RESUMO
Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.
Assuntos
Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Experimentais/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Notch/fisiologia , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patologia , Ágar , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Viral/genética , Feminino , Proteínas de Homeodomínio/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas do Leite/genética , Proteínas de Neoplasias/genética , Gravidez , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor Notch4 , Receptores Notch/química , Receptores Notch/deficiência , Receptores Notch/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Sequências Repetidas Terminais/genética , Fatores de Transcrição HES-1 , Células Tumorais Cultivadas/citologiaRESUMO
Branching morphogenesis within the peripubertal mouse mammary gland is directed by progesterone (P). A role for the homeobox-containing transcription factor, Msx2, during branching morphogenesis is suggested from its ontogenic expression profile and hormonal regulation. Herein, we define the spatio-temporal control of Msx2 expression, the regulation of its expression by P and its direct role in ductal branching morphogenesis. P induces Msx2 in the presence of estrogen (E) both in vitro and in vivo while absence of the P receptor (PR) decreased Msx2 expression. Stable transfection of PR into mouse mammary epithelial cells increased the endogenous expression of Msx2 and their ability to undergo branching morphogenesis in vitro. Furthermore, normal mammary cells stably-transfected with Msx2 demonstrated increased branching morphogenesis in vitro while transgenic mice expressing Msx2 in their mammary glands demonstrated enhanced lateral branching during early development. The action of P on branching morphogenesis appears to involve Bmp2/4. Together, these data demonstrate that P, acting through PR-A and the Bmp2/4 pathway, induces Msx2 to enhance ductal branching in the mammary glands.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Glândulas Mamárias Animais/fisiologia , Morfogênese/fisiologia , Progesterona/farmacologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Morfogênese/efeitos dos fármacos , Ovariectomia , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Receptores de Progesterona/fisiologia , Transdução de SinaisRESUMO
Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L-PRLR) and two of the three short forms (S1- and S3-PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2-PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L-PRLR mRNA. By contrast, S1-PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2-PRLR became undetectable during puberty. Stromal expression of S3-PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptores de Progesterona/metabolismo , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Transcrição Gênica/fisiologia , Tecido Adiposo/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sinergismo Farmacológico , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ovariectomia , Progesterona/farmacologia , Prolactina/farmacologia , RNA Mensageiro/análise , Receptores de Progesterona/genética , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Accompanying changes in the development and function of the mammary gland is the establishment of a vascular network of critical importance for lactogenesis and tumorigenesis. A potent angiogenic and permeability factor that regulates vascular development in association with epithelial-stromal interactions is vascular endothelial growth factor (VEGF). Analysis of VEGF transcription by RT-PCR revealed mRNA for all three VEGF isoforms (VEGF120, 164, 188) within the mammary gland of nulliparous females. During pregnancy the level of VEGF188 declined and became undetectable during lactation in association with the increased abundance of VEGF120 and VEGF164 mRNAS: All three isoforms were expressed at consistent levels within the cleared mammary fat pad throughout development. Furthermore, the presence of VEGF188 mRNA in omental adipose tissue at various stages established that VEGF188 is expressed specifically in adipose tissue within the mammary gland. Using 3T3-L1 preadipocytes it was demonstrated that VEGF188 mRNA transcription occurs as a late event during lipogenesis distinct from earlier induction of VEGF120 and VEGF164 mRNA during differentiation. In contrast, HC11 mammary epithelial cells only expressed mRNA for VEGF120 and VEGF164. Localization of VEGF mRNA and protein revealed that VEGF is expressed in stromal cells of the mammary gland in nulliparous females and then undergoes a transition to epithelial expression during lactation. By contrast, mRNA for the VEGF receptors, Flk-1 and Flt-1, localized to stromal cells within the mammary fat pad during virgin and gestational development and was expressed in the interstitial tissue basal to epithelial cells during lactation. Taken together, these results support the conclusion that VEGF is differentially transcribed by specific cell types within the mammary gland, and that under hormonal regulation it functions in an autocrine/paracrine manner.
