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The Journal of Immunology, 2003, 171: 2496-2503.
Copyright © 2003 by The American Association of Immunologists

TCR{zeta} mRNA with an Alternatively Spliced 3'-Untranslated Region Detected in Systemic Lupus Erythematosus Patients Leads to the Down-Regulation of TCR{zeta} and TCR/CD3 Complex 1

Kensei Tsuzaka2, Izumi Fukuhara, Yumiko Setoyama, Keiko Yoshimoto, Katsuya Suzuki, Tohru Abe and Tsutomu Takeuchi

Second Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The reduction or absence of TCR {zeta}-chain ({zeta}) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of {zeta} mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; {zeta}mRNA/as-3'UTR) and a reduction in the expression of {zeta} mRNA containing the wild-type 3'UTR ({zeta}mRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in {zeta} protein that have been transfected with {zeta}mRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and {zeta} 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 3'UTR mutants (MA5.8 cells transfected with {zeta}mRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of {zeta}mRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of {zeta}mRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of {zeta}mRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including {zeta} protein, in SLE T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) 3 is a systemic autoimmune disease characterized by multiorgan involvement and the abundant production of a variety of autoantibodies of unknown etiology (1, 2, 3). 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 (4). Abnormalities in peripheral blood T cells (PBTs) from SLE patients include T lymphocytopenia; low proliferative responses to lectin, anti-CD3, and anti-CD2 stimulation (5, 6); and a lower production of Th1-type cytokines, such as IL-2 (7, 8, 9). Although the costimulatory pathway is up-regulated, the TCR/CD3 pathway appears to be down-regulated (10, 11). One of the earliest biochemical events occurring after TCR engagement is phosphorylation of the TCR{zeta} ({zeta}) protein. {zeta} protein exists in the TCR/CD3 complex primarily as a disulfide-linked homodimer. {zeta} protein is thought to be coupled to the signal transduction machinery of T cells, and a reduced or aberrant expression of {zeta} may cause T cell dysfunction, loss of tolerance, or the development of autoimmunity (12). We and other groups have reported that a reduction in tyrosine phosphorylation and the diminished expression of {zeta} protein play crucial roles in the pathogenesis of SLE (13, 14, 15). Clinically, a reduction in {zeta} expression is not correlated with either the disease activity of SLE or the dose of prednisolone (16). Aberrance of a {zeta} gene promotor has been related to the decreased {zeta} expression (17). On the other hand, we and other groups have reported that SLE patients exhibit alterations in the {zeta} mRNA open reading frame (ORF) or the 3'-untranslated region (3'UTR) of {zeta} mRNA (18, 19, 20, 21). Recently, we reported an aberrant form of the {zeta} mRNA 3'UTR; this form is alternatively spliced and 562 bp shorter than the wild-type (WT) 3'UTR. The presence of this aberrant form may be related to the reduced expression of {zeta} protein in SLE patients. In SLE T cells, {zeta} mRNA containing the alternatively spliced 3'UTR ({zeta} mRNA/as-3'UTR) is predominantly expressed, and the expression of {zeta} mRNA containing the WT 3'UTR ({zeta} mRNA/w-3'UTR) is reduced (22). Furthermore, the expression of {zeta} protein is positively correlated with that of {zeta} mRNA/w-3'UTR and negatively correlated with that of {zeta} mRNA/as-3'UTR (22). To investigate the effect of {zeta} mRNA/as-3'UTR on the intracellular and cell surface expression of the {zeta} protein and TCR/CD3 complex, {zeta} cDNA containing the alternatively spliced 3'UTR ({zeta} cDNA/as-3'UTR) or {zeta} cDNA containing the WT 3'UTR ({zeta} cDNA/w-3'UTR) were transfected using a recombinant retrovirus system into murine T cell hybridomas (MA5.8) (23) deficient for {zeta} expression. Here we report that not only {zeta} protein but also TCR/CD3 complex expression were down-regulated on the cell surface of the MA5.8 mutant cells expressing {zeta} mRNA/as-3'UTR; IL-2 production was also reduced in these cells because of the reduction in {zeta} mRNA stability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and inhibition of RNA synthesis

The MA5.8 cells (lacks endogenous {zeta} expression) were provided by Dr. T. Saito (Chiba University, Chiba, Japan) and were grown in RPMI medium supplemented with 10% FCS. The NIH-3T3 cell line was purchased from American Type Culture Collection (Manassas, VA). The dual-tropic packaging cell line RetroPack PT67 (Clontech Laboratories, Palo Alto, CA) and NIH-3T3 cells were grown in DMEM supplemented with 10% FCS.

For experiments involving the inhibition of RNA synthesis, cell cultures were incubated with 4 µg/ml of actinomycin D in the culture medium. Samples were collected for up to 48 h after drug exposure.

RT-PCR

One microgram of whole mRNA was isolated from the cell samples using the Quick Prep MicromRNA Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ). The mRNA was then converted to whole cDNA by reverse transcriptase using the RETROscript First Strand Synthesis Kit (Ambion, Austin, TX). Using 5 µl of the whole cDNA as the template, specific cDNA was amplified by PCR using specific primers and Taq DNA polymerase (PerkinElmer, PE Applied Biosystems, Tokyo, Japan). 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 {zeta} cDNA, including the 3'UTR were arranged upstream of the open reading frame (5'-TCAGCCTCTGCCTCCCAGCCTCTTTCT-3', +136 to +162) and 3' end of exon 8 (5'-GCAGAGCAGAGAGCGTTTTCCATCCAT-3', +1627 to +1601) of human {zeta} mRNA (24). The primers for amplifying the ORF of human {zeta} mRNA were arranged upstream and downstream of the ORF (5'-TCAGCCTCTGCCTCCCAGCCTCTTTCT-3' (+136 to +162) and 5'-ATGCTTCATCCTGTGTCTCATAATCTG-3' (+739 to +713), respectively) (24). To amplify the murine CD3{epsilon} cDNA (25), specific primers were arranged as follows: 5'-ATCCTGTGCCTCAGCCTCCTAGCTGT-3' (+25 to +50) and 5'-ATGGGCTCATAGTCTGGGTTGGGAA-3' (+494 to +88). As a positive control, mouse and human G3PDH cDNA (983 bp of the expected size, each) were amplified by PCR using primers specific for mouse G3PDH and human G3PDH, respectively (Clontech Laboratories). PBTs were isolated from whole blood according to a previously described method (13).

Real-time PCR

For amplifying human {zeta}, murine CD3{epsilon}, and human G3PDH cDNA by real-time PCR, oligonucleotide primers and TaqMan probes were designed from the GenBank databases using Primer Express version 1.0 (PerkinElmer, PE Applied Biosystems) as described previously (26). The primers for human {zeta} were located in two different exons of each gene to avoid amplification of any contaminating genomic DNA (24): the forward primer was 5'-GCGGAGGCCTACAGTGAGATT-3' (+429 to +449; exon 6), and the reverse primer was 5'-GCATGTGAAGGGCGTCGTA-3' (+547 to +528; exon 7). The TaqMan probe was 5'-CACGATGGCCTTTACCAGGGTCTCAGT-3' (+483 to +509) 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{epsilon} cDNA were located in two different exons of each gene (25): 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, primers and the TaqMan probe for murine G3PDH cDNA were purchased from PerkinElmer, PE Applied Biosystems, and served as an internal control.

For amplifying the genomic DNA by real-time PCR, oligonucleotide primers and TaqMan probes for amplifying pDON-AI (Takara Bio, Otsu, Japan) DNA (27) integrated in the genomic DNA were designed as described above. The forward primer was 5'-ATGGATTGCACGCAGGTTCT-3' (+1624 to +1643), and the reverse primer was 5'-CATCAGAGCAGCCGATTGTCT-3' (+1710 to +1690). The TaqMan probe was 5'-TGTGCCCAGTCATAGCCGAATAGCCT-3' (+1662 to +1687). In addition, primers and the TaqMan probe for murine {beta}-actin genomic DNA were purchased from PerkinElmer, PE Applied Biosystems, and served as an internal control. Amplification and detection of specific products were conducted in an ABI PRISM 7700 sequence detection system (PerkinElmer, PE Applied Biosystems) using an amplification protocol consisting of one cycle at 95°C for 10 min, 50 cycles at 95°C for 15 s, and one cycle at 60°C for 1 min.

To prepare template DNA standards, a target DNA fragment was amplified by PCR, fused into pCRII vector using the TA Cloning Kit (Invitrogen, Carlsbad, CA), amplified, and refined. The amount of construct per well was adjusted to 10 pg and then serially diluted, yielding samples containing 1, 10-1, 10-2, 10-3, and 10-4 pg, which were used to construct standard plots.

PCR amplification of genomic DNA

Whole genomic DNA was isolated from the cells using the Wizard Genomic DNA isolation kit (Promega, Madison, WI). For amplifying pDON-AI (Takara Bio) DNA (fragment size, 494 bp) (27), primers were designed as the following: the forward primer was 5'-TAACTCCGCCCAGTTCCGCCCATT-3' (+1405 to +1428), and the reverse primer was 5'-GTAGCCGGATCAAGCGTATGCAGC-3' (+1876 to +1899).

DNA transfection and infection

The strategy used for DNA transfection and infection has been outlined in previous reports (28, 29). Full-length {zeta}cDNA was ligated into a SalI cut pDON-AI (Takara Bio). An amount equal to 10 µg of purified pDON-AI was then used to transfect 5.0 x 106 RetroPack PT67 cells using a cationic liposome kit (TransFast Transfection Reagent; Promega). After 48 h of transfection, the cells were provided with 10 ml of DMEM, and supernatant containing the same amount of the vector retrovirus was subsequently used to infect 1.0 x 107 NIH-3T3 or MA5.8 cells in the presence of 8 µg/ml of polybrene. After 24 h of incubation, G418 was added to select the infected cells, and 30 colonies obtained randomly were cultured together in RPMI.

Cell surface biotinylation, immunoprecipitation, and SDS-PAGE

Cells (1.0 x 107 cells/ml) were biotinylated in bicarbonate buffer (20 mM NaHCO3 and 150 mM NaCl) with EZ-link sulfo-NHS biotin (100 µg/ml; Pierce, Rockford, IL), as described by Bolliger et al. (29). Cells were then lysed in a cell lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% digitonin), and cleared lysates were immunoprecipitated for 2 h at 4°C with 2 µg of mouse anti-human {zeta} mAb (TIA-2; Coulter, Hialeah, FL), rabbit anti-mouse TCR{alpha} mAb (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse TCR{beta} mAb (Santa Cruz Biotechnology), goat anti-mouse CD3{epsilon} mAb (Santa Cruz Biotechnology), goat anti-mouse CD3{gamma} mAb (Santa Cruz Biotechnology), or goat anti-mouse CD3{delta} mAb (Santa Cruz Biotechnology) bound to 15 µl of equilibrated protein G-Sepharose (Amersham Pharmacia Biotech) per 5.0 x 107 cells. The resulting pellets were resuspended in a nonreducing sample buffer and loaded on a 12% SDS-PAGE.

Western blot analysis

Cells were lysed with 1 ml of lysis buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) at 4°C for 15 min and were disrupted by sonication. After centrifuging at 10,000 x g for 5 min, the supernatant was loaded onto a 15% SDS-PAGE gel. The proteins were electrophoretically blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), and the membranes were soaked at 37°C for 1 h in blocking agents (Blockace; Dainippon Pharmaceuticals, Tokyo, Japan). The blots were then probed with a mouse anti-human {zeta} mAb (TIA-2) at 16°C for 1 h. TIA-2 was visualized using peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech). Biotinylated proteins were detected using streptavidin-peroxidase (Southern Biotechnology Associates, Birmingham, AL). After washing three times, the signals were detected by chemiluminescence-enhancing reagents (Amersham Pharmacia Biotech). The treated membranes were visualized on ECL x-ray film (Amersham Pharmacia Biotech). The density of the specific bands was quantified by scanning with a Scan Jet II (HewlettPackard) and National Institutes of Health Image software (version 1.56).

Flow cytometric analysis

The methods were described by Pang et al. (16). Briefly, MA5.8 or the transfectants were stained with an FITC-conjugated Armenian hamster anti-mouse CD3{epsilon} mAb (145-2C11; Coulter) or an FITC-conjugated mouse anti-human {zeta} mAb (TIA-2; Coulter). The analysis was performed using a FACScan flow cytometer and Consort-30 software. An FITC-conjugated Armenian hamster anti-mouse IgG (Coulter) and an FITC-conjugated mouse anti-human IgG (Coulter) were used as the negative controls.

Ab stimulation and IL-2 quantification

Anti-mouse CD3 mAb (KT3; Coulter) was bound for 16 h to a 24-well, flat-bottom plate in PBS. The wells were rinsed with fresh PBS three times before 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 1, 2, 3, and 6 days after stimulation. The harvested supernatants were assayed using a standard IL-2 assay. Recombinant murine IL-2 (BD PharMingen, San Diego, CA) was used as a standard.

Statistical analysis

Statistical significance was calculated by Student’s t test for unpaired data using StatView software (version 4.5; Abacus, Berkeley, CA). A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduced expression of {zeta} protein in MA5.8 mutants expressing {zeta} mRNA/as-3'UTR

Full-length human {zeta} cDNA containing the WT 3'UTR ({zeta}cDNA/w-3'UTR; +136 to +1627 (1492 bp)) and full-length human {zeta} cDNA containing the alternatively spliced 3'UTR ({zeta}cDNA/as-3'UTR; +136 to +770, +1333 to +1627 (930 bp)), which has a 562-bp deletion in the 3'UTR, were amplified from PBTs of a normal healthy control and an SLE patient (TaS), respectively, by RT-PCR (Fig. 1). They were cloned into pDON-AI at the SalI sites. Purified pDON-AI containing the {zeta}cDNA/w-3'UTR insert, the {zeta}cDNA/as-3'UTR insert, or no DNA insert were then transfected into RetroPack PT67 cells. MA5.8 and NIH-3T3 cells were subsequently infected with the supernatants of the transfected RetroPack PT67 cells using medium containing G418, and 30 colonies obtained randomly were cultured together to construct MA5.8 mutants (WT3'UTR, AS3'UTR, and NEG, respectively) and NIH-3T3 mutants (3T3-WT3'UTR, 3T3-AS3'UTR, and 3T3-NEG, respectively).



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  		FIGURE 1. RT-PCR of human full-length {zeta} cDNA. A, Human full-length {zeta} cDNA containing WT 3'UTR ({zeta}cDNA/w-3'UTR; 1492 bp) and human full-length {zeta} cDNA containing the alternatively spliced 3'UTR ({zeta} cDNA/as-3'UTR; 930 bp) were amplified by RT-PCR from PBTs obtained from a healthy normal control and an SLE patient (TaS), respectively, and were electrophoresed on an agarose gel (1.0%). B, {zeta} mRNA/as-3'UTR is missing a 562-bp segment in the 3'UTR of the {zeta} mRNA. The arrows indicate the specific primers used to amplify the full-length {zeta}cDNA.

 
To confirm the amount of the pDON-AI DNA that was integrated into the MA5.8 mutants, pDON-AI DNA (494 bp) was amplified from MA5.8, NEG, WT3'UTR, and AS3'UTR by PCR and was analyzed in the agarose-gel electrophoresis. Also, the amount of pDON-AI DNA was quantified by real-time PCR (Fig. 2). As a result, the pDON-AI DNA bands (494 bp) from the NEG, WT3'UTR, and AS3'UTR were almost the same, while that from MA5.8 cells was not observed. To validate the real-time PCR, the standard curves for pDON-AI DNA and the murine {beta}-actin gene were constructed from the pCRII fused with pDON-AI DNA and the murine {beta}-actin genomic DNA, respectively. The critical threshold cycles (Ct) for pDON-AI and murine {beta}-actin genomic DNA were inversely proportional (correlation coefficient, 0.9945 and 0.9988, respectively) to the logarithm of the initial amount of the standard template DNA. Then, the Ct for pDON-AI and murine {beta}-actin genomic DNA in MA5.8, NEG, WT3'UTR, and AS3'UTR were measured in triplicate by real-time PCR, and the amounts of both DNA were determined from the standard curves. Statistical significance was calculated using Student’s t test. As a result, the ratios of pDON-AI DNA/{beta}-actin gene DNA among NEG (0.245 ± 0.023), WT3'UTR (0.205 ± 0.016), and AS3'UTR (0.254 ± 0.038) were similar (not significant), but were significantly (p < 0.001) higher than that in MA5.8 cells (5.18 x 10-3 ± 0.11 x 10-3). Therefore, the amounts of integrated retroviral gene were almost the same among these MA5.8 mutants.



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  		FIGURE 2. The pDON-AI gene integrated in the MA5.8 mutants. The pDON-AI fragment DNA (494 bp) was amplified by PCR from whole genomic DNA obtained from MA5.8 cells and MA5.8 mutants, followed by agarose gel electrophoresis. The standard curves for the real-time PCR of pDON-AI DNA and the murine {beta}-actin gene were constructed from pCRII fused with pDON-AI DNA (86 bp) and murine {beta}-actin genomic DNA (112 bp), respectively. The critical Ct values for pDON-AI DNA and the murine {beta}-actin genomic DNA were inversely proportional (correlation coefficient, 0.9945 and 0.9988, respectively) to the logarithm of the initial amount of the standard template DNA. The amount of pDON-AI DNA was evaluated as the ratio of pDON-AI DNA/{beta}-actin gene DNA.

 
In a Western blot analysis using an anti-human {zeta} mAb (TIA-2), the production of {zeta} monomer (18-kDa protein detected by a reducing method) and {zeta} homodimer (34-kDa protein detected by a nonreducing method) by the AS3'UTR mutants was 20–47% lower than that by the WT3'UTR mutants (Fig. 3). In the NIH-3T3 mutants, the production of both {zeta} monomer and homodimer in the 3T3-AS3'UTR mutants was 5–23% lower than that in the 3T3-WT3'UTR mutants. Therefore, we concluded that the expression of {zeta} protein was reduced in mutants containing {zeta} mRNA/as-3'UTR.



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  		FIGURE 3. Western blot analysis of human {zeta} protein expressed by MA5.8 and NIH-3T3 mutants. Cell lysates from MA5.8 and its mutants (NEG, AS3'UTR, WT3'UTR) as well as NIH-3T3 and its mutants (3T3-NEG, 3T3-AS3'UTR, 3T3-WT3'UTR) were electrophoresed on 15% SDS-polyacrylamide gels using a reducing or nonreducing method and were blotted onto a PVDF membrane. The membranes were then incubated with a mouse anti-human {zeta} 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 {zeta} monomer and 34-kDa {zeta} homodimer protein bands (indicated by the arrows) were quantified as relative OD values. The asterisks indicate the Western blot of the MA5.8 or NIH-3T3 mutants using a hamster anti-mouse {beta}-actin mAb.

 
Absence of {zeta} protein and TCR/CD3 complex expression on the cell surface of MA5.8 mutants expressing {zeta}mRNA/as-3'UTR

To investigate the expression of {zeta} protein and the TCR/CD3 complex on the cell surface, MA5.8 and its mutants were stained with an FITC-conjugated anti-mouse CD3{epsilon} mAb (145-2C11; black profiles in Fig. 4A) or an FITC-conjugated anti-human {zeta} mAb (TIA-2; black profiles in Fig. 4B) and analyzed by flow cytometry (Fig. 4). Although the expression of {zeta} protein on the cell surface of AS3'UTR mutants (mean channel fluorescence, 24.49) seemed to be up-regulated compared with that on MA5.8 (6.17) and NEG (6.47) cells, it was much lower than that on the WT3'UTR mutants (65.50). CD3{epsilon} was weakly positive on the cell surface of MA5.8 (20.25) and NEG (25.94) cells. Interestingly, the expression of CD3{epsilon} on the cell surface of AS3'UTR mutants (15.10) was as low as that on MA5.8 and NEG cells and much lower than that on the WT3'UTR mutants (38.00).



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  		FIGURE 4. Flow cytometric analysis of the MA5.8 mutants. The surface expression of the TCR/CD3 complex (A) and the {zeta} protein (B) on MA5.8 and its mutants (NEG, WT3'UTR, AS3'UTR) was quantified using FITC-conjugated anti-mouse CD3{epsilon} mAb (145-2C11; black profiles) and FITC-conjugated anti-human {zeta} mAb (TIA-2; black profiles), respectively. An FITC-conjugated Armenian hamster anti-mouse IgG (open profiles in A) or an FITC-conjugated mouse anti-human IgG (open profiles in B) was used as the negative control. The mean channel fluorescence value is indicated within the figures at the top right.

 
To confirm the cell surface expression of {zeta} protein and the TCR/CD3 complex, we subjected MA5.8 and its mutants to surface biotinylation, immunoprecipitation (IP), SDS-PAGE analysis under nonreducing conditions, and Western blot analysis (Fig. 5). IP of WT3'UTR mutants with both hamster anti-mouse CD3{epsilon} mAb (145-2C11) and mouse anti-human {zeta} mAb (TIA-2) yielded the following surface-labeled proteins: mature forms of the TCR{alpha}{beta} heterodimers ({alpha}{beta}m; 67–95 kDa), a {zeta} homodimer (34 kDa), CD3{gamma} and -{delta} (28 kDa), and CD3{epsilon} (25 kDa). These protein bands of TCR/CD3 components were confirmed by Western blot and IP of the whole cell lysate of WT3'UTR mutants with Abs against each TCR/CD3 component. IP of WT3'UTR mutants with nonspecific hamster and mouse IgG did not yield any of these proteins. Interestingly, IP of AS3'UTR mutants did not produce any labeled cell surface {zeta} proteins or TCR/CD3 complexes, except for CD3{epsilon} protein. Although the intrinsic murine CD3{epsilon} protein was observed in AS3'UTR by IP of the biotinylated cell surface protein (lane AS3'UTR in Fig. 5A), its expression was lower than that on WT3'UTR (lane WT3'UTR in Fig. 5A).



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  		FIGURE 5. A, Immunoprecipitation of cell surface TCR/CD3 complexes and {zeta} protein in MA5.8 mutants. MA5.8 and its mutants (NEG, WT3'UTR, AS3'UTR) were biotinylated and lysed in a cell lysis buffer. The cell lysates were immunoprecipitated using goat anti-mouse CD3{epsilon} mAb (145-2C11) or mouse anti-human {zeta} mAb (TIA-2) bound to protein G-Sepharose. The WT3'UTR mutants were also immunoprecipitated with a nonspecific goat IgG (IgG*1) or mouse IgG (IgG*2). The pellets were resuspended in a nonreducing sample buffer and loaded onto a 12% SDS-PAGE gel. 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 indicates the protein molecular markers. The arrow shows the protein band of CD3{epsilon}. B, Western blot and immunoprecipitation of TCR/CD3 components in WT3'UTR mutant. The cell lysates from WT3'UTR 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 {zeta} mAb (TIA-2), followed by a peroxidase-conjugated anti-mouse IgG (lane W). The cell lysates from WT3'UTR were immunoprecipitated using a goat anti-mouse CD3{epsilon} mAb (145-2C11), rabbit anti-mouse TCR{alpha} and TCR{beta} mAbs, and goat anti-mouse CD3{gamma} and CD3{delta} 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). {alpha}{beta}m, {alpha}im, and {beta}im indicate the mature forms of TCR {alpha}/{beta}-chains, the immature forms of TCR{alpha}, and the immature forms of TCR {beta}-chains, respectively.

 
Decrease in IL-2 production in MA5.8 mutants expressing {zeta}mRNA/as-3'UTR

To evaluate the physiological effect of the short 3'UTR of the {zeta} mRNA, MA5.8 mutants were stimulated with anti-mouse CD3{epsilon} mAb (145-2C11; Fig. 6). IL-2 production in WT3'UTR, NEG, or MA5.8 on day 1, 2, 4, or 6 after stimulation was compared statistically with that in AS3'UTR. IL-2 production in AS3'UTR mutants on day 1 (1.53 ± 0.09 ng/ml), day 2 (5.20 ± 0.97 ng/ml), day 3 (4.65 ± 0.54 ng/ml), and day 6 (8.75 ± 2.36 ng/ml) was significantly (p < 0.001–0.05) lower than that in WT3'UTR mutants on day 1 (4.43 ± 0.58 ng/ml), day 2 (14.32 ± 0.73 ng/ml), day 3 (13.30 ± 1.74 ng/ml), and day 6 (41.79 ± 5.47 ng/ml), respectively. Although IL-2 production in NEG seemed to increase continuously, and AS3'UTR appeared to produce lower amounts of IL-2 than NEG, the IL-2 production values of the AS3'UTR mutants on days 1–6 were not significantly different between those of NEG and MA5.8. Consequently, IL-2 production in the MA5.8 mutants expressing {zeta} mRNA/as-3'UTR was lower than usual. Unreconstituted MA5.8 has been reported to respond slightly to 145-2C11 because of residual TCR surface expression (3–8%). However, this background response is not large enough to affect our findings.



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  		FIGURE 6. IL-2 production in MA5.8 mutants after stimulation with anti-CD3{epsilon} Ab. Anti-mouse CD3{epsilon} mAb (145-2C11) was bound to a 96-well, flat-bottom plate. MA5.8 and its mutants (NEG, WT3'UTR, AS3'UTR) were then added to the wells and incubated. The culture supernatants were collected 1, 2, 3, and 6 days after stimulation and assayed using a standard IL-2 assay. IL-2 production in WT3'UTR, NEG, or MA5.8 on day 1, 2, 4, or 6 after stimulation was compared statistically with that in AS3'UTR.

 
{zeta} mRNA stability assay

To evaluate the relationship between the reduction in {zeta} protein expression and the aberrant 3'UTR forms, we examined the stability of {zeta} mRNA. WT3'UTR and AS3'UTR mutants were exposed to actinomycin D to inhibit transcription. The cell cultures were incubated with 4 µg/ml of actinomycin D, and the cells were collected 0, 6, 12, 24, and 48 h after drug exposure. One microgram of whole mRNA was isolated from the cell samples and was converted to whole cDNA by reverse transcriptase. Using 5 µl of the whole cDNA as the template, {zeta} and CD3{epsilon} cDNA were quantified by real-time PCR. To validate the real-time PCR, the standard curves for {zeta}, CD3{epsilon}, and G3PDH cDNA were constructed from the pCRII fused with the {zeta} ORF cDNA (603 bp), CD3{epsilon} cDNA (469 bp), and G3PDH cDNA (983 bp), respectively. The Ct for {zeta}, CD3{epsilon}, and G3PDH cDNA was inversely proportional (correlation coefficients were all 0.999) to the logarithm of the initial amount of the standard template DNA (Fig. 7). Then, the Ct for {zeta}, CD3{epsilon}, and G3PDH mRNA in the cell samples were measured in triplicate by real-time PCR, and the amounts of these mRNA were determined from the standard curves. Statistical significance was calculated using Student’s t test. The amount of {zeta} or CD3{epsilon} mRNA was evaluated as the relative quantity against G3PDH mRNA in the cells before treatment with actinomycin D (Fig. 8). Analysis of these mRNA by real-time PCR demonstrated that {zeta}mRNA/w-3'UTR in the WT3'UTR mutants was more stable than {zeta}mRNA/as-3'UTR in the AS3'UTR mutants during the first 6 h, while there was only a minimal difference in the stability between these two mRNA in the following 6–24 h. Also, the half-life of {zeta} mRNA/as-3'UTR in the AS3'UTR mutants (3 h) was much shorter than that of {zeta} mRNA/w-3'UTR in the WT3'UTR mutants (15 h). On the other hand, the half-life of CD3{epsilon} mRNA in the AS3'UTR mutants (2 h) was almost the same as than that in the WT3'UTR mutants (2 h). These findings suggest that the {zeta} mRNA in the MA5.8 mutant expressing {zeta} mRNA/as-3'UTR was more unstable than that in the mutant expressing {zeta} mRNA/w-3'UTR. In addition, the amount of {zeta} mRNA/as-3'UTR in the AS3'UTR (0.006 ± 0.001) was already lower than that of {zeta} mRNA/w-3'UTR in the WT3'UTR (0.012 ± 0.002) before treatment with actinomycin D. These observations demonstrated that the {zeta} mRNA was already reduced before treatment with actinomycin D.



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  		FIGURE 7. Standard curves for quantifying the amount of {zeta}, CD3{epsilon}, and G3PDH cDNA. Human {zeta} ORF cDNA (603 bp), murine CD3{epsilon} cDNA (469 bp), and murine G3PDH cDNA (983 bp) were fused with pCRII vector, respectively. Real-time PCR was performed with serial dilution (10, 1, 10-1, 10-2, 10-3, and 10-4 pg) of the plasmid DNA as the template.

 


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  		FIGURE 8. Reduction in {zeta} mRNA stability in the absence of a 562-bp portion of the 3'UTR. MA5.8 mutants (WT3'UTR and AS3'UTR) were cultured and incubated with 4 µg/ml of actinomycin D in the culture medium. Samples were collected at various time points, and the mRNA was subsequently extracted and converted to the whole cDNA. {zeta} (A) and CD3{epsilon} (B) cDNA were quantified by real-time PCR and evaluated as the ratio against G3PDH cDNA. Each horizontal solid and broken line indicates half the initial amount of {zeta} (A) or CD3{epsilon} (B) cDNA in AS3'UTR and WT3'UTR, respectively. Bars show the mean + SD or the mean - SD. *, p < 0.01; **, p < 0.001 (AS3'UTR (•) vs WT3'UTR).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We recently reported that {zeta} mRNA/as-3'UTR was predominantly expressed in SLE T cells, and that the production of {zeta} protein was positively correlated with {zeta} mRNA/w-3'UTR, but negatively correlated with {zeta} mRNA/as-3'UTR (22). Here we attempt to confirm the reduction in {zeta} protein expression in cells containing {zeta} mRNA/as-3'UTR using a recombinant retrovirus system (28, 29).

In this study IP of both the {zeta} protein and the TCR/CD3 complex in WT3'UTR mutants using anti-{zeta} mAb and anti-CD3{epsilon} mAb, respectively, demonstrated that the human {zeta} protein and the mouse TCR/CD3 complex formed a chimeric complex that was expressed on the cell surface of MA5.8 mutants expressing {zeta} mRNA/w-3'UTR. From this observation, we concluded that the TCR/CD3 complex was successfully reconstituted in MA5.8 cells by the transfer of human {zeta} mRNA/w-3'UTR, probably because of the high degree of homology (86%) between the human {zeta} protein (30) and the murine {zeta} protein (31). Göbel et al. (32) also reported reconstitution of the TCR/CD3 complex in MA5.8 cells by the transfer of chicken {zeta} mRNA.

The down-regulation of {zeta} protein in AS3'UTR and 3T3-AS3'UTR, as confirmed by Western blot analysis, suggests that the production of {zeta} protein is down-regulated when it is translated from {zeta} mRNA/as-3'UTR because of the deletion in the 3'UTR region. As the amount of the integrated pDON-AI DNA was confirmed to be the same between the AS3'UTR and WT3'UTR mutants, the difference in {zeta} protein expression in these two MA5.8 mutants was not due to a difference in the amount of the integrated recombinant retroviral DNA. However, the expression of {zeta} mRNA from pDON-AI in these MA5.8 mutants cannot be adequately addressed without an assay of transcription, such as a nuclear run-on assay. Interestingly, {zeta} protein expression was not observed in the AS3'UTR mutants when analyzed using IP, whereas it was reduced, but not deficient, when analyzed using Western blot and FACS. As the cells were permeabilized using digitonin in the FACS analysis, free intracellular {zeta} protein may have been detected using this method. Therefore, these observations might demonstrate that both the {zeta} monomer and the homodimer produced in MA5.8 mutants expressing {zeta} mRNA/as-3'UTR were only produced in small quantities, were retained in the cytoplasm, and were not expressed on the cell surface. Reportedly, only the {zeta} homodimer is capable of assembling with other TCR/CD3 complexes in the cytoplasm for expression on the cell surface (33, 34, 35). Therefore, in the MA5.8 mutants expressing {zeta} mRNA/as-3'UTR, the TCR/CD3 complex could not be expressed on the cell surface because of the reduction in expression of the {zeta} homodimer. Other groups have shown that the detergent-insoluble, membrane-associated form of {zeta} was decreased in SLE T cells (36), supporting our findings. The reduction in IL-2 production in AS3'UTR mutants revealed that the signal from the TCR was not transduced into the cytoplasm by anti-CD3{epsilon} Ab stimulation in this MA5.8 mutant. The results obtained from the MA5.8 mutants in this study may explain the mechanism behind the reduction in {zeta} protein expression in SLE T cells. In SLE T cells, the predominant expression of {zeta}mRNA/as-3'UTR form of {zeta}mRNA may lead to the down-regulation of {zeta} protein. However, one experiment that would help to prove our point that {zeta}mRNA/as-3'UTR form decreases {zeta} protein and TCR/CD3 complex would be to introduce this form of {zeta}mRNA into the cell lines already expressing {zeta} protein. This study is now underway in our laboratory.

We examined the stability of {zeta} mRNA to investigate the reduction in {zeta} protein expression in MA5.8 mutants expressing {zeta} mRNA/as-3'UTR. From our observations, {zeta} mRNA/as-3'UTR appeared to be less stable and more easily degraded than {zeta} mRNA/w-3'UTR. Therefore, it is conceivable that the reduction in {zeta} mRNA/as-3'UTR stability may lead to a reduction in the expression of the intracellular {zeta} homodimer, leading to the absence of TCR/CD3 complex expression on the cell surface. Observed lower basal levels of {zeta} mRNA/as-3'UTR in the AS3'UTR before treatment with actinomycin D than those of {zeta}mRNA/w-3'UTR in the WT3'UTR could also be due to the instability in the AS3'UTR. However, it is not uncommon that infected cells, while having identical copies of the integrated gene, nonetheless express different amounts of mRNA and proteins due to the so-called positional effects imposed by the integration site.

Observations in this study also suggest that the deleted 562-bp portion of the 3'UTR in {zeta} mRNA/as-3'UTR is critical for {zeta} mRNA stability. Gramolini et al. (37) reported that a 171-bp region in the 3'UTR of utrophin mRNA regulates utrophin mRNA stability, since the half-life of utrophin mRNA without this region is much shorter than that of the WT mRNA. The 3'UTR region of mRNA is known to control the turnover rate of presynthesized mRNAs through interactions with trans-acting factors by altering mRNA stability and affecting the transportation and localization of mRNA (38, 39, 40). mRNA 3'UTR contains cis-acting elements, i.e., adenosine-uridine (AU)-rich elements, that bind to trans-acting proteins and participate in either the stabilization or destabilization of transcripts. {zeta} mRNA contains 1055 nucleotides in its 3'UTR. Three AU-rich elements are located at positions +735, +803, and +1646 of the {zeta} mRNA. The second AU-rich domain located at position +803 may be responsible for the instability of {zeta} mRNA/as-3'UTR, because this domain is included in the deleted 562-bp portion of the {zeta} mRNA 3'UTR. The epitopes in this 562-bp region that is responsible for {zeta} mRNA stability will be determined in a project that is now in progress in our laboratory. From our observations in this study, it is possible that the decreased expression of {zeta} and TCR/CD3 complex and the aberrant signal transductions in SLE T cells could be improved if these SLE T cells are reconstructed with {zeta} mRNA/w-3'UTR. This strategy might be important for the treatment of SLE. However, decreased stability of {zeta} mRNA and reduced expression of {zeta} by the {zeta} mRNA/as-3'UTR should be confirmed by a better model using human CD4 T cells transfected with {zeta} mRNA/as-3'UTR, because the system using MA5.8 mutants of the murine cell lines may not address SLE T cells. Projects using human CD4 T cells are now underway in our laboratory.


   Acknowledgments
 
We thank Dr. T. Saito for providing MA5.8 cells.


   Footnotes
 
1 This work was supported by grants from Grant-in-Aid for Scientific Research (C); the Ministry of Education, Science, and Culture; and grants from the Ministry of Health and Welfare, Japan. Back

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 Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; AU, adenosine-uridine; Ct, threshold cycle; IP, immunoprecipitation; ORF, open reading frame; PBT, peripheral blood T cell; PVDF, polyvinylidene difluoride; UTR, untranslated region; WT, wild type. Back

Received for publication October 16, 2002. Accepted for publication July 1, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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