RESUMO
17ß-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ERα mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ERα mice did not produce normal outgrowths. Ex vivo, C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development.
Assuntos
Epitélio/metabolismo , Receptor alfa de Estrogênio/biossíntese , Glândulas Mamárias Animais/embriologia , Comunicação Parácrina/fisiologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/transplante , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lipoilação/fisiologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.
RESUMO
Oestrogen receptor α (ERα) is a transcription factor with ligand-independent and ligand-dependent activation functions (AF)-1 and -2. Oestrogens control postnatal mammary gland development acting on a subset of mammary epithelial cells (MECs), termed sensor cells, which are ERα-positive by immunohistochemistry (IHC) and secrete paracrine factors, which stimulate ERα-negative responder cells. Here we show that deletion of AF-1 or AF-2 blocks pubertal ductal growth and subsequent development because both are required for expression of essential paracrine mediators. Thirty percent of the luminal cells are ERα-negative by IHC but express Esr1 transcripts. This low level ERα expression through AF-2 is essential for cell expansion during puberty and growth-inhibitory during pregnancy. Cell-intrinsic ERα is not required for cell proliferation nor for secretory differentiation but controls transcript levels of cell motility and cell adhesion genes and a stem cell and epithelial mesenchymal transition (EMT) signature identifying ERα as a key regulator of mammary epithelial cell plasticity.
Assuntos
Epitélio/metabolismo , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Glândulas Mamárias Animais/metabolismo , Animais , Proliferação de Células , Sistema Endócrino/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Regulação da Expressão Gênica , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Fenótipo , Gravidez , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroides/metabolismo , Relação Estrutura-AtividadeRESUMO
Amphiregulin (AREG)-/- mice demonstrate impaired mammary development and form only rudimentary ductal epithelial trees; however, AREG-/- glands are still capable of undergoing alveologenesis and lactogenesis during pregnancy. Transplantation of AREG-/- mammary epithelial cells into cleared mouse mammary fat pads results in a diminished capacity for epithelial growth (â¼15%) as compared to that of wild-type mammary epithelial cells. To determine whether estrogen receptor α (ERα, also known as ESR1) and/or AREG signaling were necessary for non-mammary cell redirection, we inoculated either ERα-/- or AREG-/- mammary cells with non-mammary progenitor cells (WAP-Cre/Rosa26LacZ+ male testicular cells or GFP-positive embryonic neuronal stem cells). ERα-/- cells possessed a limited ability to grow or reprogram non-mammary cells in transplanted mammary fat pads. AREG-/- mammary cells were capable of redirecting both types of non-mammary cell populations to mammary phenotypes in regenerating mammary outgrowths. Transplantation of fragments from AREG-reprogrammed chimeric outgrowths resulted in secondary outgrowths in six out of ten fat pads, demonstrating the self-renewing capacity of the redirected non-mammary cells to contribute new progeny to chimeric outgrowths. Nestin was detected at the leading edges of developing alveoli, suggesting that its expression may be essential for lobular expansion.
Assuntos
Anfirregulina/genética , Linhagem da Célula , Reprogramação Celular , Células Epiteliais/citologia , Transdução de Sinais , Animais , Diferenciação Celular , Proliferação de Células , Transplante de Células , Córtex Cerebral/embriologia , Células-Tronco Embrionárias/citologia , Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Gravidez , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.
Assuntos
Células Epiteliais/fisiologia , Glândulas Mamárias Animais/fisiologia , Células Musculares/fisiologia , Contração Muscular/fisiologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Feminino , Lactação/efeitos dos fármacos , Lactação/fisiologia , Glândulas Mamárias Animais/diagnóstico por imagem , Camundongos , Células Musculares/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ocitocina/farmacologiaRESUMO
Over the last few years, the discovery of basal-type mammary carcinomas and the association of the regenerative potential of the mammary epithelium with the basal myoepithelial cell population have attracted considerable attention to this second major mammary lineage. However, many questions concerning the role of basal myoepithelial cells in mammary morphogenesis, functional differentiation and disease remain unanswered. Here, we discuss the mechanisms that control the myoepithelial cell differentiation essential for their contractile function, summarize new data concerning the roles played by cell-extracellular matrix (ECM), intercellular and paracrine interactions in the regulation of various aspects of the mammary basal myoepithelial cell functional activity. Finally, we analyze the contribution of the basal myoepithelial cells to the regenerative potential of the mammary epithelium and tumorigenesis.
Assuntos
Mama/citologia , Glândulas Mamárias Animais/citologia , Animais , Mama/crescimento & desenvolvimento , Mama/fisiologia , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Comunicação Celular , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Matriz Extracelular/fisiologia , Feminino , Humanos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/fisiologia , Camundongos , Mioepitelioma/etiologia , Mioepitelioma/patologia , Comunicação Parácrina , Transdução de Sinais , Células-Tronco/citologiaRESUMO
In the functionally differentiated mammary gland, basal myoepithelial cells contract to eject the milk produced by luminal epithelial cells from the body. We report that conditional deletion of a laminin receptor, α3ß1 integrin, from myoepithelial cells leads to low rates of milk ejection due to a contractility defect but does not interfere with the integrity or functional differentiation of the mammary epithelium. In lactating mammary gland, in the absence of α3ß1, focal adhesion kinase phosphorylation is impaired, the Rho/Rac balance is altered and myosin light-chain (MLC) phosphorylation is sustained. Cultured mammary myoepithelial cells depleted of α3ß1 contract in response to oxytocin, but are unable to maintain the state of post-contractile relaxation. The expression of constitutively active Rac or its effector p21-activated kinase (PAK), or treatment with MLC kinase (MLCK) inhibitor, rescues the relaxation capacity of mutant cells, strongly suggesting that α3ß1-mediated stimulation of the Rac/PAK pathway is required for the inhibition of MLCK activity, permitting completion of the myoepithelial cell contraction/relaxation cycle and successful lactation. This is the first study highlighting the impact of α3ß1 integrin signalling on mammary gland function.