RESUMO
BACKGROUND: HER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3ß1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3ß1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis. METHODS: To investigate the role of α3ß1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3ß1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3ß1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence. RESULTS: Tumor onset in mice was independent of the presence of α3ß1. In contrast, the depletion of α3ß1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3ß1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow. CONCLUSION: Downregulation of α3ß1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3ß1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3ß1 in breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Integrina alfa3beta1/deficiência , Receptor ErbB-2/genética , Animais , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Development of wound therapies is hindered by poor understanding of combinatorial integrin function in the epidermis. In this study, we generated mice with epidermis-specific deletion of α3ß1, α9ß1, or both integrins as well as keratinocyte lines expressing these integrin combinations. Consistent with proangiogenic roles for α3ß1, α3-null keratinocytes showed reduced paracrine stimulation of endothelial cell migration and survival, and wounds of epidermis-specific α3 knockout mice displayed impaired angiogenesis. Interestingly, α9ß1 in keratinocytes suppressed α3ß1-mediated stimulation of endothelial cells, and wounds of epidermis-specific α9 knockout mice displayed delayed vascular normalization and reduced endothelial apoptosis, indicating that α9ß1 cross-suppresses α3ß1 proangiogenic functions. Moreover, α9ß1 inhibited α3ß1 signaling downstream of focal adhesion kinase (FAK) autoactivation at the point of Src-mediated phosphorylation of FAK Y861/Y925. Finally, α9ß1 cross-suppressed many α3ß1-dependent genes, including the gene that encodes MMP-9, which we implicated as a regulator of integrin-dependent cross talk to endothelial cells. Our findings identify a novel physiological context for combinatorial integrin signaling, laying the foundation for therapeutic strategies that manipulate α9ß1 and/or α3ß1 during wound healing.
Assuntos
Epiderme/metabolismo , Integrina alfa3beta1/antagonistas & inibidores , Integrinas/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Cicatrização , Animais , Apoptose , Movimento Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfa3beta1/deficiência , Integrina alfa3beta1/metabolismo , Integrinas/deficiência , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Ferimentos e Lesões/sangueRESUMO
The efficient trafficking of immune cells into peripheral nonlymphoid tissues is key to enact their protective functions. Despite considerable advances in our understanding of cell migration in secondary lymphoid organs, real-time leukocyte recruitment into inflamed tissues is not well characterized. The conventional multistep paradigm of leukocyte extravasation depends on CD18 integrin-mediated events such as rapid arrest and crawling on the surface of the endothelium and transmigration through the endothelial layer. Using enhanced three-dimensional detection of fluorescent CD18 fusion proteins in a newly developed knockin mouse, we report that extravasating leukocytes (neutrophils, monocytes, and T cells) show delayed uropod detachment and become extremely elongated before complete transmigration across the endothelium. Additionally, these cells deposit CD18(+) microparticles at the subendothelial layer before retracting the stretched uropod. Experiments with knockout mice and blocking antibodies reveal that the uropod elongation and microparticle formation are the result of LFA-1-mediated adhesion and VLA-3-mediated cell migration through the vascular basement membrane. These findings suggest that uropod elongation is a final step in the leukocyte extravasation cascade, which may be important for precise regulation of leukocyte recruitment into inflamed tissues.
Assuntos
Extensões da Superfície Celular/fisiologia , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Vasculite/metabolismo , Animais , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Extensões da Superfície Celular/genética , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfa3beta1/deficiência , Integrina alfa3beta1/genética , Leucócitos/metabolismo , Leucócitos/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência/métodos , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Neutrófilos/ultraestrutura , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Linfócitos T/ultraestrutura , Migração Transendotelial e Transepitelial/genética , Vasculite/genéticaRESUMO
Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. In this study, we demonstrate that alpha3beta1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events.
Assuntos
Integrina alfa3beta1/metabolismo , Rim/embriologia , Rim/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Sobrevivência Celular , Primers do DNA/genética , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Integrina alfa3beta1/deficiência , Integrina alfa3beta1/genética , Rim/citologia , Laminina/deficiência , Laminina/genética , Laminina/metabolismo , Camundongos , Camundongos Knockout , Morfogênese , Gravidez , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteínas Wnt/genéticaRESUMO
The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.
Assuntos
Antígenos CD/metabolismo , Células Epiteliais/metabolismo , Integrina alfa3beta1/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP , Integrina alfa3beta1/deficiência , Integrina alfa3beta1/genética , Laminina/metabolismo , Substâncias Macromoleculares , Camundongos , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Receptores de Quinase C Ativada , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetraspanina 24 , Transativadores/metabolismo , beta CateninaRESUMO
alpha3beta1-integrin is abundantly expressed in the epidermis, and in mice, ablation of the alpha3 gene results in embryonic defects and perinatal lethality. To determine the role of alpha3-integrin in adult skin development, we grafted skin from newborn alpha3-integrin-deficient mice on to ICRF nu/nu recipients. We report that adult alpha3-integrin-deficient skin has severe abnormalities restricted to hair follicle morphology, which include stunted hair follicle growth, increased hair follicle fragility, aberrant pigment accumulation and formation of hair follicle clusters. These abnormalities are caused by a combination of defects in: (1) keratinocyte cytoskeletal organisation, (2) outer root sheath architecture and (3) integrity of the lamina densa. Our results indicate that alpha3beta1 is not essential for adult interfollicular epidermal differentiation, but it is required to direct several processes important in hair follicle maintenance and morphogenesis.
Assuntos
Epiderme/anormalidades , Folículo Piloso/anormalidades , Integrina alfa3beta1/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Animais Recém-Nascidos , Padronização Corporal/genética , Diferenciação Celular/genética , Epiderme/metabolismo , Epiderme/ultraestrutura , Folículo Piloso/metabolismo , Folículo Piloso/ultraestrutura , Integrina alfa3beta1/deficiência , Integrina alfa3beta1/genética , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Laminina/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Morfogênese/genética , Pigmentos Biológicos/metabolismo , Pigmentação da Pele/genética , Transplante de PeleRESUMO
Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.