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Mechanisms involved in α6β1-integrin-mediated Ca2+ signalling
Cellular Signalling 13 (2001) 895 – 899
Mechanisms involved in a6b1-integrin-mediated Ca2+ signalling Hella Scho¨ttelndreier a, Barry V.L. Potterb, Georg W. Mayra, Andreas H. Gusea,* a
Institute for Medical Biochemistry and Molecular Biology, Division of Cellular Signal Transduction, University of Hamburg, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany b Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK Received 6 April 2001; accepted 13 June 2001
Abstract Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting a6b1-integrin-mediated Ca2 + signalling. Both Ca2 + release from thapsigargin-sensitive Ca2 + stores and capacitative Ca2 + entry via Ca2 + channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of a6b1-integrin-mediated Ca2 + signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca2 + -releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Integrin; Extracellular matrix; Ca2+ signalling; Inositol 1,4,5-trisphosphate; Cyclic ADP-ribose; Capacitative Ca2+ entry; SKF 96365
1. Introduction Peripheral T-cells are activated by antigenic peptides presented on MHC molecules by antigen-presenting cells. Activation of intracellular signalling events via the T-cell receptor/CD3 (TCR/CD3) complex and additional T-cell surface receptors, such as CD4/CD8 and CD28, comprises several pathways, including tyrosine phosphorylation, activation of the MAP-kinase pathway, the PI-3-kinase pathway, and activation of Ca2 + signalling [1,2]. Once activated, T-cells express a different set of cell surface antigens, known as ‘‘activation antigens,’’ e.g. HLA DR (MHCII), CD25, CD69, and integrins [3 – 5]. Integrins facilitate binding of T-cells to other cell types, e.g. endothelial cells, which is very important to stabilise the weak, selectin-based T-cell – endothelial cell interaction [6,7]. Subsequently, activated T-cells transmigrate through the endo-
Abbreviations: [Ca2+]i, intracellular free Ca2+ concentration; cADPR, cyclic adenosine diphosphoribose; ECM, extracellular matrix; EMM, extracellular minimal medium; InsP3, D-myo-inositol 1,4,5-trisphosphate; mAb, monoclonal antibody; PBS, phosphate-buffered saline; TCR/CD3, T-cell receptor/CD3 * Corresponding author. Tel.: +49-40-42803-2828; fax: +49-40-428039880. E-mail address: [email protected] (A.H. Guse).
thelial cell layer to access area of inflammation and/or infection. During this migration, T-cells make contact with proteins of the extracellular matrix (ECM), either in the basement membrane or in the interstitium. We have previously shown that T-cells, either human Jurkat T-cells or T-cell blasts, respond to the ECM proteins laminin, fibronectin, collagen types I, IV, and VI, and tenascin by a sustained but relatively small elevation of the free cytosolic Ca2 + concentration ([Ca2 + ]i) [8]. Ca2 + signalling by these ECM proteins was shown to proceed specifically via the corresponding b1-integrins. In contrast, vitronectin via avb3-integrin did not activate Ca2 + signalling [8]. Recently, we demonstrated that enhanced physical attachment of Jurkat T-cells, which was achieved by cocoating of ECM proteins with poly-L-lysine, significantly increased the amplitude of Ca2 + signals stimulated by laminin and collagen type IV [9]. Importantly, we showed that the stimulatory effects were transmitted exclusively via the corresponding b1-integrins [9]. T-cell spreading, which was also observed on coverslips co-coated with laminin and poly-L-lysine, was mediated by protein kinase C (PKC) but not by [Ca2 + ]i [9]. Both b1-integrin-mediated Ca2 + signalling and spreading was inhibited by the tyrosine kinase inhibitor genistein [9]. However, the mechanisms involved in b1-integrin-mediated Ca2 + signalling were not investigated in detail.
0898-6568/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 1 ) 0 0 2 2 5 - X
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Here, we show that Ca2 + signalling stimulated by laminin via a6b1-integrin involves src kinases, probably p59fyn and/or p56lck, and the second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cyclic adenosine diphosphoribose (cADPR). Furthermore, we demonstrate that Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry via SKF 96365-sensitive Ca2 + channels are major components of a6b1-integrin-mediated Ca2 + signalling.
2. Materials and methods 2.1. Materials The ECM protein laminin I, isolated from EHS mouse tumour, was obtained from Life Technologies (Karlsruhe, Germany). Poly- L -lysine hydrobromide, M w 30,000 – 70,000, was purchased from Sigma (Deisenhofen, Germany). The enzyme inhibitors U73122, U73343, and thapsigargin and the inhibitors for the Ca2 + channels SKF 96365, nitrendipine, and verapamil were obtained from Calbiochem Novabiochem (Bad Soden, Germany). The cADPR antagonist 7-deaza-8-Br-cADPR was synthesized as described (Ref. [10] and references therein). All other chemicals used were of the highest purity grade available. MilliQ water (Millipore Waters, Eschborn, Germany) was used for the preparation of all buffers.
Cells were loaded with the Ca2 + -sensitive fluorophore FURA2-AM (Calbiochem) as follows: 107 cells in 1 ml complete RPMI 1640 medium were incubated with 4 mM FURA2-AM for 15 min at 37 C, diluted fourfold with complete RPMI 1640 medium and incubated for additional 15 min at 37 C. After washing the cells twice with extracellular minimal medium (EMM; 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM HEPES, 1 mM Na2HPO4, 5.5 mM glucose, pH 7.4), the cells were resuspended in 4 ml EMM. For Ca2 + measurements, 40 ml cell suspension and 60 ml EMM were added into the chamber on the coated glass coverslip and mounted on the stage of an inverted fluorescence microscope (Axiovert 100; Zeiss, Oberkochingen, Germany). The excitation wavelengths for the ratiometric analysis were 340 and 380 nm, and the emission beam was filtered at 510 nm and monitored by a CCD camera (C240077, Hamamatsu). In each experiment, the fluorescent images of an optical field containing about 20 cells for excitation at 340 and 380 nm as well as the ratio 340:380 were displayed and stored separately on hard disk. Usually, images were taken every 20 s. Offline analyses of stored image data were then carried out using the ImageMaster software (Photon Technology/PhotoMed, Wedel, Germany). So-called regions of interest were set to cover the whole area of a single cell. The numerical median ratio and the corresponding free [Ca2 + ]i for each single cell were then calculated by the software using an external calibration curve.
2.2. Cell culture Jurkat T-lymphocytes (subclone JMP) were cultured as described previously [9] in RPMI 1640 medium containing Glutamax I (Life Technologies, Eggenstein, Germany), buffered by HEPES (20 mM, pH 7.4) and supplemented with newborn calf serum (7.5%), penicillin (100 U/ml), and streptomycin (50 mg/ml; all from Life Technologies, Eggenstein, Germany). The cells were cultured at 37 C in a humidified atmosphere in the presence of 5% CO2.
3. Results Co-coatings of laminin and polycationic polyamino acids like poly-L-lysine stimulated long-lasting Ca2 + signalling in human Jurkat T-cells [9] (Fig. 1A). Since the broad-range tyrosine kinase inhibitor genistein inhibited the Ca2 + signals efficiently [9], the effect of the src kinase inhibitor PP2 was studied. PP2 dose-dependently blocked laminin-medi-
2.3. Coating of coverslips with laminin and poly-L-lysine For coating of the coverslips, O-rings were fixed onto a thin (0.1 – 0.2 mm) glass coverslip with silicon grease, serving as a small chamber. Then, laminin I (5 mg/ml) in phosphate-buffered saline (PBS; 137 mM NaCl, 7 mM Na2HPO4, 1 mM KH2PO4, 2 mM KCl, pH 7.4) or BSA (2% in A. bidest) was added to the chamber for 60 min at 37 C. After rinsing twice with cold PBS, poly-L-lysine (50 mg/ml in A. bidest) was added for additional 60 min at 37 C. Then, the coverslips were rinsed again twice with PBS. 2.4. Ratiometric Ca2+ imaging Measurement of [Ca2 + ]i was performed using a digital ratiometric imaging station as previously described [8,9].
Fig. 1. Inhibition of src tyrosine kinases inhibits a6b1-integrin-mediated Ca2 + signalling. T-lymphocytes were loaded with FURA 2-AM and added to coverslips coated with laminin/poly-L-lysine (Ln/PL). [Ca2 + ]i was measured by digital ratiometric Ca2 + imaging. (A) The cells were added directly to the coverslips or preincubated with 5 mM PP2 (20 min, RT). (B) The inhibition of Ca2 + signalling by PP2 was dose dependent. Cells were preincubated with different concentrations of PP2 (20 min, RT) and attached to Ln/PL. The [Ca2 + ]i was measured 10 min after contact of the cells to Ln/PL. Shown are mean values of [Ca2 + ]i ± S.E.M. from at least three experiments. Asterisks mark significant differences vs. Ln/PL according to student’s t test ( P > 0.95).
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Fig. 2. a6b1-integrin-mediated Ca2 + signalling consists of Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry. Cells were loaded with FURA 2-AM and (A) were added to Ln/PL-coated coverslips, (B) were preincubated with EGTA (2 mM, 10 min, RT) and added to Ln/PL coated coverslips, and (C) were preincubated with EGTA (2 mM, 10 min, RT) and thapsigargin (1 mM, 10 min, RT) and added to Ln/PL-coated coverslips. Shown are representative tracings from at least three independent experiments.
ated Ca2 + signalling (Fig. 1B), indicating that src tyrosine kinases, e.g. p59fyn and/or p56lck, are involved in the signal transmission via a6b1-integrins. Ca2 + signalling stimulated by a6b1-integrins was longlasting in the presence of extracellular Ca2 + (1 mM; Fig. 2A) but was transient when extracellular Ca2 + was complexed by EGTA (Fig. 2B). Thapsigargin is well known as inhibitor of SERCA-type Ca2 + pumps and has been often used to deplete intracellular Ca2 + stores [11– 13]. Here, we demonstrate that T-cells preincubated with thapsigargin (1 mM) did not respond to laminin in the absence of extracellular Ca2 + (Fig. 2C). This indicates that the Ca2 + stores essentially involved in a6b1-integrin-mediated Ca2 + signalling are localised in the ER. Next, we addressed the question of which of the known Ca2 + -releasing second messengers are involved in a6b1integrin-mediated Ca2 + signalling. The main candidates for such messengers in T-cells are InsP3 and cADPR [2,14,15]. Pharmacological tools to block either formation of InsP3 or
Fig. 3. Effect of PLC inhibitor U73122 on a6b1-integrin-mediated Ca2 + signalling. (A) T-lymphocytes were loaded with FURA 2-AM and attached to Ln/PL, either directly or after preincubation (10 min, RT) with 1 mM U73122 or 1 mM U73343. Shown are representative experiments of at least three independent experiments. (B) Shown are mean values of [Ca2 + ]i of Jurkat cells preincubated with different concentrations of U73122 or U73343 (10 min, RT) and then placed on to BSA/PL or Ln/PL for 10 min. Shown are mean values ± S.E.M. of at least three experiments. Asterisks mark significant differences vs. Ln/PL according to student’s t test ( P > 0.95).
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the effect of cADPR on Ca2 + release were used. The PLC inhibitor U73122 (1 mM) efficiently antagonised Ca2 + signalling stimulated by laminin, whereas the inactive control compound U73343 (1 mM) did not (Fig. 3). However, higher concentrations of the inactive control compound U73343 (2 mM) also slightly inhibited Ca 2 + signalling, indicating that above 1 mM both U73122 and U73343 exhibit unspecific inhibitory effects on Ca2 + signalling. Nevertheless, our data indicate that stimulation of a6b1-integrin involves the formation of InsP3. Recently, we have provided strong evidence for the involvement of cADPR in TCR/CD3-mediated Ca2 + signalling [16]. a6b1-integrin-mediated Ca2 + signalling in T-cells preincubated with the specific and membranepermeant cADPR antagonist 7-deaza-8-Br-cADPR [10] was also significantly reduced (Fig. 4). The specificity of 7-deaza-8-Br-cADPR was demonstrated by the fact that spreading of the cells on laminin/poly-L-lysine, which was shown to depend on PKC activation but not on Ca2 + signalling [9], was not affected (data not shown). Thus, in addition to InsP3, cADPR is also involved in Ca2 + signalling via a6b1-integrins. This is the first demonstration that integrin signalling involves cADPR-mediated Ca2 + release. As pointed out above, Ca2 + signalling via a6b1-integrins was composed both of Ca2 + release and Ca2 + entry (Fig. 2A and B). It is generally believed that capacitative Ca2 + signalling is an important Ca2 + entry mechanism in T-cells [2,17]. Stimulation of T-cells by laminin also activates capacitative Ca2 + entry, as shown in Fig. 5. Stimulation of cells in the absence of extracellular Ca2 + caused transient Ca2 + release from the stores (Fig. 5A). This resulted in Ca2 + pool depletion, since in all cells, the [Ca2 + ]i was back to basal levels after 500 s (Fig. 5A). Readdition of extracellular Ca2 + resulted in rapid and longlasting Ca2 + signalling (Fig. 5A). Evidence for the involvement of capacitative Ca2 + entry was also obtained by the pharmacological profile of the Ca2 + channel. SKF 96365
Fig. 4. Effect of cADPR-antagonist 7-deaza-8-Br-cADPR on a6b1integrin-mediated Ca2 + signalling. T-lymphocytes were loaded with FURA 2-AM and added to coverslips coated with Ln/PL directly or the cells were preincubated prior with 100 mM 7-deaza-8-Br-cADPR (30 min, RT). (A) [Ca2 + ]i was measured by ratiometric digital Ca2 + imaging. Shown are representative tracings of at least five independent experiments. (B) Shown are the mean [Ca2 + ]i ± S.E.M. 1500 s after contact of the cells to the coverslips. The asterisk marks a significant difference vs. Ln/PL according to student’s t test ( P >.99).
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Fig. 5. a6b1-integrin-mediated Ca2 + signalling and capacitative Ca2 + entry. Jurkat cells were loaded with FURA 2-AM and preincubated with 2 mM EGTA (10 min, RT) before addition to Ln/PL-coated coverslips. (A) Then, 750 mM CaCl2 was added as indicated. To investigate the Ca2 + channel(s) involved, Jurkat T-lymphocytes were stimulated by Ln/PL, and during the plateau phase of Ca2 + signalling, SKF 96365 (1 mM), verapamil (100 mM), or nitrendipine (50 mM) was added. (B) Shown are mean values of [Ca2 + ]i ± S.E.M. 300 s after addition of the inhibitor from at least three experiments. The asterisk marks a significant difference vs. Ln/PL according to student’s t test ( P >.99).
significantly inhibited Ca2 + signalling stimulated by laminin, whereas inhibitors of voltage-operated Ca2 + channels, e.g. verapamil or nitrendipine, did not (Fig. 5B).
4. Discussion In the present study, we have shown that Ca2 + signalling stimulated by laminin via a6b1-integrin was inhibited by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cADPR antagonist 7-deaza-8-Br-cADPR. In addition, we have demonstrated that a6b1-integrin-mediated Ca2 + signalling involves Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry, most likely via the capacitative mechanism. In T-cells, stimulation of a variety of cell surface receptors results in the activation of intracellular Ca2 + signalling (for review, see Ref. [2]). The TCR/CD3 complex mediates activation by antigenic peptides presented in MHC context and therefore is regarded the most important receptor for T-cell activation. The mechanisms underlying TCR/CD3 complex-mediated Ca2 + signalling have been analysed in much more detail as compared to the other Ca2 + mobilising receptors. Thus, one of our initial questions was whether integrins, and in particular a6b1-integrin, would utilise similar or distinct mechanisms of Ca2 + signalling as compared to the TCR/CD3 complex. Interestingly, Ca2 + signalling via the TCR/CD3 complex shows significant similarities to Ca2 + signalling via the a6b1-integrin, indicating that at least b1-integrins, but perhaps also other cell surface receptors, can make use of only one Ca2 + signalling machinery of the T-cell. In both cases, the TCR/CD3 complex and the a6b1integrin, we observed a complex pattern involving both Ca2 + release from intracellular stores and Ca2 + entry across the plasma membrane.
For the TCR/CD3 complex, it has been shown that the src kinases p56lck and p59fyn are among the early players in several signal transduction pathway, including activation of PLCg [18,19]. In the case of a6b1-integrin-mediated Ca2 + signalling, we observed almost complete inhibition using the src kinase inhibitor PP2 (Fig. 1), indicating a similar signalling mechanism for both receptors. Both InsP3 and cADPR have been shown to contribute to TCR/CD3 complex-mediated Ca2 + signalling, probably in a temporally coordinated fashion (for review, see Ref. [15]). In the present study, we have shown that inhibition of InsP3 formation or inhibition of cADPR binding to its receptor both resulted in partial reduction of a6b1-integrin-mediated Ca2 + signalling (Figs. 3 and 4). This reduction was comparable to data obtained for TCR/CD3 complex-mediated Ca2 + signalling [16]. Taken together, our data indicate that both the TCR/CD3 complex and the a6b1-integrin make use of major components of the same signalling machinery. This view is further confirmed at the level of Ca2 + entry. As shown for stimulation of the TCR/CD3 complex [20], stimulation of a6b1-integrin also caused a transient Ca2 + signal in the absence of extracellular Ca2 + (Fig. 5). Under these conditions, the Ca2 + pools were depleted, allowing subsequent Ca2 + entry upon readdition of extracellular Ca2 + (Fig. 5). This behaviour is typical for capacitative Ca2 + entry, which has been discussed as a major Ca2 + influx pathway for T-cells by several authors [21,22]. The Ca2 + channels involved in both a6b1-integrin- and TCR/CD3-mediated Ca2 + signalling were sensitive to the receptor-operated Ca2 + channel antagonist SKF 96365, but not to verapamil, an antagonist of voltage-operated Ca2 + channels (Fig. 5; [20]). However, a difference was observed regarding the effect of nitrendipine. This Ca2 + antagonist, known to inhibit voltage-operated Ca2 + channels at low concentrations, was shown to inhibit TCR/CD3-mediated Ca2 + signalling in the micromolar range [20,23]. However, at 50 mM nitrendipine, a concentration known to result in 70 – 80% inhibition of the TCR/CD3-mediated Ca2 + signals [20], only about 20% inhibition was observed in the case of a6b1-integrin-mediated Ca2 + signalling. This indicates that the pharmacological profile of the Ca2 + channel(s) involved is at least partially different between the TCR/CD3 complex and the a6b1-integrin. In this study, we demonstrated the role of PLC in integrin-mediated Ca2 + signalling. Consistent with our data, Ricard et al. [24] showed that an elevation of InsP3 was one of the major events in a4b1-integrin-mediated Ca2 + signalling in Jurkat T-cells. Furthermore, InsP3 production upon integrin engagement was observed also in different cellular systems. PLCg2 was tyrosine phosphorylated in neutrophils followed by production of InsP3 and Ca2 + release after stimulation of b2-integrin [25]. Wrenn et al. [26] observed tyrosine phosphorylation of PLCg1 in pancreatic acinar cells after stimulation of b1-integrin. In
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addition, PLCg1 was tyrosine phosphorylated in activated T-cells and is involved in LFA-1 (b2-integrin)-mediated Ca2 + signalling [27]. In this case, Ca2 + signalling was stimulated via the b2-chain, but also via the a-chain, though to a much lower level and only in 50% of the cells [27]. However, in Jurkat T-cells Ca2 + signalling is mainly mediated by the b-chain of the integrin, because it could almost completely be inhibited by an anti-b1-integrin antibody [9]. InsP3 was also elevated after tyrosine phosphorylation of PLCg1 in rat glomerular epithelial cells after contact to collagen mediated by a2b1-integrin [28]. In contrast, human umbilical vein endothelial cells did not respond with Ca2 + signalling upon a2b1-integrin-dependent binding to collagen but showed Ca2 + signalling mediated by avb3-integrin after binding to vitronectin [29]. This is different from the results observed in T-cells, which did not show Ca2 + signalling after adhesion to vitronectin [8]. Taken together, these results suggest that various integrins expressed in different cell types mediate Ca2 + signalling by using the InsP3 pathway. As a novel aspect of integrin-mediated Ca2 + signalling, we suggest that also cADPR is involved in this process. Experimental evidence for this was obtained by inhibition of the a6b1-integrin-mediated Ca2 + signalling by 7-deaza-8Br-cADPR. This antagonist was successfully used to block the effect of cADPR in sea urchin eggs, human T-cells, and neurons [10,16,30]. Interestingly, we found that the a6b1integrin-stimulated spreading of T-cells, which is apparently mediated by PKC [9], was totally unaffected by 7-deaza-8Br-cADPR (100 mM). This demonstrates the high specificity of this compound. A complementary approach to confirm a role of cADPR in a6b1-integrin-mediated Ca2 + signalling would be the determination of endogenous cADPR upon engagement of a6b1-integrin. Though we have developed and successfully applied an analytical HPLC system for cADPR determination [31], some theoretical considerations detracted from these experiments in the case of a6b1integrin stimulation. First, the amount of cells needed for the cADPR determination is 108 per sample. To stimulate such a big number of cells in a comparable manner by solid phase-bound laminin, very large glass plates coated with laminin would have been necessary. Secondly, one of the problems with stimulation by solid phase-bound laminin is the fact that the cells do not respond in a synchronized fashion, because the start of the signal depends on the time the cells need to fall down and touch the laminin coating. Thus, a direct determination of an a6b1-integrin-mediated increase in cADPR, and for identical reasons also in InsP3, would be practically impossible. In conclusion, we have obtained evidence for a complex regulation of a6b1-integrin-mediated Ca 2 + signalling involving src-family tyrosine kinases, Ca2 + release mediated by the second messengers InsP3 and cADPR, and Ca2 + entry via Ca2 + channels sensitive to SKF 96365. Most remarkably, we show for the first time that cADPR is involved in integrin-mediated Ca2 + signalling, thereby add-
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ing integrins to the growing list of receptors that utilise the cADPR/Ca2 + signalling system. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (grant no. Gu 360/2-4 and 2-5 to AHG and GWM) and the Wellcome Trust (051326 to BVLP and AHG and 055709 to BVLP). References [1] Berridge MJ. Crit Rev Immunol 1997;17:155 – 78. [2] Guse AH. Crit Rev Immunol 1998;18:419 – 48. [3] Waldburger JM, Masternak K, Muhlethaler-Mottet A, Villard J, Peretti M, Landmann S, Reith W. Immunol Rev 2000;178:148 – 65. [4] Algarte M, Lecine P, Costello R, Plet A, Olive D, Imbert J. EMBO J 1995;14:5060 – 72. [5] Marzio R, Mauel J, Betz-Corradin S. Immunopharmacol Immunotoxicol 1999;21:565 – 82. [6] Albelda SM, Smith CW, Ward PA. FASEB J 1994;8:504 – 12. [7] Ager A. Trends Cell Biol 1994;4:326 – 33. [8] Weismann M, Guse AH, Sorokin L, Bro¨ker B, Frieser M, Hallmann R, Mayr GW. J Immunol 1997;158:1618 – 27. [9] Scho¨ttelndreier H, Mayr GW, Guse AH. Cell Signalling 1999;11: 611 – 9. [10] Sethi JK, Empson RM, Bailey VC, Potter BVL, Galione A. J Biol Chem 1997;272:16358 – 63. [11] Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Proc Natl Acad Sci USA 1990;87:2466 – 70. [12] Zweifach A, Lewis RS. Proc Natl Acad Sci USA 1993;90:6295 – 9. [13] Premack BA, McDonald TV, Gardner P. J Immunol 1994;152: 5226 – 40. [14] Guse AH. J Mol Med 2000;78:26 – 35. [15] da Silva CP, Guse AH. Biochim Biophys Acta 2000;1498:122 – 33. [16] Guse AH, da Silva CP, Berg I, Skapenko AL, Weber K, Heyer P, Hohenegger M, Ashamu GA, Schulze-Koops H, Potter BVL, Mayr GW. Nature 1999;398:70 – 3. [17] Putney JW. Cell Calcium 1986;7:1 – 12. [18] Weiss A, Koretzky G, Schatzman RC, Kadlecek T. Proc Natl Acad Sci USA 1991;88:5484 – 8. [19] Wange RL, Samelsen LE. Immunity 1996;5:197 – 205. [20] Guse AH, de Wit C, Klokow T, Schweitzer K, Mayr GW. Cell Calcium 1997;22:91 – 7. [21] Donnadieu E, Bismuth G, Trautmann A. J Biol Chem 1992;267: 25864 – 72. [22] Zweifach A, Lewis RS. Proc Natl Acad Sci USA 1993;90:6295 – 9. [23] Dupuis G, Aoudjit F, Ricard I, Payet MD. J Leukocyte Biol 1993;53: 66 – 72. [24] Ricard I, Payet MD, Dupuis G. Eur J Immunol 1997;27:1530 – 8. [25] Hellberg C, Molony L, Zheng L, Andersson T. Biochem J 1996;317: 403 – 7. [26] Wrenn RW, Creazzo TL, Herman LE. Biochem Biophys Res Commun 1996;226:876 – 82. [27] Kanner SB, Grossmaire LS, Ledbetter JA, Damle NK. Proc Natl Acad Sci USA 1993;90:7099 – 103. [28] Cybulsky AV, Carbonetto S, Cyr M, McTravish AJ, Huang Q. Am J Physiol 1993;264:C323 – 32. [29] Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA. J Cell Biol 1993;121:163 – 70. [30] Reyes-Harde M, Empson R, Potter BV, Galione A, Stanton PK. Proc Natl Acad Sci USA 1999;96:4061 – 6. [31] da Silva CP, Potter BVL, Mayr GW, Guse AH. J Chromatogr, B 1998;707:43 – 50.
Mechanisms involved in a6b1-integrin-mediated Ca2+ signalling Hella Scho¨ttelndreier a, Barry V.L. Potterb, Georg W. Mayra, Andreas H. Gusea,* a
Institute for Medical Biochemistry and Molecular Biology, Division of Cellular Signal Transduction, University of Hamburg, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany b Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK Received 6 April 2001; accepted 13 June 2001
Abstract Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting a6b1-integrin-mediated Ca2 + signalling. Both Ca2 + release from thapsigargin-sensitive Ca2 + stores and capacitative Ca2 + entry via Ca2 + channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of a6b1-integrin-mediated Ca2 + signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca2 + -releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Integrin; Extracellular matrix; Ca2+ signalling; Inositol 1,4,5-trisphosphate; Cyclic ADP-ribose; Capacitative Ca2+ entry; SKF 96365
1. Introduction Peripheral T-cells are activated by antigenic peptides presented on MHC molecules by antigen-presenting cells. Activation of intracellular signalling events via the T-cell receptor/CD3 (TCR/CD3) complex and additional T-cell surface receptors, such as CD4/CD8 and CD28, comprises several pathways, including tyrosine phosphorylation, activation of the MAP-kinase pathway, the PI-3-kinase pathway, and activation of Ca2 + signalling [1,2]. Once activated, T-cells express a different set of cell surface antigens, known as ‘‘activation antigens,’’ e.g. HLA DR (MHCII), CD25, CD69, and integrins [3 – 5]. Integrins facilitate binding of T-cells to other cell types, e.g. endothelial cells, which is very important to stabilise the weak, selectin-based T-cell – endothelial cell interaction [6,7]. Subsequently, activated T-cells transmigrate through the endo-
Abbreviations: [Ca2+]i, intracellular free Ca2+ concentration; cADPR, cyclic adenosine diphosphoribose; ECM, extracellular matrix; EMM, extracellular minimal medium; InsP3, D-myo-inositol 1,4,5-trisphosphate; mAb, monoclonal antibody; PBS, phosphate-buffered saline; TCR/CD3, T-cell receptor/CD3 * Corresponding author. Tel.: +49-40-42803-2828; fax: +49-40-428039880. E-mail address: [email protected] (A.H. Guse).
thelial cell layer to access area of inflammation and/or infection. During this migration, T-cells make contact with proteins of the extracellular matrix (ECM), either in the basement membrane or in the interstitium. We have previously shown that T-cells, either human Jurkat T-cells or T-cell blasts, respond to the ECM proteins laminin, fibronectin, collagen types I, IV, and VI, and tenascin by a sustained but relatively small elevation of the free cytosolic Ca2 + concentration ([Ca2 + ]i) [8]. Ca2 + signalling by these ECM proteins was shown to proceed specifically via the corresponding b1-integrins. In contrast, vitronectin via avb3-integrin did not activate Ca2 + signalling [8]. Recently, we demonstrated that enhanced physical attachment of Jurkat T-cells, which was achieved by cocoating of ECM proteins with poly-L-lysine, significantly increased the amplitude of Ca2 + signals stimulated by laminin and collagen type IV [9]. Importantly, we showed that the stimulatory effects were transmitted exclusively via the corresponding b1-integrins [9]. T-cell spreading, which was also observed on coverslips co-coated with laminin and poly-L-lysine, was mediated by protein kinase C (PKC) but not by [Ca2 + ]i [9]. Both b1-integrin-mediated Ca2 + signalling and spreading was inhibited by the tyrosine kinase inhibitor genistein [9]. However, the mechanisms involved in b1-integrin-mediated Ca2 + signalling were not investigated in detail.
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Here, we show that Ca2 + signalling stimulated by laminin via a6b1-integrin involves src kinases, probably p59fyn and/or p56lck, and the second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cyclic adenosine diphosphoribose (cADPR). Furthermore, we demonstrate that Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry via SKF 96365-sensitive Ca2 + channels are major components of a6b1-integrin-mediated Ca2 + signalling.
2. Materials and methods 2.1. Materials The ECM protein laminin I, isolated from EHS mouse tumour, was obtained from Life Technologies (Karlsruhe, Germany). Poly- L -lysine hydrobromide, M w 30,000 – 70,000, was purchased from Sigma (Deisenhofen, Germany). The enzyme inhibitors U73122, U73343, and thapsigargin and the inhibitors for the Ca2 + channels SKF 96365, nitrendipine, and verapamil were obtained from Calbiochem Novabiochem (Bad Soden, Germany). The cADPR antagonist 7-deaza-8-Br-cADPR was synthesized as described (Ref. [10] and references therein). All other chemicals used were of the highest purity grade available. MilliQ water (Millipore Waters, Eschborn, Germany) was used for the preparation of all buffers.
Cells were loaded with the Ca2 + -sensitive fluorophore FURA2-AM (Calbiochem) as follows: 107 cells in 1 ml complete RPMI 1640 medium were incubated with 4 mM FURA2-AM for 15 min at 37 C, diluted fourfold with complete RPMI 1640 medium and incubated for additional 15 min at 37 C. After washing the cells twice with extracellular minimal medium (EMM; 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM HEPES, 1 mM Na2HPO4, 5.5 mM glucose, pH 7.4), the cells were resuspended in 4 ml EMM. For Ca2 + measurements, 40 ml cell suspension and 60 ml EMM were added into the chamber on the coated glass coverslip and mounted on the stage of an inverted fluorescence microscope (Axiovert 100; Zeiss, Oberkochingen, Germany). The excitation wavelengths for the ratiometric analysis were 340 and 380 nm, and the emission beam was filtered at 510 nm and monitored by a CCD camera (C240077, Hamamatsu). In each experiment, the fluorescent images of an optical field containing about 20 cells for excitation at 340 and 380 nm as well as the ratio 340:380 were displayed and stored separately on hard disk. Usually, images were taken every 20 s. Offline analyses of stored image data were then carried out using the ImageMaster software (Photon Technology/PhotoMed, Wedel, Germany). So-called regions of interest were set to cover the whole area of a single cell. The numerical median ratio and the corresponding free [Ca2 + ]i for each single cell were then calculated by the software using an external calibration curve.
2.2. Cell culture Jurkat T-lymphocytes (subclone JMP) were cultured as described previously [9] in RPMI 1640 medium containing Glutamax I (Life Technologies, Eggenstein, Germany), buffered by HEPES (20 mM, pH 7.4) and supplemented with newborn calf serum (7.5%), penicillin (100 U/ml), and streptomycin (50 mg/ml; all from Life Technologies, Eggenstein, Germany). The cells were cultured at 37 C in a humidified atmosphere in the presence of 5% CO2.
3. Results Co-coatings of laminin and polycationic polyamino acids like poly-L-lysine stimulated long-lasting Ca2 + signalling in human Jurkat T-cells [9] (Fig. 1A). Since the broad-range tyrosine kinase inhibitor genistein inhibited the Ca2 + signals efficiently [9], the effect of the src kinase inhibitor PP2 was studied. PP2 dose-dependently blocked laminin-medi-
2.3. Coating of coverslips with laminin and poly-L-lysine For coating of the coverslips, O-rings were fixed onto a thin (0.1 – 0.2 mm) glass coverslip with silicon grease, serving as a small chamber. Then, laminin I (5 mg/ml) in phosphate-buffered saline (PBS; 137 mM NaCl, 7 mM Na2HPO4, 1 mM KH2PO4, 2 mM KCl, pH 7.4) or BSA (2% in A. bidest) was added to the chamber for 60 min at 37 C. After rinsing twice with cold PBS, poly-L-lysine (50 mg/ml in A. bidest) was added for additional 60 min at 37 C. Then, the coverslips were rinsed again twice with PBS. 2.4. Ratiometric Ca2+ imaging Measurement of [Ca2 + ]i was performed using a digital ratiometric imaging station as previously described [8,9].
Fig. 1. Inhibition of src tyrosine kinases inhibits a6b1-integrin-mediated Ca2 + signalling. T-lymphocytes were loaded with FURA 2-AM and added to coverslips coated with laminin/poly-L-lysine (Ln/PL). [Ca2 + ]i was measured by digital ratiometric Ca2 + imaging. (A) The cells were added directly to the coverslips or preincubated with 5 mM PP2 (20 min, RT). (B) The inhibition of Ca2 + signalling by PP2 was dose dependent. Cells were preincubated with different concentrations of PP2 (20 min, RT) and attached to Ln/PL. The [Ca2 + ]i was measured 10 min after contact of the cells to Ln/PL. Shown are mean values of [Ca2 + ]i ± S.E.M. from at least three experiments. Asterisks mark significant differences vs. Ln/PL according to student’s t test ( P > 0.95).
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Fig. 2. a6b1-integrin-mediated Ca2 + signalling consists of Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry. Cells were loaded with FURA 2-AM and (A) were added to Ln/PL-coated coverslips, (B) were preincubated with EGTA (2 mM, 10 min, RT) and added to Ln/PL coated coverslips, and (C) were preincubated with EGTA (2 mM, 10 min, RT) and thapsigargin (1 mM, 10 min, RT) and added to Ln/PL-coated coverslips. Shown are representative tracings from at least three independent experiments.
ated Ca2 + signalling (Fig. 1B), indicating that src tyrosine kinases, e.g. p59fyn and/or p56lck, are involved in the signal transmission via a6b1-integrins. Ca2 + signalling stimulated by a6b1-integrins was longlasting in the presence of extracellular Ca2 + (1 mM; Fig. 2A) but was transient when extracellular Ca2 + was complexed by EGTA (Fig. 2B). Thapsigargin is well known as inhibitor of SERCA-type Ca2 + pumps and has been often used to deplete intracellular Ca2 + stores [11– 13]. Here, we demonstrate that T-cells preincubated with thapsigargin (1 mM) did not respond to laminin in the absence of extracellular Ca2 + (Fig. 2C). This indicates that the Ca2 + stores essentially involved in a6b1-integrin-mediated Ca2 + signalling are localised in the ER. Next, we addressed the question of which of the known Ca2 + -releasing second messengers are involved in a6b1integrin-mediated Ca2 + signalling. The main candidates for such messengers in T-cells are InsP3 and cADPR [2,14,15]. Pharmacological tools to block either formation of InsP3 or
Fig. 3. Effect of PLC inhibitor U73122 on a6b1-integrin-mediated Ca2 + signalling. (A) T-lymphocytes were loaded with FURA 2-AM and attached to Ln/PL, either directly or after preincubation (10 min, RT) with 1 mM U73122 or 1 mM U73343. Shown are representative experiments of at least three independent experiments. (B) Shown are mean values of [Ca2 + ]i of Jurkat cells preincubated with different concentrations of U73122 or U73343 (10 min, RT) and then placed on to BSA/PL or Ln/PL for 10 min. Shown are mean values ± S.E.M. of at least three experiments. Asterisks mark significant differences vs. Ln/PL according to student’s t test ( P > 0.95).
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the effect of cADPR on Ca2 + release were used. The PLC inhibitor U73122 (1 mM) efficiently antagonised Ca2 + signalling stimulated by laminin, whereas the inactive control compound U73343 (1 mM) did not (Fig. 3). However, higher concentrations of the inactive control compound U73343 (2 mM) also slightly inhibited Ca 2 + signalling, indicating that above 1 mM both U73122 and U73343 exhibit unspecific inhibitory effects on Ca2 + signalling. Nevertheless, our data indicate that stimulation of a6b1-integrin involves the formation of InsP3. Recently, we have provided strong evidence for the involvement of cADPR in TCR/CD3-mediated Ca2 + signalling [16]. a6b1-integrin-mediated Ca2 + signalling in T-cells preincubated with the specific and membranepermeant cADPR antagonist 7-deaza-8-Br-cADPR [10] was also significantly reduced (Fig. 4). The specificity of 7-deaza-8-Br-cADPR was demonstrated by the fact that spreading of the cells on laminin/poly-L-lysine, which was shown to depend on PKC activation but not on Ca2 + signalling [9], was not affected (data not shown). Thus, in addition to InsP3, cADPR is also involved in Ca2 + signalling via a6b1-integrins. This is the first demonstration that integrin signalling involves cADPR-mediated Ca2 + release. As pointed out above, Ca2 + signalling via a6b1-integrins was composed both of Ca2 + release and Ca2 + entry (Fig. 2A and B). It is generally believed that capacitative Ca2 + signalling is an important Ca2 + entry mechanism in T-cells [2,17]. Stimulation of T-cells by laminin also activates capacitative Ca2 + entry, as shown in Fig. 5. Stimulation of cells in the absence of extracellular Ca2 + caused transient Ca2 + release from the stores (Fig. 5A). This resulted in Ca2 + pool depletion, since in all cells, the [Ca2 + ]i was back to basal levels after 500 s (Fig. 5A). Readdition of extracellular Ca2 + resulted in rapid and longlasting Ca2 + signalling (Fig. 5A). Evidence for the involvement of capacitative Ca2 + entry was also obtained by the pharmacological profile of the Ca2 + channel. SKF 96365
Fig. 4. Effect of cADPR-antagonist 7-deaza-8-Br-cADPR on a6b1integrin-mediated Ca2 + signalling. T-lymphocytes were loaded with FURA 2-AM and added to coverslips coated with Ln/PL directly or the cells were preincubated prior with 100 mM 7-deaza-8-Br-cADPR (30 min, RT). (A) [Ca2 + ]i was measured by ratiometric digital Ca2 + imaging. Shown are representative tracings of at least five independent experiments. (B) Shown are the mean [Ca2 + ]i ± S.E.M. 1500 s after contact of the cells to the coverslips. The asterisk marks a significant difference vs. Ln/PL according to student’s t test ( P >.99).
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Fig. 5. a6b1-integrin-mediated Ca2 + signalling and capacitative Ca2 + entry. Jurkat cells were loaded with FURA 2-AM and preincubated with 2 mM EGTA (10 min, RT) before addition to Ln/PL-coated coverslips. (A) Then, 750 mM CaCl2 was added as indicated. To investigate the Ca2 + channel(s) involved, Jurkat T-lymphocytes were stimulated by Ln/PL, and during the plateau phase of Ca2 + signalling, SKF 96365 (1 mM), verapamil (100 mM), or nitrendipine (50 mM) was added. (B) Shown are mean values of [Ca2 + ]i ± S.E.M. 300 s after addition of the inhibitor from at least three experiments. The asterisk marks a significant difference vs. Ln/PL according to student’s t test ( P >.99).
significantly inhibited Ca2 + signalling stimulated by laminin, whereas inhibitors of voltage-operated Ca2 + channels, e.g. verapamil or nitrendipine, did not (Fig. 5B).
4. Discussion In the present study, we have shown that Ca2 + signalling stimulated by laminin via a6b1-integrin was inhibited by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cADPR antagonist 7-deaza-8-Br-cADPR. In addition, we have demonstrated that a6b1-integrin-mediated Ca2 + signalling involves Ca2 + release from thapsigargin-sensitive Ca2 + stores and Ca2 + entry, most likely via the capacitative mechanism. In T-cells, stimulation of a variety of cell surface receptors results in the activation of intracellular Ca2 + signalling (for review, see Ref. [2]). The TCR/CD3 complex mediates activation by antigenic peptides presented in MHC context and therefore is regarded the most important receptor for T-cell activation. The mechanisms underlying TCR/CD3 complex-mediated Ca2 + signalling have been analysed in much more detail as compared to the other Ca2 + mobilising receptors. Thus, one of our initial questions was whether integrins, and in particular a6b1-integrin, would utilise similar or distinct mechanisms of Ca2 + signalling as compared to the TCR/CD3 complex. Interestingly, Ca2 + signalling via the TCR/CD3 complex shows significant similarities to Ca2 + signalling via the a6b1-integrin, indicating that at least b1-integrins, but perhaps also other cell surface receptors, can make use of only one Ca2 + signalling machinery of the T-cell. In both cases, the TCR/CD3 complex and the a6b1integrin, we observed a complex pattern involving both Ca2 + release from intracellular stores and Ca2 + entry across the plasma membrane.
For the TCR/CD3 complex, it has been shown that the src kinases p56lck and p59fyn are among the early players in several signal transduction pathway, including activation of PLCg [18,19]. In the case of a6b1-integrin-mediated Ca2 + signalling, we observed almost complete inhibition using the src kinase inhibitor PP2 (Fig. 1), indicating a similar signalling mechanism for both receptors. Both InsP3 and cADPR have been shown to contribute to TCR/CD3 complex-mediated Ca2 + signalling, probably in a temporally coordinated fashion (for review, see Ref. [15]). In the present study, we have shown that inhibition of InsP3 formation or inhibition of cADPR binding to its receptor both resulted in partial reduction of a6b1-integrin-mediated Ca2 + signalling (Figs. 3 and 4). This reduction was comparable to data obtained for TCR/CD3 complex-mediated Ca2 + signalling [16]. Taken together, our data indicate that both the TCR/CD3 complex and the a6b1-integrin make use of major components of the same signalling machinery. This view is further confirmed at the level of Ca2 + entry. As shown for stimulation of the TCR/CD3 complex [20], stimulation of a6b1-integrin also caused a transient Ca2 + signal in the absence of extracellular Ca2 + (Fig. 5). Under these conditions, the Ca2 + pools were depleted, allowing subsequent Ca2 + entry upon readdition of extracellular Ca2 + (Fig. 5). This behaviour is typical for capacitative Ca2 + entry, which has been discussed as a major Ca2 + influx pathway for T-cells by several authors [21,22]. The Ca2 + channels involved in both a6b1-integrin- and TCR/CD3-mediated Ca2 + signalling were sensitive to the receptor-operated Ca2 + channel antagonist SKF 96365, but not to verapamil, an antagonist of voltage-operated Ca2 + channels (Fig. 5; [20]). However, a difference was observed regarding the effect of nitrendipine. This Ca2 + antagonist, known to inhibit voltage-operated Ca2 + channels at low concentrations, was shown to inhibit TCR/CD3-mediated Ca2 + signalling in the micromolar range [20,23]. However, at 50 mM nitrendipine, a concentration known to result in 70 – 80% inhibition of the TCR/CD3-mediated Ca2 + signals [20], only about 20% inhibition was observed in the case of a6b1-integrin-mediated Ca2 + signalling. This indicates that the pharmacological profile of the Ca2 + channel(s) involved is at least partially different between the TCR/CD3 complex and the a6b1-integrin. In this study, we demonstrated the role of PLC in integrin-mediated Ca2 + signalling. Consistent with our data, Ricard et al. [24] showed that an elevation of InsP3 was one of the major events in a4b1-integrin-mediated Ca2 + signalling in Jurkat T-cells. Furthermore, InsP3 production upon integrin engagement was observed also in different cellular systems. PLCg2 was tyrosine phosphorylated in neutrophils followed by production of InsP3 and Ca2 + release after stimulation of b2-integrin [25]. Wrenn et al. [26] observed tyrosine phosphorylation of PLCg1 in pancreatic acinar cells after stimulation of b1-integrin. In
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addition, PLCg1 was tyrosine phosphorylated in activated T-cells and is involved in LFA-1 (b2-integrin)-mediated Ca2 + signalling [27]. In this case, Ca2 + signalling was stimulated via the b2-chain, but also via the a-chain, though to a much lower level and only in 50% of the cells [27]. However, in Jurkat T-cells Ca2 + signalling is mainly mediated by the b-chain of the integrin, because it could almost completely be inhibited by an anti-b1-integrin antibody [9]. InsP3 was also elevated after tyrosine phosphorylation of PLCg1 in rat glomerular epithelial cells after contact to collagen mediated by a2b1-integrin [28]. In contrast, human umbilical vein endothelial cells did not respond with Ca2 + signalling upon a2b1-integrin-dependent binding to collagen but showed Ca2 + signalling mediated by avb3-integrin after binding to vitronectin [29]. This is different from the results observed in T-cells, which did not show Ca2 + signalling after adhesion to vitronectin [8]. Taken together, these results suggest that various integrins expressed in different cell types mediate Ca2 + signalling by using the InsP3 pathway. As a novel aspect of integrin-mediated Ca2 + signalling, we suggest that also cADPR is involved in this process. Experimental evidence for this was obtained by inhibition of the a6b1-integrin-mediated Ca2 + signalling by 7-deaza-8Br-cADPR. This antagonist was successfully used to block the effect of cADPR in sea urchin eggs, human T-cells, and neurons [10,16,30]. Interestingly, we found that the a6b1integrin-stimulated spreading of T-cells, which is apparently mediated by PKC [9], was totally unaffected by 7-deaza-8Br-cADPR (100 mM). This demonstrates the high specificity of this compound. A complementary approach to confirm a role of cADPR in a6b1-integrin-mediated Ca2 + signalling would be the determination of endogenous cADPR upon engagement of a6b1-integrin. Though we have developed and successfully applied an analytical HPLC system for cADPR determination [31], some theoretical considerations detracted from these experiments in the case of a6b1integrin stimulation. First, the amount of cells needed for the cADPR determination is 108 per sample. To stimulate such a big number of cells in a comparable manner by solid phase-bound laminin, very large glass plates coated with laminin would have been necessary. Secondly, one of the problems with stimulation by solid phase-bound laminin is the fact that the cells do not respond in a synchronized fashion, because the start of the signal depends on the time the cells need to fall down and touch the laminin coating. Thus, a direct determination of an a6b1-integrin-mediated increase in cADPR, and for identical reasons also in InsP3, would be practically impossible. In conclusion, we have obtained evidence for a complex regulation of a6b1-integrin-mediated Ca 2 + signalling involving src-family tyrosine kinases, Ca2 + release mediated by the second messengers InsP3 and cADPR, and Ca2 + entry via Ca2 + channels sensitive to SKF 96365. Most remarkably, we show for the first time that cADPR is involved in integrin-mediated Ca2 + signalling, thereby add-
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ing integrins to the growing list of receptors that utilise the cADPR/Ca2 + signalling system. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (grant no. Gu 360/2-4 and 2-5 to AHG and GWM) and the Wellcome Trust (051326 to BVLP and AHG and 055709 to BVLP). References [1] Berridge MJ. Crit Rev Immunol 1997;17:155 – 78. [2] Guse AH. Crit Rev Immunol 1998;18:419 – 48. [3] Waldburger JM, Masternak K, Muhlethaler-Mottet A, Villard J, Peretti M, Landmann S, Reith W. Immunol Rev 2000;178:148 – 65. [4] Algarte M, Lecine P, Costello R, Plet A, Olive D, Imbert J. EMBO J 1995;14:5060 – 72. [5] Marzio R, Mauel J, Betz-Corradin S. Immunopharmacol Immunotoxicol 1999;21:565 – 82. [6] Albelda SM, Smith CW, Ward PA. FASEB J 1994;8:504 – 12. [7] Ager A. Trends Cell Biol 1994;4:326 – 33. [8] Weismann M, Guse AH, Sorokin L, Bro¨ker B, Frieser M, Hallmann R, Mayr GW. J Immunol 1997;158:1618 – 27. [9] Scho¨ttelndreier H, Mayr GW, Guse AH. Cell Signalling 1999;11: 611 – 9. [10] Sethi JK, Empson RM, Bailey VC, Potter BVL, Galione A. J Biol Chem 1997;272:16358 – 63. [11] Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Proc Natl Acad Sci USA 1990;87:2466 – 70. [12] Zweifach A, Lewis RS. Proc Natl Acad Sci USA 1993;90:6295 – 9. [13] Premack BA, McDonald TV, Gardner P. J Immunol 1994;152: 5226 – 40. [14] Guse AH. J Mol Med 2000;78:26 – 35. [15] da Silva CP, Guse AH. Biochim Biophys Acta 2000;1498:122 – 33. [16] Guse AH, da Silva CP, Berg I, Skapenko AL, Weber K, Heyer P, Hohenegger M, Ashamu GA, Schulze-Koops H, Potter BVL, Mayr GW. Nature 1999;398:70 – 3. [17] Putney JW. Cell Calcium 1986;7:1 – 12. [18] Weiss A, Koretzky G, Schatzman RC, Kadlecek T. Proc Natl Acad Sci USA 1991;88:5484 – 8. [19] Wange RL, Samelsen LE. Immunity 1996;5:197 – 205. [20] Guse AH, de Wit C, Klokow T, Schweitzer K, Mayr GW. Cell Calcium 1997;22:91 – 7. [21] Donnadieu E, Bismuth G, Trautmann A. J Biol Chem 1992;267: 25864 – 72. [22] Zweifach A, Lewis RS. Proc Natl Acad Sci USA 1993;90:6295 – 9. [23] Dupuis G, Aoudjit F, Ricard I, Payet MD. J Leukocyte Biol 1993;53: 66 – 72. [24] Ricard I, Payet MD, Dupuis G. Eur J Immunol 1997;27:1530 – 8. [25] Hellberg C, Molony L, Zheng L, Andersson T. Biochem J 1996;317: 403 – 7. [26] Wrenn RW, Creazzo TL, Herman LE. Biochem Biophys Res Commun 1996;226:876 – 82. [27] Kanner SB, Grossmaire LS, Ledbetter JA, Damle NK. Proc Natl Acad Sci USA 1993;90:7099 – 103. [28] Cybulsky AV, Carbonetto S, Cyr M, McTravish AJ, Huang Q. Am J Physiol 1993;264:C323 – 32. [29] Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA. J Cell Biol 1993;121:163 – 70. [30] Reyes-Harde M, Empson R, Potter BV, Galione A, Stanton PK. Proc Natl Acad Sci USA 1999;96:4061 – 6. [31] da Silva CP, Potter BVL, Mayr GW, Guse AH. J Chromatogr, B 1998;707:43 – 50.