Design and synthesis of rho kinase inhibitors (III)

The structure-activity relationship of Rho kinase inhibitors bearing an isoquinoline scaffold was studied. N-(1-Benzyl-3-pyrrolidyl)-N-(5-isoquinolyl)amine analogues were optimized with respect to their inhibitory potencies for the enzyme and for chemotaxis. The potent analogues were further evaluated by an ex vivo test in which the selected compounds were orally administered to rats, and the Rho kinase inhibitory potency observed in the rat serum was evaluated 3h after the administration. Compound 23g showed a high level of Rho kinase inhibitory activity in the rat serum and was stable in an in vitro metabolic test using a microsomal cytochrome preparation. The (R)-isomer of 23g displayed a higher level of inhibitory potency than the (S)-isomer in a cell-free kinase assay and in the cell migration assay (IC(50)(ENZ)=25 nM and IC(50)(MCP)=1 microM). The (R)-isomer successfully inhibited the phosphorylation of MBS (myosin-binding subunit) in cells.     

Design and synthesis of Rho kinase inhibitors (I)

Several structurally unrelated scaffolds of the Rho kinase inhibitor were designed using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict the structure-activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of higher potency and selectivity. We identified several potent platforms for developing the Rho kinase inhibitors, namely, pyridine, 1H-indazole, isoquinoline, and phthalimide.     

Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis

Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated beta1 integrin-dependent, but not beta2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.     

Identification of TRIO-GEFD1 chemical inhibitors using the yeast exchange assay

BACKGROUND INFORMATION: Rho GTPases are involved in many biological processes and participate in cancer development. Their activation is catalysed by exchange factors [RhoGEFs (Rho GTPase guanine nucleotide-exchange factor)] of the Dbl family. RhoGEFs display proto-oncogenic features, thus appearing as candidate targets for anticancer drugs. Dominant-negative Rho GTPase mutants have been widely used to block RhoGEF signalling. However, these tools suffer from limitations, due to the high number of RhoGEFs and the complex mechanisms that control Rho GTPase activation. RESULTS: RhoG-T17N is a poor inhibitor of its exchange factor TRIO-GEFD1 (first exchange domain of the exchange factor TRIO) in vivo: although it binds to TRIO-GEFD1, RhoG-T17N does not block the downstream signalling. Using the yeast exchange assay, we show that in the presence of TRIO-GEFD1, RhoG-T17N can bind to its effectors, which illustrates how negative mutants may produce misleading interpretations and emphasizes the need for new types of RhoGEF inhibitors. In that prospect, we adapted the yeast exchange assay method to identify RhoGEF inhibitors. Using this novel approach, we screened a 3500-chemical-compound library and identified a potential inhibitor of TRIO-GEFD1. This molecule inhibited TRIO-GEFD1 in vitro. Among the chemical analogues of this compound, we identified two molecules with better inhibitory activity. The three TRIO-GEFD1 inhibitors had no effect on ARHGEF17 and ARNO [ARF (ADP-ribosylation factor) nucleotide-binding-site opener], two exchange factors for RhoA and Arf1 respectively. CONCLUSIONS: The development of RhoGEF inhibitors appears as a valuable tool for the study of Rho GTPase signalling pathways. The yeast exchange assay adaptation we present here is suitable to screen for chemical or peptide libraries and identify candidate inhibitors.     

Involvement of the Rho-kinase/myosin light chain kinase pathway on human monocyte chemotaxis induced by ATL-1, an aspirin-triggered lipoxin A4 synthetic analog

Lipoxins (LX) are arachidonic acid metabolites able to induce monocyte chemotaxis in vitro and in vivo. Nonetheless, the signaling pathways mediating this process are yet unclear. In this study, we have investigated the mechanisms associated with human monocyte activation in response to 15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATL-1), a stable 15-epi-LXA4 analog. Our results demonstrate that ATL-1-induced monocyte chemotaxis (10-300 nM) is inhibited by pertussis toxin, suggesting an effect via the G-protein-linked LXA4 receptor. Monocytes stimulated with the analog presented an increased ERK-2 phosphorylation, which was reduced by PD98059, a selective inhibitor of the MEK 1/2 pathway. After exposure of the cells to ATL-1, myosin L chain kinase (MLCK) phosphorylation was evident and this effect was inhibited by PD98059 or Y-27632, a specific inhibitor of Rho kinase. In addition, Y-27632 abolished ERK-2 activation, suggesting that the MAPK pathway is downstream of Rho/Rho kinase in MLCK activation induced by ATL-1. The specific MLCK inhibitor ML-7, as well as Y-27632, abrogated monocyte chemotaxis stimulated by the analog, confirming the central role of the Rho kinase/MLCK pathway on ATL-1 action. Together, these results indicate that ATL-1 acts as a potent monocyte chemoattractant via Rho kinase and MLCK. The present study clarifies some of the mechanisms involved on the activation of monocytes by LXs and opens new avenues for investigation of these checkpoint controllers of inflammation.     

Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation

The inflammatory response is one of the most dramatic examples of directed cell movement in nature. Inflammation is triggered at the site of injury and results in the migration of immune cells to the site to protect the host from infection. We have devised an in vivo inflammation assay using translucent zebrafish embryos, which allow live imaging and pharmacological manipulation of macrophage chemotaxis to wounds inflicted with a laser. Using this assay, we test the role of the microtubule cytoskeleton in macrophage chemotaxis in vivo using nocodazole to disrupt microtubule polymerization. We find that de-stabilisation of microtubules with nocodazole impairs macrophage recruitment to wounds, but that addition of the Rho kinase inhibitor Y-27632 suppresses these effects and restores the recruitment of macrophages to wounds. Taken together, these results suggest that destabilizing microtubules activates Rho kinase and that this increase in Rho kinase activity interferes with leukocyte recruitment in vivo.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.     

Inhibition of Rho modulates cytokine-induced prostaglandin E2 formation in renal mesangial cells

Stimulation of rat mesangial cells for 24 h with interleukin-1beta (IL- 1beta) plus forskolin (Fk) leads to a marked increase in prostaglandin E2 (PGE2) synthesis. This effect is further enhanced by the small G-protein Rho inhibitor toxin A. A similar increase in PGE2 formation is obtained with Y27632, a Rho-dependent kinase inhibitor, and with lovastatin, a hydroxymethylglutaryl-coenzyme A inhibitor which depletes cells from geranylgeranyl moieties and thus blocks Rho activation. In parallel to the increased PGE2 synthesis, a potentiation of IL-1beta-induced secretory group IIA phospholipases A2 (sPLA2-IIA) protein expression also occurs by Rho inhibition. However, only toxin A triggers an increased sPLA2-IIA activity consistent with the elevated levels of protein expression, whereas Y27632 and lovastatin rather reduced IL-1beta-induced sPLA2-IIA activity. In vitro activity studies reveal that Y27632 and lovastatin can directly block sPLA2-IIA enzyme activity in a concentration-dependent manner. Interestingly, in the absence of IL-1beta/Fk stimulation and the lack of sPLA2-IIA protein expression, all Rho inhibitors exert a small but significant increase in PGE2 formation suggesting that additional PLA2s or downstream enzymes like cyclooxygenases or prostaglandin synthases may be activated by Rho inhibitors. Western blot analyses of toxin A-, Y27632- and lovastatin-stimulated cells reveal that the cytosolic group IV PLA2 (cPLA2) and the cytosolic PGE2 synthase (cPGES), but not the sPLA2-IIA, cyclooxygenase-2 or the microsomal PGE2 synthase (mPGES), are upregulated compared to unstimulated cells. Furthermore, the Rho inhibitors induced arachidonic acid release from intact cells which is blocked by the cPLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP). In summary, these data show that inhibition of the small G-protein Rho, either by toxin A, lovastatin, or Y27632, exert a dual effect on mesangial cells: (i) in the absence of an inflammatory stimulus it activates the constitutive cPLA2 and cPGE2 synthase and generates low amount of PGE2. (ii) In the presence of inflammatory cytokines it potentiates sPLA2-IIA expression and subsequent PGE2 formation. In addition, we identified lovastatin and Y27632 as direct inhibitors of sPLA2-IIA in a cell-free system.     

Heme induces neutrophil migration and reactive oxygen species generation through signaling pathways characteristic of chemotactic receptors

Hemolysis or extensive cell damage can lead to high concentrations of free heme, causing oxidative stress and inflammation. Considering that heme induces neutrophil chemotaxis, we hypothesize that heme activates a G protein-coupled receptor. Here we show that similar to heme, several heme analogs were able to induce neutrophil migration in vitro and in vivo. Mesoporphyrins, molecules lacking the vinyl groups in their rings, were not chemotactic for neutrophils and selectively inhibited heme-induced migration. Moreover, migration of neutrophils induced by heme was abolished by pretreatment with pertussis toxin, an inhibitor of Galpha inhibitory protein, and with inhibitors of phosphoinositide 3-kinase, phospholipase Cbeta, mitogen-activated protein kinases, or Rho kinase. The induction of reactive oxygen species by heme was dependent of Galpha inhibitory protein and phosphoinositide 3-kinase and partially dependent of phospholipase Cbeta, protein kinase C, mitogen-activated protein kinases, and Rho kinase. Together, our results indicate that heme activates neutrophils through signaling pathways that are characteristic of chemoattractant molecules and suggest that mesoporphyrins might prove valuable in the treatment of the inflammatory consequences of hemorrhagic and hemolytic disorders.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

Benzimidazole- and benzoxazole-based inhibitors of Rho kinase

Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral bioavailability.     

Signaling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line.

The adaptor protein Crk has been reported to associate with focal adhesions and is thought to be involved in integrin-mediated signaling pathway. However, the precise mechanism of Crk-dependent regulation of cytoskeleton still remains under investigation. In this study, we have established a v-Crk-inducible cell line in rat fibroblasts 3Y1 cells and found that v-Crk activated Rho and induced actin stress fiber formation. In addition to the induction of tyrosine-phosphorylation of p130(Cas) and paxillin, we demonstrated that v-Crk induced threonine-phosphorylated bands sized at 72/78 kDa found specifically in 3Y1 cells. Both of the inhibitors of Rho and Rho-associated kinase, C3 and Y27632, respectively, inhibited these v-Crk-induced biochemical effects. Although v-Crk-induced cells exhibited a decrease of cell motility, integrin stimulation recovered the suppression of motility. Furthermore, v-Crk enhanced motility in chemotactic assay toward fibronectin with additional activation of Rho and the increase of levels of CD44 cleavage. These results suggest that v-Crk activated Rho and induced actin stress fiber formation and CD44 cleavage leading to the regulation of cell motility.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Myosin light chain phosphorylation-dependent modulation of volume-regulated anion channels in macrovascular endothelium

The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML-9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1(191-210), an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.     

Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca(2+)] (27). This Ca(2+)-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca(2+) sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used alpha-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca(2+)-desensitizing hypoxic relaxation.     

The involvement of lipid rafts in epidermal growth factor-induced chemotaxis of breast cancer cells

Metastasis is the major cause of morbidity and mortality in cancer. Recent studies reveal a role of chemotaxis in cancer cell metastasis. Epidermal growth factor receptors (EGFR) have potent chemotactic effects on human breast cancer cells. Lipid rafts, organized microdomain on plasma membranes, regulate the activation of many membrane receptors. In the current study, we investigated the role of lipid rafts in EGFR-mediated cancer cell chemotaxis. Our confocal microscopy results suggested that EGFR co-localized with GM1-positive rafts. Disrupting rafts with methyl-beta-cyclodextrin (mbetaCD) inhibited EGF-induced chemotaxis of human breast cancer cells. Supplementation with cholesterol reversed the inhibitory effects. Pretreatment with mbetaCD also impaired directional migration of cells in an in vitro "wound healing" assay, EGF-induced cell adhesion, actin polymerization, Akt phosphorylation and protein kinase Czeta (PKCzeta) translocation. Taken together, our study indicated that integrity of lipid rafts was critical in EGF-induced chemotaxis of human breast cancer cells.