RESUMO
Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.
Assuntos
Integrinas/fisiologia , Lactação , Glândulas Mamárias Animais/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Citoesqueleto/fisiologia , Progressão da Doença , Feminino , Deleção de Genes , Hormônios/fisiologia , Integrina alfa3/fisiologia , Integrina alfa6/fisiologia , Integrina beta1/fisiologia , Integrina beta4/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Mutantes , Mutação , Células-Tronco Neoplásicas/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Ovário/fisiologia , Fenótipo , Gravidez , Prenhez , Prognóstico , Ligação Proteica , Multimerização ProteicaRESUMO
Stem cells (SCs) drive mammary development, giving rise postnatally to an epithelial bilayer composed of luminal and basal myoepithelial cells. Dysregulation of SCs is thought to be at the origin of certain breast cancers; however, the molecular identity of SCs and the factors regulating their function remain poorly defined. We identified the transmembrane protein podoplanin (Pdpn) as a specific marker of the basal compartment, including multipotent SCs, and found Pdpn localized at the basal-luminal interface. Embryonic deletion of Pdpn targeted to basal cells diminished basal and luminal SC activity and affected the expression of several Wnt/ß-catenin signaling components in basal cells. Moreover, Pdpn loss attenuated mammary tumor formation in a mouse model of ß-catenin-induced breast cancer, limiting tumor-initiating cell expansion and promoting molecular features associated with mesenchymal-to-epithelial cell transition. In line with the loss-of-function data, we demonstrated that mechanistically Pdpn enhances Wnt/ß-catenin signaling in mammary basal cells. Overall, this study uncovers a role for Pdpn in mammary SC function and, importantly, identifies Pdpn as a new regulator of Wnt/ß-catenin signaling, a key pathway in mammary development and tumorigenesis.
Assuntos
Glicoproteínas de Membrana/metabolismo , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Carcinogênese/metabolismo , Técnicas de Cultura de Células , Citometria de Fluxo , Imunofluorescência , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via de Sinalização Wnt/genéticaRESUMO
BACKGROUND: The adult mammary epithelium is composed of basal and luminal cells. The luminal lineage comprises two major cell populations, positive and negative for estrogen and progesterone receptors (ER and PR, respectively), both containing clonogenic progenitor cells. Deregulated ER/PR- luminal progenitor cells are suspected to be at the origin of basal-type triple-negative (TNBC) breast cancers, a subtype frequently associated with loss of P53 function and MET signaling hyperactivation. Using mouse models, we recently reported that p53 restricts luminal progenitor cell amplification whereas paracrine Met activation stimulates their growth and favors a luminal-to-basal switch. Here, we analyzed how these two critical pathways interact to control luminal progenitor function. METHODS: We have (i) established and analyzed the gene expression profile of luminal progenitors isolated by ICAM-1, a robust surface marker we previously identified; (ii) purified luminal progenitors from p53-deficient and p53-proficient mouse mammary epithelium to compare their functional and molecular characteristics; and (iii) analyzed their response to HGF, the major Met ligand, in three-dimensional cultures. RESULTS: We found that luminal progenitors, compared to non-clonogenic luminal cells, overexpress Trp53 and numerous p53 target genes. In vivo, loss of Trp53 induced the expansion of luminal progenitors, affecting expression of several important p53 target genes including those encoding negative regulators of cell cycle progression. Consistently, p53-deficient luminal progenitors displayed increased proliferative and self-renewal activities in culture. However, they did not exhibit perturbed expression of luminal-specific markers and major regulators, such as Hey1, Elf5, and Gata3. Moreover, although expressing Met at higher level than p53-proficient luminal progenitors, p53-deficient luminal progenitors failed to acquire basal-specific features when stimulated by HGF, showing that p53 promotes the plastic behavior of luminal progenitors downstream of Met activation. CONCLUSIONS: Our study reveals a crosstalk between Met- and p53-mediated signaling pathways in the regulation of luminal progenitor function. In particular, it shows that neither p53 loss alone nor p53 loss combined with Met signaling activation caused an early detectable cell fate alteration in luminal progenitors. Conceivably, additional events are required to confer basal-specific characteristics to luminal-derived TNBCs.
Assuntos
Plasticidade Celular/fisiologia , Glândulas Mamárias Animais/citologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/fisiologia , Células Epiteliais/fisiologia , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The seventh annual meeting of the European Network of Breast Development and Cancer Laboratories, held in Weggis, Switzerland, in April 2015, was focused on techniques for the study of normal and cancer stem cells, cell fate decisions, cancer initiation and progression.
Assuntos
Neoplasias da Mama/patologia , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Animais , Diferenciação Celular/fisiologia , Progressão da Doença , Feminino , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
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.
Assuntos
Epitélio/fisiologia , Integrina alfa3beta1/metabolismo , Glândulas Mamárias Animais/citologia , Células Musculares/fisiologia , Animais , Células Cultivadas , Deleção de Genes , Integrina alfa3beta1/genética , Camundongos , Contração Muscular , Relaxamento MuscularRESUMO
Integrins are ubiquitously expressed major cell surface receptors for extracellular matrix. Integrin interaction with their extracellular ligands triggers activation of the intracellular signaling pathways that control cell shape, motility, proliferation, survival, cell-type-specific gene expression. In this review, we summarize recent studies analyzing contribution of integrins to the control of the mammary morphogenesis and differentiation, function and maintenance of mammary stem and progenitor cells and resume the data from mouse models revealing the contribution of the integrin-mediated signaling to mammary tumorigenesis.
Assuntos
Integrinas/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Mammary epithelium comprises a layer of luminal cells and a basal myoepithelial cell layer. Both mammary epithelial compartments, basal and luminal, contain stem and progenitor cells, but only basal cells are capable of gland regeneration upon transplantation. Aberrant expansion of stem/progenitor cell populations is considered to contribute to breast tumorigenesis. Germline deletions of p53 in humans and mice confer a predisposition to tumors, and stem cell frequency is abnormally high in the mammary epithelium of p53-deficient mice. However, it is unknown whether stem/progenitor cell amplification occurs in both, basal and luminal cell populations in p53-deficient mammary tissue. We used a conditional gene deletion approach to study the role of p53 in stem/progenitor cells residing in the mammary luminal and basal layers. Using two- and three-dimensional cell culture assays, we showed that p53 loss led to the expansion of clonogenic stem/progenitor cells in both mammary epithelial cell layers. Moreover, following p53 deletion, luminal and basal stem/progenitor cells acquired a capacity for unlimited propagation in mammosphere culture. Furthermore, limiting dilution and serial transplantation assays revealed amplification and enhanced self-renewal in the basal regenerating cell population of p53-deficient mammary epithelium. Our data suggest that the increase in stem/progenitor cell activity may be, at least, partially mediated by the Notch pathway. Taken together, these results strongly indicate that p53 restricts the propagation and self-renewal of stem/progenitor cells in both layers of the mammary epithelium providing further insight into the impact of p53 loss in breast cancerogenesis.
Assuntos
Compartimento Celular , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Contagem de Células , Proliferação de Células , Células Clonais , Células Epiteliais/metabolismo , Feminino , Deleção de Genes , Humanos , Integrases/metabolismo , Queratina-5/genética , Camundongos , Regiões Promotoras Genéticas/genética , Receptores Notch/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Basal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Recent studies have linked activation of the Wnt/ß-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNßcat previously generated by our team present a constitutive activation of Wnt/ß-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNßcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers. METHODS: Microarray analysis was used to compare K5ΔNßcat mouse tumors to human breast tumors, mammary cancer cell lines and the tumors developed in other mouse models. Cre-Lox approach was employed to delete Myc from the mammary basal cell layer of K5ΔNßcat mice. Stem cell amplification in K5ΔNßcat mouse mammary epithelium was assessed with 3D-culture and transplantation assays. RESULTS: Histological and microarray analyses of the mammary lesions of K5ΔNßcat females revealed their high similarity to a subset of basal-like human breast tumors with squamous differentiation. As in human basal-like carcinomas, the Myc pathway appeared to be activated in the mammary lesions of K5ΔNßcat mice. We found that a basal cell population with stem/progenitor characteristics was amplified in K5ΔNßcat mouse preneoplastic glands. Finally, the deletion of Myc from the mammary basal layer of K5ΔNßcat mice not only abolished the regenerative capacity of basal epithelial cells, but, in addition, completely prevented the tumorigenesis. CONCLUSIONS: These results strongly indicate that ß-catenin-induced stem cell amplification and tumorigenesis rely ultimately on the Myc pathway activation and reinforce the hypothesis that basal stem/progenitor cells may be at the origin of a subset of basal-like breast tumors.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Células-Tronco Neoplásicas/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , Deleção de Sequência , Células Tumorais Cultivadas , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
The mammary epithelium comprises two major cell lineages: basal and luminal. Basal cells (BCs) isolated from the mammary epithelium and transplanted into the mouse mammary fat pad cleared from the endogenous epithelium regenerate the mammary gland, strongly suggesting that the basal epithelial compartment harbors a long-lived cell population with multipotent stem cell potential. The luminal cell layer is devoid of the regenerative potential, but it contains cells with clonogenic capacity, the luminal progenitors. Mammary BCs and luminal progenitors express high levels of the transcription factor Myc. Here, we show that deletion of Myc from mammary basal epithelial cells led to impaired stem cell self-renewal as evaluated by limiting dilution and serial transplantation assays. Luminal progenitor population was significantly diminished in mutant epithelium suggesting control by the BC layer. Colony formation assay performed with isolated BCs showed that clonogenic capacity was abolished by Myc deletion. Moreover, transplanted BCs depleted of Myc failed to produce epithelial outgrowths. Stimulation with ovarian hormones estrogen (E) and progesterone (P) partially rescued the repopulation capacity of Myc-depleted BCs; however, the Myc-deficient mammary epithelium developed in response to E/P treatment lacked stem and progenitor cells. This study provides the first evidence that in the mammary gland, Myc has an essential nonredundant function in the maintenance of the self-renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and is required downstream from ovarian hormones, for the control of mammary stem and progenitor cell functions.
Assuntos
Glândulas Mamárias Animais/citologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Feminino , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Transgênicos , Células-Tronco/citologiaRESUMO
The European Network for Breast Development and Cancer (ENBDC) meeting on 'Methods in Mammary Gland Development and Cancer' has become an annual international rendezvous for scientists with interests in the normal and neoplastic breast. The fourth meeting in this series, held in April in Weggis, Switzerland, focused on proteomics, epigenetics, symmetric division, and metastasis.
Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Epigenômica/métodos , Glândulas Mamárias Humanas/metabolismo , Proteômica/métodos , Animais , Divisão Celular , Feminino , Humanos , Metástase NeoplásicaRESUMO
The orthotopic transplantation assay has provided important insights into mammary development, stem cell function, and tumorigenesis. Technically, it consists in grafting mammary tissue fragments, organoids, mammospheres, or isolated cells into the fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed, thereby allowing growth and differentiation of mammary epithelial cells in their physiological environment. Here, we describe how is conducted transplantation of epithelial fragments and cells isolated from mouse mammary glands, report the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells, and highlight the strengths and limitations of this in vivo grafting assay.
Assuntos
Células Epiteliais , Glândulas Mamárias Animais , Animais , Diferenciação Celular , Células Epiteliais/transplante , Epitélio/fisiologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Células-TroncoRESUMO
The second meeting of the European Network for Breast Development and Cancer (ENBDC) on 'Methods in Mammary Gland Development and Cancer' was held in April 2010 in Weggis, Switzerland. The focus was on genomics and bioinformatics, extracellular matrix and stroma-epithelial cell interactions, intravital imaging, the search for metastasis founder cells and mouse models of breast cancer.
Assuntos
Neoplasias da Mama , Mama , Glândulas Mamárias Animais , Neoplasias Mamárias Animais , Neoplasias Mamárias Experimentais , Animais , Mama/embriologia , Mama/crescimento & desenvolvimento , Mama/patologia , Feminino , Genômica , Humanos , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Metástase NeoplásicaRESUMO
The bilayered mammary epithelium comprises a luminal layer of secretory cells and a basal layer of myoepithelial cells. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells; however, their molecular characteristics and differentiation pathways are largely unknown. BC44 mammary epithelial cells in culture, display phenotypic characteristics of basal epithelium, i.e., express basal cytokeratins 5 and 14 and P-cadherin, but no smooth muscle markers. In vivo, after injection into the cleared mammary fat pad, these cells gave rise to bilayered, hollow, alveolus-like structures comprising basal cells expressing cytokeratin 5 and luminal cells positive for cytokeratin 8 and secreting beta-casein in a polarized manner into the lumen. The persistent stimulation of EGF receptor signaling pathway in BC44 cells in culture resulted in the loss of the in vivo morphogenetic potential and led to the induction of active MMP2, thereby triggering cell scattering and motility on laminin 5. These data (a) suggest that BC44 cells are capable of asymmetric division for self-renewal and the generation of a differentiated progeny restricted to the luminal lineage; (b) clarify the function of EGF in the control of the BC44 cell phenotypic plasticity; and (c) suggest a role for this phenomenon in the mammary gland development.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Células-Tronco/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/transplante , Proteínas da Matriz Extracelular/metabolismo , Feminino , Citometria de Fluxo , Integrinas/metabolismo , Glândulas Mamárias Animais/anatomia & histologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fatores de TempoRESUMO
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
To investigate the mechanisms through which p130Cas adaptor protein is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone. Mammary epithelial cells isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. Consistently with the double-transgenic mice model, p130Cas is overexpressed in a significant subset of human breast cancers and high levels of p130Cas in association with HER2 expression correlate with elevated proliferation. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a putative target for clinical therapy.
Assuntos
Transformação Celular Neoplásica/patologia , Proteína Substrato Associada a Crk/fisiologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Receptor ErbB-2/fisiologia , Animais , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular , Transformação Celular Neoplásica/genética , Proteína Substrato Associada a Crk/biossíntese , Proteína Substrato Associada a Crk/genética , Feminino , Genes erbB-2 , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Transgênicos , Interferência de RNA , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Transdução de Sinais , Regulação para CimaRESUMO
P-cadherin expression is restricted to the basal layer of stratified epithelia including that of the mammary gland. Although evidence for an important role of P-cadherin in mammary morphogenesis and tumorigenesis is increasing, the mechanisms that regulate its expression are poorly understood. We show that in basal mammary epithelial cells, beta-catenin is associated with the P-cadherin promoter and activates its expression independently of LEF/TCF in a cell-type specific manner. Down-regulation of endogenous beta-catenin levels by RNA interference technique inhibited P-cadherin promoter activity. In vivo, in skin and mammary gland of mutant mice, activation of beta-catenin signalling correlates with up-regulation of P-cadherin expression. These data suggest that beta-catenin-dependent modulation of P-cadherin expression can contribute to the establishment of the basal phenotype.
Assuntos
Caderinas/genética , Glândulas Mamárias Animais/metabolismo , beta Catenina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Núcleo Celular/metabolismo , Primers do DNA/genética , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Pele/metabolismo , Fatores de Transcrição TCF/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
To study the mechanism of beta1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of beta1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-beta1-cyto). Mammary glands from MMTV-beta1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of beta1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in beta-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-kappaB, a negative regulator of STAT5 signaling. This is the first study demonstrating in vivo that cell-extracellular matrix interactions involving beta1-integrins play an important role in the control of milk gene transcription and in the maintenance of the mammary epithelial cell differentiated state.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/fisiologia , Integrina beta1/metabolismo , Lactação/fisiologia , Glândulas Mamárias Animais/fisiologia , Proteínas Proto-Oncogênicas , Animais , Apoptose/fisiologia , Antígenos CD4/genética , Antígenos CD4/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Feminino , Genes Reporter , Integrina beta1/genética , Janus Quinase 2 , Glândulas Mamárias Animais/citologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , NF-kappa B/metabolismo , Gravidez , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT5 , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismoRESUMO
The postnatal mammary morphogenesis comprises two steps, first, formation of a system of branching ducts at puberty and second, alveologenesis during pregnancy. The mammary epithelium is organized as a bilayer, composed of two cellular types, basal myoepithelial and luminal epithelial. The remarkable regenerative properties revealed in serial transplantation experiments suggest that the adult mammary epithelium harbors stem cells. Various strategies including analysis of DNA label-retaining cells, transgenic approach, and in vivo transplantation assay, have been used to isolate and characterize murine mammary stem and progenitor cells. Their molecular characteristics remain to be defined precisely but notable progress have been already made in the enrichment and identification of these cells. Current studies favor the hypothesis of a basal-type mammary stem cells expressing high levels of alpha 6, beta1 and beta 3 integrin chains, the major receptors of extracellular matrix proteins. Luminal-type progenitors may participate in the establishment of the bilayered alveolar epithelium during pregnancy.
Assuntos
Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Envelhecimento , Animais , Feminino , Integrinas/genética , Integrinas/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Morfogênese , Maturidade SexualRESUMO
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
In this issue, Tosoni et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505037) report that cell fate determinant and tumor suppressor Numb imposes asymmetric cell divisions in mammary stem cells by regulating p53. Numb thereby restricts mammary stem cell expansion and controls the proliferation and lineage-specific characteristics of their progeny.