ABSTRACT
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.
Subject(s)
Mammary Glands, Animal/embryology , Mammary Neoplasms, Experimental/genetics , Neoplasm Proteins/physiology , Proto-Oncogene Proteins/physiology , Receptors, Notch/physiology , Adenocarcinoma, Papillary/genetics , Adenocarcinoma, Papillary/pathology , Agar , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Proteins/metabolism , Cell Transformation, Viral/genetics , Female , Homeodomain Proteins/metabolism , Mammary Neoplasms, Experimental/pathology , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Knockout , Mice, Nude , Milk Proteins/genetics , Neoplasm Proteins/genetics , Pregnancy , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Receptor, Notch4 , Receptors, Notch/chemistry , Receptors, Notch/deficiency , Receptors, Notch/genetics , Recombinant Fusion Proteins/physiology , Repressor Proteins/genetics , Terminal Repeat Sequences/genetics , Transcription Factor HES-1 , Tumor Cells, Cultured/cytologyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , Homeodomain Proteins/genetics , Mammary Glands, Animal/physiology , Morphogenesis/physiology , Progesterone/pharmacology , Animals , Female , Gene Expression Regulation/drug effects , Mammary Glands, Animal/drug effects , Mice , Mice, Inbred BALB C , Morphogenesis/drug effects , Ovariectomy , Polymerase Chain Reaction , RNA/genetics , RNA/isolation & purification , Receptors, Progesterone/physiology , Signal TransductionABSTRACT
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.
