HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/9/1577 00-0733fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SAVIGNAC, M. Articles by PELLETIER, L. Search for Related Content PubMed PubMed Citation Articles by SAVIGNAC, M. Articles by PELLETIER, L.

Protein kinase C-mediated calcium entry dependent upon dihydropyridine sensitive channels: a T cell receptor-coupled signaling pathway involved in IL-4 synthesis¹

MAGALI SAVIGNAC^*,2, ABDALLAH BADOU^*,2, MARC MOREAU, CATHERINE LECLERC, JEAN-CHARLES GUÉRY, PIERRE PAULET^*, PHILIPPE DRUET^*, JEANNIE RAGAB-THOMAS and LUCETTE PELLETIER^*3

^* INSERM U28,
INSERM U326 and the ‘Institut Fédératif de Recherche’ IFR 30, 31059 Toulouse Cedex France; and
CNRS, UMR 5547, Université Paul Sabatier, 31062 Toulouse, France

³Correspondence: INSERM U28, Pavillon Lefebvre, Place du Dr Baylac, 31059 Toulouse Cedex, France. E-mail: Lucette.Pelletier{at}purpan.inserm.fr

SPECIFIC AIM

CD4+T helper (Th) cells include effector Th1 cells, which produce interleukin 2 (IL-2) and interferon (IFN-), and IL-4-producing Th2 ceIls; these subsets have distinct functions and it has been suggested that low T cell receptor (TCR) –peptide interactions favor Th2 cell differentiation. Since signaling pathways involved in IL-4 production are poorly known, we investigated by which pathway IL-4 is produced after weak TCR engagement.

PRINCIPAL FINDINGS

1. IL-4 production on weak TCR engagement does not require a full pattern of tyrosine phosphorylation
The 2G12.1 T cell hybridoma produced a different set of cytokines depending on the strength of TCR engagement; when cells were stimulated on plates coated with 0.03 µg/ml anti-TCR mAb (defined as weak TCR stimulation), only IL-4 was produced, whereas these cells synthesized both IL-4 and IFN- when stimulated on plates coated with 1.6 µg/ml anti-TCR mAb (defined as strong TCR stimulation). We first studied the pattern of tyrosine phosphorylation by immunoblotting the lysates of cells after weak or strong TCR engagement with the 4G10 anti-phosphotyrosine mAb. The profile of tyrosine phosphorylation induced by strong TCR engagement did not differ from the one described in the literature, whereas the intensity of phosphorylated bands and their number were reduced upon weak TCR engagement (Fig. 1A ). In these conditions, the src kinase p56^lck appeared to be tyrosine phosphorylated, but there was no phosphorylation of the tyrosine kinase ZAP-70 (Fig. 1B ), the adapter molecule SLP-76, or phospholipase C (PLC)1 (Fig. 1C ).

View larger version (35K): [in this window] [in a new window]   Figure 1. Weak TCR stimulation induces an incomplete pattern of tyrosine phosphorylation. A) 2G12. 1 T cells were unstimulated (Ct) or stimulated for 5 min on plates coated with 0.03 or 1.6 µg/ml anti-TCR mAb. Cell lysates from 2G12.1 T cells were subjected to 7.5% SDS-PAGE and immunoblotted with the 4G10 anti-phosphotyrosine mAb. p56lck, ZAP-70, SLP-76, and PLC were identified by stripping the membranes and reblotting with the corresponding specific antibodies. The asterisk indicates the only band that persisted in lysates from cells treated with the src kinase inhibitor PP2, suggesting that this band was not specific. B) Tyrosine phosphorylation was assessed as in panel A by blotting the membrane with the 4G10 mAb (left), stripping the membrane, and reblotting with the anti-ZAP-70 mAb (right). C) PLC1 was immunoprecipitated and probed with 4G10 mAb or anti-PLC1 antibodies. D) The calcium response in 2G12.1 T cells loaded with indo1-AM and stimulated ([]) or not () on plates coated with 0.03 µg/ml anti-TCR mAb for various times.

2. Weak TCR stimulation induces a tyrosine kinase-dependent calcium entry necessary for IL-4 synthesis
A sustained calcium signal was induced by weak TCR stimulation in 2G12.1 T cells loaded with Indo1-AM. PP2 (10 µM), an inhibitor of src kinases, abolished any tyrosine phosphorylation, reduced the calcium response by 65% (P<0.005, n=5), and abolished IL-4 synthesis. Removal of extracellular calcium or BAPTA/AM, a chelator of intracellular calcium, suppressed the calcium signal and IL-4 secretion induced by weak TCR stimulation. Cyclosporin A, an inhibitor of calcineurin, also abolished IL-4 production. These data indicate that src kinase activation induced by weak TCR engagement provokes a Ca²⁺ entry resulting in IL-4 production.

3. Protein kinase C controls a dihydropyridine sensitive calcium response that could occur through L-type-related calcium channels
HgCl₂, a chemical that triggers a polyclonal activation of Th2 cells and autoimmunity in Brown-Norway rats, used a signaling pathway implying a protein kinase C (PKC) -dependent influx of Ca²⁺ through L type calcium channels (LTCC) and resulting in IL-4 gene transcription by some T cells. We investigated whether such a pathway could be activated upon weak TCR stimulation in 2G12.1 T cells. By using primers specific for the 1 subunits of the LTCC, we showed that 2G12.1T cells expressed 1LTCC mRNA. LTCC are known to bind dihydropyridine and 2G12.1 T cells were specifically labeled with ST-Bodipy dihydropyridine (Fig. 2A ). The LTCC agonist or PMA, a PKC activator, induced an entry of calcium that was antagonized by R(+)-BayK 8644, a LTCC blocker (Fig. 2B ). S(-)-BayK 8644 (10 µM) per se triggered IL-4 gene expression after 1 h of stimulation as assessed by real-time quantitative PCR. In addition, PMA-induced IL-4 secretion was also blocked by R(+)-BayK 8644 (Fig. 2C ). Ro-31–82220, a PKC inhibitor, or R(+)-BayK 8644 diminished the calcium response and IL-4 production induced by weak TCR engagement (Fig. 2B , C and not shown). PKC was translocated to the cell membrane as soon as 1 min after stimulation (Fig. 2D , right), indicating that PKC was actually recruited at the cell membrane upon weak TCR stimulation.

View larger version (22K): [in this window] [in a new window]   Figure 2. Weak TCR stimulation induces a calcium response. A) 2G12.1 T cells were labeled with ST-bodipy dihydropyridine (left), and this staining was abolished by preincubation with an excess of unlabeled agonist LTCC, S(-)-Bay K 8644 (right). B) The calcium response in 2G12.1 T cells loaded with fluo3-AM and stimulated with the LTCC agonist S(-)-Bay K 8644 (BayK-, 10 µM), PMA (10 ng/ml), or microsphere-bound anti-TCR mAb in the presence or absence of the LTCC antagonist R(+)-Bay K 8644 (Bay K+, 10 µM) or of the PKC inhibitor Ro-31–8220 (RO, 2.5 µM). C) IL-4 production by cells stimulated with PMA or with microsphere-bound anti-TCR mAb for 24 h in the presence or absence of BayK+ (10 µM), cyclosporin A (CsA, 0.1 µg/ml) or RO (2.5 µM). D) Translocation of PKC in 2G12.1 T cells that were unstimulated (left) or stimulated for 1 min (right) on plates coated with 0.03 µg/ml anti-TCR mAb, as visualized by confocal microscopy after staining with fim-1, a fluorescent probe that binds the catalytic site of PKC.

4. TCR-induced IL-4 synthesis is dependent on a dihydropyridine sensitive Ca²⁺ response in Th2 cells
The conalbumin specific D10.G4.1Th2 clone was also specifically labeled with ST-Bodipy dihydropyridine. PMA induced IL-4 production, which was abolished by the PKC inhibitor, the LTCC blocker, and cyclosporin A. R(+)-BayK 8644 partially inhibits TCR-dependent IL-4 production. This shows that the pathway contributes to TCR-induced IL-4 synthesis. In addition, S(-)-BayK 8644 elicits a calcium response in transgenic D011.10 T cells differentiated in Th2 cells, but not in T cells differentiated in Th1 cells, suggesting that a dihydropyridine sensitive calcium response may be induced in Th2 cells only.

CONCLUSIONS

TCR engagement triggers activation of tyrosine kinases of the src and syk family. This results in a cascade of tyrosine phosphorylation leading to activation of several downstream signaling pathways. Among them, phospholipase C is activated, which is essential to generate a calcium response and PKC activation. However, this scheme does not account for IL-4 production, which is still observed in the absence of optimal conditions of stimulation. We show here that tyrosine phosphorylation in 2G12.1 T cells is gradual, depends on the strength of TCR engagement, and controls the type of cytokines produced. Upon weak TCR stimulation, the intensity of tyrosine phosphorylation and the diversity of phosphorylated proteins were reduced as compared to strong stimulation. For example, p56^lck was only faintly phosphorylated and ZAP-70, SLP-76, or PLC1 phosphorylation was undetectable. However, tyrosine kinases are involved in controlling the calcium response and IL-4 production induced by weak TCR engagement, since PP2 abolished TCR-induced tyrosine phosphorylation and suppressed both the calcium response and IL-4 production induced by TCR engagement. The calcium-dependent transcription factor NFAT that plays an essential role in IL-4 gene transcription is likely to be activated as a consequence of the TCR-induced calcium response since cyclosporin A, which blocks calcineurin, a Ca²⁺-dependent phosphatase responsible for nuclear translocation of NFAT, abolished IL-4 expression. The original finding of this study is that weak TCR stimulation recruits PKC, which allows a dihydropyridine-sensitive entry of calcium, resulting in IL-4 production. It is likely that the channels involved are L-type calcium-related channels. These conclusions are schematized in Fig. 3 . The arguments that support the existence of such a pathway are 1) 2G12.1 T cells express LTCC and the cells specifically bind dihydropyridine, which characterizes LTCC. An agonist of LTCC induces a calcium signal and IL-4 gene expression in 2G12.1 T cells; 2) PMA, a PKC activator, induces a calcium response and IL-4 production that were markedly reduced by an inhibitor of PKC or by an antagonist of LTCC; and 3) TCR-induced calcium signal or IL-4 production was suppressed by the PKC inhibitor or the LTCC antagonist. Finally, the expression of LTCC is not restricted to the T cell hybridoma since functional LTCC are also found in the D10.G4.1 Th2 clone.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic representation of the signaling pathway involved in IL-4 production on weak TCR stimulation in the 2G12.1 T cell hybridoma. Depending on the strength of TCR engagement, TCR stimulation induces a tunable activation of downstream signaling pathways. Weak TCR stimulation, although unable to activate all the TCR-associated signaling pathways, induces a PKC-dependent calcium response through L type calcium channels (LTCC), resulting in IL-4 production. Strong TCR stimulation is required for IFN- to be produced and amplifies IL-4 synthesis. Tyr-P = tyrosine phosphorylation; PKC = protein kinase C; PLC = phospholipase C; LTCC = L type calcium channels

How TCR is coupled to PKC remains to be elucidated. PLC1-dependent PKC activation seems unlikely, since weak TCR stimulation does not trigger detectable tyrosine phosphorylation of PLC1. Other PLC may be involved. Alternatively, src kinase might interact directly with PKC as recently shown for PKC and src in T lymphocytes. Finally, LTCC have been shown to contribute to the Ca²⁺ signal induced by stimulation through B cell receptor in B cells, and a crucial role for cGMP suggests a possible involvement for cGMP-dependent protein kinase. Such a possibility remains to be explored in T lymphocytes.

Identification of a new signaling pathway involved in IL-4 production could have therapeutic consequences in the treatment of Th2-dependent diseases by blocking this pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0733fje ; to cite this article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0733fje

² M.S. and A.B. contributed equally to this work.

This article has been cited by other articles:

				B. Gomes, M. D. Cabral, A. Gallard, M. Savignac, P. Paulet, P. Druet, B. Mariame, M. Moreau, C. Leclerc, J.-C. Guery, et al. Calcium Channel Blocker Prevents T Helper Type 2 Cell-mediated Airway Inflammation Am. J. Respir. Crit. Care Med., June 1, 2007; 175(11): 1117 - 1124. [Abstract] [Full Text] [PDF]

				A. Badou, M. K. Jha, D. Matza, W. Z. Mehal, M. Freichel, V. Flockerzi, and R. A. Flavell Critical role for the beta regulatory subunits of Cav channels in T lymphocyte function PNAS, October 17, 2006; 103(42): 15529 - 15534. [Abstract] [Full Text] [PDF]

				B. Gomes, M. Savignac, M. D. Cabral, P. Paulet, M. Moreau, C. Leclerc, R. Feil, F. Hofmann, J.-C. Guery, G. Dietrich, et al. The cGMP/Protein Kinase G Pathway Contributes to Dihydropyridine-sensitive Calcium Response and Cytokine Production in TH2 Lymphocytes J. Biol. Chem., May 5, 2006; 281(18): 12421 - 12427. [Abstract] [Full Text] [PDF]

				C. M. Berchtold, K.-S. Chen, S. Miyamoto, and M. N. Gould Perillyl Alcohol Inhibits a Calcium-Dependent Constitutive Nuclear Factor-{kappa}B Pathway Cancer Res., September 15, 2005; 65(18): 8558 - 8566. [Abstract] [Full Text] [PDF]

				L. Stokes, J. Gordon, and G. Grafton Non-voltage-gated L-type Ca2+ Channels in Human T Cells: PHARMACOLOGY AND MOLECULAR CHARACTERIZATION OF THE MAJOR {alpha} PORE-FORMING AND AUXILIARY {beta}-SUBUNITS J. Biol. Chem., May 7, 2004; 279(19): 19566 - 19573. [Abstract] [Full Text] [PDF]

				M. Savignac, B. Gomes, A. Gallard, S. Narbonnet, M. Moreau, C. Leclerc, P. Paulet, B. Mariame, P. Druet, A. Saoudi, et al. Dihydropyridine Receptors Are Selective Markers of Th2 Cells and Can Be Targeted to Prevent Th2-Dependent Immunopathological Disorders J. Immunol., May 1, 2004; 172(9): 5206 - 5212. [Abstract] [Full Text] [PDF]

				S. Weiss, T. Doan, K. E. Bernstein, and N. Dascal Modulation of Cardiac Ca2+ Channel by Gq-activating Neurotransmitters Reconstituted in Xenopus Oocytes J. Biol. Chem., March 26, 2004; 279(13): 12503 - 12510. [Abstract] [Full Text] [PDF]

				M. F. Kotturi, D. A. Carlow, J. C. Lee, H. J. Ziltener, and W. A. Jefferies Identification and Functional Characterization of Voltage-dependent Calcium Channels in T Lymphocytes J. Biol. Chem., November 21, 2003; 278(47): 46949 - 46960. [Abstract] [Full Text] [PDF]

				Y. Blumenstein, N. Kanevsky, G. Sahar, R. Barzilai, T. Ivanina, and N. Dascal A Novel Long N-terminal Isoform of Human L-type Ca2+ Channel Is Up-regulated by Protein Kinase C J. Biol. Chem., January 25, 2002; 277(5): 3419 - 3423. [Abstract] [Full Text] [PDF]

				S. Meehan, A. J. Wu, E. C. Kang, T. Sakai, and I. S. Ambudkar Interferon-gamma induces a decrease in the intracellular calcium pump in a human salivary gland cell line Am J Physiol Cell Physiol, December 1, 1997; 273(6): C2030 - C2036. [Abstract] [Full Text] [PDF]

				R. Zanner, G. Hapfelmeier, M. Gratzl, and C. Prinz Intracellular signal transduction during gastrin-induced histamine secretion in rat gastric ECL cells Am J Physiol Cell Physiol, February 1, 2002; 282(2): C374 - C382. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/9/1577 00-0733fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SAVIGNAC, M. Articles by PELLETIER, L. Search for Related Content PubMed PubMed Citation Articles by SAVIGNAC, M. Articles by PELLETIER, L.