Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Current data indicates that feeding dairy calves more nutrients in early life allows them to produce more milk in the future. Mechanisms responsible are poorly understood. Thirty-six Holstein heifer calves were fed either a restricted (R; 20.2% crude protein [CP], 19.8% fat, dry matter (DM) basis, fed at 0.44 kg/hd/d, DM basis) or an enhanced (EH; 28.9% CP, 26.2% fat, DM basis, fed at 1.08 kg/hd/day, DM basis) milk replacer (MR) and given either a placebo or estradiol (E2) implant to assess differential responses to E2. Our underlying hypothesis was that calves fed more nutrients are better able to respond to mammogenic stimuli and will have a more developed mammary gland as a result of imposed treatments. Enhanced-fed calves grew at a faster rate, were heavier at weaning, and had more functional mammary tissue (i. e., parenchyma; PAR) mass in the mammary gland at weaning (7.3-fold). Additionally, biochemical composition of the PAR was not impacted by the dietary treatments imposed. Furthermore, EH-fed calves had an increase in the number of actively dividing cells throughout the mammary PAR as well as increased intensity of estrogen receptor expression in the population of cells expressing the estrogen receptor. Enhanced-fed calves had an up-regulation of genes and pathways in the PAR related to metabolism, cellular signaling, and cellular growth. When given E2, EH-fed calves experienced the greatest overall mammary gland development and had the greatest PAR mass without compromised composition. When comparing EH- and R-fed calves given E2, differential expression of genes and pathways related to cell growth, cell signaling, and metabolism was observed. In summary, data indicates that enhanced feeding of calves in early life allows increased responsiveness to mammogenic stimuli and a corresponding increase in mammary development. We suggest that this may at least partly explain the improved future milk production in calves fed in this manner. / Ph. D.

No description available.

While a great deal is known about the changing hormonal environment and the structural development of the mammary gland from pregnancy to lactation, very little is known about the molecular mechanisms governing differentiation of the mammary epithelium into a milk-secreting phenotype. It is important to acknowledge the diversity among the mammary glands of different species in order to better understand applications in human health and the dairy industry. In this study, we examined global protein expression during two states of differentiation in mammary epithelial cells from two species: in vitro proliferating and differentiated MAC-T cells (a bovine immortal cell-line), and primary mammary epithelial cells isolated from pregnant and lactating mice. When comparing the lists of proteins that differed in abundance in the two experiments, we observed many similarities in proteins related to structural dynamics and mRNA processing within these two mammary epithelial cell types. Intriguingly, we observed several differences in the regulation of metabolic proteins, highlighting the distinct pathways by which different species probably metabolize energy and synthesize milk components.

Die Familie der ErbB-Rezeptor-Tyrosinkinasen besteht aus vier Mitgliedern, dem EGF-Rezeptor (ErbB-1), ErbB-2, ErbB-3 und ErbB-4. ErbB-Rezeptoren spielen eine wichtige Rolle bei der Entwicklung des Nervensystems, des Herzens und der Brustdrüsen. Ein Teil dieser Differenzierungsvorgänge läßt sich in vitro nachvollziehen: so ist zum Beispiel die Aktivierung des ErbB-2 Rezeptors ausreichend für alveoläre Morphogenese der Brustdrüsenepithelzellinie EpH4. Intrazelluläre Moleküle, die dieses ErbB2-Signal übertragen, sind allerdings noch unbekannt. Mit Hilfe eines neuen, modifizierten Hefe-2-Hybrid-Systems wurde in der vorliegenden Arbeit Vav2 als neuer Interaktionspartner von ErbB-2 identifiziert. Vav2 assoziiert mit aktiviertem ErbB-2 über eine SH2-Domäne. Die Interaktion ist direkt und ist von zwei Phosphotyrosinen in ErbB-2 abhängig. Vav2 kann den GDP/GTP-Austausch bei GTPasen der Rho-Familie vermitteln. Dadurch kann der Umbau des Zytoskeletts und Veränderungen der Transkription sowie Zelltransformation induziert werden. In einem dreidimensionalen Zellkultursystem kann aktiviertes Vav2 in EpH4 Zellen die Bildung von alveolären Zellaggregaten induzieren. In diesen Alveolen umgibt eine Schicht polarisierter milchproduzierender Zellen ein zentrales Lumen. Diese Vav2-vermittelte Morphogenese ist abhängig von der katalytischen GDP/GTP-Austausch Aktivität von Vav2. Katalytisch-inaktives Vav2 kann die morphogenetische Aktivität von ErbB-2 in EpH4-Zellen verhindern, ohne die mitogene Aktivität von ErbB-2 zu beeinflussen. Vav2 ist mit ErbB-2 coexprimiert und interagiert mit dem Rezeptor in Brustdrüsenzellen schwangerer Mäuse. Diese Untersuchungen deuten darauf hin, dass Vav2 eine wichtige Funktion bei der durch ErbB-2 induzierten alveolären Morphogenese der Brustdrüse spielt. / The ErbB receptor tyrosine kinases constitute a subfamily of four structurally related members, the EGF receptor (ErbB-1), ErbB-2, ErbB-3 and ErbB-4. ErbB receptor tyrosine kinases are critical for embryonic development of central and peripheral neural structures and heart. In addition, ErbB receptors play an important role in the postnatal development of the mammary gland. Previous studies showed that activated ErbB-2 receptor induces alveolar morphogenesis of EpH4 mammary epithelial cells that are cultured on a three-dimensional matrix (termed Matrigel). However, the downstream signaling proteins that mediate this biological activity of ErbB-2 were unknown. In this work, Vav2 was identified as a direct interaction partner of tyrosine-phosphorylated ErbB receptors using the yeast two-hybrid system. Vav2 is a member of a family of guanine nucleotide exchange factors that induce cytoskeletal rearrangements, transcriptional alterations, and have oncogenic potential when activated. To test the ability of Vav2 to mediate morphogenic signals of ErbB-2, EpH4 cells overexpressing Vav2 protein were cultured on Matrigel. Indeed, Vav2 induces alveolar morphogenesis of EpH4 cells when activated either by oncogenic mutation or tyrosine phosphorylation by ErbB-2. The morphogenic activity of Vav2 requires the Dbl homology domain, which mediates GDP/GTP exchange. Dominant-negative Vav2 specifically blocks the morphogenic signals of ErbB-2 in EpH4 cells without interfering with ErbB2-induced mitogenesis. Importantly, Vav2 is co-expressed and interacts with ErbB-2 in the mammary glands of pregnant mice. Taken together, these results point to Vav2 as a candidate to mediate ErbB-2 signals for alveolar morphogenesis in vivo, which is a relevant step in the development of the mammary gland during pregnancy.