STAT5 in regulation of chronic leukemia K562 cell proliferation: Inhibitory effect of WHI-P131

In this study, we examined the role of JAK/STAT signaling in the regulation of chronic leukemia K562 cell proliferation. STAT3 and STAT5 tyrosine phosphorylation was used as a marker of the activation status of STAT proteins. We demonstrated that, in growing cultures of K562 cells, both STAT3 and STAT5 are constitutively activated. To determine the significance of STAT activity in maintaining the high level of K562 proliferation, we tested two JAK inhibitors, AG-490 (JAK2 and JAK3 inhibitor) and WHI-P131 (a new specific JAK3 inhibitor). We showed that, during the prolonged cultivation (48 h) of K562 cells with AG-490 or WHI-P131, the cells remained viable. It was found that treatment with WHI-P131 (30–100 μM) decreased tyrosine phosphorylation of STAT5 and did not affect the high level of STAT3 phosphorylation. In proliferating K562 cells, AG-490 (25–50 μM) did not influence on STAT3 and STAT5 phosphorylation. The flow cytometry analysis revealed a dose-dependent decrease in G1 and S phases and an increase in G2/M phases in WHI-P131-treated K562 cells and no changes in cell cycle structure in AG-490-treated cultures. Thus, our findings indicate the preferential role of STAT5 (not constitutively active STAT3) in the proliferation of leukemia K562 cells and demonstrate the specificity of WHI-P131 inhibitory effect; unlike other JAK drugs that stimulate apoptosis and decrease proliferation, WHI-P131 prevents K562 cells growth by arresting in G2/M phases of the cell cycle.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.     

Oncostatin M induces proliferation of human adipose tissue-derived mesenchymal stem cells

Interleukin-6 (IL-6) subfamily of cytokines, including oncostatin M (OSM), leukemia inhibitory factor (LIF), and IL-6, has been implicated in a variety of physiological responses, such as cell growth, differentiation, and inflammation. In the present study, we demonstrated that both OSM and LIF stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hATSCs), however, IL-6 had no effect on cell proliferation. OSM treatment induced phosphorylation of ERK, and pretreatment with U0126, a MEK inhibitor, prevented the OSM-stimulated proliferation of hATSCs, suggesting that the MEK/ERK pathway is involved in the OSM-induced proliferation. Treatment with OSM also induced phosphorylation of JAK2 and JAK3, and pretreatment of the cells with WHI-P131, a JAK3 inhibitor, but not with AG490, a JAK2 inhibitor, attenuated the OSM-induced proliferation of hATSCs. Furthermore, OSM treatment elicited phosphorylation of STAT1 and STAT3, and pretreatment with WHI-P131 specifically prevented the OSM-induced phosphorylation of STAT1, without affecting the OSM-induced phosphorylation of ERK and STAT3. These results suggest that two separate signaling pathways, such as MEK/ERK and JAK3/STAT1, are independently involved in the OSM-stimulated proliferation of hATSCs.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

STAT5 signaling in expression of the α-subunit of interleukin-2 receptor in human blood lymphocytes

A comparative study of STAT3 and STAT5 activity (assessed by tyrosine phosphorylation level) and the expression of an α-subunit of the interleukin-2 receptor (examined by cytophotometric evaluation of CD25 cell number) during phytohemaglutinin (PHA)-induced proliferation of human blood lymphocytes (HBLs) has been carried out. It was found that the level of STAT3 phosphorylation was high both in resting and competent HBLs and remained unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen either in resting or competent HBL. In the presence of PHA, STAT5 phosphorylation was observed no earlier than in 2–5 h; maximal phosphorylation was detected after 24 h. In competent HBLs, exogenous IL-2 induced high phosphorylation of STAT5 in 30 min that was retained for the next 24–48 h. Alterations in the level of tyrosine phosphorylation of STAT5 correlated with CD25 expression. WHI-P131, a JAK3 kinase inhibitor, prevents STAT5 activation, CD25 surface expression, and lymphocyte proliferation. It is concluded that JAK3/STAT5 signaling via an IL-2 receptor is necessary to support the long-term expression of a high-affinity αβγ_c-receptor of IL-2 and HBL optimal proliferation.     

EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway,but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line

The number of epidermal growth factor receptors (EGFRs) and their ligands are highly expressed in malignant tumor cells. The EGF signaling pathway is also activated in up to one-third of patients with breast cancer. In this study, we investigated the novel function of the JAK3 inhibitor, WHI-P131, on EGF-induced MMP-9 expression and the regulatory mechanism of EGF-induced MMP-9 expression in SKBR3 cells. We observed that EGF increased MMP-9 mRNA and protein expression in a dose-dependent manner. EGF also induced the phosphorylation of EGFR, ERK, and STAT-3, and these effects were inhibited by the EGFR inhibitor, AG1478. To investigate the involvement of the STAT-3 pathway on EGF-induced MMP-9 expression, we pretreated SKBR3 cells with JAK1, JAK2, and JAK3 inhibitors prior to EGF treatment. The results showed that the JAK3 inhibitor, WHI-P131, as well as JAK3 siRNA transfection, but not the JAK1 and JAK2 inhibitors, significantly decreased EGF-induced MMP-9 expression. In addition, EGF-induced STAT-3 phosphorylation was only inhibited by WHI-P131. We then transfected cells with adenoviral STAT-3 (Ad-STAT-3), followed by treatment with EGF. Interestingly, EGF-induced MMP-9 expression was decreased by Ad-STAT-3 overexpression in a dose-dependent manner, while it was significantly increased by STAT-3 siRNA transfection. Our results also showed that basal levels of MMP-9 expression were significantly increased by constitutive active-MEK (CA-MEK) overexpression. EGF-induced ERK phosphorylation was prevented by WHI-P131, but not by JAK1 and JAK2 inhibitors. On the other hand, EGF-induced MMP-9 expression was decreased by the MEK1/2 inhibitor, UO126. Therefore, for the first time, we suggest that the JAK3 inhibitor, WHI-P131, inhibits EGF-induced STAT-3 phosphorylation as well as ERK phosphorylation. The JAK3/ERK pathway may play an important role in EGF-induced MMP-9 expression in SKBR3 cells.     

Oncostatin M decreases adiponectin expression and induces dedifferentiation of adipocytes by JAK3- and MEK-dependent pathways

Adiponectin, an adipokine secreted from adipocytes, plays a crucial role in the regulation of glucose and lipid metabolism. In the present study, we examine the role of the IL-6 family of cytokines in the expression of adiponectin in human adipocytes derived from human adipose tissue-derived stromal cells. Oncostatin M (OSM), but not IL-6, attenuated the expression level of adiponectin dose- and time-dependently, and the inhibitory effect of OSM on adiponectin expression was as potent as that of TNF-alpha. The OSM-induced down-regulation of adiponectin expression was correlated with the down-regulation of PPARgamma2 and lipoprotein lipase, markers for adipogenic differentiation, and depletion of intracellular lipid droplets, suggesting dedifferentiation of adipocytes in response to OSM. OSM induced phosphorylation of STAT1, and treatment of adipocytes with JAK3 inhibitor WHI-P131 or MEK inhibitor U0126, but not with JAK2 inhibitor AG490, prevented the activation of STAT1. Furthermore, the OSM-induced suppression of adiponectin expression and dedifferentiation of adipocytes were ameliorated by WHI-P131 or U0126, but not by AG490. These results suggest that OSM inhibits adiponectin expression by inducing dedifferentiation of adipocytes through signaling pathways involving JAK3 and MEK, but not JAK2.     

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.     

Complex effects of different green tea catechins on human platelets

Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre-incubation with EGCG concentration-dependently inhibited thrombin-induced aggregation and phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP-76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase-dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3' position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2' position (epigallocatechin) did not.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Akt activation in platelets depends on Gi signaling pathways

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.     

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).     

Targeting Janus kinase 3 in mast cells prevents immediate hypersensitivity reactions and anaphylaxis.

Janus kinase 3 (JAK3), a member of the Janus family protein-tyrosine kinases, is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcepsilonRI cross-linking. Selective inhibition of JAK3 in mast cells with 4-(4'-hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline) (WHI-P131) blocked the phospholipase C activation, calcium mobilization, and activation of microtubule-associated protein kinase after lgE receptor/FcepsilonRI cross-linking. Treatment of IgE-sensitized rodent as well as human mast cells with WHI-P131 effectively inhibited the activation-associated morphological changes, degranulation, and proinflammatory mediator release after specific antigen challenge without affecting the functional integrity of the distal secretory machinery. In vivo administration of the JAK3 inhibitor WHI-P131 prevented mast cell degranulation and development of cutaneous as well as systemic fatal anaphylaxis in mice at nontoxic dose levels. Thus, JAK3 plays a pivotal role in IgE receptor/FcepsilonRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.     

The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.     

Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.

STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.