Volume expansion stimulates p72(syk) and p56(lyn) in skate erythrocytes

Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with 32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety.     

The activation pathway of the volume-sensitive organic osmolyte channel in Xenopus laevis oocytes expressing skate anion exchanger 1 (AE1)

When swollen, skate red blood cells increase permeability and allow efflux of a number of solutes, including taurine. Hypoosmosis-induced taurine permeability appears to involve the red cell anion exchanger. However, three isoforms have been cloned from these cells. Therefore, to determine the ability of the individual isoform skate anion exchanger 1 (skAE1) to mediate hypoosmosis-induced taurine permeability as well as associated regulatory events, skAE1 was expressed in Xenopus oocytes. This study focused on investigating the role of tyrosine kinases and lipid rafts in the regulation of the channel. The results showed that tyrosine kinase inhibitors and lipid raft-disrupting agents inhibited the volume-sensitive organic osmolyte channel while protein tyrosine phosphatase inhibitors activated the channel in oocytes expressing skAE1. To study the role of lipid rafts in the activation of the volume-sensitive organic osmolyte channel, the cellular localization of skAE1 was investigated. Also, the role of tyrosine kinases was investigated by examining the tyrosine phosphorylation state of skAE1. Hypoosmotic stress induced mobilization of skAE1 into light membranes and the cell surface as well as tyrosine phosphorylation of skAE1. These events are involved in the activation of the volume-sensitive organic osmolyte channel in Xenopus oocytes expressing skAE1.     

The Anion Exchanger as an Osmolyte Channel in the Skate Erythrocyte

Regulatory volume decrease occurs in fish erythrocytes by the release of osmolytes via a channel involving the anion exchanger (AE), also known as band 3. This review focuses primarily on work done on red blood cells from the skate (Raja erinacea) to further understand the activation, regulation and identification of this osmolyte channel. A model is proposed in which the reduction in intracellular ionic strength that occurs with increased cell volume may change the interaction between AE dimers and other cytoplasmic proteins (band 4.1 and ankyrin), promoting the formation of a tetrameric osmolyte channel. Phosphorylation by two tyrosine kinases, p72syk and p56lyn, is linked to this oligomerization. The skate AE has been recently cloned, resulting in three different isoforms, one of which, skAE1, when expressed in Xenopus oocytes, demonstrates taurine transport.     

The Activation Pathway of the Volume-Sensitive Organic Osmolyte Channel in Xenopus laevis Oocytes Expressing Skate Anion Exchanger 1 (AE1)

When swollen, skate red blood cells increase permeability and allow efflux of a number of solutes, including taurine. Hypoosmosis-induced taurine permeability appears to involve the red cell anion exchanger. However, three isoforms have been cloned from these cells. Therefore, to determine the ability of the individual isoform skate anion exchanger 1 (skAE1) to mediate hypoosmosis-induced taurine permeability as well as associated regulatory events, skAE1 was expressed in Xenopus oocytes. This study focused on investigating the role of tyrosine kinases and lipid rafts in the regulation of the channel. The results showed that tyrosine kinase inhibitors and lipid raft-disrupting agents inhibited the volume-sensitive organic osmolyte channel while protein tyrosine phosphatase inhibitors activated the channel in oocytes expressing skAE1. To study the role of lipid rafts in the activation of the volume-sensitive organic osmolyte channel, the cellular localization of skAE1 was investigated. Also, the role of tyrosine kinases was investigated by examining the tyrosine phosphorylation state of skAE1. Hypoosmotic stress induced mobilization of skAE1 into light membranes and the cell surface as well as tyrosine phosphorylation of skAE1. These events are involved in the activation of the volume-sensitive organic osmolyte channel in Xenopus oocytes expressing skAE1.     

Membrane trafficking factors are involved in the hypotonic activation of the taurine channel in the little skate (Raja erinacea) red blood cell

In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmotic-induced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The second-messenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002, which decreased the hypoosmotic-stimulated taurine uptake by 28%. The state of actin is also involved, as the actin filament depolymerizer latrunculin B decreased hypoosmotic-stimulated taurine uptake by approximately 40%. Although hypoosmotic conditions did not stimulate changes in the distribution of actin between filamentous and globular forms, latrunculin stimulated a decrease in filamentous actin and increase in globular actin in both isoosmotic and hypoosmotic conditions. Disruptors of other potential cytoskeletal factors (myosin, kinesin, dynein, and microtubules) did not affect taurine uptake. The present results suggest that the exocytosis of skAE1 stimulated by hyposmotic-induced cell volume expansion requires activation of PI3 kinase and is regulated by the state of actin filaments.     

A role for PLCgamma2 in platelet activation by homocysteine

The aim of this study was to examine the homocysteine effect on phospholipase Cgamma2 (PLCgamma2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCgamma2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor-mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N-acetyl-L-cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCgamma2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCgamma2, suggesting that thromboxane A(2) could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N-acetyl-L-cysteine of 35% or 50% respectively, while the PLCgamma2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCgamma2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A(2).     

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias)

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.     

Hypotonicity-induced exocytosis of the skate anion exchanger skAE1: role of lipid raft regions

Upon hypotonic volume expansion, skate erythrocytes lose solutes via a pathway that requires participation of anion exchangers (AEs). Three skate AE isoforms (skAEs) are expressed, and at least skAE1 has been shown to mediate this effect when expressed in oocytes. Under isoosmotic conditions, only a small fraction of skAE1 is expressed on the external plasma membrane. Under these conditions, a portion of skAE1 may be found in non-ionic detergent-insoluble regions. However, the detergent-insoluble material is found intracellularly. Cellular volume expansion by hypoosmotic volume expansion but not volume expansion by isoosmotic medium by permeant solutes (ethylene glycol, diethyl urea, or ammonium chloride) stimulates the appearance of skAE1 in the external plasma membrane, and a significant portion of this is found in detergent-insoluble regions. Upon hypoosmotic volume expansion nearly half of the skAE1 is found as oligomers. SkAE1 in these detergent-insoluble fractions is highly tyrosine phosphorylated. These data suggest that volume expansion by hypoosmotic medium stimulates movement of skAE1 from an intracellular pool contained in detergent-insoluble lipid rafts to the plasma membrane. This skAE1 associates to form oligomers that could be involved in the solute efflux that occurs upon volume expansion.     

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.     

Volume expansion stimulates monoubiquitination and endocytosis of surface-expressed skate anion-exchanger isoform

In hyposmotic medium, skate erythrocytes swell and then lose taurine and other solutes with obligate water to achieve a regulatory volume decrease (RVD) over a 90-min period. The skate erythrocyte anion-exchanger isoform 1 (skAE1) participates in the RVD, and increased surface expression after hyposmolality-induced volume expansion occurs within 5 min but decreases to baseline within 120 min. The subsequent fate of skAE1 is the focus of these studies. SkAE1 sent to the surface becomes monoubiquitinated, a modification that is present while skAE1 is associated with clathrin and Rab5 but is removed before skAE1 is passed to the Rab4 compartment. Endocytosis of skAE1 involves clathrin-mediated internalization. Surface plasma membrane skAE1 forms tetramers and demonstrates increased tyrosine phosphorylation, and both of these processes decrease before skAE1 appears in the Rab5 compartment. Volume expansion-stimulated surface skAE1 comes from an intracellular pool in a buoyant membrane fraction resistant to nonionic detergent extraction (DRM), and the amount of skAE1 increases in this buoyant DRM fraction on the surface. Clathrin heavy chain is found largely in the erythrocyte DRM, but in dense, rather than buoyant, fractions. Rab5- and Rab4-containing membranes are largely detergent soluble, suggesting that as skAE1 is passed to clathrin and then to Rab5 compartments, the membrane microdomain composition changes. The present studies demonstrate that skAE1, which appears on the surface after hyposmolality-induced volume expansion, is monoubiquitinated, a modification that may serve as a signal for removal of skAE1 from the surface. This modification is eliminated after clathrin-mediated removal of skAE1 in a membrane domain containing Rab5, potentially permitting recycling and reuse of skAE1.     

Effector-induced Syk-mediated phosphorylation in human erythrocytes

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.     

Tyrosine phosphorylation and association of FcgammaRII and p72(Syk) are not limited to the FcgammaRII signalling pathway

The tyrosine kinase p72(Syk) plays a critical role in platelet signal transduction. It associates with the platelet receptor for the Fc domain of IgGs, FcgammaRII, following stimulation by FcgammaRII cross-linking. Here, we show that p72(Syk) and FcgammaRII tyrosine phosphorylation and association occured following platelet stimulation by: (1) two monoclonal antibodies, which form a bridge between a target antigen and FcgammaRII, and (2) the G-protein-coupled receptor agonist thrombin. The kinetics of the p72(Syk)/FcgammaRII association depended on the signalling pathway (i.e., the antigen targeted or the thrombin receptor). We established a direct relationship between the level of FcgammaRII phosphorylation and the detection of its association with p72(Syk). Inhibition of p72(Syk) by piceatannol resulted in partial or total inhibition of FcgammaRII phosphorylation, after immunological activation or addition of thrombin, respectively, suggesting that p72(Syk) participates in FcgammaRII phosphorylation. The results provide evidence that p72(Syk)/FcgammaRII association is not restricted to immunological activation.     

Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα

Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα.     

Differential effects of quercetin and resveratrol on Band 3 tyrosine phosphorylation signalling of red blood cells

The protective effects of eating fruits and vegetables in the prevention of several degenerative pathologies have been attributed at least in part to the antioxidant and anti-inflammatory properties of polyphenols. In this study, we investigated the effects of two polyphenols, quercetin and resveratrol, on red blood cell Band 3 tyrosine phosphorylation signalling activated by peroxynitrite. Peroxynitrite is a physiological oxidant scavenged largely by the erythrocyte and formed by the reaction between nitrogen monoxide and superoxide anion. Quercetin and its structurally analogous (+)-catechin inhibited the peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation. Quercetin was found to downregulate the activity of syk, which is upstream in the Band 3 tyrosine phosphorylation cascade, and partially prevented peroxynitrite-mediated phosphotyrosine phosphatase inhibition. Resveratrol and hydroxytyrosol, unexpectedly, amplified peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family. The present results clearly indicate that polyphenols may activate cell transduction pathways in different and sometimes opposite ways.     

CR3, FcgammaRIIA and FcgammaRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca(2+), phospholipase D and tyrosine phosphorylation.

Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcgammaRIIA and FcgammaRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcgammaRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcgammaRIIA Pansorbins induced an even weaker signal. However, FcgammaRIIA induced strong phosphorylation of p72(syk) whereas FcgammaRIIIB induced only a very weak p72(syk) phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72(syk) was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72(syk) that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72(syk) phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcgammaRIIA and FcgammaRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72(syk) is an early signal in the cascade induced by FcgammaRIIA but not by CR3.     

Expression of the Skate (<Emphasis Type="Italic">Raja erinacea</Emphasis>) AE1 Osmolyte Channel in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes: Monovalent Cation Permeability

The aim of this study was to express the cloned skate anion exchanger 1 (skAE1) in Xenopus oocytes and determine whether the differences in monovalent cation permeabilities in hypotonically stimulated skate and trout erythrocytes could be due to differences in the presence or absence of intracellular channel regulators between the two species or in the intrinsic permeability properties of the channels themselves. The expressed protein (skAE1) was inserted into the oocyte cell membrane and facilitated both Cl⁻ exchange and taurine transport. Expression of skAE1 in oocytes showed similar monovalent cation permeabilities as previously reported for skate erythrocytes and different from both trout erythrocytes and trAE1 expressed in Xenopus oocytes. These results show that the skAE1 expressed in oocytes functions in a manner similar to that of the osmolyte channel in hypotonically activated skate erythrocytes and supports the hypothesis that differences in the monovalent cation permeabilities of the osmolyte channels in skate and trout RBCs resides in the differences in permeability properties of the channels between the two species.This revised version was published online in August 2005 with a corrected cover date.     

The lectin wheat germ agglutinin stimulates a protein-tyrosine kinase activity of p72syk in porcine splenocytes

We previously reported a molecular cloning of porcine gene syk encoding a non-receptor type 72-kDa protein-tyrosine kinase and a generation of anti-CPTK40 antibodies which could immunoprecipitate the activity of p72syk (Taniguchi et al. (1991) J. Biol. Chem. 266, 15790-15796). In this study, we have demonstrated that wheat germ agglutinin caused an increase in the autophosphorylation activity of p72syk which preceded an increase of protein-tyrosine phosphorylation observed at the same time in porcine splenocyte. The increase of p72syk activity was dose dependent and was inhibited by the coexistence of N-acetyl-D-glucosamine. Upon stimulation by the combination of 4 beta-phorbol 12-myristate 13-acetate and ionophore A23187, we could not detect the increase of activity of p72syk suggesting that the activation of p72syk was independent of protein kinase C and calcium ions.     

Piceatannol prevents lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nuclear factor (NF)-kappaB activation by inhibiting IkappaB kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.     

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.