Margaret C. Neville

Hormonal regulation of mammary differentiation and milk secretion

The endocrine system coordinates development of the mammary gland with reproductive development and the demand of the offspring for milk. Three categories of hormones are involved. The levels of the reproductive hormones, estrogen, progesterone, placental lactogen, prolactin, and oxytocin, change during reproductive development or function and act directly on the mammary gland to bring about developmental changes or coordinate milk delivery to the offspring. Metabolic hormones, whose main role is to regulate metabolic responses to nutrient intake or stress, often have direct effects on the mammary gland as well. The important hormones in this regard are growth hormone, corticosteroids, thyroid hormone, and insulin. A third category of hormones has recently been recognized, mammary hormones. It currently includes growth hormone, prolactin, PTHrP, and leptin. Because a full-term pregnancy in early life is associated with a reduction in breast carcinogenesis, an understanding of the mechanisms by which these hormones bring about secretory differentiation may offer clues to the prevention of breast cancer.

Tight Junction Regulation in the Mammary Gland

Tight junctions form a narrow, continuous sealthat surrounds each endothelial and epithelial cell atthe apical border, and act to regulate the movement ofmaterial through the paracellular pathway. In the mammary gland, the tight junctions of thealveolar epithelial cells are impermeable duringlactation, and thus allow milk to be stored betweennursing periods without leakage of milk components from the lumen. Nonetheless mammary epithelial tightjunctions are dynamic and can be regulated by a numberof stimuli. Tight junctions of the mammary gland fromthe pregnant animal are leaky, undergoing closure around parturition to become the impermeabletight junctions of the lactating animal. Milk stasis,high doses of oxytocin, and mastitis have been shown toincrease tight junction permeability. In general changes in tight junction permeability in themammary gland appear to be the results of a state changeand not assembly and disassembly of tight junctions.Both local factors, such as intramammary pressure and TGF-beta, and systemic factors, such asprolactin, progesterone, and glucocorticoids, appear toplay a role in the regulation of mammary tightjunctions. Finally, the tight junction state appears to be closely linked to milk secretion. Anincrease in tight junction permeability is accompaniedby decrease in the milk secretion rate, and conversely,a decrease in tight junction permeability is accompanied by an increase in the milk secretionrate.

Mammary physiology and milk secretion

The presence of drugs or other potentially toxic materials in milk is an obvious public health risk, especially to infants and neonates. There is also increasing concern that human breast cancer is principally epigenetic in origin and results from environmentally produced lesions. Little is known about the mechanisms by which toxic substances enter milk or mammary tissue but knowledge of these processes is important to toxicologists and researchers involved in drug design and metabolism. Five general pathways have been described for transport of proteins, lipids, ions, nutrients and water into milk. Four of these pathways are transcellular, involving transport across at least two membrane barriers; the fifth is paracellular and allows direct exchange of interstitial and milk components. Solute transport by these pathways is mediated by a diverse, and complex array of transport and secretory processes that are regulated by hormonal, developmental, and physiological factors. Current research is beginning to define the mechanisms underlying some of these processes, however the regulation and coordination of solute transport mechanisms remains poorly understood. In this article we review our current understanding of the normal solute transport and secretory processes involved in milk production, and discuss potential regulatory mechanisms.

Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis!

The transition from pregnancy to lactation is a critical event in the survival of the newborn since all the nutrient requirements of the infant are provided by milk. While milk contains numerous components, including proteins, that aid in maintaining the health of the infant, lactose and milk fat represent the critical energy providing elements of milk. Much of the research to date on mammary epithelial differentiation has focused upon expression of milk protein genes, providing a somewhat distorted view of alveolar differentiation and secretory activation. While expression of milk protein genes increases during pregnancy and at secretory activation, the genes whose expression is more tightly regulated at this transition are those that regulate lipid biosynthesis. The sterol regulatory element binding protein (SREBP) family of transcription factors is recognized as regulating fatty acid and cholesterol biosynthesis. We propose that SREBP1 is a critical regulator of secretory activation with regard to lipid biosynthesis, in a manner that responds to diet, and that the serine/threonine protein kinase Akt influences this process, resulting in a highly efficient lipid synthetic organ that is able to support the nutritional needs of the newborn.

Nuclear import of PKCδ is required for apoptosis: identification of a novel nuclear import sequence

We have shown previously that protein kinase Cδ (PKCδ) is required for mitochondrial‐dependent apoptosis. Here we show that PKCδ is imported into the nucleus of etoposide‐treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C‐terminus of PKCδ. Mutation of the caspase cleavage site of PKCδ inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCδ catalytic fragment (CFδ) in transfected cells results in nuclear localization and apoptosis. We show that the PKCδ NLS is required for nuclear import of both full‐length PKCδ and CFδ, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFδ prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase‐negative catalytic fragment (KN‐CFδ) protects cells from etoposide‐induced apoptosis. Mutation of the NLS blocks the ability of KN‐CFδ to protect against etoposide‐induced apoptosis. These results indicate that PKCδ regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.

Functional Development of the Mammary Gland: Use of Expression Profiling and Trajectory Clustering to Reveal Changes in Gene Expression During Pregnancy, Lactation, and Involution

To characterize the molecular mechanisms by which progesterone withdrawal initiates milk secretion, we examined global gene expression during pregnancy and lactation in mice, focusing on the period around parturition. Trajectory clustering was used to profile the expression of 1358 genes that changed significantly between pregnancy day 12 and lactation day 9. Predominantly downward trajectories included stromal and proteasomal genes and genes for the enzymes of fatty acid degradation. Milk protein gene expression increased throughout pregnancy, whereas the expression of genes for lipid synthesis increased sharply at the onset of lactation. Examination of regulatory genes with profiles similar or complementary to those of lipid synthesis genes led to a model in which progesterone stimulates synthesis of TGF-β, Wnt 5b, and IGFBP-5 during pregnancy. These factors are suggested to repress secretion by interfering with PRL and IGF-1 signaling. With progesterone withdrawal, PRL and IGF-1 signaling are activated, in turn activating Akt/PKB and the SREBPs, leading to increased lipid synthesis.

Lactogenesis: The Transition from Pregnancy to Lactation

The most important factors in initiation of the cascade of changes in the mammary epithelium that constitute lactogenesis stage II seem to be a prepared mammary epithelium, progesterone withdrawal, maintained plasma prolactin (in most species), and removal of milk from the breast within an undefined interval after birth. Although the molecular mechanisms by which prolactin regulates milk protein synthesis are the subject of intense and productive studies, the specific mechanisms by which progesterone and milk removal interact with the mammary epithelial cell at parturition have not been studied, perhaps because no in vitro model system exists that mimics lactogenesis stage II, or because of the complexity of the changes that must be coordinated during this process, or because of a lack of general understanding of the complex progression of changes in the function of the breast as it goes from the quiescent state of pregnancy to the active secretory state of lactation. With new technologies designed to investigate the biology of complex systems arising from the growing knowledge of the genome of human and animal species and the growing availability of animal and tissue culture models for these processes, physicians can expect a rapid increase in the molecular understanding of lactogenesis in the near future. These fundamental studies must be coupled with good prospective clinical studies if physicians are to obtain a useful, comprehensive understanding of lactogenesis in women.

Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor αvβ3, and CD91. In addition, MEC, like macrophages, produced TGFβ in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.

The calcium-sensing receptor regulates mammary gland parathyroid hormone–related protein production and calcium transport

The transfer of calcium from mother to milk during lactation is poorly understood. In this report, we demonstrate that parathyroid hormone-related protein (PTHrP) production and calcium transport in mammary epithelial cells are regulated by extracellular calcium acting through the calcium-sensing receptor (CaR). The CaR becomes expressed on mammary epithelial cells at the transition from pregnancy to lactation. Increasing concentrations of calcium, neomycin, and a calcimimetic compound suppress PTHrP secretion by mammary epithelial cells in vitro, whereas in vivo, systemic hypocalcemia increases PTHrP production, an effect that can be prevented by treatment with a calcimimetic. Hypocalcemia also reduces overall milk production and calcium content, while increasing milk osmolality and protein concentrations. The changes in milk calcium content, milk osmolality, and milk protein concentration were mitigated by calcimimetic infusions. Finally, in a three-dimensional culture system that recapitulates the lactating alveolus, activation of the basolateral CaR increases transcellular calcium transport independent of its effect on PTHrP. We conclude that the lactating mammary gland can sense calcium and adjusts its secretion of calcium, PTHrP, and perhaps water in response to changes in extracellular calcium concentration. We believe this defines a homeostatic system that helps to match milk production to the availability of calcium.