RESUMO
Infectious agents are often considered potential triggers of chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. Here we show that following control of acute infection of mice with the myotropic Colombiana strain of Trypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Lesions occurred in many but not all organs and tissues, with skeletal muscle arteries being the most severely affected, and were associated with myositis, atrophy, paresis/paralysis, and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80(+)macrophages, and T. cruzi tetramer-specific CD8(+) T lymphocytes capable of producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) but not interleukin-17 (IL-17). T. cruzi-specific IgG was detected in sera from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus,T. cruzi infection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific type I immune responses.
Assuntos
Doença de Chagas/patologia , Paralisia/parasitologia , Trypanosoma cruzi , Vasculite/patologia , Vasculite/parasitologia , Animais , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Regulação da Expressão Gênica/fisiologia , Membro Posterior/patologia , Linfócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Paralisia/patologia , Parasitemia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vasculite/imunologiaRESUMO
AIMS: Atypical chemokine receptor 1 (Ackr1; previously known as the Duffy antigen receptor for chemokines or Darc) is thought to regulate acute inflammatory responses in part by scavenging inflammatory CC and CXC chemokines; however, evidence for a role in chronic inflammation has been lacking. Here we investigated the role of Ackr1 in chronic inflammation, in particular in the setting of atherogenesis, using the apolipoprotein E-deficient (ApoE(-/-)) mouse model. METHODS AND RESULTS: Ackr1(-/-)ApoE(-/-) and Ackr1(+/+)ApoE(-/-) littermates were obtained by crossing ApoE(-/-) mice and Ackr1(-/-) mice on a C57BL/6J background. Ackr1 (+/+)ApoE(-/-)mice fed a Western diet up-regulated Ackr1 expression in the aorta and had markedly increased atherosclerotic lesion size compared with Ackr1(-/-)ApoE(-/-) mice. This difference was observed in both the whole aorta and the aortic root in both early and late stages of the model. Ackr1 deficiency did not affect serum cholesterol levels or macrophage, collagen or smooth muscle cell content in atherosclerotic plaques, but significantly reduced the expression of Ccl2 and Cxcl1 in the whole aorta of ApoE(-/-) mice. In addition, Ackr1 deficiency resulted in a modest decrease in T cell subset frequency and inflammatory mononuclear phagocyte content in aorta and blood in the model. CONCLUSIONS: Ackr1 deficiency appears to be protective in the ApoE knockout model of atherogenesis, but it is associated with only modest changes in cytokine and chemokine expression as well as T-cell subset frequency and inflammatory macrophage content.
Assuntos
Aorta , Aortite , Apolipoproteínas E , Aterosclerose , Receptores de Superfície Celular , Animais , Feminino , Transferência Adotiva , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Aortite/genética , Aortite/imunologia , Aortite/metabolismo , Aortite/patologia , Aortite/prevenção & controle , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Dieta Ocidental , Modelos Animais de Doenças , Sistema do Grupo Sanguíneo Duffy/genética , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Placa Aterosclerótica , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Fatores de TempoRESUMO
AMD3100 (plerixafor), is a specific CXCR4 antagonist approved by the FDA for mobilizing hematopoietic stem cells from bone marrow to blood for transplantation in cancer. AMD3100 also mobilizes most mature leukocyte subsets to blood; however, their source and trafficking potential have not been fully delineated. Here, we show that a single injection of AMD3100 10 mg/kg into C57Bl/6 mice rapidly mobilizes (peak â¼ 2.5 h) the same leukocyte subsets to blood as in humans. Using this model, we found that AMD3100 mobilization of neutrophils, lymphocytes, and monocytes to blood is not reduced by splenectomy or by blockade of lymphocyte egress from lymph node with FTY720, but is coupled to (i) reduced content of each of these cell types in the bone marrow; (ii) reduced T-cell numbers in thymuses; (iii) increased lymphocytes in lymph nodes; and (iv) increased neutrophil and monocyte content in the lung. Direct intrathymic labeling showed that AMD3100 selectively mobilizes naïve thymic CD4(+) and CD8(+) T cells to blood. Finally, AMD3100-induced neutrophil mobilization to blood did not reduce neutrophil trafficking to thioglycollate-inflamed peritoneum. Thus, AMD3100 redistributes lymphocytes, monocytes, and neutrophils from primary immune organs to secondary immune organs, peripheral tissues, and blood, without compromising neutrophil trafficking to inflamed sites.
Assuntos
Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Receptores CXCR4/antagonistas & inibidores , Animais , Benzilaminas , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Movimento Celular/imunologia , Ciclamos , Feminino , Imunofenotipagem , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Contagem de Leucócitos , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Fenótipo , Baço/efeitos dos fármacos , Baço/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.
Assuntos
Candida albicans/fisiologia , Candidíase Invasiva/imunologia , Rim/imunologia , Macrófagos/fisiologia , Receptores de Quimiocinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transferência Adotiva , Animais , Apoptose , Receptor 1 de Quimiocina CX3C , Candida albicans/imunologia , Candida albicans/ultraestrutura , Candidíase Invasiva/patologia , Movimento Celular , Quimiocina CCL2/fisiologia , Quimiocina CX3CL1/fisiologia , Feminino , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/imunologia , Humanos , Hifas/ultraestrutura , Rim/microbiologia , Rim/patologia , Ativação de Macrófagos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Monócitos/microbiologia , Monócitos/fisiologia , Países Baixos , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único , Quimera por Radiação , Receptores CCR2/fisiologia , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Fatores de Risco , Organismos Livres de Patógenos Específicos , Estados UnidosRESUMO
AIMS: Recent evidence suggests that both Ccr7 and its ligands, Ccl19 and Ccl21, are present in mouse and human atherosclerotic plaques; however, the role of Ccr7 in atherogenesis is still controversial. Here, we addressed this question by using the classic apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: Ccr7(-/-)ApoE(-/-) double knockout mice and Ccr7(+/+)ApoE(-/-) littermates were generated and maintained on a high-fat Western diet for 8 weeks to induce atherosclerosis. Ccr7(-/-)ApoE(-/-) mice showed an ~80% increase in atherosclerotic lesion size in the whole aorta and a two-fold increase in the aortic root compared with Ccr7(+/+)ApoE(-/-) mice. Ccr7(-/-)ApoE(-/-) mice had increased T cells in the blood, bone marrow, and spleen, as well as in atherosclerotic lesions. Competitive repopulation experiments revealed that T cells from Ccr7(-/-)ApoE(-/-) mice migrated poorly into lymph nodes but better into mouse aortas compared with T cells from Ccr7(+/+)ApoE(-/-) mice. Transplantation of the bone marrow from Ccr7(-/-)ApoE(-/-) mice into lethally irradiated Ccr7(+/+)ApoE(-/-) mice resulted in ~60% more atherosclerotic lesions compared with Ccr7(+/+)ApoE(-/-) donor bone marrow, suggesting that exacerbation was mediated by a Ccr7(+) bone marrow-derived cell(s). Furthermore, in Ccr7(-/-)ApoE(-/-) mice the serum level of IL-12 was markedly increased, whereas the level of transforming growth factor beta (TGF-ß) was significantly decreased, suggesting an imbalance of T cell responses in these mice. CONCLUSION: Our data suggest that genetic deletion of Ccr7 exacerbates atherosclerosis by increasing T cell accumulation in atherosclerotic lesions.
Assuntos
Aterosclerose/fisiopatologia , Deleção de Genes , Receptores CCR7/deficiência , Receptores CCR7/genética , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Transplante de Medula Óssea/patologia , Modelos Animais de Doenças , Feminino , Interleucina-12/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR7/fisiologia , Linfócitos T/patologia , Fator de Crescimento Transformador beta/sangueRESUMO
Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.
Assuntos
Apolipoproteínas B/fisiologia , Lipoproteínas/fisiologia , Neovascularização Fisiológica , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Sequência de Aminoácidos , Animais , Apolipoproteína C-II/fisiologia , Proteínas de Bactérias/genética , Proteínas de Transporte/fisiologia , Células Cultivadas , Humanos , Lipoproteínas LDL/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/análise , Peixe-ZebraRESUMO
RATIONALE: The chemokine receptor Ccr6 is a G-protein-coupled receptor expressed on various types of leukocytes identified in mouse atherosclerotic lesions. Recent evidence suggests that both CCR6 and its ligand CCL20 are also present in human atheroma; however, their functional roles in atherogenesis remain undefined. OBJECTIVE: Our objective was to delineate the role of Ccr6 in atherogenesis in the apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: Both Ccr6 and Ccl20 are expressed in atherosclerotic aorta from ApoE(-/-) mice. Aortic lesion area in Ccr6(-/-)ApoE(-/-) mice was â¼40% and â¼30% smaller than in Ccr6(+/+)ApoE(-/-) mice at 16 and 24 weeks of age, respectively. Transplantation of bone marrow from Ccr6(-/-) mice into ApoE(-/-) mice resulted in â¼40% less atherosclerotic lesion area than for bone marrow from Ccr6(+/+) mice; lesions in Ccr6(-/-)ApoE(-/-) mice had 44% less macrophage content than lesions in Ccr6(+/+)ApoE(-/-) mice. Ccr6 was expressed on a subset of primary mouse monocytes. Accordingly, Ccl20 induced chemotaxis of primary monocytes from wild-type but not Ccr6(-/-) mice; moreover, Ccl20 induced monocytosis in ApoE(-/-) mice in vivo. Consistent with this, we observed 30% fewer monocytes in circulating blood of Ccr6(-/-)ApoE(-/-) mice, mainly because of fewer CD11b(+)Ly6C(high) inflammatory monocytes. CONCLUSIONS: Ccr6 promotes atherosclerosis in ApoE-deficient mice, which may be due in part to Ccr6 support of normal monocyte levels in blood, as well as direct Ccr6-dependent monocyte migration.
Assuntos
Aorta/imunologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Deleção de Genes , Receptores CCR6/deficiência , Animais , Antígenos Ly/sangue , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Transplante de Medula Óssea , Antígeno CD11b/sangue , Linhagem Celular , Quimiocina CCL20/metabolismo , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Feminino , Contagem de Leucócitos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Receptores CCR6/genética , Fatores de TempoRESUMO
High expression of 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been detected in various invasive cancers. In the current study, we investigated its role in cancer cell migration and experimental metastasis. Down-regulation of PDK1 expression by small interference RNA markedly inhibited spontaneous migration and epidermal growth factor (EGF)-induced chemotaxis of human breast cancer cells. The defects were rescued by expressing wild-type PDK1. PDK1-depleted cells showed impaired EGF-induced actin polymerization and adhesion, probably due to a decrease in phosphorylation of LIM kinase/cofilin and integrin beta1. Confocal microscopy revealed that EGF induced cotranslocation of PDK1 with Akt and protein kinase Czeta (PKCzeta), regulators of LIM kinase, and integrin beta1. Furthermore, PDK1 depletion dampened EGF-induced phosphorylation and translocation of Akt and PKCzeta, suggesting that Akt and PKCzeta functioned downstream of PDK1 in the chemotactic signaling pathway. In severe combined immunodeficiency mice, PDK1-depleted human breast cancer cells formed more slowly growing tumors and were defective in extravasation to mouse lungs after i.v. injection. Our results indicate that PDK1 plays an important role in regulating the malignant behavior of breast cancer cells, including their motility, through activation of Akt and PKCzeta. Thus, PDK1, which increases its expression in cancer cells, can be used as a target for the development of novel therapies.
Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Actinas/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Regulação para Baixo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Metastasis is the major cause of mortality in lung cancer. Chemotaxis plays a vital role in cancer cell metastasis. In the current study, we reported that epidermal growth factor (EGF) induced a robust chemotaxis of A549 and H1299 cells, two representative human non-small cell lung cancer (NSCLC) cells. Chelerythrine chloride, an inhibitor of all protein kinase C (PKC) isozymes, significantly reduced the chemotactic capacity of NSCLC cells while inhibitors of classical or novel PKC isozymes, such as Gö6976, calphostin C, or Gö6850, showed no effect, which suggested that atypical PKC might be involved in the chemotactic process of NSCLC cells. EGF-elicited translocation and phosphorylation of atypical PKCzeta, indicating that EGF could activate PKCzeta. Treatment with a PKCzeta specific inhibitor, a myristoylated pseudosubstrate, blocked the chemotaxis in a dose-dependent manner, further confirming that atypical PKCzeta was required for NSCLC chemotaxis. Mechanistic studies suggested that PKCzeta was regulated by phosphatidylinositol 3 kinase (PI3K)/Akt. Furthermore, PKCzeta-mediated chemotaxis by regulating actin polymerization and cell adhesion. Taken together, our study suggested that PKCzeta was required in NSCLC cell chemotaxis, thus could be used as a target to develop anti-lung cancer metastasis therapies.
Assuntos
Quimiotaxia/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase C/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Regulação para Baixo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores CXCR4/fisiologiaRESUMO
Protein kinase Czeta PKCzeta mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the research for its upstream regulator, we investigated the role of Akt2 in chemotaxis and metastasis of human breast cancer cells. Reduction of Akt2 expression by siRNA inhibited chemotaxis of MDA-MB-231, T47D, and MCF7 cells, three representative human breast cancer cells. Expression of a wild type Akt2 in siRNA transfected cells rescued the phenotype. EGF-induced integrin beta1 phosphorylation was dampened, consistent with defects in adhesion. Phosphorylation of LIMK and cofilin, a critical step of cofilin recycle and actin polymerization, was also impaired. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during chemotaxis. Depletion of Akt2 by siRNA impaired the activation of PKCzeta while inhibition of PKCzeta did not interfere with EGF induced phosphorylation of Akt. Furthermore, EGF induced co-immunoprecipitation between PKCzeta and Akt2, but not Akt1, suggesting that a direct interaction between PKCzeta and Akt2 in chemotaxis. Protein levels of integrin beta1, LIMK, cofilin, and PKCzeta didn't alter, suggesting that Akt2 does not regulate the expression of these signaling molecules. In a Severe Combine Immunodeficiency mouse model, Akt2 depleted MDA-MB-231 cells showed a marked reduction in metastasis to mouse lungs, demonstrating the biological relevancy of Akt2 in cancer metastasis in vivo. Taken together, our results suggest that Akt2 directly mediates EGF-induced chemotactic signaling pathways through PKCzeta and its expression is critical during the extravasation of circulating cancer cells.
Assuntos
Neoplasias da Mama/enzimologia , Quimiotaxia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Adesão Celular , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNARESUMO
Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Quimiotaxia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Metástase Neoplásica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.