The Small GTPase Rap1 Is Activated by Turbulence and Is Involved in Integrin {alpha}IIb{beta}3-mediated Cell Adhesion in Human Megakaryocytes*

Kim M. T. de Bruyn {ddagger}, Fried J. T. Zwartkruis {ddagger}, Johan de Rooij {ddagger} §, Jan-Willem N. Akkerman ¶ || and Johannes L. Bos {ddagger} **

From the {ddagger}Department of Physiological Chemistry and Centre for Biomedical Genetics and the Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

Received for publication, November 26, 2002 , and in revised form, March 18, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrin-mediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin {alpha}IIb{beta}3, in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by {alpha}-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls {alpha}IIb{beta}3-mediated cell adhesion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The small GTPase Rap1 is a molecular switch that cycles between an inactive GDP and active GTP-bound conformation. The protein can be activated by a plethora of stimuli (16), indicating that Rap1 activation is a common event in signaling. In general this activation is mediated by second messengers such as cAMP, calcium ions, and diacylglycerol; and a number of guanine nucleotide exchange factors have been identified to mediate this activation (7). In human platelets, Rap1 is abundantly expressed and is rapidly activated by a large variety of agonists including {alpha}-thrombin, thromboxane A2, epinephrine, platelet-activating factor, ADP, and the phorbol ester TPA1 (1). At least two different signaling pathways are involved in this activation, one mediated by Gq, phospholipase C, and calcium and one mediated by protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) (1, 810). In platelets, Rap1 activation is rapidly inhibited by agents that increase the intracellular cAMP concentration, like for instance prostaglandin I2 (1). Activation of Rap1 is prior to platelet activation, suggesting a key role of Rap1 in this process. After platelet activation Rap1 relocalizes to the actin cytoskeleton (8, 11, 12). In the control of Rap1 in platelets, RapGAP, which can interact with the active {alpha} subunit of the heterotrimeric G-protein Gz, plays a role as well (13).

In a variety of cell lines, Rap1 was found to be involved in the regulation of integrin-mediated cell adhesion. Rap1 controls T-cell receptor-, CD31-, and CD98-induced activation of {alpha}L{beta}2 (1418) but is also required for Mn2+- or activating antibody-induced {alpha}L{beta}2-mediated adhesion (19). Also T-cells from transgenic mice expressing active Rap1 from a T-cell-specific promoter showed increased integrin-mediated cell adhesion (17). In macrophages Rap1 regulates {alpha}M{beta}2-mediated phagocytosis (18, 20), and in a variety of cell types Rap1 was found to regulate {beta}1 integrins as well (19, 21). The mechanism of this activation is still unclear and may involve an increase in both integrin clustering and integrin affinity. In Drosophila, Rap1 was found to control cell morphology polarity (22) and, recently, to regulate the even distribution of adherence junctions (23).

The role of Rap1 in the regulation of integrins with {beta}1 or {beta}2 subunits raised the question of whether Rap1 may regulate the activation of integrin {alpha}IIb{beta}3. Proper control of this integrin forms a key step in platelet adhesion and aggregation. Because platelets are not suitable for genetic interference studies, we have chosen human megakaryocytes, platelet precursor cells expressing {alpha}IIb{beta}3. In these studies we have noted that these cells are highly sensitive to mild disturbances such as tumbling tubes to mix added growth factors. This turbulence-induced effect was observed in several other hematopoietic cell lines as well. In addition we observed that inhibition of Rap1 strongly reduced cell adhesion to fibrinogen, the counter-receptor of {alpha}IIb{beta}3. This inhibition was partially rescued by treatment with the phorbol ester TPA but not by {alpha}-thrombin. From these results we conclude that in megakaryocytes turbulence is a mechanism to activate Rap1 and that Rap1 is involved in the control of {alpha}IIb{beta}3-mediated cell adhesion to fibrinogen.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The following stimuli and inhibitors were used at the indicated concentrations unless stated otherwise: 8-bromo-cyclic AMP (Biolog Life Science Institute, 1 mM), forskolin (ICN, 20 µM) BAPTA-AM (Molecular Probes, Eugene, OR, 25 µM), Roche 31-8220 (Biomol, Plymouth, PA, 5 µM), GF 109203X (Biomol, 5 µM), staurosporine (Sigma, 200 nM), jasplakinolide (Molecular Probes, 5 µM), cytochalasin D (Biomol, 5 µM), latrunculin A (Molecular Probes, 5 µM), latrunculin B (Molecular Probes 5 µM), LY294002 (Biomol, 10 µM), wortmannin (Sigma, 100 nM), H-89 (Biomol, 10 µM), PP1 (Biomol, 50 µM), genistein (Biomol, 100 µM), SB 203580 (Biomol, 10 µM), rapamycin (Biomol, 10 nM), thrombin (Sigma, 1unit/ml), and TPA (Sigma, 100 ng/ml).

Cell Culture, Cell Lines, and Transfection—MEG-01 cells (24) were grown at 37 °C in RPMI 1640 (Invitrogen) supplemented with 20% heat-inactivated (30 min at 56 °C) fetal bovine serum. DAMI cells (25) were grown in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated horse serum. CHRF-288-11 cells (26) were cultured in Fischer's medium enriched with 20% heat-inactivated horse serum. The Jurkat T-cell line JHM1 2.2 was provided by Dr. D. Cantrell (Imperial Cancer Research Fund, London) with kind permission of Dr. A. Weiss (University of California at San Francisco). These Jurkat cells were grown as described earlier (14). AZU II cells were kindly provided by Dr. M. van de Wetering (Department of Immunology, University Medical Center Utrecht, The Netherlands). They were grown in RPMI 1640 supplemented with 10% fetal calf serum. The erythroleukemic K562 cells, a gift from Dr. C. G. Figdor, were grown in 75% RPMI 1640 and 25% Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. THP-1 cells were kindly provided by Dr C. J. Heijnen (Department of Immunology, University Medical Centre Utrecht, The Netherlands) and were maintained like the Jurkat cells but in the presence of 4x 10–4% mercaptoethanol. All cell types were cultured in the presence of 0.05% glutamine, penicillin, and streptomycin in a humidified atmosphere with 5% CO2. For electroporation, DAMI cells (1.2x 107 cells/ml in 0.4 ml of complete medium) were pulsed at 250 V and 960 microfarad with 5 µg of thymidine kinase-luciferase plasmid DNA, construct plasmid as indicated in the figure legends, and added vector plasmid to keep the DNA amounts constant. Subsequently, 24 h after transfection, cells were transferred to serum-free medium and used 42–48 h after transfection. The constructs used for electroporation have been described previously (19).

Platelet Isolation—Donors claimed not to have taken any medication during the preceding 10 days. After informed consent was obtained, freshly drawn venous blood from healthy volunteers was collected into 0.1 volume of 130 mM trisodium citrate. Citrated blood was centrifuged (150 x g, 15 min at 20 °C), and subsequently the platelet-rich plasma was supplemented with prostaglandin I2 (10 ng/ml) and ACD (2.5 g of trisodium citrate, 1.5 g of citric acid, and 2.0 g of D-glucose in 100 ml of distilled H2O) for acidification to pH 6.5, thereby preventing platelet activation during further isolation. Next, platelets were purified from platelet-rich plasma by centrifugation (330 x g, 15 min at 20 °C). The platelet pellet was resuspended in HEPES-tyrode buffer (10 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH2PO4, 1.7 mM MgCl2, 11.9 mM NaHCO2, pH 7.4) containing 5 mM D-glucose at 2x 108 platelets/ml.

Turbulence-induced Stimulation—Cells were incubated overnight in serum-free medium. Next, cells were resuspended at 5x 106 cells/ml (MEG-01) 10x 106 cells/ml (DAMI, CHRF-288-11) or 25x 106 cells/ml (Jurkat, AZU II, K562, THP-1) in medium. 200 µl of cell suspension or 500 µl of platelet suspension (2x 108 platelets/ml) in Eppendorf tubes was incubated in a 37 °C water bath for 30 min. A sample was mixed gently, i.e. five times up and down, and incubated at 37 °C for the indicated periods of time. Subsequently, cells were lysed for 15 min at 4 °C by the addition of ice-cold lysis buffer (10% glycerol, 1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 1 µM leupeptin, 0.1 µM aprotinin), and lysates were cleared by centrifugation at maximal speed in an Eppendorf centrifuge for 10 min at 4 °C.

Measurement of Activation of Rap1, Ras, and Ral—The GTP-bound form of Rap1 was isolated using RalGDS-RBD as an activation-specific probe and quantified by Western blotting using anti-Rap1 antibody as described previously (1). Similarly, RasGTP and RalGTP were determined using Raf1RBD and RalBP as activation-specific probes (27, 28).

Adhesion Assay—Adhesion assays were performed and analyzed as described extensively (14). Briefly, cells were transiently transfected with a luciferase expression construct in the presence or absence of additional expression constructs as indicated. Cells were harvested, washed, and resuspended in TSM buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) containing 0.5% BSA at a concentration of 5x 105 cells/ml. Coating of 96-well plates was performed as described previously (19) with some minor modifications. Greiner Microlon 96-well plates that had been coated overnight at 4 °C with different concentrations of fibrinogen (Chromogenix AB, Mölndal, Sweden) in sodium bicarbonate buffer (50 µl per well) were washed with TSM buffer and blocked for 30 min at 37 °C with 5% BSA/TSM. After washing the plate again with TSM buffer, 50 µl of 0.5% BSA/TSM was added to each well containing a stimulating agent or not. Subsequently, 50 µl of cell suspension was added per well, after which the cells were spun down for 1 min at 200 rpm in a Heraeus Sepatech Megafuge 1.0. Cells were allowed to adhere for 30 min at 37 °C, and nonadherent cells were removed with warmed 0.5% BSA/TSM. Adherent cells were lysed and subjected to a luciferase assay as described previously (29). Expression of transfected constructs was confirmed by immunoblotting of total cell lysates. Bound cells were calculated, and numbers were corrected for transfection efficiency and nonspecific effects of constructs by measuring luciferase activity of total input cells ([counts in cells bound/counts in total input cells]x 100%).

Western Blotting—Western blotting of all protein samples was carried out using polyvinylidene difluoride membranes. The antibodies used for protein detection were rabbit polyclonal anti-Rap1 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Ras (Transduction Laboratories). Phosphospecific antibodies against ERK1 and –2, protein kinase B (PKB)/Akt substrate, and S6 were all purchased from Cell Signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Turbulence-induced Rap1 Activation—In our analysis of Rap1 activation in MEG-01 cells, we noted that elevated Rap1GTP levels already occurred in the absence of the stimulating agents, solely because of the gentle mixing procedure. This effect was rapid, as it occurred within 30 s after tumbling the tube and was sustained for at least 5 min (Fig. 1a). Addition of the phorbol ester TPA, a strong inductor of Rap1 in platelets, did not induce or hardly induced a further activation of Rap1 (data not shown). Also cAMP-elevating stimuli, like forskolin, which was previously shown to activate Rap1 in MEG-01 cells, did not further increase the Rap1GTP level (data not shown). A similar turbulence-induced activation was observed in a number of other hematopoietic cell lines, such as K562 (erythroleukemia), Jurkat (T-lymphocytes), AZU II (B-lymphocytes), and THP-1 (monocytes) (Fig. 1b). Also, in two other megakaryocytic cell lines, DAMI and CHRF-288-11, turbulence induced Rap1 activation (Fig. 1d). This activation was neither enhanced nor inhibited by forskolin, which is an activator of adenylate cyclase, resulting in elevation of the intracellular cAMP level. However, TPA has an additive effect on CHRF-288-11 cells. Importantly, mixing-induced stress did not induce Rap1 activation in platelets (Fig. 1c). From these results we conclude that in megakaryocytes and in a number of other hematopoietic cell lines, but not in platelets, mild mixing is sufficient to strongly induce Rap1 activation.



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FIG. 1.
Activation of Rap1 in MEG-01 cells is induced by turbulence. a, serum-starved MEG-01 cells were subjected to turbulence induced by mixing stress as described under "Experimental Procedures," and after the indicated time points cells were lysed and Rap1GTP determined using the activation-specific probe assay. WB, Western blot. b, K562, Jurkat, Azu II, and THP-1 cells were subjected to mixing-induced turbulence, and Rap1 activation was measured as described in a. c, the megakaryoblastic cell lines DAMI and CHRF-288-11 were subjected to mixing-induced turbulence as indicated in a. In addition, the cells were treated with forskolin (20 µM) and the phorbol ester TPA (100 ng/ml) at the indicated time points. Platelets were either stimulated with {alpha}-thrombin (1 unit/ml) or subjected to mixing-induced turbulence, and the levels of RapGTP were determined at the indicated time points (b, basal, untreated control). Rap1 indicates total Rap1 in the lysates to demonstrate equal input per sample. Rap1GTP indicates Rap1GTP levels determined by the pull-down assay.

 

To investigate the activation pathway responsible for mix turbulence-induced Rap1 activation, we investigated the effect of several pharmacological inhibitors. Most of the inhibitors used had been shown to affect activation of Rap1. In certain cell types, an increase in [Ca2+]i was demonstrated to be necessary or even sufficient to induce Rap1 activation and could be blocked by BAPTA-AM (1, 4). Pretreatment of either MEG-01 or DAMI cells with a combination of BAPTA-AM and EGTA did not inhibit turbulence-induced Rap1GTP increase (Fig. 2a). PKC inhibitors (Roche 31-8220, GF 109203X, staurosporine) were described as blocking block Rap1 activation in various cell types (4, 6, 8); however, they also did not inhibit mixing-induced elevation of active Rap1 (Fig. 2b) in these megakaryocytic cell lines. Also PI3K and PKA were implicated in the Rap1 activation processes (8, 9, 30). However, inhibitors of PI3K activity (LY294002 or wortmannin) or PKA (H-89) did not interfere with the turbulence-induced activation (Fig. 2c). The contribution of Src and p38 MAPK was proposed (4, 30, 31), but inhibitors of these proteins (PP1/genistein and SB 203580, respectively) showed no effect on mixing-induced Rap1 activation (Fig. 2d). Apparently, turbulence-induced Rap1 activation is not mediated by one of these previously described pathways. Finally, we tested whether the effect is direct or mediated by changes in the actin cytoskeleton. However, no role was found for actin cytoskeleton dynamics, as neither actin polymer stabilizing (jasplakinolide) nor dissociating (cytochalasin D, latrunculin A and B) agents seemed to affect this Rap1 activation (Fig. 2e). The effectiveness of the pharmacological inhibitors used was tested in parallel experiments (data not shown). From these results we conclude that stress applied by mixing activates Rap1 rather directly, presumably through a cell surface receptor.



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FIG. 2.
Effect of pharmacological inhibitors on the turbulence-induced Rap1 activation. MEG-01 and DAMI cells were serum-starved overnight, after which inhibitors were added at the indicated concentrations at 30 min prior to the induction of mixing-induced stress. During this period cells were kept at 37 °C. Cells were subjected to mixing-induced turbulence followed by incubation for the indicated periods of time (a) or for 1 min (b–e), and Rap1GTP was determined. a, cells were preincubated with 25 µM BAPTA-AM and 5 mM EGTA. b, cells were incubated with the PKC inhibitors Roche 31-8220, GF 109202X, staurosporine, or EDTA, used at the indicated concentrations. c, cells were incubated with the PI3K inhibitors LY294002 (LY) (10 µM) and wortmannin (W) (100 nM) and the PKA inhibitor H-89 (10 µM). The effect of the inhibitors on mixing-induced stress is in duplicate. d, cells were incubated with the Src inhibitor PP1 (50 µM), the tyrosine kinase inhibitor genistein (G) (100 µM), and the p38 MAPK inhibitor SB 203580 (SB) (10 µM). The effect of the inhibitors on mixed-induced stress is in duplicate. e, cells were incubated with indicated concentrations of inhibitors of actin dynamics, i.e. jasplakinolide (jaspl), cytochalasin D (cytoD), latrunculin A and B (latA and latB) (b, basal, untreated control). Rap1 indicates total Rap1 in the lysates to demonstrate equal input per sample. Rap1GTP indicates Rap1GTP levels determined by the pull-down assay.

 

Turbulence-induced Activation of Ras and Phosphorylation of Ribosomal Protein S6 —To study whether turbulence induces additional signal transduction pathways, we determined the activation of both the small GTPases Ras and Ral and of ERK in MEG-01 cells (Fig. 3a). Interestingly, Ras was strongly activated but at a later time point than Rap1. Two downstream targets of Ras, Ral and ERK, were not activated. Using a phosphospecific antibody recognizing phosphorylated targets of PKB we observed phosphorylation of several proteins in lysates of mixed MEG-01 cells (Fig. 3b). However, we did not find activation of PKB itself using phosphospecific antibodies (data not shown), but we did observe phosphorylation of the ribosomal protein S6, which was sensitive to the mTOR inhibitor rapamycin and the PI3K inhibitor LY294002 (Fig. 3C). This suggests that p70 S6 kinase is responsible for the phosphorylation of S6. Rapamycin- and LY294002-sensitive turbulence-induced S6 phosphorylation was also observed in Jurkat cells (data not shown). Interestingly, shear stress-induced activation of p70 S6 kinase was demonstrated previously (32). From these results we conclude that mix stress induces activation of several signaling pathways, one of which is the activation of Rap1. However, activation of Rap1 appears to be a particularly fast response.



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FIG. 3.
Turbulence-induced activation of Ras. MEG-01 cells were subjected to mixing-induced stress. a, effect of mix stress on the activation of Ras, Ral and ERK. Activation of Ras and Ral was measured using activation-specific probes. Activation of ERK was measured using a phosphospecific antibody (pERK) that recognizes the active form of both ERK1 and ERK2. Top panel, total Rap1 in the lysates to demonstrate equal input per sample. WB, Western blot. Lane b, basal, untreated control. b, phosphorylated proteins recognized by a phospho-PKB substrate-specific antibody. Lane M, marker. c, ribosomal S6 protein recognized by antibody specific for phosphorylated S6. The PI3K inhibitor LY294002 (25 µM) and mTOR inhibitor rapamycin (10 nM) were added 30 min prior to mixing-induced stress as described in the Fig. 2 legend. Lower panel of each pair, total Rap1 in the lysates to demonstrate equal input per sample.

 

Inhibition of Rap1 Abolishes {alpha}IIb{beta}3-mediated Cell Adhesion—In contrast to platelets, which do not bind to fibrinogen in the resting state, we observed that DAMI cells adhere spontaneously to fibrinogen (Fig. 4), indicating that the integrin {alpha}IIb{beta}3 is (partially) active. To investigate whether Rap1 is involved in this process, we transfected DAMI cells with two different inhibitors of Rap1 signaling, i.e. Rap1GAP, which lowers the level of Rap1GTP, and RalGDS-RBD, which binds with high affinity to Rap1GTP. Cells were allowed to adhere to 96-well plates coated with different concentrations of fibrinogen, the counter-receptor of {alpha}IIb{beta}3. We observed that the concentration-dependent adhesion is strongly inhibited by Rap1GAP and is significantly inhibited by RalGDS-RBD, indicating that active Rap1 is required for {alpha}IIb{beta}3-mediated cell adhesion. Introduction of Rap1V12 had little effect on basal level of adhesion, suggesting that the endogenous level of Rap1GTP is already sufficiently high to support cell adhesion. Interestingly, whereas {alpha}-thrombin only marginally affects basal level of adhesion, TPA is a strong inductor of cell adhesion. Introduction of Rap1V12 did not significantly increase cell adhesion under these conditions. The reduction in basal adhesion after expression of Rap1GAP and RalGDS-RBD was also observed when cells were stimulated with {alpha}-thrombin. However, a much weaker effect of Rap1GAP and RalGDS-RBD was observed after TPA-induced cell adhesion, showing that TPA-induced adhesion is partially independent of Rap1.



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FIG. 4.
Rap1 involved in {alpha}IIb{beta}3-mediated cell adhesion DAMI cells were cotransfected with 5 µg of pG3-TK luciferase reporter plasmid and empty pMT2-SM-HA vector (vector), HA-RapGAP I (RapGAP) (20 µg), RapV12 (20 µg), or HA-RalGDS-RBD (RBD) (20 µg). After 42 h, the cells were allowed to adhere to immobilized fibrinogen for 30 min at 37 °C. The percentage of cells bound was determined by the fraction of luciferase activity bound, as described under "Experimental Procedures." 96-Well plates were coated with the indicated concentration of fibrinogen. Where indicated, the cells were treated with {alpha}-thrombin (1 unit/ml) or TPA (100 ng/ml).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this article we show that in a variety of hematopoietic suspension cells, mixing-induced turbulence results in a clear Rap1 activation. The presence of additional stimuli like forskolin, TPA, or thrombin did not accomplish a clear additional increase in the Rap1GTP level (except in CHRF-288-11 cells). The effect was rather dramatic and was observed even when cell suspensions were either tumbled once up and down or gently stirred with a pipette tip. The mechanism by which mix stress activates Rap1 is still unclear, despite our attempts to find a clue about the initial signaling events. We have tried to interfere using a variety of inhibitors previously described to inhibit Rap1 activation, such as the inhibitors for phospholipase C, PKC and PI3K, and chelators of calcium ions. We have also tested inhibitors of the cytoskeleton or integrin-based cell-cell contacts. However, none of the inhibitors tested so far showed an inhibitory effect on mix stress-induced Rap1 activation in either MEG-01 or DAMI cells.

Signaling induced by turbulence is probably most similar to signaling induced by mechanical and shear stress, with the first induced by changes in cell shape and the second by a fluid flow. For both stimuli, knowledge of the receptors that trigger the events is still elusive, although cell surface receptors and alterations in the cytoskeleton are most likely involved (3337). Both stimuli trigger a variety of signaling pathways, including the activation of Ras and p70 S6 kinase, as we observe for mix stress, resulting in changes in cell structure and function. Previously, activation of Rap1 was observed after stretching adherent fibroblastic L-929 cells (38). This implies that activation of Rap1 by external stress is not restricted to hematopoietic suspension cells.

One of the most prominent effects of active Rap1 in mammalian cells is the regulation of integrin-mediated cell adhesion. Adhesion mediated by integrins containing either {beta}1 or {beta}2 subunits has been shown to be regulated by Rap1. This regulation results in an increased binding of integrins to soluble counter-receptors or increased cell adhesion to immobilized receptors. Megakaryocytes, like platelets, express {alpha}IIb{beta}3, which allows binding to the counter-receptor fibrinogen. Interestingly, megakaryocytes already adhere to fibrinogen in the absence of a stimulus, and treatment with {alpha}-thrombin, a very potent activator of {alpha}IIb{beta}3 on platelets, only marginally increased the adhesion. This adhesion was dependent on fibrinogen, and increasing the concentration of fibrinogen also increased basal and {alpha}-thrombin-induced cell adhesion. Importantly, both basal and thrombin-induced cell adhesion was inhibited by the introduction of Rap1GAP or RalGDS-RBD, inhibitors of Rap1 signaling. This shows that Rap1 is required for {alpha}IIb{beta}3-mediated cell adhesion and suggests that Rap1 regulates {alpha}IIb{beta}3 activity. The inhibitory effect of Rap1GAP and RalGDS-RBD on basal adhesion indicates that in resting cells a basal level of Rap1 is sufficient to sustain cell adhesion. However, considering the stress induced by plating cells, it is likely that the basal level of Rap1 is elevated by mixing-induced stress and thus contributes to {alpha}IIb{beta}3-mediated cell adhesion. While this manuscript was in preparation, Bertoni et al. (39) reported that Rap1 regulates the affinity of {alpha}IIb{beta}3 in primary mouse megakaryocytes. In their study basal adhesion was low and insensitive to expression of Rap1GAP, whereas {alpha}-thrombin strongly stimulated cell adhesion, which was further augmented by expression of RapV12 and inhibited by RapGAP (39); however, they did not measure the level of Rap1GTP in these cells. Apparently, these mature megakaryocytes are less sensitive to mix stress-induced Rap1 activation than the DAMI cells, which represents a more immature megakaryoblast.

What could be the reason for cells to activate Rap1 in response to extracellular applied stress? The most likely explanation would be that when cells are subject to detaching forces, they respond by increased binding to their support. Alternatively, Rap1 regulates integrin dynamics to allow cells to respond to changes in shape without cell rupture. Interestingly, Rap1 activation is induced by a large variety of stimuli and is thus a very common event. In addition, Rap1 appears to modulate all integrins that have been tested thus far, indicating that Rap1 regulates a process that is required for the activation of all integrins. Elucidation of this process is currently one of the great challenges in cell biology.


    FOOTNOTES
 
* This work was supported by Grant 98.122 from the Netherlands Heart Association (to K. M. T. de B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Back

|| Supported by the Netherlands Thrombosis Foundation. Back

** To whom correspondence should be addressed. Tel.: 31-30-253-89-77; Fax: 31–30-253-90-35; E-mail: j.l.bos{at}med.uu.nl.

1 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PKB, protein kinase B; PKA, cAMP-dependent protein kinase; BSA, bovine serum albumin; RBD, Ras-binding domain. Back


    ACKNOWLEDGMENTS
 
We thank our colleagues in the Department of Physiological Chemistry for continuous support and discussions.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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