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The Journal of Immunology, 2000, 164: 5590-5595.
Copyright © 2000 by The American Association of Immunologists

Molecular Basis for Functional Maturation of Thymocytes: Increase in c-fos Translation with Positive Selection1

Satoshi Nunomura, Takehito Sato and Sonoko Habu2

Department of Immunology, Tokai University School of Medicine, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the process of positive selection, immature CD4+8+ double positive (DP) thymocytes expressing TCR reactive to self-MHC by appropriate avidity develop into mature thymocytes. Positive selection involves not only down-regulation of either CD4 or CD8 but also acquisition of immunocompetent potential such as cell proliferation and cytokine production. To understand the molecular basis for such functional maturation during the positive selection process, we examined whether nonselected DP, selected DP, and CD4+8- single positive thymocytes possess the activation potential for signaling pathways from mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) to AP-1. In response to stimulation, a marked induction of c-Fos protein expression as well as cell proliferation is detected only in CD4+8- single positive cells but not in selected and nonselected DP cells, though mitogen-activated protein kinase activities and c-fos transcripts are equally induced. In the presence of proteasome inhibitors, c-Fos protein became detectable in selected DP cells but still not in nonselected DP cells, suggesting that DP cells receiving positive selection signals acquire the capacity to translate the c-fos gene, but it may not be sufficiently high to overcome the degradation of c-Fos protein. These data indicate that the translating ability of the c-fos gene is up-regulated in the thymic positive selection process, from nonselected DP to CD4+8- single positive cells through positively selected DP cells. The distinguished responsiveness to stimulation in thymocytes with and without positive selection may be a result in part of the distinct regulation of the c-fos gene at the translational level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mature-type T cells develop in the thymus from CD4-8- double negative (DN)3 cells through immature CD4+CD8+ double positive (DP) cell stage (1). DP thymocytes expressing the TCR become CD4+8- single positive (SP) (CD4SP) or CD4-8+ SP (CD8SP) cells when they interact with MHC molecules expressed on thymic stromal cells by appropriate avidity, a process termed positive selection (2). During this process, thymocytes stop expressing either CD4 or CD8 surface molecules (3), and at the same time they acquire similar functional potentials as those of mature peripheral T cells: CD4SP thymocytes are able to proliferate as well as peripheral T cells and produce several cytokines such as IL-2 by responding to mitogenic stimulation, but DP cells show little response to stimulation (4, 5). The acquisition of such functional capability of CD4SP cells is called "functional maturation" (6). In the past years, a large number of studies (7, 8, 9, 10) have contributed to determining the correlation between maturation stage and surface markers during the intrathymic developmental pathway, but little has been clarified about the molecular mechanism of this functional maturation.

AP-1 is known to play a pivotal role in proliferation as well as IL-2 production and is also known to be activated by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) (11). These kinases phosphorylate transcription factors such as Elk-1 and c-Jun and then the phosphorylated forms can induce transcription of c-fos, c-jun, fosB, and junB (12, 13), products that generate the AP-1 complex as homo- or heterodimer (14, 15). Therefore, in DP thymocytes that cannot proliferate by stimulation, the expression of AP-1 and/or a series of intracellular molecules engaged in AP-1 activation may be suppressed. In fact, Chen et al. and other investigators showed that the binding activity of AP-1 is induced after stimulation in CD4SP cells but not in DP cells (16, 17, 18).

To confirm which molecules substantially regulate AP-1 activation in DP thymocytes, we used thymocytes of TCR transgenic (Tg) mice with selecting and nonselecting MHC. The former contains a large number of selected CD4SP and DP thymocytes, whereas the latter contains nonselected DP cells but not SP cells (19). We discovered that the translation of c-Fos protein is not induced in nonselected DP thymocytes but becomes inducible in postselected DP and CD4SP thymocytes, despite the fact that the activation of mitogen-activated protein (MAP) kinases and the transcription of fos and jun family were comparably detectable. These data indicate that the translational regulation of c-Fos protein is altered during the course of thymocyte functional maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PMA and ionomycin (IM) were purchased from Sigma (St. Louis, MO). The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I were purchased from Calbiochem (San Diego, CA). MAP kinase kinase inhibitor, PD98059, was purchased from Calbiochem. PE-anti-mouse CD4 was purchased from Caltag (San Francisco, CA). Biotin-anti-mouse CD69 was purchased from PharMingen (San Diego, CA). Biotin-anti-mouse CD5 was purchased from Becton Dickinson (Mountain View, CA). Biotin-anti-mouse TCR-V{alpha}3 (1H9; Ref. 20), biotin-anti-mouse TCR-ß (H57-597), biotin-anti-mouse heat-stable Ag (J11D), and FITC-anti-mouse CD8{alpha} (53-6.7) were prepared in our laboratory.

Mice

MHC class II-restricted, OVA-specific-TCR-Tg (I-Ad, OVA323–339 specific) mice generated in our laboratory (OVA23-3; Ref. 19) were back-crossed with BALB/cA (H-2d) or C57BL/6 (H-2b). Selecting and nonselecting MHC background mice were designed as Tg-Posi and Tg-Neut, respectively. Tg-Neut mice were further back-crossed onto recombinase-activating gene 2 (RAG2) knockout mice, which were from the C57BL/6 background (19, 21). c-fos knockout mice were kindly provided by Dr. T. Tokuhisa (Chiba University School of Medicine, Chiba, Japan). Mice were used at the age of 4–8 wk.

Preparation of thymocytes

Thymocytes from Tg-Posi and Tg-Neut were isolated, filtered through a nylon mesh, and CD4-CD8- DN cells were removed by anti-mouse CD4 magnetic beads and DETACHa beads (Dynal, Olso, Norway). For cell sorting experiments, total thymocytes of Tg-Posi were stained with the PE-anti-CD4 and FITC-anti-CD8 Abs and then sorted on a FACStarPlus flow cytometer (Becton Dickinson).

Cell proliferation assay

Thymocytes (5 x 105/well) were cultured for 36 h in RPMI 1640 medium containing 10% FCS, PMA, and IM (0–0.5 ng/ml and 1.0 µg/ml, respectively) in the absence or presence of 50 µM PD98059, pulsed with [3H]thymidine (1 µCi/well), and harvested 12 h later. The amount of 3H incorporated into the cells was measured by liquid scintillation counter.

In vitro kinase assay

Kinase assays were performed as described (20). Cell extracts in lysis buffer (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 1 mM Na3VO4, 0.1 mM EGTA, 1 mM DTT, 0.1 M NaF, 1% Triton-X-100, 1 mM PMSF, 20 mg/ml aprotinin, and 20 ng/ml leupeptin) were mixed with 4 µg anti-ERK1 or anti-JNK1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA)-conjugated protein G Sepharose beads and rotated at 4°C for 3 h in a microfuge tube. The immunoprecipitate was washed with lysis buffer. Washed immune complexes were resuspended in kinase buffer (50 mM Tris-HCl, pH7.4, 10 mM MgCl2, 2 mM EGTA, 1 mM DTT, 2 mM ATP, 1 mCi [{gamma}-32P]ATP) together with myelin basic protein (MBP) and GST-c-Jun as substrates for ERK and JNK, respectively. After kinase reaction at 30°C for 30 min (JNK) or at room temperature for 10 min (ERK), we stopped the reaction with the addition of 2x Laemmli buffer and resolved it with 12% SDS-PAGE and performed autoradiography.

RNase protection assay

Thymocytes were collected at certain time points after PMA/IM stimulation, and the total cellular RNA was immediately prepared. Protection assay was performed by using a RiboQuant multiprobe kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions. Two micrograms of total RNA from stimulated thymocytes was hybridized with 2 x 105 cpm of 32P-labeled RNA probe overnight. The RNA probes were generated from m-fos/jun multiprobe template set. After hybridization, single-strand RNA was digested with 20 g/ml RNase A plus 2 g/ml RNase T1 at 37°C for 1 h. Digested samples were electrophoresed through a 6% denaturing acrylamide gel and autoradiographed. The fos and jun transcripts were identified by the length of the respective fragments. RNA loading was estimated by measuring the intensities of protected fragments representing a housekeeping gene (GAPDH).

RT-PCR

The mRNA was isolated from thymocytes using micro-Fast Track ver 2.0 (Invitrogen, Carlsbad, CA). cDNA was synthesized from mRNA by 200 U of Moloney murine leukemia virus reverse transcriptase, 2.5 mM dNTP, and 1.0 mM oligo(dT) primer. The cDNA was then used as the template of the PCR. The open reading frame of c-fos and ß2-microglobulin (ß2m) were amplified using 5'-CCGAATTCTTCCCCAAACTTCGACC-3' and 5'-TAGAATTCGGCTGCCTTGCCTTCTC-3' (c-fos), 5'-ATGGCTCGCTCGGTGACCCTAG-3' and 5'-TCATGATGCTTGATCACATGTCTCG-3' (ß2m). The PCR parameters were 94°C for 30 s, 60°C for 1 min, and 72°C for 2 min with 30 cycles, and then 10 min elongation at 72°C. The PCR products were separated on a 1% agarose gel and detected by ethidium bromide staining.

Immunoblotting

Nuclear extracts were obtained from a small number of cells (1 x 106 to 5 x 106) by using a previously reported procedure (22). The concentration of nuclear proteins was confirmed by the Bradford assay (Bio-Rad, Hercules, CA). Cytosolic extracts were prepared as described in an in vitro kinase assay. Nuclear or cytosolic proteins were mixed with an equal volume of 2x Laemmli buffer. The samples were boiled, separated on 10% SDS-PAGE, and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with anti-ERK1, anti-JNK1, anti-c-Fos, anti-ß-actin (Santa Cruz Biotechnology), anti-c-Jun, and anti-eukaryotic initiation factor 4E (eIF-4E; Transduction Laboratories, Lexington, KY). Signals were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, U.K.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferative potential of thymocytes from TCR-Tg mice with selecting or nonselecting MHC

Mature T cells can proliferate in response to a variety of stimulations, such as by Ags and cytokines. Such proliferative responsiveness is known to be absent in immature DP thymocytes but is obtained in postselected mature thymocytes. To clarify the molecular basis of how the proliferative potential is acquired in the positive selection process, we used TCR-Tg mice with selecting (d-haplotype) or nonselecting (b-haplotype) MHC, named Tg-Posi and Tg-Neut mice, respectively (Fig. 1GoA). The reactivity of the TCR is I-Ad restricted OVA323–339 specific. Tg-Posi thymocytes contain significant number of CD4SP cells but Tg-Neut thymocytes do not contain SP cells (Fig. 1GoA). Tg-Neut mice used in this study were back-crossed onto RAG2-deficient mice to exclude the contamination of endogenous TCR-expressing thymocytes. Almost all DP and CD4 SP cells in Tg-Posi express V{alpha}3 (Fig. 1GoB), indicating that there are few cells that do not express transgene-derived TCR. In DP cells in Tg-Posi, TCR-ß, TCR-{alpha}, CD69, and CD5 were expressed at a higher level than those in RAG2-/- Tg-Neut (Fig. 1GoB). Such characters are not distinct from those of other previously reported TCR-Tg mice (23, 24, 25). These findings suggest that DP cells in Tg-Posi have already received positive selection signals via transgene-derived TCR, whereas those in RAG2-/- Tg-Neut are not.



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  		FIGURE 1. Surface phenotypes of Tg-Posi and Tg-Neut thymocytes. A, CD4/CD8 expression. B, TCR-ß, TCR-V{alpha}3, CD69, CD5, and heat stable Ag (HSA) expressions on CD4SP (Tg-Posi CD4SP), selected DP (Tg-Posi DP), and nonselected DP (RAG2-/- Tg-Neut DP) cells.

 
To compare selected and nonselected thymocytes, CD4SP and/or DP cells of these mice were positively purified with anti-CD4 mAb conjugated microbeads to deplete CD4-CD8- (DN) cells. DN-depleted thymocytes of Tg-Posi contained 50–70% DP and 30–45% CD4SP cells, and those of Tg-Neut contained >95% DP cells (Table IGo). As expected, when the thymocytes were stimulated with PMA/IM, the proliferation was markedly induced in a dose-dependent manner in Tg-Posi thymocytes, but was almost undetectable in Tg-Neut thymocytes (Fig. 2GoA).


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  		Table I. The proportion of subsets in DN-depleted thymocytes used in this study1

 


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  		FIGURE 2. Thymocyte proliferation requires MAP kinase activation. A, Thymocytes from Tg-Posi or RAG2-/- Tg-Neut mice were stimulated (5 x 105/well) with indicated concentrations of PMA and IM in the presence or absence of PD98059. Thymocyte proliferation was measured by [3H]thymidine incorporation. B, PD98059 inhibits ERK activation. Tg-Posi thymocytes were stimulated with PMA/IM in the presence or absence of PD98059. ERK activities were determined by in vitro kinase assay with MBP as substrate. The fold activation was calculated using a densitometer.

 
MAP kinase activity is inducible in both nonselected and selected thymocytes

For cell proliferation, MAP kinases are known to play an important role in peripheral T cells. We examined whether the activation of MAP kinases is required for the cell proliferation of Tg-Posi thymocytes. As shown in Fig. 2GoA, when Tg-Posi thymocytes were cultured with PMA/IM in the presence of MAP kinase kinase inhibitor, PD98059 (26), the cell proliferation was decreased to 20% that of cultured thymocytes without the inhibitor. In these thymocytes, the kinase activity of ERK, one of the MAP kinases, was also decreased to around 30% (Fig. 2GoB).

This result raised a question whether nonselected thymocytes lacking proliferative ability fail to activate MAP kinases such as ERK and JNK. Then, we performed an in vitro kinase assay of ERK and JNK in both Tg-Posi and Tg-Neut thymocytes using the specific substrates MBP and GST-c-Jun, respectively. The thymocytes were lysed after stimulation with PMA/IM for different durations and were immunoprecipitated with anti- ERK or JNK Ab. The ERK and JNK activities reached a maximum at 15 min at the same level in both Tg-Posi and Tg-Neut thymocytes (Fig. 3Go). Though the increased kinase activity declined a little sooner in Tg-Neut thymocytes than in Tg-Posi thymocytes, this difference does not seem to be sufficient to explain their different proliferative potential to PMA/IM as shown in Fig. 2GoA.



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  		FIGURE 3. Kinetics of ERK and JNK activation in Tg-Posi and RAG2-/- Tg-Neut thymocytes. A, Thymocytes from Tg-Posi and RAG2-/- Tg-Neut were stimulated with PMA/IM and then harvested at the indicated times (min). Activation of ERK and JNK was determined by in vitro kinase assay with MBP or GST-c-Jun as substrate, respectively. B, The expressions of ERK and JNK protein in Tg-Posi and RAG2-/- Tg-Neut thymocytes were determined by Western blot analysis.

 
Inducibility of fos and jun transcription is also found in selected and nonselected thymocytes

AP-1 is a set of heterodimers composed of the Fos and Jun families. Activated ERK and JNK phosphorylate transcription factors such as Elk1, c-Fos, and c-Jun, and consequently fos and jun family genes are induced to express mRNA and the encoding proteins. Based on this process, it is possible that fos and/or jun family genes are not expressed in Tg-Neut thymocytes, even if the MAP kinase activation is sufficiently inducible.

To examine this possibility, we first examined the transcriptional kinetics of fos and jun family genes in the stimulated thymocytes in an RNase protection assay (Fig. 4Go). A protection assay showed that the transcripts of these protooncogenes including c-fos, fosB, fra1, fra2, c-jun, junB, and junD reached a maximum level at 30 min after stimulation and then declined until 180 min in both Tg-Posi and Tg-Neut thymocytes, and there was no significant difference of transcription and degradation rate between these mice.



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  		FIGURE 4. Kinetics of fos and jun transcripts induction in Tg-Posi and RAG2-/- Tg-Neut thymocytes. Thymocytes from Tg-Posi and RAG2-/- Tg-Neut were stimulated with PMA/IM. Cells were harvested at the indicated time points, and total cellular RNA was analyzed for fos and jun family transcript levels by RNase protection assay. The housekeeping gene encoding GAPDH was used as positive control.

 
c-Fos protein is inducible in selected thymocytes but not in nonselected thymocytes

We next compared the expression level of c-Fos and c-Jun proteins between Tg-Posi and Tg-Neut thymocytes. In the thymocytes stimulated with PMA/IM, c-Jun protein was equally expressed in both Tg-Posi and Tg-Neut mice at 180 min, but c-Fos protein was almost undetectable in Tg-Neut, whereas it was highly expressed in Tg-Posi mice (Fig. 5GoA). With c-Fos protein, we performed a time course analysis and showed the larger amount at 90 min and the decreased one at 180 min in Tg-Posi mice (Fig. 5GoB). In a shorter exposure, a strong signal of Tg-Posi at 90 min became clear to be multiple bands (data not shown), among which the band with slowest mobility correspond to a single band detected at 180 min. As c-Fos protein has been reported to show multiple electrophoretic mobility because of the distinct phosphorylation status and the less phosphorylated form is unstable (27, 28), c-Fos protein is considered to be degraded according to the magnitude of phosphorylation during the latter 90 min. However, its expression was almost undetectable at either 90 or 180 min in Tg-Neut mice (Fig. 5GoB). This was supported by the EMSA result of stimulated thymocytes in which the DNA binding activity of AP-1 was detectable in Tg-Posi but not in Tg-Neut mice (data not shown). Because the c-fos and c-jun genes were similarly transcribed in the thymocytes of both mice (Fig. 4Go), one explanation for the results is that the transcript of c-fos gene is not translated in thymocytes without receiving positive selection signals.



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  		FIGURE 5. Impaired induction of c-Fos protein expression in RAG2-/- Tg-Neut thymocytes. A, Thymocytes from Tg-Posi and RAG2-/- Tg-Neut were stimulated with PMA/IM for 180 min. Expressions of c-Fos and c-Jun protein were determined by Western blot analysis. B, Thymocytes from Tg-Posi and RAG2-/- Tg-Neut were stimulated with PMA/IM for 0, 90, and 180 min. Expression of c-Fos protein was determined by Western blot analysis. Similar results were obtained in three independent experiments. C, The specificity of anti-c-Fos Ab used in this study was confirmed by Western blot analysis. Spleen cells from Tg-Posi and c-Fos-/- mice stimulated for 90 min were lysed, electrophoresed, and blotted. There were strong signals that can be seen in Tg-Posi but not in c-Fos-/- mice.

 
As the thymus of Tg-Posi contains DP cells and CD4SP cells, we tried to determine the developmental stage, either DP or CD4SP, at which c-fos mRNA is translated in stimulated thymocytes. Isolated CD4SP cells, postselected DP cells, and preselected DP cells from Tg mice were cultured with PMA/IM for 90 min and were analyzed for c-fos transcript and c-Fos protein. Western blot analysis showed a high expression of c-Fos protein only in Tg-Posi CD4SP cells, and its expression was very low in DP thymocytes of both Tg-Posi and Tg-Neut mice (Fig. 6GoA). However, RT-PCR analysis showed fully induced transcripts of c-fos in all the above three thymocytes (Fig. 6GoB). These results enable us to lead two possible interpretations; c-Fos protein translation is inhibited in DP thymocytes, or c-Fos protein is easily degraded in DP thymocytes.



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  		FIGURE 6. A, CD4SP cells from Tg-Posi thymocytes and DP cells from Tg-Posi or Tg-Neut thymocytes were purified using FACStar and then stimulated with PMA/IM for 90 min. c-Fos protein expression of each subset was determined as shown in Fig. 5Go. Similar results were obtained in three independent experiments. B, mRNA from stimulated cells were prepared and transcripts of c-fos and ß2m were determined by semiquantitative RT-PCR. C, c-Fos translation was detected in CD4SP and selected DP cells of Tg-Posi thymocytes but not in nonselected DP cells of RAG2-/- Tg-Neut. The purity of Tg-Posi DP cells was >99% (upper panel). After the stimulation in the presence of proteasome inhibitors (10 µM), lysates were blotted, and c-Fos protein expression was determined (bottom panel). The addition of 3% CD4SP cells to nonselected DP cells did not enhance the band intensity, indicating that the increased expression in Tg-Posi DP cells did not result from contamination of CD4SP cells. Similar results were obtained in three independent experiments.

 
c-Fos expression is differentially regulated at the translational level during the positive selection process

It is known that c-Fos protein is sensitive to and is easily degraded by 26S proteasome in a ubiquitin-dependent or -independent manner. Thus, the amount of c-Fos protein we estimated by Western blot may not reflect the substantially synthesized protein. To clarify this issue, we examined the protein amounts of c-Fos induced in the stimulated thymocytes in the presence of proteasome inhibitors (proteasome inhibitor I, MG132, and lactacystin). Western blotting revealed that in the presence of the inhibitors, c-Fos protein was detected at high and low levels in CD4SP and DP cells of Tg-Posi, respectively, but was almost undetectable in Tg-Neut DP cells (Fig. 6GoC). This finding suggests that the reason for the undetectable level of c-Fos protein induced in Tg-Neut DP cells was poor translation, not increased degradation of the protein.

It is also notable that c-Fos protein in Tg-Posi DP cells became detectable in the presence of inhibitors, suggesting that c-fos gene translation is initiated at the DP stage of thymocytes receiving positive selection signals. To rule out the possibility that contaminating mature CD4SP cells result in increased intensity of c-Fos in Tg-Posi DP cells, we examined whether the addition of 3% CD4SP cells to Tg-Neut DP cells affect the intensity of blotting bands (Fig. 6GoC). The purity of sorted Tg-Posi DP cells was 99.8%, whereas that of Tg-Neut DP cells was 97.5%. As shown in lane 4, the addition of small number of CD4SP cells did not alter the band intensity, indicating that the increased c-Fos expression in Tg-Posi DP cells is not an experimental artifact.

The translational process is regulated by the combination of a variety of factors. Among them, 5' cap binding protein eIF-4E is reported to be important as the rate-limiting factor in translation initiation (29). Then, we analyzed the expression of eIF-4E protein in CD4SP and DP cells of Tg-Posi and Tg-Neut DP cells. The eIF-4E protein was highly expressed in both selected thymocytes, CD4SP and DP cells in Tg-Posi, but was expressed at a much lower level in nonselected DP thymocytes (Fig. 7Go). This differential expression level was correlated with the ability of c-Fos translation.



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  		FIGURE 7. The expression of translation initiation factor 4E (eIF-4E) of nonselected DP thymocytes was lower than those of CD4SP and selected DP thymocytes. Each subset of thymocytes was prepared as in Fig. 6Go. The expressions of eIF-4E (bottom panel) and ß-actin (upper panel), as positive control, were determined by Western blot analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DP thymocytes receiving positive selection signals via TCR not only lose either CD4 or CD8 expression but also acquire T cell functional potential such as cell growth and cytokine production. The latter phenomenon is called "functional maturation" in the positive selection process, but relatively little is known about the molecular events regarding how "functional maturation" is acquired. In this study, we found that c-Fos protein, which is important for cell proliferation and cytokine production as a component of the AP-1 complex, becomes inducible to be expressed in CD4SP thymocytes but is not yet inducible in preselected DP thymocytes because of its lack of translating ability. Thus, this suggests that the positive selection signaling induces the potential to translate particular genes related to cell proliferation, resulting in functional maturation of thymocytes, and this translation is suppressed at preselected DP cells despite the fact that c-fos transcription is inducible in both selected and nonselected thymocytes.

MAP kinase family proteins, upstream of AP-1, are activated when cells are stimulated. Among MAP kinases, ERK and JNK are known to enhance the transcription activity of Elk-1 and c-Jun by their phosphorylation, and consequently the expression of fos and jun family genes are induced (30). Because all of the Fos and Jun family members can compose AP-1 as a heterodimer, reduction of ERK and JNK activity may suppress AP-1 formation and activation, resulting in a decrease in cell proliferation.

Nonselected DP thymocytes did not show cell proliferation after mitogenic stimulation, but activation of both ERK and JNK in these cells was inducible to the same extent as in selected thymocytes (Fig. 3Go). Moreover, the transcription levels of fos and jun family genes were also similar in both selected and nonselected thymocytes (Fig. 4Go). Despite the substantial mRNA expression, c-Fos protein was hardly inducible to express in nonselected DP thymocytes, although it was highly inducible in selected thymocytes. In line with this observation, AP-1 DNA binding activity was not detectable in nonselected DP thymocytes when analyzed by EMSA (data not shown). Taken together, it is suggested that positive selection signals induce the expression potential of the c-fos gene at the posttranscription level. Currently, Chen et al. reported that c-fos mRNA induction is impaired in DP cortical thymocytes. At the moment, we do not have any good explanation of this discrepancy from the result (31). Other AP-1 components such as FosB, c-Jun, JunB, and JunD were not sufficiently inducible to estimate the expression difference between nonselected DP and selected thymocytes, although it may be determined by using more sensitive detection tools in the future.

Several factors regulating mRNA stability, protein degradation, and translation may be involved in posttranscriptional regulation. c-fos mRNA is known to be typical short-lived RNA (32). As shown in the protection assay (Fig. 4Go), c-fos mRNA newly induced by stimulation was equally degraded until 180 min in both nonselected DP thymocytes and selected thymocytes (Fig. 4Go and data not shown). Therefore, it seems likely that c-Fos protein expression in the selected thymocytes is not caused by the decreased sensitivity to mRNA degradation. It is also known that c-Fos protein is short-lived and undergoes its degradation mainly by proteasome in a ubiquitin-dependent or -independent manner (33, 34, 35). As shown in Fig. 6GoC, in the presence of proteasome inhibitors, the amount of c-Fos protein was still very low in the nucleus of nonselected DP thymocytes, although it was increased in selected thymocytes. c-Fos protein is newly synthesized in the cytoplasm and is thought to translocate into the nucleus afterward. As far as we examined, cytoplasmic c-Fos was also lower in DP cells than in SP cells (data not shown). Taken together, it may be ruled out that c-Fos protein in nonselected DP thymocytes is undetectable because of either protein degradation or decreased translocation from cytoplasm into nucleus. However, we would not deny the possible mechanism that degradation of c-Fos protein is down-regulated during thymocyte development, e.g., de-ubiquitinating enzymes, which may influence c-Fos expression, are activated in selected thymocytes.

A small increase of c-Fos protein was detected in the presence of proteasome inhibitor in the stimulation of selected DP thymocytes but not in nonselected DP cells, despite the fact that c-fos mRNA was equally induced in both DP cells (Fig. 6Go, B and C). This implies that c-Fos translation ability is acquired in the DP cell stage immediately after receiving positive selection, because DP cells of Tg-Posi expressed high TCR, CD5, and CD69, which mark thymocytes receiving positive selection signals (Fig. 1GoB). However, in the absence of proteasome inhibitors, c-Fos protein was almost undetectable even in selected DP cells, presumably because c-Fos’ synthesizing ability cannot overcome degradation. Alternatively, degradation activity of the protein may not be fully down-regulated during DP stages.

It is known that the translation process is mainly controlled at the initiation level and that translation initiation factor eIF-4E, which binds to the cap structure of mRNA, plays a key role in this process. Overexpression of eIF-4E in NIH3T3 cells results in accelerated cell growth and malignant transformation (36, 37). eIF-4E expression increases when peripheral T cells are activated (38, 39), which may allow T cells to proliferate and to produce cytokines. It is notable that the expression level of eIF-4E is higher in postselected DP and CD4SP cells than in preselected DP cells (Fig. 7Go), suggesting that positive selection signaling via TCR induces eIF-4E expression, as do TCR-mediated signals in peripheral T cells. It is known that 4E-binding protein-1/2 (4E-BP1/2) binds to eIF-4E and inhibits its binding to the cap structure (40). We found that 4E-BP1 is expressed at a higher level in nonselected DP cells than in selected thymocytes at the mRNA level (data not shown), which is consistent with findings previously reported (41). The reciprocal expression pattern of eIF-4E and 4E-BP in the two DP cells is correlated with the translational activity of each subset. Further analysis using gene-manipulated animals or cells is required for elucidating the precise role of such initiation-regulating molecules in thymocyte functional maturation.

To examine this issue, we have already established a novel retrovirus-mediated gene transfer system using reaggregation culture with thymic stromal-thymocyte interaction, which we will be using for future investigations.


   Acknowledgments
 
We thank N. Hamamura and Y. Okada for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture, Japan and a grant-in-aid from Kanagawa Academy of Science and Technology Research Grants and Core Research for Engineering, Science, and Technology. Back

2 Address correspondence and reprint requests to Dr. Sonoko Habu, Department of Immunology, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-1193, Japan. Back

3 Abbreviations used in this paper: DN, double negative; SP, single positive; CD4SP, CD4+8- SP; CD8SP, CD4-8+ SP; DP, double positive; IM, ionomycin; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; Tg, transgenic; eIF-4E, eukaryotic initiation factor 4E; MBP, myelin basic protein; ß2m, ß2-microglobulin; RAG2, recombinase-activating gene 2. Back

Received for publication November 4, 1999. Accepted for publication March 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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