TG1019/OXE, a Galpha(i/o)-protein-coupled receptor, mediates 5-oxo-eicosatetraenoic acid-induced chemotaxis

We have previously identified a Galpha(i/o)-protein-coupled receptor (TG1019/OXE) using 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) as its ligand. We investigated signal transduction from TG1019 following stimulation with 5-oxo-ETE and role of TG1019 in 5-oxo-ETE-induced chemotaxis, using Chinese hamster ovary cells expressing TG1019 (CHO/TG1019 cells). 5-Oxo-ETE induced intracellular calcium mobilization and rapid activation of MEK/ERK and PI3K/Akt pathways in CHO/TG1019 cells. CHO/TG1019 cells stimulated with 5-oxo-ETE and other eicosanoids exhibited chemotaxis with efficacies related to agonistic activity of each eicosanoid for TG1019. Pretreatment of the cells with pertussis toxin, a phospholipase C (PLC) inhibitor (U73122) or a PI3K inhibitor (LY294002), markedly suppressed 5-oxo-ETE-induced chemotaxis, whereas pretreatment with a MEK inhibitor (PD98059) had no significant effect on the chemotaxis. Our results show that TG1019 mediates 5-oxo-ETE-induced chemotaxis and that signals from TG1019 are transduced via Galpha(i/o) protein to PLC/calcium mobilization, MEK/ERK, and PI3K/Akt, among which PLC and PI3K would play important roles in the chemotaxis.     

Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3′-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P_1–3. We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P₁ in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P₁ internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P₁ internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P₁ internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.     

Protein-tyrosine phosphatase alpha regulates stem cell factor-dependent c-Kit activation and migration of mast cells

The role of protein-tyrosine phosphatase alpha (PTPalpha) in mast cell function was investigated in tissues and cells from PTPalpha-deficient mice. Bone marrow-derived mast cells (BMMCs) lacking PTPalpha exhibit defective stem cell factor (SCF)-dependent polarization and migration. Investigation of the molecular basis for this reveals that SCF/c-Kit-stimulated activation of the Fyn tyrosine kinase is impaired in PTPalpha(-/-) BMMCs, with a consequent inhibition of site-specific c-Kit phosphorylation at tyrosines 567/569 and 719. Although c-Kit-mediated activation of phosphatidylinositol 3-kinase and Akt is unaffected, profound defects occur in the activation of downstream signaling proteins, including mitogen-activated protein kinases and Rho GTPases. Phosphorylation and interaction of Fyn effectors Gab2 and Shp2, which are linked to Rac/JNK activation in mast cells, are impaired in PTPalpha(-/-) BMMCs. Thus, PTPalpha is required for SCF-induced c-Kit and Fyn activation, and in this way regulates a Fyn-based c-Kit signaling axis (Fyn/Gab2/Shp2/Vav/PAK/Rac/JNK) that mediates mast cell migration. These defective signaling events may underlie the altered tissue-resident mast cell populations found in PTPalpha(-/-) mice.     

Pinoresinol-4,4′-di-O-β-d-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4′-di-O-β-d-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 μM PDG resulted in strong stimulation of MEF cell migration and the EC₅₀ was about 2 μM. Pretreatment with pertussis toxin (PTX), an inhibitor of G_i protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G_i-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10 μM), which is a selective antagonist for LPA₁ and LPA₃ receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.     

Clusterin stimulates the chemotactic migration of macrophages through a pertussis toxin sensitive G-protein-coupled receptor and Gβγ-dependent pathways

Clusterin induces the expression of various chemotactic cytokines including tumor necrosis factor-α (TNF-α) in macrophages and is involved in the cell migration. According to the results of this study, clusterin induced the directional migration (chemotaxis) of macrophages based on a checkerboard analysis. The chemotactic activity of clusterin was prevented by pretreatment with pertussis toxin (PTX), indicating that the G_αi/o-protein coupled receptor (GPCR) was involved in the chemotactic response of clusterin. Clusterin-stimulated chemotaxis was abrogated in a dose-dependent manner by pretreatment with gallein (a G_βγ inhibitor), indicating the involvement of G_βγ released from the GPCR. In addition, inhibitors of phospholipase C (PLC, U73122) and phosphoinositide 3-kinase (PI3K, LY294002), the key targets of G_βγ binding and activation, suppressed chemotactic migration by clusterin. The phosphorylation of Akt induced by clusterin was blocked by pretreatment with gallein or LY294002 but not with U73122, indicating that G_βγ released from the PTX-sensitive G_i protein complex activated PLC and PI3K/Akt signaling pathways separately. The activation of cellular MAP kinases was essential in that their inhibitors blocked clusterin-induced chemotaxis, and G_βγ was required for the activation of MAP kinases because gallein reduced their phosphorylations induced by clusterin. In addition, the inflammation-induced migration of macrophages was greatly reduced in clusterin-deficient mice based on a thioglycollate-induced peritonitis model system. These results suggest that clusterin stimulates the chemotactic migration of macrophages through a PTX-sensitive GPCR and G_βγ-dependent pathways and describe a novel role of clusterin as a chemoattractant of monocytes/macrophages, suggesting that clusterin may serve as a molecular bridge between inflammation and its remodeling of related tissue by recruiting immune cells.     

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

Suppression of phospholipase Cγ1 phosphorylation by cinnamaldehyde inhibits antigen-induced extracellular calcium influx and degranulation in mucosal mast cells

Antigen-IgE-mediated mucosal mast-cell activation is critical in the development of food allergies. Cinnamaldehyde, a major constituent of Cinnamomi cortex, dose-dependently inhibited the antigen-IgE-induced degranulation of mucosal-type bone-marrow derived mast cells (mBMMCs) and RBL-2H3 cells. Cinnamaldehyde also suppressed the elevation of the intracellular Ca(2+) level that is induced by the extracellular Ca(2+) influx in antigen-IgE-stimulated mBMMCs. Furthermore, tyrosine phosphorylation of phospholipase C (PLC) γ1, which is a crucial activation switch for the intracellular Ca(2+) mobilization in mast cells, was attenuated by cinnamaldehyde. Together, our results demonstrated that cinnamaldehyde suppressed the intracellular Ca(2+) mobilization and the degranulation of mucosal mast cells by inhibiting the activity of the IgE receptor-PLCγ-Ca(2+) influx pathway. These findings suggest that cinnamaldehyde may have therapeutic potential in mucosal mast cell-related allergic diseases, such as food allergies.     

Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca²⁺]_i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca²⁺]_i increases at active zones, or a calcium ionophore to raise [Ca²⁺]_i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca²⁺]_i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.     

Phospholipase cbeta is critical for T cell chemotaxis

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.     

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background

The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.

Methodology/Principal Findings

We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P₃ using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.

Conclusion/Significance

These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.     

Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells

The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcepsilonRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcepsilonRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCgamma1 and PLCgamma2 and calcium responses. Thus, Vav1 is essential to FcepsilonRI-mediated activation of PLCgamma and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCgamma-activated calcium signals.     

The major outer sheath protein of Treponema denticola selectively inhibits Rac1 activation in murine neutrophils

Treponema denticola major outer sheath protein (Msp) inhibits neutrophil chemotaxis in vitro , but key regulatory mechanisms have not been identified. Because the Rac small GTPases regulate directional migration in response to chemoattractants, the objective was to analyse the effects of Msp on formyl -methionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil polarization and Rac activation in murine neutrophils. Msp pretreatment of neutrophils inhibited both polarization and chemotactic migration in response to fMLP. Activation of small GTPases was measured by p21 binding domain (PBD) pulldown assays, followed by Western analysis, using monoclonal anti-Rac1, anti-Rac2, anti-cdc42 and anti-RhoA antibodies. Enriched native Msp selectively inhibited fMLP-stimulated Rac1 activation in a concentration-dependent manner, but did not affect Rac2, cdc42 or RhoA activation. Murine neutrophils transfected with vectors expressing fluorescent probes PAK-PBD-YFP and PH-AKT-RFP were used to determine the effects of Msp on the localization of activated Rac and PI3 kinase products. Real-time confocal images showed that Msp inhibited the polarized accumulation of activated Rac and PI3-kinase products upon exposure to fMLP. The findings indicate that T. denticola Msp inhibition of neutrophil polarity may be due to the selective suppression of the Rac1 pathway.     

Regulation of Rac1 and Cdc42 activation in thrombin- and collagen-stimulated CHRF-288-11 cells

Rac1 and Cdc42 are members of the Rho family of small GTPases and have been shown to promote the formation of lamellipodia and filopodia at the leading edge of motile cells and affect cell migration. In this study the authors have investigated the activation of Rac1 and Cdc42 by thrombin or collagen using the megakaryocytic cell line, CHRF-288-11. Maximal activation of Rac1 by thrombin or collagen was observed at 3 and 1 min, respectively. Similar results were obtained for the activation of Cdc42 by thrombin or collagen. The activation of Rac1 by thrombin and collagen was inhibited by the calmodulin-specific inhibitor, W7. However, W7 had no effect on the activation of Cdc42 by thrombin or collagen. The less potent calmodulin inhibitor, W5, did not have any effect on Rac1 or Cdc42 activation by thrombin or collagen. Transient over-expression of calmodulin in CHRF cells increased the basal and thrombin-mediated activation of Rac1 when compared to control but had no effect on the basal and thrombin-mediated activation of Cdc42 when compared to control. The results suggest that calmodulin regulates the activation of Rac1 in CHRF-288-11 cells in a manner similar to that in the human platelets.     

The Rac/Cdc42 guanine nucleotide exchange factor beta1Pix enhances mastoparan-activated Gi-dependent pathway in mast cells

Carbachol stimulates granule exocytosis, phospholipase C (PLC), and phospholipase D (PLD) in RBL-2H3hm1 mast cells by a mechanism that involves Galphaq. However, mastoparan stimulates the same responses through Gi protein. Both Gi and Galphaq pathways are suppressed by Clostridium difficile toxin B, suggesting that Rac and Cdc42 small GTPases are also involved. Over-expression of beta1Pix, a guanine nucleotide exchange factor for Rac and Cdc42, enhances mastoparan-but not carbachol-induced hexosaminidase secretion and PLC and PLD activation. Furthermore, cells expressing beta1Pix exhibit elevated levels of mastoparan-stimulated IP3 production. Taken together, these findings implicate beta1Pix in regulating hexoasaminidase secretion and IP3 production in early stage upon mastoparan stimulation.     

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.