Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Endocytic prolactin routes to the secretory pathway in lactating mammary epithelial cells

Plasma-borne prolactin is carried from blood to milk by transcytosis across the mammary epithelial cell through the endocytic and secretory pathways. To determine the precise route of prolactin endocytosis, intracellular transport of biotinylated prolactin was monitored, in parallel with endocytosis of fluorescein isothiocyanate-conjugated dextran and IgG, by using pulse-chase experiments in lactating mammary fragments and in enzymatically dissociated acini. Biotinylated prolactin was sorted to vesiculo-tubular organelles whereas dextran was very rapidly carried to the lumen and IgG remained accumulated in the basal region of cells. To determine whether prolactin uses routes into and across the Golgi and trans-Golgi network, localisation of biotinylated prolactin was combined with the immunofluorescence detection of caseins and, respectively, p58 and TGN38. Biotinylated prolactin strongly colocalised with caseins during a chase but not all or only very little with p58 and TGN38. To characterise the organelles involved in transcytosis, gold-labelled prolactin, experimentally accumulated in late endosomes and which recovered a normal transport, was localised by electron microscopy. In mammary fragments incubated at low temperature, and in mammary fragments from rats fed with a lipid-deprived diet, transport of gold-labelled prolactin was restored by increasing the temperature and by adding arachidonic acid, respectively. These data demonstrate that a sorting occurs very rapidly between prolactin, dextran and IgG. They suggest that prolactin may reach the biosynthetic pathway after direct fusion between multivesicular bodies and secretory vesicles.     

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.     

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.     

Autocrine-paracrine inhibition of growth hormone and prolactin production by GH3 cell-conditioned medium

Summary In previous work we have shown that perifused GH₃ cells exhibit spontaneously accelerating growth hormone (GH) and prolactin (PRL) secretory rates. This behavior contrasts with GH and PRL secretion rates that are decreasing or stable over the same 3-d period in static cell culture. We now report that GH₃ cells maintained in serum-supplemented medium produce an autocrine-paracrine factor(s) which inhibits GH secretion in plate culture; PRL release is frequently reduced as well. The inhibitory effect of conditioned medium on GH secretion was concentration dependent, whereas PRL release was stimulated at low and inhibited at high concentrations over the same range. Extensive dialysis of conditioned medium using membranes with a molecular weight cut-off of 12 000–14 000 did not remove GH inhibition but produced a retentate that stimulated PRL secretion. Heat-inactivation of conditioned medium did not abolish inhibition of GH release but did remove the PRL-stimulatory effect. IGF-I added to fresh culture medium did not reproduce the GH-inhibitory effects of conditioned medium. We conclude that GH₃ cell secretory behavior in perifusion and plate culture systems may be partially explained by the production of an autocrine-paracrine factor: its accumulation in plate culture inhibits GH and PRL secretion whereas its removal, by perifusing medium, allows GH and PRL secretion to accelerate. Supported by grant DK33388 to M. E. S. from the National Institute of Health, Bethesda, MD, and in part by the Medical Research Service of the Veterans Administration, Washington, DC.     

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.     

Temperature dependence of prolactin endocytosis and casein exocytosis in epithelial mammary cells

As previously reported in epithelial mammary cells of lactating rabbit, prolactin exerts a stimulatory effect on casein secretion. After binding to a membrane receptor, the complex hormone-receptor is internalized in mammary cells. Peptide hormone action involves the generation of second messengers. These second messengers can be emitted as soon as hormone is linked to the membrane receptor. However, it is not excluded that endocytosis and transfer of prolactin inside the cell take part in the emission of second messenger and related secretory response. In order to precise intracellular transport pathways in the lactating mammary cell, we have examined the effects of reduced temperature on the one hand on prolactin endocytosis, on the other hand on casein secretion and on the stimulating effect of prolactin on casein secretion. Endocytosed prolactin was cytochemically localized mainly on the plasma membrane at 4 degrees C. At 25 degrees C, the hormone accumulated, during 60 min, in endosomes and multivesicular bodies. At 37 degrees C, prolactin was detectable after 15 and 30 min inside the cells and disappeared after 60 min. Transport and exocytosis of secretory proteins were only partly inhibited at 25 degrees C as attested by autoradiography localization and biochemical assays of newly synthesized caseins. However, at 25 degrees C, prolactin was no more able to stimulate casein exocytosis. These results show that intracellular transport of prolactin and secretagogue effect of the hormone does not proceed at 25 degrees C. However, secretory mechanisms of the cell are always able to be stimulated by exogenous arachidonic acid at this temperature. Low temperature appears as a good means to study intracellular transport in the mammary cell.     

Adenoviral vector mediates high expression levels of human growth hormone in the milk of mice and goats

The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest.     

Prolactin and growth hormone regulate adiponectin secretion and receptor expression in adipose tissue

Adiponectin is a hormone secreted from adipose tissue, and serum levels are decreased with obesity and insulin resistance. Because prolactin (PRL) and growth hormone (GH) can affect insulin sensitivity, we investigated the effects of these hormones on the regulation of adiponectin in human adipose tissue in vitro and in rodents in vivo. Adiponectin secretion was significantly suppressed by PRL and GH in in vitro cultured human adipose tissue. Furthermore, PRL increased adiponectin receptor 1 (AdipoR1) mRNA expression and GH decreased AdipoR2 expression in the cultured human adipose tissue. In transgenic mice expressing GH, and female mice expressing PRL, serum levels of adiponectin were decreased. In contrast, GH receptor deficient mice had elevated adiponectin levels, while PRL receptor deficient mice were unaffected. In conclusion, we demonstrate gene expression of AdipoR1 and AdipoR2 in human adipose tissue for the first time, and show that these are differentially regulated by PRL and GH. Both PRL and GH reduced adiponectin secretion in human adipose tissue in vitro and in mice in vivo. Decreased serum adiponectin levels have been associated with insulin resistance, and our data in human tissue and in transgenic mice suggest a role for adiponectin in PRL and GH induced insulin resistance.     

SOCS3 promotes apoptosis of mammary differentiated cells

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).     

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 up-regulated 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.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways

In mammary epithelial cells, milk lipids and proteins are synthesised in the same compartment, the endoplasmic reticulum. Lipids, carried through the cytoplasm, associate with the apical membrane which then pinches off and releases the lipid globule. Proteins, carried through membrane compartments are released in the lumen after fusion of secretory vesicles with the apical membrane. These processes assure a relatively constant composition of milk but it is not known whether lipid and protein secretion are linked. The protein composition of the milk fat globule membrane and the stimulatory effects of prolactin and oxytocin on lipid and protein secretion suggest that these processes are coupled and co-regulated. However, it is possible to observe a dissociation between the formation and the secretion of the two constituents, during differentiation and in various experimental conditions, and this suggests that coupling is not strictly required.     

The mRNA encoding a parathyroid hormone-like peptide is produced in mammary tissue in response to elevations in serum prolactin

During lactation, a dramatic rise in serum PRL stimulates milk production, resulting in a substantial rise in calcium mobilization from gut and bone. We found that the production of a newly characterized calcium-mobilizing PTH-like peptide (PTH-LP) by mammary tissue was tightly linked to lactation, suggesting a possible role for PRL in the expression of PTH-LP. Here it is shown that suckling results in both an elevation in serum PRL and the appearance of PTH-LP mRNA in mammary tissue. Bromocriptine, a potent inhibitor of PRL secretion, blocked the suckling-associated rise in serum PRL and the subsequent induction of PTH-LP mRNA in mammary gland. Furthermore, injection of PRL dramatically induced PTH-LP mRNA in unsuckled puerperal glands, but not in glands on day 21 of pregnancy. Thus, the correlation between serum levels of PRL and the expression of PTH-LP mRNA in mammary tissue extends the role of PRL in milk production and suggests a possible mechanism for the PRL effects on calcium metabolism.     

Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland

The occurrence of phosphorylated secretory proteins such as caseins and vitellogenin and the recent characterization of phosphorylated proteoglycans, in the xylose and protein core, has raised the question of where in the cell and how this phosphorylation occurs. Previous studies have described a casein kinase activity in the lumen of the Golgi apparatus and this organelle as the site of xylose addition to the protein core of proteoglycans. We now report the translocation in vitro of ATP into the lumen of rat liver and mammary gland Golgi vesicles which are sealed and have the same membrane topographical orientation as in vivo. The entire ATP molecule was translocated into the lumen of the Golgi vesicles; this was established by using ATP radiolabeled with tritium in the adenine and gamma-32P. Translocation was temperature dependent and saturable, with an apparent Km of 0.9 microM and Vmax of 58 pmol/mg protein/min. Preliminary evidence suggests that translocation of ATP into the vesicles' lumen is coupled to exit of AMP from the lumen. Following translocation of ATP into the lumen of the vesicles, proteins were phosphorylated.