Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway.

ARTICLE

INTRODUCTION

The association of the Src family protein tyrosine kinase (PTK) Lck with the interleukin-2 receptor (IL-2R) beta-chain has been reported by several groups [1, 2]. Lck interacts with the acidic domain of IL-2R beta-chain, and this interaction is necessary for Lck activation upon IL-2 stimulation [1-6]. The serine-rich region of IL-2R beta-chain is also necessary for Lck activation and the induction of the nuclear proto-oncogenes c-fos and c-jun [2, 7-9]. However, the actual contribution of Lck to IL-2-induced proliferation and cell survival is still unclear. An IL-2R beta-chain mutant lacking the acidic region is able to transmit IL-2-induced cell proliferation in BAF-B03 cells, indicating that Lck does not play a vital role in IL-2-mediated B cell proliferation. However, in this cell system, a constitutively active form of Lck (LckF505) was able to cooperate with either c-Myc or Bcl-2 in the induction of cell proliferation, suggesting that Lck has some potential for regulating growth of B cells [10, 11]. Furthermore, BAF-B03/LckF505 cells showed prolonged cell survival upon growth factor deprivation (EGF), suggesting that Lck might be involved in the regulation of B cell survival as well [11].

Also, little is known about the role of Lck in the regulation of growth and survival of T cells. An Lck negative variant of the murine IL-2-dependent cytotoxic T cell line, CTLL-2, showed only a modestly decreased proliferative response to IL-2, suggesting that Lck ­ also in T cells ­ might not play an essential role in IL-2-induced growth [12]. It is, however, an open question whether other members of the Src kinase family substitute for Lck when Lck is absent. Upon IL-2 stimulation, only Lck is activated among the Src kinases family in T cells, however, at least Fyn and Lyn were activated by IL-2-in an Lck-deficient pro-B-cell line, suggesting that other members of the Src kinase family can potentially take over Lck functions in IL-2R signaling [13-15]. Since Lyn is not expressed and Src is expressed only negligibly, or not at all, in normal T cells, the best candidate for Lck substitution, in Lck-negative cell lines, is Fyn [16].

One of the pathways involved in mitogen-mediated proliferation is the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway. It is believed that the MAPK pathway is activated by IL-2 through an activation of the p21 Ras pathway (3,17-19). A study using transfected fibroblasts showed that the acidic region of the IL-2R beta-chain is indispensable for MAPK phosphorylation, suggesting that Lck might play a role in IL-2-mediated MAPK activation [20]. Here, we investigated the role of Lck in IL-2-induced MAPK activation, proliferation, and cell survival in human T cells. Taking advantage of an Lck negative T cell line and the newly developed inhibitor of the Src kinase family, PP1 [21], we also investigated the role of other members of the Src kinase family when Lck is absent.

METHODS

Cells

A tumor T cell line (MyLa2059) was established from a plaque biopsy specimen from a patient with mycosis fungoides as described in detail elsewhere [22, 23]. The tumor cell line used in this study proliferated beyond 1,000 population doublings. MyLa2059 and Jurkat T leukemia cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 100 mug/ml penicillin and 100 mug/ml streptomycin. Antigen specific, human, CD4⁺ T cell lines were obtained from healthy donors, and have been described in detail previously [24].

Antibodies and other reagents

Phospho-specific p44/42 (MAPK/Erk) mAb recognizing only activated Erk was from New England Biolabs (Beverly, MA, USA). Phospho-specific tyrosine mAb (4G10) was from Upstate Biotechnology (Lake Placid, NY, USA). Anti-Erk2 pAb and anti-Lck mAb were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human IL-2 (generous gift from Craig W. Reynolds, National Cancer Institute, Bethesda, MD) was used at 500 U/ml unless otherwise indicated. The Src kinase family inhibitor PP1, was from Biomol (Plymouth Meeting, PA, USA).

Immunoprecipitation and Western blotting

Following treatment with or without rIL-2 and with or without PP1, the cells (2 x 10⁶ MyLa2059 or 1 x 10⁷ CD4⁺ T cells) were rapidly pelleted, and the reaction stopped by lysing the cells in ice-cold lysis buffer (1% NP-40, 20 mM Tris-HCL pH 8.0, 137 mM NaCl, 10% glycerol with the following inhibitors: 1 mM PMSF, 5 mM EDTA, 1 mM Na₃VO₄, 10 mug/ml aprotinin, 10 mul/ml IAA, 10 mM NaF). Total cell lysate proteins were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were evaluated using enhanced chemiluminescence (ECL), stripped, and reprobed according to the manufacturer´s manual (Amersham).

Isolation of RNA and RT-PCR

Total cellular RNA from MyLa2059 cells and Jurkat cells was isolated by a modification of the procedure of Sambrook et al. [25, 26]. Purified RNA was reverse transcribed using cloned Moloney murine leukemia virus reverse transcriptase and either the Lck specific "downstream2" primer 5´-CTTGTCGAATTCGAAGTCCCGGACCGACAGTGA-3´ (incl. EcoRI site), the Lck specific "downstream4" primer 5´-CTTGTCGAATTCTCAAGGCTGAGGCTGGTACTG-3´ (incl. EcoRI site), or the control CD3-gamma specific "downstream" primer 5´-TGAGTCGAATTCGGATCCTCAATTCCTCCTCAACTG-3´ (incl. EcoRI and BamHI sites). The resulting cDNA was amplified by PCR using recombinant Taq DNA polymerase and either the Lck specific "upstream2" primer 5´-CTTCATTCTAGACTCCCGGGCTGGGCAGGGACC-3´ (incl. XbaI site), the Lck specific "upstream4" primer 5´-CTTCATTCTAGATTCACCATCAAGTCAGATGTG-3´ (incl. XbaI site), or the control CD3-g specific "upstream" primer 5´-CAAGGACCCTGCAGGGATGTATCA-3´. This potentially creates fragments with the size of 555 (up/down 2), 306 (up/down 4), or 339 (CD3-gamma) nucleotides. The samples were analysed in a 1% agarose gel.

Plasmids

The cDNA for Lck was cloned into the pEF vector as described previously [27].

Stable transfection

MyLa2059 cells (20 x 10⁶) were transfected with 25 mug Lck cDNA in pEF vector by electroporation at 960 FF and 240 V. Two days after electroporation, cells were pelleted and resuspended in normal growth medium with the addition of 1.5 mg/ml Geneticin (G-418, Gibco) to a concentration of 5 x 10⁴ cells/ml. 200 mul was added to flat-bottom, microtiter wells (NUNC, Denmark). Media including G-418 was added on days 5 and 10. Surviving transfectants were grown in normal growth medium with the addition of 1 mg/ml G-418 and tested for Lck expression by Western blot.

Proliferation assay

10⁴ human CD4⁺ T cells were incubated for 1, 3, or 5 days as triplicates, with or without PP1 (10 muM) and with or without 500 U/ml IL-2, in flat-bottom, microtiter wells (Falcon 3072, BD) in culture medium with 10% heat-inactivated, filtered, pooled human serum. Twelve hours before harvest, [³H]thymidine [1.0 muCi per well (1 muCi = 37 kBq)] was added, the cells harvested onto glass fiber filters, and [³H]thymidine incorporation was measured.

Quantification of apoptotic cells

Apoptotic cells were quantified by the use of the DNA binding dye 7-amino-actinomycin D (7-AAD ­ Sigma). 10⁶ cells were stimulated with 1) increasing concentrations of inhibitor (PP1) and with or without IL-2 for 3 days or 2) with increasing concentration of IL-2 and with or without PP1 for 3 days, harvested, washed once with PBS, resuspended in 0.5 ml 0.05% saponin in PBS, and pelleted. The cells were resuspended in 0.5 ml 4 mug/ml 7-AAD in 0.05% saponin in PBS and incubated at room temperature, in the dark. Untreated cells were used to set gates for viable cells with 2N-4N DNA, for apoptotic cells with subnormal DNA content, and for exclusion of debris.

RESULTS

Inhibition of proliferation, but not survival signals, by the Src kinase family inhibitor PP1

Human CD4⁺ T cell lines were incubated with or without IL-2 and/or PP1 for up to 5 days. The proliferation was measured at days 1, 3, and 5, by adding [³H]thymidine twelve hours before the cells were harvested. As expected, IL-2-induced an almost exponential growth of IL-2 responsive CD4⁺ T cells (Figure 1A). The Src kinase inhibitor, PP1, induced a substantial inhibition of IL-2-driven proliferation. Thus, PP1 induced approximately 50% inhibition of [³H]thymidine uptake in all of the five CD4⁺ T cell lines tested (Figure 1A, and data not shown). The inhibition was concentration-dependent reaching a maximal effect (i.e. 50% inhibition) at PP1 concentrations at and above10 muM (Figure 1A and data not shown), which was in accordance with the reported concentrations required for maximal inhibition of Lck activity in intact T cells [21].

Besides the proliferative signals, IL-2 triggers anti-apoptotic (survival) signals. Therefore, we investigated whether PP1 inhibited IL-2-mediated T cell survival or whether apoptosis/survival and proliferation were regulated differently by PP1. As shown in Figure 1B, approximately 75% apoptosis was observed in T cell cultures incubated for 48 hours without IL-2. As expected [28], IL-2-induced a drop in apoptosis to levels below 50% (Figure 1B), i.e. IL-2-induced a 40-50% rescue from apoptosis in these CD4⁺ T cell lines. Addition of PP1 had no effect on the level of apoptosis in either medium or IL-2-treated T cells (Figure 1B). Since low concentrations of IL-2 can induce cell survival without proliferation, we addressed whether cell survival at suboptimal IL-2 concentrations was sensitive to inhibition by PP1. Accordingly, cells were incubated with or without PP1 and increasing concentrations of IL-2 prior to flow cytometric analysis of cell survival. As shown in Figure 1C, PP1 had no effect on IL-2-mediated cell survival ­ even at concentrations (20 U/ml) which induce cell survival and where the proliferation was substantially reduced (Figure 1C and data not shown). As Lck is the principal members of the Src kinase family activated upon IL-2 stimulation, these findings suggest that Lck is involved in the IL-2-induced proliferation, but not cell survival, of human CD4⁺ T cells. The tumor T cell line (MyLa2059) proliferate independently of IL-2, thus we cannot use this cell line to support the above results seen in normal T cells.

PP1 has little effect on IL-2-induced MAPK activation

As MAPK activation is important for induction of proliferation by IL-2, we investigated whether PP1 would have the same effect on MAPK activation as on proliferation. CD4⁺ T cells were incubated with 0, 2, or 10 muM PP1 over-night followed by 5 min of stimulation with medium or IL-2. As seen in Figure 2 (top panel), 2 muM PP1 had no effect on IL-2-induced MAPK phosphorylation/activation, whereas 10 muM weakly inhibited the MAPK activation. The control blot (middle panel) showed the same amount of Erk protein in every sample, and the phospho-specific tyrosine blot (bottom panel) showed inhibition of several IL-2-induced phospho-tyrosine proteins by PP1 (10 muM more effectively than 2 muM), indicating that PP1 was working satisfactorily. To exclude the possibility that residual activity of Lck caused the MAPK activation, we performed similar experiments with PP1 at concentrations up to 50 muM, but even at these supraoptimal concentrations, PP1 had no effect on MAPK activation (data not shown). Taken together, the findings suggest that the inhibition of proliferation by PP1 is not caused by inhibition of MAPK.

The mycosis fungoides tumor T cell line, MyLa2059, is Lck negative

Upon testing the mycosis fungoides tumor T cell line, MyLa2059, we were unable to detect Lck protein by Western blotting (Figure 3A, lane 2), and therefore we investigated whether any Lck RNA was expressed at all in this line. Total RNA was purified followed by RT-PCR with the relevant primers (see methods). As shown in Figure 3B, both Jurkat and MyLa2059 cells expressed CD3-gamma chain RNA (lane 3 and 6) (339 bp fragment). By using two different primer sets we looked for Lck RNA, and two bands at the correct size appeared when testing the Jurkat cells (lane 1 and 2) (555 and 306 bp fragments), but not when testing the MyLa2059 cells (lane 4 and 5). This indicates that the MyLa2059 cell line does not express any Lck RNA.

Lck is not essential for IL-2-induced MAPK activation in MyLa2059 cells

To further investigate whether Lck is necessary for IL-2-induced MAPK activation, we took advantage of the Lck negative MyLa2059 cell line. Non-malignant CD4⁺ T cells and MyLa2059 cells were stimulated with medium or IL-2 for up to 20 min and cell lysates were tested for phospho-specific p44/42 (Erk) by Western blotting. In CD4⁺ T cells, the MAPK activation peaked at 3 min, whereas the activation peaked at 5 min using MyLa2059 cells (Figure 4A upper panel, lane 3 and 10). Furthermore, at all other time intervals the kinetics in the MyLa2059 cells were sligthly delayed compared with CD4⁺ T cells. As seen from the bottom panel, the Erk level is comparable in all lanes. This indicates that Lck is not essential for IL-2-induced MAPK activation in MyLa2059 cells. To investigate whether other Src kinase family members had substituted for Lck and possibly caused the delay in the kinetics of MAPK activation, MyLa2059 cells were incubated with medium or PP1 overnight, followed by medium or IL-2 stimulation for 5 min (Figure 4B). Phospho-specific p44/42 blot showed that PP1 had no effect on MAPK activation, suggesting that Lck and other Src kinases are not essential for IL-2-induced MAPK activation in MyLa2059 cells, which agrees with the results shown in Figure 2. Furthermore, the phosphotyrosine blot (lower panel) showed that IL-2-induced tyrosine phosphorylation of the proteins around 40 kDa, 55 kDa, 85 kDa, and 110 kDa was unaffected by PP1, which was not the case for normal T cells (Figure 2, lower panel). This suggests that Fyn or another Src kinase family members might not substitute for Lck in MyLa2059 cells and that a PP1 insensitive kinase might partly substitute for Lck in MyLa2059 cells.

Reintroducing Lck into MyLa2059 cells had no effect on MAPK activation

To investigate whether the delay in MAPK activation observed in MyLa2059 cells was due to the Lck deficiency, Lck was stably transfected into MyLa2059 cells (MyLa2059-Lck). MyLa2059 and MyLa2059-Lck cells were stimulated with medium or IL-2 for 5 min and the cell lysate was tested for the presence of Lck by Western blotting. As shown in Figure 5A, Lck was only expressed in MyLa2059-Lck cells, and furthermore, the slowly migrating Lck was dominant after IL-2 stimulation. Next, we compared the kinetics of MAPK activation between MyLa2059 and MyLa2059-Lck cells. Again the cells were stimulated with IL-2 for up to 20 min, and the cell lysate was subjected to phospho-specific p44/42 by Western blotting. The blot showed no difference in MAPK activation between MyLa2059 and MyLa2059-Lck cells, indicating that the delay in MAPK activation observed in MyLa2059 cells was not caused by Lck deficiency (Figure 5B).

DISCUSSION

Among the Src kinase family, mainly Lck and Fyn are expressed in T cells, and only Lck seems to be involved in IL-2R signaling. It has been suggested that Lck is involved in IL-2-induced proliferation and cell survival, but studies with Lck negative cell lines or cell lines where the acidic domain of the IL-2R beta-chain (Lck-binding) has been removed, argue against a role for Lck in IL-2-induced proliferation. Thus, we decided to look further into the role of Lck in IL-2-induced proliferation and cell survival using human CD4⁺ T cell lines from healthy donors. When using the Src kinase family inhibitor, PP1, the IL-2-induced proliferation was reduced by up to 50% in CD4⁺ T cells, indicating that Lck is somehow involved in IL-2-induced proliferation. The inhibition of proliferation by PP1 was concentration-dependent, reaching a maximal effect at 10 muM, suggesting that 10 muM PP1 is enough to block the Src kinase family activity. When looking at cell survival, IL-2-induced a 40-50% rescue from apoptosis in CD4⁺ T cells at a concentration of 500 U/ml. Unexpectedly, PP1 had no effect on cell survival at any IL-2 concentration, indicating that Lck is not involved in cell survival of non-cancerous CD4⁺ T cells. This does not agree with findings in pre-B cell lines by Miyazaki et al., but they used a BAF-B03 derived cell line with stable expression of epidermal growth factor receptor (EGFR) and an active form of Lck [11]. Miyazaki et al. observed that BAF-B03 cells deprived of EGF survived for a longer time compared with the same cell line without active Lck, suggesting that Lck activation results in the suppression of apoptosis. The two distinct results may be explained by the different cell types and different signaling pathways, or by the fact that we have used a "negative" approach using an inhibitor of Lck and other members of the Src kinase family, whereas Miyazaki et al. used a "positive" approach with transfection of a constitutivly active Lck.

As MAPK activation is important for cell proliferation, and an association between Lck and MAPK upon IL-2 stimulation has been reported [29], we investigated whether the involvement of Lck in proliferation was mediated through the MAPK pathway. Our results showed only a slight inhibition of IL-2-induced MAPK activation when CD4⁺ T cells were pre-incubated with PP1. The phosphotyrosine blot showed that PP1 did not block IL-2-induced protein phosphorylation of all the induced proteins. To investigate whether residual Lck activity was responsible for MAPK activation, we included higher concentrations of PP1, but even at supraoptimal concentrations PP1 had no or little effect on MAPK activation in human T cells. In order to confirm this observation in another system, we took advantage of the tumor T cell line, MyLa2059, which we found was Lck negative. When comparing the MAPK activation in CD4⁺ T cells and MyLa2059 cells, the activation in MyLa2059 cells was a little delayed, but otherwise normal, indicating that Lck is not a prerequisite for IL-2-induced MAPK activation in human T cells. As Fyn was activated by IL-2-in an Lck-deficient pro-B-cell line, the delay could be explained by Fyn substituting for Lck [13]. To address this, we tested the MAPK activation in MyLa2059 cells after pre-incubation with PP1, and found that PP1 had no effect on IL-2-induced MAPK activation. Furthermore, the phosphotyrosine blot of MyLa2059 cells (Figure 4B, lower panel) showed that among the IL-2 phosphotyrosine-induced proteins, only the proteins around 150 kDa were inhibited by PP1. The proteins at about 40 kDa, 55 kDa, 85 kDa, and 110 kDa were unaffected by PP1, which was not the case for normal T cells (Figure 2, lower panel), suggesting that PP1-sensitive members of the Src kinase family (incl. Fyn) are not substituting for Lck in IL-2-induced MAPK activation. We also investigated whether the delay in IL-2-induced MAPK activation seen in MyLa2059 cells compared to normal T cells, was Lck-dependent. Thus, a MyLa2059 cell line was stably transfected with Lck. However, the MAPK activation was unchanged compared to non-transfected MyLa2059 cells. This supports the conclusion above that Lck is not involved in IL-2-induced MAPK activation in T cells.

The pathways leading to IL-2-induced proliferation in T cells are very obscure. Miyazaki et al. suggested that activated Lck could promote cell cycle progression in the absence of cytokines when coexpressed with either c-Myc or Bcl-2 [11]. This indicates that Lck might work through pathways involving c-Myc and/or Bcl-2, but the exact nature of these pathways have still to be elucidated.

CONCLUSION

In conclusion, our results indicate that Lck is involved in IL-2-induced proliferation, but not cell survival, in human T cells through a MAPK-independent pathway.

Abbreviations
MAPK Mitogen activated protein kinase
IL-2 Interleukin-2
PTK Protein tyrosine kinase
EGF Epidermal growth factor

Acknowledgements. We are very grateful to Gitte Kølander for technical assistance. This work was supported in part by the University of Copenhagen (Ph.D. programme for JB), the Danish Medical Research Council, the Danish Biotechnological Center for Cellular Communication (CCC), the Danish Allergy Research Center, the Novo Nordic Foundation, the Danish Medical Association Research Foundation (Lægeforeningens Forskningsfond), the Weimann Foundation (MN grant), the Beckett Foundation, the Danish Cancer Research Foundation (Dansk Kræftforsknings fond), the Carlsberg Foundation, and the Danish Cancer Society (Kræftens Bekæmpelse).

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