INTRODUCTION

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Alternative pre-mRNA splicing is a process by which multiple functionally distinct proteins may be encoded from a single gene through the variable inclusion of individual exons. In general, variable exon inclusion is regulated by proteins which bind to sequences within the regulated gene and influence the recognition of splice sites by the basic splicing machinery, or spliceosome (for recent reviews, see references 1, 4, 6, and 18). Frequently, alternative splicing is regulated in a tissue- or developmental stage-specific fashion through the activity of tissue-specific factors (17, 60). In addition, there have been several documented examples of exon inclusion being regulated in response to extracellular stimuli, including stimulation of the T-cell receptor (TCR) (10, 21, 47, 51, 61, 65). However, the mechanisms by which such signaling events can influence pre-mRNA splicing are largely unexplored.

Engagement of the TCR by ligand initiates a signal transduction cascade within the T cell which ultimately results in a number of morphological and functional changes in the cell (11, 62). One such change is a dramatic alteration in the cell surface expression of isoforms of the transmembrane protein tyrosine phosphatase CD45 (for reviews see references 57 and 58). The gene encoding CD45 encompasses 33 exons. Most of these exons, including those which encode the intracellular phosphatase domains, are constitutively included in the mature mRNA. However, three of the exons which encode part of the extracellular domain (exons 4, 5, and 6) are variably excluded from the mRNA. The peptide sequences encoded by the CD45 variable exons are rich in O-linked glycosylation sites; thus, a change in inclusion of these exons from the mRNA results in a dramatic change in the size and structure of the resulting CD45 protein (33).

No ligand has yet been identified for CD45; however, the use of chimeric molecules has demonstrated that activity of the phosphatase domain of CD45 is influenced by homodimerization, suggesting that ligand binding might regulate CD45 phosphatase activity (14, 31). Moreover, the largest and smallest CD45 isoforms have been shown to differ in the ability to associate with the TCR (26, 27). In T cells, CD45 functions to maintain Lck in an active conformation by removing an inhibitory phosphate from this kinase (34, 36, 37, 48, 50). Because Lck function is critical for early events in T-cell development and for activation, CD45 phosphatase activity is likewise required for both activation and development (7, 20, 22-24, 52). Thus, although the functional consequence of alternative isoform expression of CD45 is not yet clear, it is likely that the various isoforms differentially influence T-cell function due to a difference in their abilities to interact with ligand, with one another, or with the TCR.

CD45 is expressed in all nucleated hematopoietic cell types. Whereas CD45 surface expression changes frequently in T cells during development and upon activation, B cells only ever express the largest CD45 isoforms (58). The difference in CD45 surface expression between B cells and thymocytes (which express predominantly the smallest CD45 isoforms) has clearly been shown to be a result of regulated alternative splicing. These two cell types, and cell lines derived from each, show a marked difference in their expression of CD45 mRNA variants (39, 53, 54). Furthermore, this difference in mRNA expression requires sequences within and flanking the variable exons and is mediated by some, yet undefined, cell-specific factors (39, 42, 54, 59). An additional conclusion from these studies is that although exons 4, 5, and 6 are all variably included in CD45 mRNA, only the inclusion of exons 4 and 6 appears to be tightly regulated. Inclusion of exon 5, by contrast, is most likely a stochastic event.

Despite the progress in understanding the tissue-specific regulation of CD45 expression, characterization of activation-mediated changes in CD45 isoform expression in T cells has been significantly more limited. One reason for the limited understanding of activation-induced changes in CD45 expression is that all previous studies have analyzed primary T cells, which are not easily propagated or transfected and do not represent a homogeneous population (2, 5, 28, 40). In this report we describe a cell line-based assay which faithfully reproduces the activation-induced alternative splicing of CD45 which is observed in primary T cells. Using this cell line, we show that activation-induced exclusion of the CD45 variable exons is mediated via a protein kinase C (PKC)-dependent signaling pathway, which can be mimicked by constitutive activation of Ras. We rule out the possibility that CD45 alternative splicing is a general result of stimulation of PKC by demonstrating that treatment of several B cell lines with phorbol myristate acetate (PMA) has no effect on CD45 splicing, indicating some level of specificity to the response in T cells. In addition, synthesis of an activation-specific factor(s) is required for CD45 regulation, as indicated by the inhibition of the activation-induced alternative splicing of CD45 by treatment with cycloheximide. Last, we demonstrate that sequences within and flanking exon 4, which have previously been shown to be important for the cell-type-specific regulation of CD45 splicing, are similarly sufficient for mediating activation-induced splicing. In the future, this cell line should allow for a more detailed mechanistic study of CD45 splicing than is possible with primary cells, thereby leading to a greater understanding of how T-cell activation, and signaling events in general, may regulate alternative splicing.

	MATERIALS AND METHODS

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PBL isolation. Primary blood lymphocytes (PBLs) enriched for CD4⁺ CD3⁺ T cells were isolated from 100 ml of human blood as previously described (55). Total PBLs were first isolated by diluting the blood with an equal volume of phosphate-buffered saline (PBS) and spinning through a Ficoll gradient. After two washes with PBS, the PBLs were incubated for 30 min at 4°C in PBS plus antibodies specific for CD16, CD19, CD11b, CD45R0, CD8 (all purchased from Becton Dickinson, Mountain View, Calif.), CD14 (3C10 hybridoma from the American Type Culture Collection [ATCC]), MHCII (IVA12 hybridoma from ATCC), and erythrocytes (10F7 hybridoma from ATCC). Cells were pelleted, washed once in PBS, and then incubated again for 30 min at 4°C in PBS plus 1 ml of magnetic beads coated with sheep anti-mouse immunoglobulin G (IgG) antibodies (Dynal Inc., Great Neck, N.Y.). Following incubation, the sample was exposed to a magnet for several minutes to isolate the beads. The remaining cells were removed and saved for further use. Flow cytometry of the resulting cell population indicated that approximately 80% of the remaining cells expressed CD3 and CD4.

JSL1 isolation. Jurkat cells were plated in 96-well plates at concentrations of 1, 5, and 15 cells/ml and allowed to grow for 2 weeks. Wells which contained single colonies were expanded, and cells were analyzed by flow cytometry for expression of CD3 and CD45RA. All clones exhibited good expression of CD3. Four clones which expressed the highest levels of CD45RA were grown in medium with or without PMA plus ionomycin (100 ng/ml and 1 µM, respectively), and RNA was harvested and analyzed by reverse transcription (RT)-PCR. At least two of these clones (JSL1 and JSL4) showed a change in CD45 isoform expression in response to stimulation. Flow cytometry was performed as described previously (31), using indicated antibodies from Becton Dickinson.

Cell culture and stimulations. JSL1, Raji, Daudi, and Ramos cells were maintained in RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin, and streptomycin. The PBLs were cultured in similar medium except that fetal calf serum was added to 10%. All cells were grown at 37°C in the presence of 5% CO₂. For stimulations, cells were diluted to 3 × 10⁵ cells/ml and incubated in medium alone or with the specified additions for the times indicated.

RT-PCR assay. Total RNA from PBLs and transfected JSL1 cells was harvested by using an RNeasy kit (Qiagen, Valencia, Calif.) following the standard protocol. Total RNA from all other cells was isolated by using RNAzol (Tel-Test, Friendswood, Tex.) according to the included protocol. For RT-PCR analysis of endogenous CD45, 1.5 µg of total RNA was heated to 90°C in the presence of 1 ng of RT primer (Fig. 1A), 300 mM NaCl, 10 mM Tris (pH 7.5), and 2 mM EDTA and allowed to cool to 43°C. This annealed reaction was diluted into an RT mix containing final concentrations of 10 mM Tris (pH 7.5), 6 mM MgCl₂, 10 mM dithiothreitol, 50 mM NaCl, and 1 mM deoxynucleoside triphosphates, and incubation was continued at 43°C for 30 min. The RT reaction was stopped by boiling samples for 5 min and then placing them on ice. For PCR, a third of the RT reaction mixture was diluted into a PCR mix containing final concentrations of 1.5 mM MgCl₂, 10 mM Tris (pH 8), 50 mM KCl, 0.2 mM deoxynucleoside triphosphates, 20 ng of RT primer, and 10 ng each of primers V and C (Fig. 1A) and overlaid with mineral oil. PCR was done by heating samples to 94°C for 2 min followed by 20 cycles of 1 min at 94°C, 1 min at 70°C, and 2 min at 72°C. RT-PCR analysis of minigene RNA was done similarly except that primers MG5 and MG3, which anneal in expressed vector sequences which flank the 5' and 3' ends of the minigene, respectively, replaced primers V and RT, and primer MT, which anneals to exon 7, replaced primer C. Also, PCR was limited to 16 cycles. All of the above conditions were determined empirically to give a signal which was linear with respect to input RNA. Following completion of PCR, the reaction products were extracted with phenol-chloroform-isoamyl alcohol and ethanol precipitated in the presence of glycogen as carrier. The resulting pellets were resuspended in formamide loading buffer and resolved on a 6% denaturing polyacrylamide gel.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Detection of CD45 mRNA in resting and stimulated PBLs reveals an activation-dependent change in mRNA isoform expression. (A) Schematic of the CD45 gene (top) and predicted RT-PCR products (bottom). Exons are designated by boxes, and introns are designated by lines. The annealing sites for the primers used for RT-PCR are indicated in the CD45 gene. Primer RT was used in the RT step, and all three primers (V, C, and RT) were used for PCR. (B) Representative autoradiogram of an RT-PCR assay. Total RNA was harvested from CD4+ CD3+-enriched PBLs which either were freshly isolated (day 0) or had been cultured for the time indicated in either medium alone or medium supplemented with PHA (1 mg/ml). Primers V and C were both 5' end labeled with 32P, such that following PCR the products could be separated on a denaturing polyacrylamide gel and visualized by autoradiography. The identity of each product was confirmed by sequencing. The asterisk indicates a product we believe to be R45, but we have been unable to confirm this inference due to contamination with R56. (C) Quantitation of three experiments with T-cell-enriched PBLs. The R56/T and R0/T ratios are calculated for each condition and compared to these ratios at day 0, set as 100%. Quantitation of all RT-PCR assays was done with a PhosphorImager.

Plasmids and reagents. MG4 was synthesized by ligating three fragments containing (i) exons 2 and 3 fused together with 130 bp of downstream intron sequence, (ii) exon 4 and flanking sequence, and (iii) exon 7 and flanking sequence and then inserting into pGem7Z. The above fragments were generated by PCR using pSV-MiLCA2 (a kind gift of H. Saito) (54) as a template. The full minigene was then removed from pGem7Z and inserted into the neomycin-resistant expression vector pAWneo3 (14). MG4M was made from MG4 by using a QuickChange kit (Stratagene, La Jolla, Calif.) to engineer the point mutation. pEF-TacT was made by insertion of a truncated version of CD25 (TT-T [29]) into the expression plasmid pEFBos (35). AP1-Luc and RasV12 constructs were described previously (12, 49). The inhibitor R0-31-8220 was purchased from Calbiochem (La Jolla, Calif.).

Transfections and luciferase assay. Transfections were done as previously described (64). For stable cell lines expressing minigenes, cells were recovered for 3 days in RPMI 1640-10% serum, then serially diluted into medium containing 2 mg of geneticin per ml, and grown for an additional 2 to 3 weeks; 24 geneticin-resistant clones were then expanded further and analyzed for minigene expression by RT-PCR. For RasV12 transfections and purification, 100 × 10⁶ cells distributed in five cuvettes were transfected for each sample. Each cuvette also contained 20 µg of pEF-TacT, 10 µg of AP1-Luc, and given amounts of RasV12. Following transfection, each sample was pooled and recovered in RPMI 1640-10% serum. For purification, transfected cells were harvested, washed in PBS, and incubated with unconjugated CD25-specific antibody (Becton Dickinson). Cells were then washed again and incubated with magnetic beads coated with sheep anti-mouse IgG antibodies (Dynal). Following incubation, the sample was exposed to a magnet for several minutes to isolate the beads, and bound cells were lysed and RNA was harvested as described above. Flow cytometry indicated lysed cells contained over 90% CD25⁺ cells. The luciferase assay was done 12 h posttransfection as described previously (64).

Western blotting. Erk blotting was done as described previously (66), using rabbit polyclonal anti-Erk (25).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

CD45 mRNA expression is altered by activation of primary lymphocytes. We first wanted to analyze the CD45 mRNA profile from resting and stimulated primary T cells, in order to confirm that we could detect an activation-dependent change in CD45 splicing. To directly assay the expression of the endogenous CD45 spliced variants, we used a sensitive RT-PCR assay in which the RT reaction was followed by a limiting number of PCR cycles, such that the resulting signal was linear with respect to input RNA (see the legend to Fig. 1 and Materials and Methods). Consistent with previous reports (38, 39), we reproducibly detect four or five distinct CD45 mRNA isoforms in human cells (Fig. 1A and B). The identity of these isoforms has been confirmed by subcloning and sequencing the individual RT-PCR products. To normalize the expression levels of each isoform, we used an internal control (T) which is representative of the level of total spliced CD45 mRNA. Although we are unable to quantitate the absolute amount of each splice variant by this assay, we are able to quantitate the variation of a given mRNA species, relative to product T, under different conditions.

A novel system for studying activation-induced CD45 alternative splicing. Understanding the mechanisms involved in the activation-induced change in CD45 splicing ideally requires the ability to observe and study CD45 alternative splicing in a homogeneous population of cells which are easily maintained in culture, expanded readily, are capable of being transfected. However, no T-cell line has been reported to show a change in CD45 splice variants upon stimulation. This is in part because most T-cell lines constitutively express a CD45 mRNA profile which resembles an immature thymocyte or activated primary cell (i.e., they express primarily the R0 isoform, with little or no detectable R56 and R456 isoforms) (41, 53). To isolate a cell line which could be used to study the activation-dependent alternative splicing of CD45, we performed a limiting dilution of T lymphoma-derived Jurkat cells to isolate individual clones. Jurkat cells are an attractive basis for a model system because they are well characterized and resemble naive primary T cells in their response to stimulation (63). As a population, Jurkat cells primarily express the smaller isoforms of CD45; however, studies have indicated that in vivo some T cells undergo spontaneous interconversion from expression of smaller to larger CD45 isoforms (3, 40). Therefore, we reasoned that a subpopulation of Jurkat cells may express the larger isoforms of CD45. Following limiting dilution, we screened 30 individual clones by flow cytometry for surface expression of CD45RA isoforms (proteins encoded by mRNAs containing exon 4). Six clones showed a marked increase in RA expression over that of the starting population of Jurkat cells (Fig. 2A and data not shown). We then assayed for CD45 mRNA expression in four of these six RA⁺ clones by RT-PCR. Figure 2B shows the CD45 mRNA profile of one of the clones that we identified, designated JSL1 (for Jurkat splicing line 1), which expressed relatively high levels of the larger CD45 isoforms relative to R0. This cell line has now undergone multiple freeze-thaw cycles and been maintained in culture up to several months at a time without any significant change in CD45 isoform expression or response to stimulation (data not shown).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   CD45 pre-mRNA undergoes alternative splicing upon treatment of the JSL1 cell line with PMA. (A) Fluorescence histogram following staining of total Jurkat or JSL1 cells with either IgG-fluorescein isothiocyanate FITC (filled area) or anti-CD45RA-FITC (gray line) antibodies. The IgG-FITC provides a negative control for nonspecific fluorescence. Ten thousand events of each cell type were analyzed by flow cytometry. (B) Representative RT-PCR assay of JSL1 cells which were treated for the indicated time with medium alone or medium supplemented with PMA (100 ng/ml) or ionomycin (Iono; 1 µM). (C) Effect of multiple treatments on R0 expression in JSL1 cells. For these experiments, the concentrations used were 100 ng/ml for PMA, 1 µM for ionomycin, 1 mg/ml for PHA, and 1 ng/ml for Fas. Quantitation was done as described for Fig. 1, and results are summarized from at least three independent experiments for each condition. The effect of these treatments on the expression of the larger isoforms (namely R456) was reciprocal to their effect on R0 expression but is not shown for simplicity. (D) Quantitation of isoform expression (as described in the legend to Fig. 1) upon stimulation of JSL1 or JSL4 cells with PMA for 0, 1, or 2 days. Isoform expression is presented relative to that in JSL1 cells at time zero and is a summary of at least three independent experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Protein synthesis is required for the PMA-induced alternative splicing of CD45. (A) CD45 isoform expression following treatment of JSL1 cells with PMA (2 ng/ml) for 24 h in the presence or absence of cycloheximide (cyclohex. 50 µM) during the first 12 h of stimulation. Quantitation was done as described for Fig. 1, and results represent two independent experiments. (B) Erk phosphorylation following PMA induction of JSL1 cells in the presence or absence of cycloheximide. Cells were preincubated with cycloheximide for 15 min, and then PMA was added as for panel A. An aliquot of cells was harvested at 10 min and lysed in the presence of phosphatase inhibitors. Total lysate was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a membrane, and probed with anti-Erk antibody.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Inhibition of PKC blocks stimulation-induced alternative splicing of CD45 in both PBLs and JSL1 cells. (A) Quantitation of the relative expression of R0 and R456 in JSL1 cells following stimulation with PMA (2 ng/ml) for 48 h in the absence or presence of an increasing concentration of the general PKC inhibitor R0-31-8220. Quantitation is as described in the legend to Fig. 1 and is representative of four independent experiments. (B) Erk phosphorylation in JSL1 cells upon stimulation in the absence or presence of R0-31-8220. Following stimulation of JSL1 cells for eventual analysis of RNA, an aliquot of cells was analyzed as described for Fig. 3B. PKC is known to be required for PMA induction of Erk phosphorylation; therefore, this blot serves as a positive control for R0-31-8220 activity. (C) RT-PCR assay of RNA harvested from CD4+ CD3+-enriched PBLs which were treated for 48 h with PHA (1 mg/ml) in the absence or presence of R0-31-8220. (D) Quantitation of the experiment shown in panel C.

Activated Ras is sufficient to mediate CD45 alternative splicing. One of the best-characterized effectors of PKC isoforms in T cells is the small GTPase Ras (8, 15). Therefore, we were interested in studying more directly the potential involvement of Ras in the induction of CD45 alternative splicing. Ras is active when bound to GTP, and thus constitutive activation of Ras can be conferred by mutations which dramatically reduce its GTPase activity. One such mutation is a substitution at residue 12 of valine for glycine (RasV12) (44). To determine whether Ras activation is sufficient to induce a switch in CD45 splicing in JSL1 cells, we transfected unstimulated JSL1 cells with a RasV12 expression construct. Because only a small percentage of cells take up the DNA upon transfection, we purified those cells which expressed RasV12 before analyzing the RNA. To accomplish this, we took advantage of the observation that when cells are transfected simultaneously with multiple plasmids, there is a direct correlation between expression of each construct in any given cell. Therefore, we cotransfected the RasV12 plasmid with a construct expressing the extracellular and transmembrane domains of CD25 (TacT). Two days posttransfection, those cells which express CD25 on their surface are assumed to also express RasV12 and were readily purified with anti-CD25 antibody and magnetic beads prior to lysing and RNA harvest (see Materials and Methods). Additionally, the cells were cotransfected with a reporter construct in which the luciferase gene is driven by an AP1-dependent promoter (AP1-Luc). This reporter construct has previously been demonstrated to express the luciferase enzyme in Jurkat cells in response to PMA treatment or Ras activation (49). As shown in Fig. 5B, luciferase expression in the transfected cells was indeed induced by both PMA and cotransfection with RasV12, thus confirming that RasV12 was expressed and active.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Expression of constitutively active Ras is sufficient to induce alternative splicing of CD45. (A) Quantitation of the relative expression of R0 and R456 in JSL1 cells 50 h after transfection with RasV12. Amount of RasV12 expression plasmid included in each transfection per 20 × 106 cells is indicated. PMA was added 6 h after transfection to cells transfected with empty vector. At 50 h posttransfection, cells were harvested and RNA was isolated from purified transfected cells (see Materials and Methods). Quantitation is as described for Fig. 1 and is representative of three independent experiments. (B) Activation of an AP1-luciferase reporter construct by PMA or cotransfected RasV12. The luciferase assay was performed as described in Materials and Methods. The relative expression of luciferase is given as a percentage of that induced by treatment of the cells with PMA.

PMA does not induce CD45 alternative splicing in B-cell lines. The activation of PKC and/or Ras potentially has many effects on cellular metabolism and gene regulation. Therefore, we wanted to examine the specificity of the PMA-induced alternative splicing of CD45 and exclude the possibility that treatment with PMA was affecting CD45 splicing due to a change in the activity of some component of the general splicing machinery. Primary B cells are known to express predominantly the largest CD45 protein isoform (encoded by R456), and this expression is unaffected by developmental or activation state (58). Therefore, it is likely that B cells differ from T cells in their expression of some CD45 regulatory factor(s). Consistent with the in vivo observations, at least three cultured B-cell lines express solely the R456 and R56 isoforms (Fig. 6A and data not shown). In marked contrast to JSL1 cells, treatment of each of these cells lines with PMA did not cause any downregulation of R456 or R56, nor could the R0 isoform be detected (Fig. 6A and B). This was despite the ability of PMA to upregulate expression of the activation marker CD69 in all of the cell lines tested (Fig. 6C and data not shown). Thus, although we do not yet know the nature of the critical differences between the B cells and JSL1 cells, these results indicate that PMA does not alter CD45 splicing as a result of a nonspecific change in the general splicing machinery.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   A cell-specific factor(s) is required for the PMA-induced alternative splicing of CD45. (A) Representative RT-PCR assay comparing CD45 expression in JSL1 cells and Raji cells upon stimulation with PMA (100 ng/ml) or in medium alone (). (B) Quantitation of R456 expression in JSL1 cells and three B-cell-derived cell lines (Raji, Daudi, and Ramos-Lanier) following treatment with PMA (100 ng/ml). Quantitation is as described for Fig. 1 and represents two to three independent experiments. Expression of R0 was not quantitated because no R0 mRNA could be detected in any of the B-cell lines under either unstimulated or stimulated conditions. (C) Fluorescence histogram following staining of JSL1 or Raji cells grown for 5 h in the absence (filled area) or presence (black line) of PMA. Cells were stained with anti-CD69-fluorescein isothiocyanate (FITC) antibodies. Ten thousand events of each cell type were analyzed by flow cytometry. CD69 upregulation is a marker for activation of both B and T cells.

Sequences involved in regulating the splicing of CD45 variable exon 4. Previous studies have demonstrated that sequences within and immediately flanking regulated exons 4 and 6 are necessary and sufficient to mediate their tissue-specific isoform expression (inclusion in B cells and exclusion in thymocytes) (54, 59). To examine if these same sequences were involved in activation-dependent splicing regulation, we made a minigene in which exon 4 was flanked by exons 3 and 7. We included in this construct the intron sequences flanking exon 4 which were previously determined to be minimally required for tissue-specific regulation (Fig. 7A and reference 54). When this minigene was stably integrated into JSL1 cells, we observed that in the majority of the clones isolated, approximately 50% of the minigene mRNA contained exon 4. More importantly, upon treatment of these clones with PMA, we observed a significant shift toward exon exclusion (Fig. 7B [first four lanes] and C). When we tested minigenes which contained more of the intron sequences which endogenously flank exon 4, isoform expression and regulation were essentially the same as observed in MG4 (data not shown). Therefore, we conclude that the sequences contained in MG4 are sufficient to mediate both tissue-specific and activation-induced alternative splicing of exon 4. Moreover, since the minigene used here is expressed from a heterologous promoter, these results demonstrate that activation-induced splicing regulation does not require transcription from the endogenous CD45 promoter.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   Sequences within and flanking exon 4 are necessary and sufficient for activation-induced splicing regulation. (A) A schematic of the minigene used. Exons are designated by boxes, and introns are designated by lines. Bold lines represent intron sequences which are identical to the endogenous gene; thin lines represent heterologous sequence added in the construction of the minigene. Numbers above intron and exon sequences indicate nucleotide length. MG4 and MG4M are identical in all respects except the C77G mutation. (B) RT-PCR analysis of stable integrants of minigenes MG4 and MG4M in JSL1 cells. Two clones harboring MG4 (A and B) were isolated in parallel with three MG4M clones (A, B, and C), and all were grown in the absence () or presence (+) of PMA before harvesting for RNA. Overall expression of the minigene is monitored by product MT and appears to decrease somewhat upon stimulation. (C) Quantitation of minigene expression from three independent experiments. (D) RT-PCR analysis of the endogenous CD45 expressed in the same RNA samples as used for panel B. (E) Quantitation of the endogenous CD45 isoforms from two independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Upon stimulation, naive T cells undergo numerous changes in gene expression that allow the activated cell to function in an immune response. Among these changes is the activation-induced switch in the expression of CD45 mRNA isoforms. To begin to understand the mechanisms responsible for this regulated splicing event, we have developed a cell line-based model system which mimics the alternative splicing of CD45 which we and others (28) observe in primary T cells. There are several lines of evidence that JSL1 cells represent a good model for the activation-dependent alternative splicing of CD45. First, the time course and extent of isoform change upon stimulation are highly similar between JSL1 cells and primary T cells. Second, the PKC inhibitor R0-31-8220 blocked CD45 alternative splicing at similar concentrations in both cell types. Finally, a point mutation which has been shown to increase CD45 exon 4 inclusion in primary T cells has a similar effect in JSL1 cells. Therefore, at least some components of both the initial signaling events, and the direct regulation of CD45, are conserved between primary T cells and JSL1 cells.

Requirements for activation-induced alternative splicing of CD45. One of the surprising findings in this study is that at least 24 h are needed from the time of activation to the detection of a significant change in CD45 mRNA expression. This is consistent with the observation that a significant change in CD45 surface expression first occurs 2 to 3 days following stimulation (2, 5, 40) and with the time course observed for at least one other signaling-induced alternative splicing event (51). However, many other changes in gene expression, such as induction of interleukin-2 transcription and alternative splicing of CD44 (see below for further discussion), are observed within 6 to 7 h of T-cell activation. Since the half-life of each of the CD45 mRNA variants is on the order of 3 to 4 h (reference 13 and data not shown), the lag time observed for the CD45 isoform change is not due to slow turnover of RNA. Rather, it appears that multiple events are likely required to initiate alternative splicing. Consistent with this interpretation, the addition of cycloheximide to JSL1 cells during the first 12 h of exposure to PMA blocks alternative splicing almost entirely. Thus, we propose a model in which an initial PKC/Ras-dependent pathway results in the induction of some activation-specific protein that in turn either directly or indirectly regulates CD45 pre-mRNA.

Comparison with other examples of activation-induced splicing in T cells. Several genes besides CD45 have been described to undergo alternative splicing upon T-cell activation. These include Fas, a novel death domain-containing protein of unknown function called Lard, and the cell surface molecule CD44, which is involved in cell adhesion and trafficking (21, 30, 46, 47). Similar to what we have observed for CD45, alternative splicing of endogenous CD44, and/or a minigene containing a single CD44 variable exon, can be induced in a murine T-lymphoma cell line by treatment with a phorbol ester or by activated Ras but not by ionomycin (21). However, there are several notable differences in the induction of the two splicing events. First, activation of T cells induces inclusion of the CD44 variable exons, in contrast to the induced exclusion of CD45 variable exons (21, 46). Moreover, alternative splicing of the CD44 minigene occurs within 7 h of treatment with PMA and protein synthesis is not required, as indicated by the lack of a block in alternative splicing upon addition of cycloheximide (21). Likewise, alternative splicing of Fas in PBLs is initiated during the first 6 to 12 h of activation (30). Therefore, although there may be some overlap in the pathways which mediate alternative splicing of CD45, CD44, and Fas, the difference in the kinetics of these events suggests that there are at least two pathways by which T-cell activation can induce changes in splicing regulatory factors.