RESUMO
Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.
Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibronectinas/farmacologia , Laminina/farmacologia , Glândulas Mamárias Animais/citologia , Metaloproteinase 3 da Matriz/farmacologia , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Espaço Extracelular/metabolismo , Feminino , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Camundongos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Estresse Mecânico , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is a form of epithelial plasticity implicated in fibrosis and tumor metastasis. Here we show that the mechanical rigidity of the microenvironment plays a pivotal role in the promotion of EMT by controlling the subcellular localization and downstream signaling of Rac GTPases. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against EMT, whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Rac1b, a highly activated splice variant of Rac1 found in tumors, localizes to the plasma membrane in cells cultured on stiff substrata or in collagen-rich regions of human breast tumors. At the membrane, Rac1b forms a complex with NADPH oxidase and promotes the production of reactive oxygen species, expression of Snail, and activation of the EMT program. In contrast, soft microenvironments inhibit the membrane localization of Rac1b and subsequent redox changes. These results reveal a novel mechanotransduction pathway in the regulation of epithelial plasticity via EMT.
Assuntos
Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , NADPH Oxidases/metabolismo , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Fenômenos Biomecânicos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Membrana Celular/metabolismo , Movimento Celular , Microambiente Celular , Feminino , Adesões Focais/metabolismo , Humanos , Integrina beta1/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Espécies Reativas de OxigênioRESUMO
Epithelial-mesenchymal transition (EMT) is a phenotypic change in which epithelial cells detach from their neighbors and become motile. Whereas soluble signals such as growth factors and cytokines are responsible for stimulating EMT, here we show that gradients of mechanical stress define the spatial locations at which EMT occurs. When treated with transforming growth factor (TGF)-beta, cells at the corners and edges of square mammary epithelial sheets expressed EMT markers, whereas those in the center did not. Changing the shape of the epithelial sheet altered the spatial pattern of EMT. Traction force microscopy and finite element modeling demonstrated that EMT-permissive regions experienced the highest mechanical stress. Myocardin-related transcription factor (MRTF)-A was localized to the nuclei of cells located in high-stress regions, and inhibiting cytoskeletal tension or MRTF-A expression abrogated the spatial patterning of EMT. These data suggest a causal role for tissue geometry and endogenous mechanical stresses in the spatial patterning of EMT.
