Loss of protein kinase C delta alters mammary gland development and apoptosis.

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) -/- mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ -/- mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ -/- mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ -/- mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ -/- mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.

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 alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta 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.

Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretion.

Xanthine oxidoreductase (XOR), a key enzyme of purine metabolism, has been implicated in the secretion of the milk fat droplet in lactating mammary epithelial cells, possibly through structural interactions with other milk fat globule proteins including butyrophilin (Btn) and adipophilin (ADPH). To help determine the mechanism by which XOR is regulated, we examined the expression and localization of XOR in the non-secretory states of late pregnancy and induced involution compared with the state of active secretion. XOR mRNA levels started to increase at mid-pregnancy, turned sharply upwards at the onset of lactation and decreased rapidly with forced involution, indicating transcriptional control of the enzyme level by differentiation and secretory function. During pregnancy and involution the enzyme was diffusely distributed in the cytoplasm, but moved rapidly to the apical membrane of the cells when secretion was activated, where it colocalized with both Btn and ADPH, similar to the situation in the milk fat globule itself. Size-exclusion chromatography of solubilized milk fat globule membrane proteins showed that XOR formed a sulphydryl-bond-dependent complex with Btn and ADPH in the milk fat globule membrane. XOR returned to a diffuse cytoplasmic localization shortly after induced involution, while Btn remained localized to the apical membrane, suggesting that localization of XOR is not dependent on the presence of Btn in the apical membrane. Our findings indicate that the expression and membrane association of XOR in the mammary gland are tightly regulated by secretory activity, and suggest that the apical membrane association of XOR regulates the coupling of lipid droplets to the apical plasma membrane during milk lipid secretion.

Hormonal regulation of tight junction closure in the mouse mammary epithelium during the transition from pregnancy to lactation.

Closure of the tight junctions of the mammary epithelium has been shown to accompany the onset of copious milk secretion or lactogenesis, stage 2, in both goats and humans. Here we use injection of [(14)C]sucrose and FITC-albumin (fluorescein isothiocyanate-albumin) into the mammary duct to follow the course of tight junction closure during lactogenesis in mice. To examine the hormonal changes responsible, we ovariectomized day 16 or 17 pregnant mice and found that closure followed ovariectomy with a mean delay of 13.6+/-1.5 (s.e.m. ) h. That progesterone withdrawal is the trigger for closure was shown by the finding that injection of progesterone within 4 h of ovariectomy delayed closure and that closure occurred after injection of the progesterone antagonist RU 486 in intact late pregnant mice. Endocrine ablation studies showed that low to moderate concentrations of corticosterone and either placental lactogen or prolactin are necessary for tight junction closure triggered by progesterone withdrawal. Thus the hormonal requirements for tight junction closure are similar to those shown by other investigators to promote lactogenesis, stage 2. Further, the tight temporal control of tight junction permeability suggests that ovariectomy of the late pregnant mouse may be a good model for molecular studies of the lactogenic switch.

A lipoprotein-containing particle is transferred from the serum across the mammary epithelium into the milk of lactating mice.

To investigate the role of low-density lipoprotein (LDL) in the delivery of cholesterol to the mammary gland during pregnancy and lactation, we examined the distribution of radioactivity from (125)I-tyramine cellobiose-LDL injected into the tail vein of female mice at various stages of the reproductive cycle. Changes in the proportion of isotope taken up by the mammary gland largely reflected the increased weight of the gland in pregnancy and lactation. In addition, during lactation, radioactivity was found in the milk and was associated with a protein of the molecular weight of apoB-100. Quantitatively similar results were obtained with mice homozygous for disruption of the LDL receptor gene (LDLR null). Analysis of endogenous lipoproteins showed that the milk lipoprotein particles were denser than the corresponding serum lipoproteins and largely depleted of triglyceride and cholesterol. Using fluorescence microscopy we visualize the sorting of apoB protein from the LDL lipid phase at the basal surface of the mammary epithelial cell of both wild-type and LDLR-null mice. Our findings provide evidence that the mammary epithelium of the lactating mouse is able to take up LDL from the plasma by a non-LDLR-mediated process. An apoB-containing particle from which the cholesterol has been removed is transferred into milk.

Does human lactoferrin in the milk of transgenic mice deliver iron to suckling neonates?

Lactoferrin is an iron-binding glycoprotein abundantly present in human milk, and has been postulated both to increase and to decrease intestinal iron absorption. To examine this problem, the interaction of milk iron with pup hemoglobin was studied in controls and in transgenic mice overexpressing human lactoferrin in their milk (2 lines expressing 12 mg/mL and 4 mg/mL, respectively). At day 14 of gestation, pregnant mice were switched from a diet of commercial chow containing iron at 300 mg/kg to diets containing 5, 15, or 50mg iron/kg; controls continued on chow. Nontransgenic pups were cross-fostered to transgenic dams to ensure that any results found in the pups were the effect of milk components. The hemoglobin level in the blood of 10-day-old suckling neonates was measured and calculated as total hemoglobin per pup. The total hemoglobin levels were lower in the pups receiving milk high in human lactoferrin, but the difference reached significance (P < 0.02) only at the highest level of dietary iron. Our findings do not support the hypothesis that lactoferrin functions as an intestinal iron scavenger, at least at high doses.

Vesicular transport of soluble substances into mouse milk.

Utilizing a novel protocol to study transport of substances into mouse milk in situ, we have shown that many "fluid-phase" markers are taken up by mammary epithelial cells and deposited in milk. Since the tight junctions are closed and impermeable even to small molecules, extra-alveolar substances (those not synthesized by the alveolar cells) must be transported into the milk by the epithelial cells themselves. The markers we have used include dextran, lucifer yellow dye, horseradish peroxidase, and albumin. Using these markers and immunostaining for endogenous proteins, we have visualized transcytotic vesicles involved in transporting these markers to milk.

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.

The effect of serum iron concentration on iron secretion into mouse milk.

1. The concentration of iron in mouse milk is approximately 3 times that of the serum. Although there is clear evidence for the presence of the transferrin receptor in the rodent mammary gland, the precise mechanisms of iron transfer into milk are not known. 2. Milk iron was linearly related to the serum iron:transferrin ratio in lactating mice whose serum iron ranged from 8 to 66 microM. 3. Increasing the iron binding capacity of the milk by 340 microM by targeting the lactoferrin transgene to the mammary gland did not alter the relation between milk iron and the serum iron:transferrin ratio. 4. The steady-state distribution ratio of 125I-transferrin between plasma and milk was about 0.2, indicating that transcytosed transferrin contributed a maximum of 6% of the milk iron. 5. Fluorescently labelled transferrin incubated with the in situ gland localized mainly near the basal surface of the mammary alveolar cells. 6. These experiments provide evidence that the initial and rate-limiting step in the transfer of iron into milk is binding to a basal transferrin receptor. 7. A theoretical model of the relation between milk and serum iron suggests that the affinity of apotransferrin for the basal recycling system may be higher than observed in many other cell types.

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells.

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is upregulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.

Tight junction regulation in the mammary gland.

Tight junctions form a narrow, continuous seal that surrounds each endothelial and epithelial cell at the apical border, and act to regulate the movement of material through the paracellular pathway. In the mammary gland, the tight junctions of the alveolar epithelial cells are impermeable during lactation, and thus allow milk to be stored between nursing periods without leakage of milk components from the lumen. Nonetheless mammary epithelial tight junctions are dynamic and can be regulated by a number of stimuli. Tight junctions of the mammary gland from the pregnant animal are leaky, undergoing closure around parturition to become the impermeable tight junctions of the lactating animal. Milk stasis, high doses of oxytocin, and mastitis have been shown to increase tight junction permeability. In general changes in tight junction permeability in the mammary gland appear to be the results of a state change and not assembly and disassembly of tight junctions. Both local factors, such as intramammary pressure and TGF-beta, and systemic factors, such as prolactin, progesterone, and glucocorticoids, appear to play a role in the regulation of mammary tight junctions. Finally, the tight junction state appears to be closely linked to milk secretion. An increase in tight junction permeability is accompanied by decrease in the milk secretion rate, and conversely, a decrease in tight junction permeability is accompanied by an increase in the milk secretion rate.

The mammary fat pad.

The mammary fat pad is essential for development of the mammary epithelium, providing signals that mediate ductal morphogenesis and, probably, alveolar differentiation. The "cleared" fat pad is often used as a transplantation site. Considering the crucial role of the fat pad, its properties have received relatively little attention from researchers in the field. Some of the questions whose investigation is pertinent to understanding both normal mammary development and carcinogenesis are outlined in this commentary in the spirit of stimulating enquiry into this important subject. It is clear from a brief perusal of the available literature that until studies are specifically designed to clearly differentiate between functional effects of the fibrous and the adipose stroma, more substantive information about their differential effects on mammary development and tumorigenesis will not be forthcoming.

Studies in human lactation 3: molybdenum and nickel in human milk during the first month of lactation.

Molybdenum and nickel were measured in 62 and 46 samples, respectively, of human milk collected from 13 women between delivery and 38 d postpartum. Trace elements were analyzed by graphite-furnace atomic absorption spectrophotometry, with standard additions to whole milk without pretreatment. Molybdenum levels (mean +/- SD) fell from 15.0 +/- 6.1 ng/mL on day 1 to an apparently constant level of 1-2 ng/mL by 1 mo. Nickel concentrations did not change with time; the overall mean was 1.2 +/- 0.4 ng/mL. Intakes of the elements by infants were calculated with test-weighing data on the fully breast-fed infants of these same mothers. By 1 mo, the average daily intake of molybdenum was 1.5 micrograms; that of nickel was 0.8 microgram.

Measurement of steady state protein degradation in cultured human muscle cells.

Double label techniques for measurement of protein turnover in cultured cells are described. In the isotope withdrawal method protein in cultured muscle is labeled with two isotopes of the same amino acid for 24 to 100 h, followed by exposure to fresh medium containing one isotope only at the same specific activity for an additional 24 to 48 h. In the isotope addition method the order of addition of single and double-labeled media is reversed. After incubation the ratio of the two isotopes in the cell protein is a function of the incubation time and the degradation rate constant KD; KD can readily be calculated using a graphical or iterative method. In mixed cultures of human muscle with initial incubation ranging 24 to 159 h, the KD's obtained from various incubation times were similar. Both the isotope withdrawal and the isotope addition methods gave a KD value of 0.018 h-1 similar to values obtained by two different single isotope methods which monitor the appearance of free isotope in the medium of previously labeled cells. There were no differences of KD values obtained in cultures of muscle from normal patients and those with denervation, inflammatory myopathies, or nonspecific myopathic biopsy changes. When proteins were separated by gel electrophoresis, those of molecular weight greater than 60,000 had higher average KD values as compared to lower molecular weight proteins. The double isotope labeling method has the advantage of being easily applied to cultures with small numbers of cells and is potentially useful in obtaining the degradation rates of individual cellular proteins. The major disadvantages are (1) in their present form the methods can be used only in steady state cultures and (2) they require rather long (24 h) labeling times.

Secretion of calcium into milk: review.

Milk calcium exists in bound and ionized forms. Bound calcium is associated both with casein micelles and complexed to citrate and phosphate. Ionized calcium in milk is 1 to 4 millimolar, at least 1000 times its postulated concentration in the mammary alveolar cell. For this reason active transport mechanisms are necessary for transfer of this nutrient to the lumen of the mammary alveolus. Evidence that the major active transport system is a calcium adenosine triphosphatase residing in the membrane of the Golgi secretory vesicle is summarized. This adenosine triphosphatase appears to be activated by calcium concentrations in the micromolar range, to require magnesium ions, and to operate by phosphorylation of a 100,000 dalton enzyme intermediate. Metabolic processes are required to maintain a low concentration of calcium within the cytosol of the mammary alveolar cell. Because no evidence for sodium/calcium exchange could be found in the mammary gland of the lactating mouse, we suggest that these processes operate through a calcium adenosine triphosphatase in the basolateral membrane of the cell. Decreased calcium in the alveolar lumina decreased the integrity of the barrier between blood and milk. It is postulated from observations in other secretory systems that an increase in cystolic activity calcium may play a role in lactogenesis.

ATP-dependent calcium transport by a Golgi-enriched membrane fraction from mouse mammary gland.

Crude particulate preparations from the mammary glands of lactating mice were shown to transport calcium against a concentration gradient in the presence of ATP and mitochondrial inhibitors. Density gradient centrifugation with both sucrose and Percoll gradients indicated the presence of ATP-dependent transport in more than one membrane fraction. A Golgi-enriched membrane fraction possessed the highest specific activity of calcium transport. Digitonin, which increases the permeability of plasma membranes to calcium, did not affect this process. The Golgi fraction contained a 100,000 Dalton protein whose phosphorylation by gamma-[32P]-ATP was enhanced by a micromolar concentrations of free calcium. The phosphorylation was acid-stable and hydroxylamine-sensitive. These properties suggest that Golgi membranes in an activity secreting mammary epithelium possess a calcium transport system which resembles the calcium ATPase present in the sarcoplasmic reticulum of skeletal muscle.