubiquitin-proteasome pathway, as shown by Tsokos and colleagues (47), could also contribute to decrease expression of in SLE T cells.
Several reports have been made on the relationship between exon deletion or exon skipping and the down-regulation of protein expression. Leitner et al. (48) reported that exon 3 skipping in (6R)-5,6,7,8-tetrahydro-L-biopterin mRNA in human monocytes/macrophages leads to the down-regulation of protein synthesis. Krummheuer et al. (49) also demonstrated that an alternative splicing pattern in HIV type 1 mRNA, resulting in the production of exon 2 as the leader exon, stimulates protein synthesis in HIV type 1 viruses. Exon-deletion or exon-skipping in mRNA has also been reported to be correlated with mRNA instability. Schwarze et al. (50) reported that frameshift mutations producing a premature termination codon in exon 6, 9, or 27 of type III procollagen mRNA leads to a reduction in both mRNA stability and protein synthesis. Kawamoto (51) demonstrated that the nucleotide region from +62 to +166, representing exon 1, of the nonmuscle myosin H chain-A gene could up-regulate its protein synthesis by affecting the pretranslational steps (transcriptional and mRNA stability). In contrast, parathyroid hormone-related protein mRNA containing exon 7 and exon 8 was reported to be stable, whereas that including exon 9 was unstable (52). From our observations in the present study, the deleted 36-bp portion representing exon 7 in mRNA appears to be critical for mRNA stability and may be correlated with the down-regulation of and the TCR/CD3 complex in SLE T cells. Actually, cell surface expression of TCR/CD3 complex including was reduced in T cells of the two SLE patients (patients HE and KS), who were lacking of exon 7 portion in their mRNA (14). As we have found mRNA/exon 7(–) in only 2 of 21 lupus patients, it will be important to determine how frequent this mutation occurs in the T cells of a large population and how it contributes to abnormal T cell functions, such as cytotoxicity. Previously, we reported that the expressions of and the TCR/CD3 complex were down-regulated on the cell surface of MA5.8 mutant cells
expressing mRNA containing an alternatively spliced 3'-untranslated region because of a reduction in mRNA stability (36). SLE T cells bear both the WT and the splice variant of mRNA (22). Thus, it would be interesting to compare the combined effect of transfection of both WT and the splice variant form of mRNA on IL-2 production and cell surface expression of TCR/CD3 complex to explore whether these splice variants of mRNA are more dominant. This project is now underway in our laboratory. Taken together, the reduced stability of mRNA produced by aberrant mRNA forms might be crucial to explaining the reduced expression of seen in SLE T cells.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Prof. Takashi Saito (Chiba University) for providing the MA5.8 cells.
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 Grants-in-Aid for Scientific Research (C), the Ministry of Education, Science, and Culture, Japan.
2 Address correspondence and reprint requests to Dr. Kensei Tsuzaka, Second Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Kamoda 1981, Kawagoe, Saitama 350-8550, Japan. E-mail address: kentsu{at}saitama-med.ac.jp
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; Ct, threshold cycle; ER, endoplasmic reticulum; IP, immunoprecipitation; ORF, open reading frame; PBT, peripheral blood T cell; PVDF, polyvinylidene difluoride; WT, wild type; , TCR protein.
Received for publication February 27, 2004. Accepted for publication January 12, 2005.
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The reduction or absence of TCR -chain () expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of mRNA that lacks the 36-bp exon 7 ( mRNA/exon 7(–)) and is accompanied by the down-regulation of protein in T cells from SLE patients. In this study, we show that EX7– mutants (MA5.8 cells deficient in protein that have been transfected with mRNA/exon 7(–)) exhibit a reduction in the expression of TCR/CD3 complex and protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab, compared with that in wild-type (WT) mutants (MA5.8 cells transfected with the WT mRNA). Furthermore, real-time PCR analyses demonstrated that mRNA/exon 7(–) in EX7– mutants was easily degraded compared with mRNA by the WT mutants. Pulse-chase experiment showed protein produced by this EX7– mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of mRNA/exon 7(–) might also be responsible for the reduced expression of the TCR/CD3 complex, including protein, in SLE T cells.
lntroduction
Systemic lupus erythematosus (SLE)3 is an autoimmune disease of unknown etiology (1, 2, 3). The disease is characterized by a large number of immunological abnormalities that appear to result from defects in T cells, B cells, and monocytes (4, 5). T cells are considered to be central to the pathogenesis of SLE because a dysfunction in their regulatory action may be responsible for the altered immune responses and overproduction of pathogenic autoantibodies (6). Abnormalities in peripheral blood T cells (PBTs) from SLE patients include T lymphocytopenia, low proliferative responses to lectin, anti-CD3 and anti-CD2 stimulation (7, 8), and a lower production of Th-1 type cytokines, such as IL-2 (9, 10, 11). Although the costimulatory pathway is up-regulated, the TCR/CD3 pathway appears to be down-regulated (12, 13). We and other groups have reported that a reduction in tyrosine phosphorylation and the diminished expression of TCR protein () play crucial roles in the pathogenesis of SLE (14, 15, 16). Clinically, a reduction in expression is not correlated with either the disease activity of SLE or the dose of prednisolone (17). In contrast, we and other groups have reported alterations in the mRNA open reading frame (ORF) or the 3'-untranslated region of mRNA in T cells from SLE patients (18, 19, 20, 21, 22). has crucial roles in signal transduction through the TCR/CD3 complex (23, 24, 25) and in the efficient transport of the assembled TCR complexes to the cell surface (26). is composed of three ITAM domains that are sufficient to couple chimeric receptors to early and late signaling events (24, 25, 27, 28, 29, 30, 31, 32). The mutation of tyrosines within the ITAM or the nonphosphorylated and monophosphorylated motif abrogates the signal transduction ability (32, 33), suggesting that these tyrosines and their phosphorylation have crucial roles in protein function. Furthermore, contains the GTP/GDP binding site, which is located immediately before the third ITAM (34). We have previously reported that 14 of 21 patients with SLE had decreased expression of in PBTs. And 2 of the 14 SLE patients were lacking of the exon 7 portion of mRNA (14). Exon 7 of mRNA spans the GTP/GDP binding site proximal to the third ITAM. Thus, the aberrant protein lacking exon 7 may result in skewed signal transduction, without efficient interaction with Shc and/or GTP/GDP. However, the involvement of mRNA with an altered ORF, specifically in exon 7, in the decreased expression of in SLE T cells has not been previously reported. To investigate the effect of mRNA lacking exon 7 on the intracellular and cell surface expression of the protein and TCR/CD3 complex, cDNA lacking exon 7 ( cDNA/exon 7(–)) or wild-type (WT) cDNA were transfected using a recombinant retrovirus system into murine T cell hybridomas (MA5.8) (35) deficient for expression. In this study, we report that not only the expression of protein, but also the expression of the TCR/CD3 complex, was down-regulated on the cell surface of the MA5.8 mutant cells expressing mRNA/exon 7(–) because of a reduction in mRNA stability.
Materials and Methods
Cell lines and inhibition of RNA synthesis
The MA5.8 cells (lacking endogenous expression) were kindly provided by Prof. Takashi Saito (Chiba University, Chiba, Japan), and the RetroPackPT67 (BD Clontech) was used as the dualtropic packaging cell line.
For experiments involving the inhibition of RNA synthesis, cell cultures were incubated with 4 μg/ml actinomycin D in the culture medium. Samples were collected for up to 48 h after drug exposure.
DNA transfection and infection
The DNA transfection and infection protocols have been previously described (36). Full-length WT human cDNA ( cDNA; +136+1627 (1492 bp)) and human cDNA lacking exon 7 ( cDNA/exon 7(–); +136+564, +600+1627 (1456 bp)) were amplified from the PBTs of a normal healthy control and an SLE patient (KS), respectively, using RT-PCR (Fig. 1). Each cDNA was ligated into a SalI-cut pDON-AI (Takara Bio), and each of the pDON-AI construct was sequenced in both directions to verify the nucleotide sequences. Purified pDON-AI containing the WT cDNA insert, the cDNA/exon 7(–) insert, or without any DNA insert were then transfected into 5.0 x 106 RetroPackPT67 cells using a cationic liposome kit (TransFast Transfection Reagent; Promega). Forty-eight hours after transfection, 10 ml of DMEM was added to the cells, and supernatant containing the same amount of the vector retrovirus was subsequently used to infect 1.0 x 107 MA5.8 cells in the presence of 8 μg/ml polybrene. After 24 h of incubation, G418 was added to select the infected cells, and 30 random colonies were selected and cultured together to construct MA5.8 mutants (WT, EX7–, and NEG, respectively).
FIGURE 1. RT-PCR of human cDNA lacking exon 7. A, WT human full-length cDNA ( cDNA) (1492 bp) and human cDNA lacking exon 7 ( cDNA/exon 7(–)) (1456 bp) were amplified by RT-PCR from PBTs obtained from a healthy normal control and an SLE patient (KS), respectively, and electrophoresed on an agarose gel (1.0%). B, mRNA/exon 7(–) is lacking the 36-bp region corresponding to exon 7. The arrows indicate the specific primers used to amplify the full-length cDNA.
RT-PCR
Whole mRNA was isolated from the cell samples and the mRNA was converted to whole cDNA by reverse transcriptase, according to a previously described method (18). Using 5 μl of the whole cDNA as the template, specific cDNA was amplified by PCR using specific primers and TaqDNA polymerase (Applied Biosystems). The PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles. The primers for amplifying the human full-length cDNA were arranged upstream of the ORF 5'-TCAGCCTCTGCCTCCCAGCCTCTTTCT-3' (+136 to +162) and 3' end of exon 8 5'-GCAGAGCAGAGAGCGTTTTCCATCCAT-3' (+1627 to +1601) of human mRNA (37). To amplify the murine CD3 cDNA (25), specific primers were arranged as follows: 5'-ATCCTGTGCCTCAGCCTCCTAGCTGT-3' (+25 to +50) and 5'-ATGGGCTCATAGTCTGGGTTGGGAA-3' (+494 to +488). As a positive control, murine -actin cDNA was amplified by PCR using the following primers: 5'-GGCCAACCGTGAAAAGATGA-3' (+419 to +438) and 5'-CACGCTCGGTCAGGATCTTC-3' (+669 to +650). PBTs were isolated from whole blood according to a previously described method (18).
Real-time PCR
The primers for human were located in two different exons of each gene to avoid the amplification of any contaminating genomic DNA (37): the forward primer was 5'-TGCTGGATCCCAAACTCTGC-3' (+254 to +272) (exon 3) and the reverse primer was 5'-CCCGGCCACGTCTCTTG-3' (+434 to +449) (exon 5). The TaqMan probe was 5'-ATGGAATCCTCTTCATCTATGGTGTCATTCTCAC-3' (+284 to +317) and had a fluorescent reporter dye (FAM) covalently linked to its 5'-end and a downstream quencher dye (TAMRA) linked to its 3'-end. The primers for murine CD3 cDNA were located in two different exons of each gene (38): the forward primer was 5'-GGACAGTGGCTACTACGTCTGCTA-3' (+307 to +330) (exon 4) and the reverse primer was 5'-TGATGATTATGGCTACTGCTGTCA-3' (+423 to +400) (exon 7). The TaqMan probe was 5'-CACCTCCACACAGTACTCACACACTCGA-3' (+400 to +373). In addition, the forward primer of 5'-GGCCAACCGTGAAAAGATGA-3' (+419 to +438) and the reverse primer of 5'-CACGCTCGGTCAGGATCTTC-3' (+669 to +650) for the murine -actin cDNA were designed in exon 3 and exon 4, respectively. The TaqMan probe for the murine -actin cDNA was 5'-TTTGAGACCTTCAACACCCCAGCCA-3' (+450 to +474).
Amplification and detection of specific products were performed using an ABI PRISM 7700 sequence detection system (Applied Biosystems) according to a previously described amplification protocol (36). To prepare a template DNA standard, a target DNA fragment was amplified by PCR and was fused into pCRII vector by using TA cloning kit (Invitrogen Life Technologies), amplified, and refined. The amount of standard DNA construct per well was adjusted to 10 pg and then serially diluted, yielding samples containing 10, 1, 10–1, 10–2, and 10–3 pg, which were then used to construct standard plots.
Cell surface biotinylation, immunoprecipitation (IP), and SDS-PAGE
Cells (1.0 x 107 cells/ml) were biotinylated in bicarbonate buffer to label the cell surface proteins using a previously described method (36). Cells were then lysed, and the cleared lysates were immunoprecipitated for 2 h at 4°C with 2 μg of mouse anti-human mAb (TIA-2) (Coulter Immunology), rabbit anti-mouse TCR mAb (Santa Cruz Biotechnology), rabbit anti-mouse TCR mAb (Santa Cruz Biotechnology), goat anti-mouse CD3 mAb (Santa Cruz Biotechnology), goat anti-mouse CD3 mAb (Santa Cruz Biotechnology), or goat anti-mouse CD3 mAb (Santa Cruz Biotechnology) bound to 15 μl of equilibrated protein G-Sepharose (Amersham Biosciences). The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE.
Western blot analysis
Cells were lysed with the lysis buffer and disrupted by sonication according to a previously described method (17). After centrifuging at 10,000 x g for 5 min, the supernatant was loaded on a 15% SDS-PAGE gel using a reducing method. The proteins were electrophoretically blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore), and the membranes were soaked at 37°C for 1 h in blocking agents (Blockace; Dainippon Pharmaceuticals). The blots were then probed with a mouse anti-human mAb (TIA-2) at 16°C for 1 h. TIA-2 was visualized using a peroxidase-conjugated anti-mouse IgG (Amersham Biosciences). Biotinylated proteins were detected using streptavidin-peroxidase (Southern Biotechnology Associates). After washing three times, the signals were detected by chemiluminescence-enhancing reagents (Amersham Biosciences). The treated membranes were visualized on ECL x-ray film (Amersham Biosciences). The density of the specific bands was quantified as index by scanning with a Scan Jet II (Hewlett Packard) and National Institutes of Health Image Software (version 1.56).
Flow cytometric analysis
The flow cytometric analysis procedure has been previously described (17). Briefly, MA5.8 or the transfectants were stained with a FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (Coulter Immunology) or an FITC-conjugated mouse anti-human mAb (TIA-2) (Coulter Immunology). The analysis was performed using a FACScan flow cytometer and consort-30 software. An FITC-conjugated Armenian hamster anti-mouse IgG (Coulter Immunology) and an FITC-conjugated mouse anti-human IgG (Coulter Immunology) were used as negative controls.
Pulse-chase experiment
Cells (3.0 x 107) were collected and washed twice with PBS. Cells were labeled in methionine-free RPMI 1640 medium (Sigma-Aldrich) containing 0.21 mCi of ProMix [35S]methionine in vitro cell labeling mix (Amersham Biosciences). Five hours later, the medium was removed, cells were washed three times with PBS and were chased with RPMI 1640 medium containing methionine for 0, 2, and 4 h. Cells were then washed three times with PBS and incubated for 15 min with 200 μl of lysis buffer. The cell lysates were centrifuged at 10,000 x g for 5 min. The supernatant was retained for protein assay using the BCA protein kit (Pierce). Equal amounts of proteins for each condition were then immunoprecipitated. Protein bands were detected by autoradiography using BAS5000 system (Fuji Photo Film).
Intracellular staining for , CD3, and endoplasmic reticulum (ER)
Cells were resuspended in PBS, laid on poly-L-lysine-coated slides for 15 min at 37°C, and fixed for 10 min with 4% paraformaldehyde, and permeabilized for 10 min at room temperature with washing buffer (HEPES-buffered PBS containing 0.1% Triton X-100). Cells were then stained with FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (BD Pharmingen) in PBS containing 1% BSA, a mouse anti-human mAb (6B10.2) (Santa Cruz Biotechnology), followed by Alexa Fluor 568 goat anti-mouse IgG (H+L) (Molecular Probes), and a rabbit polyclonal Ab to calreticulin (Novus Biologicals) followed by Alexa Fluor 647 F(ab')2 of goat anti-rabbit IgG (H+L) (Molecular Probes). The samples were mounted in DakoCytomation Fluorescent Mounting Medium (DakoCytomation) and were examined using a Leica TCS SP2 confocal microscope (Leica Camera).
Ab stimulation and IL-2 quantification
Anti-mouse CD3 mAb (KT3) (Coulter Immunology) was bound for 16 h to a 24-well, flat-bottom plate in PBS. The wells were rinsed with fresh PBS three times before the addition of the cells. Fifty microliters of transfected cells (1.0 x 106 cells/ml) were added to each well and incubated at 37°C in 7.0% CO2. Culture supernatants were harvested, and their aliquots were collected and frozen at 1, 2, and 3 days after stimulation. The harvested supernatants were assayed using a standard IL-2 assay. Recombinant murine IL-2 (BD Pharmingen) was used as a standard.
Statistical analysis
Statistical significance was calculated using the Student’s t test for unpaired data using Statview software (version 4.5; Abacus). A value of p < 0.05 was considered statistically significant.
Results
Western blot analysis of protein in MA5.8 mutants expressing mRNA/exon 7(–)
In a Western blot analysis using an anti-human mAb (TIA-2), the production of the exon 7-deleted (16 kDa) by the EX7– mutants was only 9.3% (index ratio, 1.8:19.3) of the WT (18 kDa) produced by the WT mutants (Fig. 2). Therefore, we concluded hat the expression of protein was reduced in mutants containing mRNA/exon 7(–).
FIGURE 2. Western blot analysis of human expressed by MA5.8 mutants. Cell lysates from MA5.8 and its mutants (NEG, WT, and EX7–) were electrophoresed on 15% SDS-polyacrylamide gels using a reducing method and blotted onto a PVDF membrane. The membranes were then incubated with a mouse anti-human mAb (TIA-2) followed by a peroxidase-conjugated anti-mouse IgG. After treatment with chemiluminescence-enhancing reagents, the membranes were visualized on ECL x-ray films, and the densities of the 18-kDa WT protein ([wild-type]) and the 17-kDa short-form exon 7-deleted protein ([exon 7(–)]) bands (indicated by the arrows) were quantified as index. *, Western blot of the MA5.8 mutants using a hamster anti-mouse -actin mAb.
Analysis of protein and TCR/CD3 complex on the cell surface of MA5.8 mutants expressing mRNA/exon 7(–) by FACS and IP
To investigate the expression of protein and the TCR/CD3 complex on the cell surface, MA5.8 and its mutants were stained with an FITC-conjugated anti-mouse CD3 mAb (145-2C11) or an FITC-conjugated anti-human mAb (TIA-2) and analyzed by flow cytometry (Fig. 3). This experiment was performed in triplicate. Although the expression of protein on the cell surface of EX7– mutants (mean channel fluorescence value, 34.48 ± 5.07 (mean ± SD)) was significantly (p < 0.05) up-regulated, compared with the MA5.8 cells (12.52 ± 1.50) and the NEG (13.78 ± 1.58) mutants, it was significantly (p < 0.05) lower than that of the WT mutants (112.19 ± 18.27). The CD3 expression level on the EX7– mutants (18.44 ± 2.50) was significantly (p < 0.05) higher than those on the MA5.8 cells (7.00 ± 1.00) and the NEG mutants (7.49 ± 1.25). However, it was significantly (p < 0.05) lower than that on the WT mutants (41.74 ± 6.51).
FIGURE 3. Flow cytometric analysis of the MA5.8 mutants. The surface expression of the TCR/CD3 complex and the protein on MA5.8 and its mutants (NEG, WT, and EX7–) was quantified as the mean channel fluorescence value using FITC-conjugated anti-mouse CD3 mAb (145-2C11) and FITC-conjugated anti-human mAb (TIA-2), respectively. Each experiment was performed in triplicate. The mean channel fluorescence value of the EX7– mutants was compared with that of MA5.8 and the other MA5.8 mutants (MA5.8, NEG, and WT), respectively. Statistical significance was calculated using Student’s t test. Bars show the mean ± SD.
To confirm the cell surface expression of protein and the TCR/CD3 complex, we subjected MA5.8 and its mutants to surface biotinylation, IP, SDS-PAGE analysis under nonreducing conditions, and Western blot analysis (Fig. 4). IP of the WT mutants with both hamster anti-mouse CD3 mAb (145-2C11) and mouse anti-human mAb (TIA-2) yielded the following surface labeled proteins: mature forms of the TCR heterodimers (m) (67–95 kDa), a homodimer (34 kDa) (indicated as the open arrowheads), CD3 (26 kDa), CD3 (28 kDa), and CD3 (23 kDa), indicating that TCR/CD3 complex is producing on the WT mutants. The protein bands of the TCR/CD3 components were confirmed by the Western blot and IP of a whole cell lysate of the WT mutants with Abs against each TCR/CD3 component. IP of the WT mutants with nonspecific hamster and mouse IgG did not yield any of these proteins (data not shown). Interestingly, IP of the EX7– mutants with anti-CD3 mAb or anti-human mAb demonstrated a reduced expression of the cell surface short homodimer (32 kDa) (indicated as the closed arrowheads) as well as a decreased expression of the cell surface CD3 and CD3 accompanied with the absence of CD3, indicating the down-regulation of TCR/CD3 complex on the cell surface of EX7– mutants.
FIGURE 4. A, Western blot and IP of TCR/CD3 components in WT mutant. The cell lysates from WT mutants were electrophoresed on 12% SDS-polyacrylamide gels using a nonreducing method and were blotted onto a PVDF membrane. The membranes were then incubated with a mouse anti-human mAb (TIA-2) followed by a peroxidase-conjugated anti-mouse IgG (lane W). The cell lysates from WT were immunoprecipitated using a goat anti-mouse CD3 mAb (145-2C11), rabbit anti-mouse TCR and TCR mAbs, and goat anti-mouse CD3 and CD3 mAbs bound to protein G-Sepharose. The pellets were electrophoresed on 12% SDS-polyacrylamide gels using a nonreducing method and were blotted onto a PVDF membrane. The membranes were then incubated with the Ab against each TCR/CD3 component, followed by a peroxidase-conjugated anti-goat or anti-rabbit IgG (lane IP). m, im, and im indicate the mature forms of the TCR -chains, the immature forms of TCR, and the immature forms of the TCR -chains, respectively. B, IP of cell surface TCR/CD3 complexes and protein in MA5.8 and its mutants. MA5.8 and its mutants (NEG, EX7–, and WT) were biotinylated and lysed in a cell lysis buffer. The cell lysates were immunoprecipitated using goat anti-mouse CD3 mAb (145-2C11) or mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE. Biotinylated proteins were blotted onto PVDF membranes and detected using streptavidin-peroxidase. After treatment with chemiluminescence-enhancing reagents, the membranes were visualized on ECL x-ray films. M, The protein molecular markers. The open and closed arrowheads indicate the protein bands of the WT (34-kDa) and the exon 7-deleted short-form (32-kDa) homodimer, respectively.
Confocal microscopic analysis of MA5.8 cells
To explore the intracellular localization of TCR/CD3 complex including in the MA5.8 mutants, WT and EX7– mutants were fixed, permeabilized, and stained with anti-, anti-CD3, and anti-calreticulin Abs, respectively (Fig. 5). The staining of the WT mutant with 6B10.2 (anti-, red in Fig. 5) and 145-2C11 (anti-CD3, green in Fig. 5) observed in a confocal microscopy showed the ring-shaped pattern (indicated as the white arrows), indicating the cell surface expression of TCR/CD3 complex including in the WT mutants. In contrast, the staining pattern of the EX7– mutants with 6B10.2 (anti-) or 145-2C11 (anti-CD3) was similar to that with anti-calreticulin Ab (indicated as the yellow arrows), indicating the staining of the ER detected by anti-calreticulin (blue in Fig. 5) as well as some weak dots on the cell surface (indicated as the yellow arrow), indicating low expression of the original TCR/CD3 complex of MA5.8 cells. From these observations, most of the TCR/CD3 complex in the EX7– mutants could be retained in the ER.
FIGURE 5. Intracellular staining for , CD3, and ER. The EX7– and WT mutants were laid on poly-L-lysine-coated slides, fixed with 4% paraformaldehyde, and permeabilized with washing buffer. Cells were then stained with FITC-conjugated Armenian hamster anti-mouse CD3 mAb (145-2C11) (green color), a mouse anti-human mAb (6B10.2), followed by Alexa Fluor 568 goat anti-mouse IgG (H+L) (red color), and a rabbit polyclonal Ab to calreticulin followed by Alexa Fluor 647 F(ab')2 of goat anti-rabbit IgG (H+L) (blue color). The samples were mounted and were examined using a confocal microscope. White arrows show the ring-shaped pattern of the WT mutants with 6B10.2 and 145-2C11, whereas yellow arrows indicate the cytoplasmic pattern of the EX7– mutants with 6B10.2, 145-2C11, and anti-calreticulin Ab.
Decrease in IL-2 production in MA5.8 mutants expressing mRNA/exon 7(–)
To evaluate the effect of exon 7 deletion in mRNA, MA5.8 mutants were stimulated with anti-mouse CD3 mAb (145-2C11) (Fig. 6). IL-2 production in the WT, NEG, or MA5.8 mutants on day 1, 2, or 3 after stimulation was compared statistically with that in the EX7– mutants. IL-2 production in the EX7– mutants on day 1 (1.50 ± 2.12 ng/ml), day 2 (3.00 ± 4.24 ng/ml), and day 3 (4.50 ± 0.71 ng/ml) was significantly (p < 0.01) lower than that in the WT mutants on day 1 (54.00 ± 0.00 ng/ml), day 2 (81.50 ± 3.54 ng/ml), and day 3 (89.50 ± 2.12 ng/ml), respectively. Consequently, IL-2 production in the MA5.8 mutants expressing mRNA/exon 7(–) was lower than that in the MA5.8 mutants expressing WT mRNA.
FIGURE 6. IL-2 production in MA5.8 mutants after stimulation with anti-CD3 Ab. Anti-mouse CD3 mAb (145-2C11) was bound to a 96-well, flat-bottom plate. MA5.8 and its mutants (NEG, WT, and EX7–) were then added to the wells and incubated. The culture supernatants were collected 1, 2, and 3 days after stimulation and assayed using a standard IL-2 assay. Each experiment was performed in triplicate. IL-2 production in WT, NEG, or MA5.8 on day 1, 2, or 3 after stimulation was compared statistically with that in EX7– mutants by using Student’s t test. Bars show the mean ± SD.
mRNA stability assay
To evaluate the relationship between the reduction in protein expression and exon 7 deletion, we examined the stability of mRNA. WT and EX7– mutants were exposed to actinomycin D to inhibit transcription. The cell cultures were incubated with 4 μg/ml actinomycin D, and the cells were collected at 0, 6, 12, 24, and 48 h after drug exposure. One microgram of whole mRNA was isolated from the cell samples and converted to whole cDNA by reverse transcriptase. Using 5 μl of the whole cDNA as the template, , CD3, and -actin cDNA in the WT or the EX7– mutants were quantified by real-time PCR. To validate the real-time PCR, the standard curves for human , murine CD3, and murine -actin gene were constructed from the pCRII fused with cDNA (1492 bp), CD3 cDNA (470 bp), and -actin cDNA (250 bp), respectively. The critical threshold cycle (Ct) for , CD3, and -actin cDNA was inversely proportional (correlation coefficient of all three genes was 0.999) to the logarithm of the initial amount of the standard template DNA
(Fig. 7). Then the Ct for these cDNA were measured by the real-time PCR. These cDNA were measured by three separate experiments, and the statistical significance was calculated using the Student’s t test. As a result, demonstrated in Table I and Fig. 8A, the transcript of CD3 and -actin in the two MA5.8 mutants was gradually degraded over time after the treatment of actinomycin D. Decrease in the expression of these mRNA itself was not affected following the transfection of mRNA because there was no observed difference in the protein expression of and CD3 of the WT or EX7– mutants at 0, 24, and 48 h after the transfection by Western blot (data not shown). However, there seemed to be a difference in the kinetics of mRNA stability between the WT and EX7– mutants because the transcripts of -actin in the WT mutants were easily degraded compared with the EX7– mutants at 6 h after the actinomycin D treatment. The amount of or CD3 transcript was evaluated as the relative quantity against -actin cDNA (Table I and Fig. 8B). As a result, the relative amount of mRNA in the EX7– mutants (0.008 ± 0.001) was already significantly (p < 0.01) lower than that in the WT mutants (0.014 ± 0.002) before the actinomycin D treatment. And the relative amount of the WT mRNA in the WT mutants increased constantly, while that of the mRNA/exon 7(–) in the EX7– mutants did not change and was significantly (p < 0.01) lower than that of the WT transcript over time (Fig. 8Bi). In contrast, relative amount of CD3 mRNA in both MA5.8 mutants was almost the same at 0, 12, and 48 h after the actinomycin D treatment. From these observations, we can conclude that the mRNA/exon 7(–) in the EX7– mutants was less stable than the WT mRNA in the WT mutants. In contrast, the stability of the CD3 mRNA was similar in the EX7– mutants and the WT mutants (Fig. 8Bii).
FIGURE 7. Standard curves for quantifying the amount for , CD3, and -actin cDNA. Human ORF cDNA (1492 bp), murine CD3 cDNA (470 bp), and murine -actin cDNA (250 bp) were fused with pCRII vector, respectively. Real-time PCR was performed with the serial dilution (10, 1, 10–1, 10–2, and 10–3 pg) of the plasmid DNA as the template to estimate the critical Ct.
Table I. mRNA stability assay for the EX7– or WT mutants
FIGURE 8. Reduction in mRNA stability in the absence of the 36-bp portion of exon 7. MA5.8 mutants (WT and EX7–) were cultured and incubated with 4 μg/ml actinomycin D in the culture medium. Samples were collected at various time points, and the mRNA was subsequently extracted and converted to whole cDNA. A, Using 5 μl of the whole cDNA as the template, , CD3, and -actin cDNA in the WT (i) or the EX7– mutants (ii) were quantified by real-time PCR. Each experiment was performed in triplicate. B, The amount of (i) or CD3 (ii) mRNA expression was evaluated as the relative quantity against -actin cDNA in WT (?) or EX7– mutants (). Bars show the mean ± SD. *, p < 0.01; **, p < 0.001 of EX7– vs WT.
Pulse-chase experiment using MA5.8 mutants
To confirm whether decreased stability of the mRNA/exon 7(–) in the EX7– mutants could be related to the reduced amount of protein, we compared the production of in the WT and EX7– mutants. After labeling with [35S]methionine in methionine-free medium for 5 h, WT and EX7– mutants were chased with the complete medium for 0, 2, and 4 h. The cell lysate was then incubated with mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE followed by the autoradiography. As shown in Fig. 9, short homodimer (32 kDa) produced by the EX7– mutants (indicated as the closed arrowheads) was gradually decreased over time, whereas the expression level of the WT homodimer (34 kDa) produced by the WT mutants (indicated as the open arrowheads) did not change even after 4 h from chasing. From these observations, reduced mRNA/exon 7(–) due to altered transcription and/or mRNA stability could lead to decreased protein formation; as degradation of mRNA occurs, less protein is made.
FIGURE 9. Pulse-chase experiment of MA5.8 mutants. A total of 3.0 x 107 of MA5.8 mutants (WT and EX7–) was collected and washed twice with PBS. Cells were labeled in methionine-free RPMI 1640 medium containing ProMix [35S]methionine in vitro cell labeling mix. Five hours later, the medium was removed, and cells were chased with RPMI 1640 medium containing methionine for 0, 2, and 4 h. Cells were then washed with PBS and incubated for 15 min with lysis buffer. The cell lysate was then incubated with mouse anti-human mAb (TIA-2) bound to protein G-Sepharose. The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE. Protein bands were detected by autoradiography using BAS5000 system. M, The protein molecular markers of 14C-methylated proteins.
Discussion
We previously reported that mRNA/exon 7(–), a splice variant of mRNA, was detected in SLE T cells (14). The cytoplasmic domain of is sufficient for coupling to receptor-associated signal transduction (25). This cytoplasmic domain contains three ITAM domains that, when phosphorylated, serve as docking sites for signaling proteins like ZAP70, actin, PI3K, and Shc. In particular, PI3K preferentially binds to ITAM1 (39), whereas Shc and the actin cytoskeleton interact predominantly with ITAM3 (40, 41). The ITAM sequence alone is sufficient to couple chimeric receptors to early and late signaling events (27). Mutations at tyrosines within the ITAM or nonphosphorylated and monophosphorylated motifs abrogate the signal transduction ability (32), suggesting a crucial role for phosphorylation of tyrosines. The GTP/GDP binding site, a glycine-rich sequence of GxxxxGKGxxGxxxG, is a unique portion that has the capacity to bind GTP/GDP, but not GMP or ATP, and is responsible for the G protein signaling pathway (38).
The mRNA mutation of the exon 7 deletion found in SLE patients influenced the ITAM3 domain and the GTP/GDP binding site, two regions that are critical for signal transduction involving the protein. In this study, we attempted to confirm that a reduction in protein expression occurs in cells containing mRNA/exon 7(–) using a recombinant retrovirus system described by Bolliger et al. (42) and Weissman et al. (43).
The down-regulation and smaller size of the protein in EX7–, as confirmed by the Western blot analysis, suggests that the production of the smaller protein is down-regulated when it is translated from mRNA/exon 7(–) because of the exon 7 deletion. These observations were also confirmed by IP and FACS analyses. Reportedly, TCR/CD3 complexes cannot be expressed on the cell surface without binding to the homodimer in the cytoplasm (44, 45, 46). Therefore, in the MA5.8 mutants expressing mRNA/exon 7(–), the TCR/CD3 complex might be down-regulated on the cell surface because of the reduction in the expression of homodimer in IP using biotinylated cell surface proteins. Confocal microscopic analysis also revealed reduced cell surface expression of the protein and the retention of TCR/CD3 complex in the cytoplasm of the EX7– mutant. Other groups have shown that the expression of the detergent-insoluble membrane-associated form of was reduced in SLE T cells (47), supporting our results. The reduction in IL-2 production in the EX7– mutants revealed that the signal from the TCR was not transduced into the cytoplasm by anti-CD3 Ab stimulation in this MA5.8 mutant. The results obtained using the MA5.8 mutants in this study may explain the mechanism behind the reduction in protein expression in SLE T cells.
We examined the stability of mRNA to investigate the reduction in protein expression in the MA5.8 mutants expressing mRNA/exon 7(–). From our observations, mRNA/exon 7(–) in the EX7– mutants appeared to be less stable and more easily degraded than the WT mRNA in the WT mutants. In pulse-chase experiment, protein produced by the EX7– mutants was gradually decreased while the expression level of the protein by the WT mutants did not change over time. From these observations, unstable mRNA/exon 7(–) in the EX7– mutants could be related to the reduced amount of protein. Therefore, it is conceivable that a reduction in mRNA/exon 7(–) stability may lead to a reduction in the expression of homodimer, leading to the absence of TCR/CD3 complex expression on the cell surface. The lower basal levels of mRNA/exon 7(–) that were observed in the EX7– mutants before the treatment of actinomycin D, compared with that of the WT mRNA in WT, may also be caused by mRNA instability in the EX7– mutants. Moreover, other reasons for reduced protein including increased degradation by a ubiquitin-proteasome pathway, as shown by Tsokos and colleagues (47), could also contribute to decrease expression of in SLE T cells.
Several reports have been made on the relationship between exon deletion or exon skipping and the down-regulation of protein expression. Leitner et al. (48) reported that exon 3 skipping in (6R)-5,6,7,8-tetrahydro-L-biopterin mRNA in human monocytes/macrophages leads to the down-regulation of protein synthesis. Krummheuer et al. (49) also demonstrated that an alternative splicing pattern in HIV type 1 mRNA, resulting in the production of exon 2 as the leader exon, stimulates protein synthesis in HIV type 1 viruses. Exon-deletion or exon-skipping in mRNA has also been reported to be correlated with mRNA instability. Schwarze et al. (50) reported that frameshift mutations producing a premature termination codon in exon 6, 9, or 27 of type III procollagen mRNA leads to a reduction in both mRNA stability and protein synthesis. Kawamoto (51) demonstrated that the nucleotide region from +62 to +166, representing exon 1, of the nonmuscle myosin H chain-A gene could up-regulate its protein synthesis by affecting the pretranslational steps (transcriptional and mRNA stability). In contrast, parathyroid hormone-related protein mRNA containing exon 7 and exon 8 was reported to be stable, whereas that including exon 9 was unstable (52). From our observations in the present study, the deleted 36-bp portion representing exon 7 in mRNA appears to be critical for mRNA stability and may be correlated with the down-regulation of and the TCR/CD3 complex in SLE T cells. Actually, cell surface expression of TCR/CD3 complex including was reduced in T cells of the two SLE patients (patients HE and KS), who were lacking of exon 7 portion in their mRNA (14). As we have found mRNA/exon 7(–) in only 2 of 21 lupus patients, it will be important to determine how frequent this mutation occurs in the T cells of a large population and how it contributes to abnormal T cell functions, such as cytotoxicity. Previously, we reported that the expressions of and the TCR/CD3 complex were down-regulated on the cell surface of MA5.8 mutant cells
expressing mRNA containing an alternatively spliced 3'-untranslated region because of a reduction in mRNA stability (36). SLE T cells bear both the WT and the splice variant of mRNA (22). Thus, it would be interesting to compare the combined effect of transfection of both WT and the splice variant form of mRNA on IL-2 production and cell surface expression of TCR/CD3 complex to explore whether these splice variants of mRNA are more dominant. This project is now underway in our laboratory. Taken together, the reduced stability of mRNA produced by aberrant mRNA forms might be crucial to explaining the reduced expression of seen in SLE T cells.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Prof. Takashi Saito (Chiba University) for providing the MA5.8 cells.
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 Grants-in-Aid for Scientific Research (C), the Ministry of Education, Science, and Culture, Japan.
2 Address correspondence and reprint requests to Dr. Kensei Tsuzaka, Second Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Kamoda 1981, Kawagoe, Saitama 350-8550, Japan. E-mail address: kentsu{at}saitama-med.ac.jp
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; Ct, threshold cycle; ER, endoplasmic reticulum; IP, immunoprecipitation; ORF, open reading frame; PBT, peripheral blood T cell; PVDF, polyvinylidene difluoride; WT, wild type; , TCR protein.
Received for publication February 27, 2004. Accepted for publication January 12, 2005.
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