Review: the cellular mechanisms underlying mammary tissue plasticity during lactation in ruminants.

The mammary tissue is characterized by its capacity to adapt in response to a wide variety of changing conditions. This adaptation capacity is referred to as the plasticity of mammary tissue. In dairy ruminants, lactation is challenged by modifications that can either be induced on purpose, such as by modifying management practices, or occur involuntarily, when adverse environmental constraints arise. These modifications can elicit both immediate changes in milk yield and composition and carryover effects that persist after the end of the challenge. This review focuses on the current knowledge concerning the cellular mechanisms underlying mammary tissue plasticity. The main mechanisms contributing to this phenomenon are changes in the activity and number of mammary epithelial cells (MECs). Changes in the number of these cells result from variations in the rates of cell proliferation and death as well as changes in the rate MEC exfoliation. The number of MECs also depends on the number of resident adult mammary stem cells and their progenitors, which can regenerate the pools of the various mammary cells. Several challenges, including changes in milking frequency, changes in level of feed supply and hormonal manipulations, have been shown to modulate milk yield together with changes in mammary cell activity, turnover and exfoliation. Epigenetic changes may be an additional mechanism of adaptation. Indeed, changes in DNA methylation and reductions in milk yield have been observed during once-daily milking and during mastitis in dairy cows and may affect cell activity persistently. In contrast to what has been assumed for a long time, no carryover effect on milk yield were observed after feed supply challenges in dairy cows and modification of milking frequency in dairy goats, even though the number of mammary cells was affected. In addition, mammary tissue plasticity has been shown to be influenced by the stage of lactation, health status and genetic factors. In conclusion, the cellular mechanisms underlying mammary tissue plasticity are diverse, and the mammary tissue either does or does not show elastic properties (with no permanent deformation), in response to environmental changes.

Lipid-deprived diet perturbs O-glycosylation of secretory proteins in rat mammary epithelial cells.

Nutrition modulates both production and composition of milk. Milk composition was studied in rats chronically fed a diet without additional lipids, and therefore eating only traces of the recommended supply of essential polyunsaturated fatty acid. Despite a large decrease in milk-protein synthesis, only protein composition, but not protein concentration, was found to change in the milk of rats following a lipid-deprived diet. Correlatively, we observed a substantial increase in the lactose concentration of milk. Analysis of milk proteins by two-dimensional electrophoresis demonstrated that the relative proportion of the various molecular forms of κ-casein, an O-glycosylated protein, was modified in the milk of rats receiving the lipid-deprived diet. In tissues, differences in the two-dimensional pattern of κ-casein between control and lipid-deprived rats were similar, if not identical. In contrast to κ-casein, the molecular forms of α-lactalbumin, an N-glycosylated protein, were not affected by the diet. These data provide evidence that O-glycosylation of milk proteins in the secretory pathway of mammary epithelial cells is modulated by the lipid content of experimental diets.

Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells.

Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines. Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells. Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein. Butanol interfered with both the endoplasmic reticulum to Golgi complex transport of the caseins and secretory vesicle formation from the trans-Golgi network. In contrast, the transport of whey acidic protein to the Golgi was less affected. Activation of protein kinase C enhanced the overall secretion of both markers and interestingly, this stimulation of secretion was maintained for whey acidic protein in the presence of butanol. Transphosphatidylation assays demonstrated the existence of a constitutive phospholipase D activity which was stimulated by the activation of protein kinase C. We conclude that phospholipase D plays a role in casein transport from the endoplasmic reticulum to the Golgi and in the secretory vesicle formation from the trans-Golgi network. Moreover, our results suggest a differential requirement for phospholipase D in the secretion of caseins and that of whey acidic protein.

Polymeric IgA are sulfated proteins.

The main sulfated proteins secreted by rabbit mammary gland tissue had M(r) of approximately 67 000, 63 000 and 23 000, and one component which most likely corresponded to proteoglycans had a diffuse electrophoretic mobility (M(r)200 000). The sulfate groups in the 67-63 kDa proteins were mostly linked to carbohydrates. These proteins and the 23 kDa protein were co-purified and identified to heavy chains of immunoglobulin A (IgA) and J chain, respectively. Sulfation of alpha-chains also occurred in rat mammary and rabbit lacrimal glands. We conclude that polymeric IgA which are produced by plasma cells and released in secretion fluids after transcytosis through epithelia are sulfated.

Alpha(S1)-casein is required for the efficient transport of beta- and kappa-casein from the endoplasmic reticulum to the Golgi apparatus of mammary epithelial cells.

In lactating mammary epithelial cells, interaction between caseins is believed to occur after their transport out of the endoplasmic reticulum. We show here that, in alpha(S1)-casein-deficient goats, the rate of transport of the other caseins to the Golgi apparatus is highly reduced whereas secretion of whey proteins is not significantly affected. This leads to accumulation of immature caseins in distended rough endoplasmic reticulum cisternae. Casein micelles, nevertheless, were still observed in secretory vesicles. In contrast, no accumulation was found in mammary epithelial cells which lack beta-casein. In mammary epithelial cells secreting an intermediate amount of alpha(S1)-casein, less casein accumulated in the rough endoplasmic reticulum, and the transport of alpha(S1)-casein to the Golgi occurred with kinetics similar to that of control cells. In prolactin-treated mouse mammary epithelial HC11 cells, which do not express alpha(S)-caseins, endoplasmic reticulum accumulation of beta-casein was also observed. The amount of several endoplasmic reticulum-resident proteins increased in conjunction with casein accumulation. Finally, the permeabilization of rough endoplasmic reticulum vesicles allowed the recovery of the accumulated caseins in soluble form. We conclude that optimal export of the caseins out of the endoplasmic reticulum is dependent upon alpha(S1)-casein. Our data suggest that alpha(S1)-casein interacts with the other caseins in the rough endoplasmic reticulum and that the formation of this complex is required for their efficient export to the Golgi.

Brefeldin A differently affects basal and prolactin-stimulated milk protein secretion in lactating rabbit mammary epithelial cells.

When lactating mammary epithelial cells were treated with prolactin in vitro, numerous small vesicles rapidly accumulated in the Golgi area, and secretion of milk proteins increased. The effects of brefeldin A on these intracellular events were investigated. As observed by electron microscopy, stacks of the median Golgi were not altered after incubation in the presence of 50 nM brefeldin A but were dissociated when the drug concentration was > or = 500 nM. Small vesicles did not accumulate in the Golgi area when mammary cells were incubated in medium containing both prolactin and brefeldin A, whatever the concentration of the latter. Immunofluorescence experiments showed that 50 nM brefeldin A did not modify the localization of the CTR 433 median Golgi protein, but it induced redistribution of trans-Golgi network-associated proteins such as TGN38, AP-1 adaptor and clathrin. These effects occurred in the presence of brefeldin A plus prolactin. Pulse-chase experiments showed that brefeldin A concentrations > or = 100 nM induced the intracellular accumulation of milk proteins, provoked the appearance of immature forms of caseins, and inhibited milk protein secretion. In contrast, concentrations of brefeldin A of < or = 50 nM did not affect basal casein secretion but inhibited the secretagogue effect of prolactin. These data show not only that several biochemical events in the transport of milk proteins which are sensitive to different brefeldin A concentrations occur in lactating mammary epithelial cells, but also that it is possible to inhibit a hormonal stimulus in a selective manner, while the machinery responsible for basal secretion is still active.

The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-Golgi network.

Chromogranin B (secretogranin I), a protein sorted to secretory granules in many endocrine cells and neurons, undergoes selective aggregation during the sorting process in the trans-Golgi network. Reduction of the single, highly conserved intramolecular disulfide bond of chromogranin B by exposure of intact PC12 cells to the thiol reducing agent dithiothreitol has previously been shown to cause its missorting to the constitutive pathway of secretion. Using saponin perforation of membrane vesicles in aggregative buffer mimicking the milieu in the lumen of the trans-Golgi network (pH 6.4, 10 mM calcium), we show here that treatment with dithiothreitol does not prevent the aggregation of chromogranin B in this compartment. This implies that the loop in the chromogranin B polypeptide that is formed by the disulfide bond has a critical role in the membrane recognition of aggregated chromogranin B during secretory granule formation.

Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond.

[Mechanism of sorting of secretory proteins and formation of secretory granules in neuroendocrine cells]. / Mécanisme de tri des protéines sécrétoires et formation des granules de sécrétion dans les cellules neuroendocrines.

We have investigated the mechanism by which constitutive and regulated secretory proteins are sorted in the trans Golgi network (TGN). Although this mechanism is believed to involved the selective aggregation of regulated secretory proteins, the factors responsible for the formation of these aggregates, which can be detected by electron microscopy as electron dense material in the lumen of the TGN, were unknown. The mechanism involved in the sorting of these aggregates to secretory granules is also poorly understood. Concerning the first point, the study of the aggregation of two regulated secretory proteins, chromogranin B and secretogranin II (granins) in the neuroendocrine cell line PC12 showed that a decrease in pH and an increase in calcium concentration in the TGN as compared to the more proximal compartments of the secretory pathway are sufficient to trigger the selective aggregation of the granins in this compartment. Results on the aggregation of the granins in the TGN of the pituitary GH4C1 cells in which the level of synthesis of regulated secretory proteins can be manipulated also suggested a role of protein concentration in the aggregation process. Finally, since granins aggregates in the TGN of PC12 cells largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers, we concluded that the selective aggregation of regulated secretory proteins, triggered by the specific luminal milieu of the TGN, is a crucial step in their segregation from constitutive secretory proteins. Concerning the second point, we studied the role of the single, highly conserved disulfide bond in chromogranin B on its sorting to secretory granules. We showed that reduction of this disulfide bond by incubation of intact PC12 cells with the membrane permeable thiol reducing agent dithiothreitol (DTT) causes the missorting of chromogranin B to the constitutive secretory pathway. This treatment only slightly decreased the intracellular storage of secretogranin II, which lacks cysteine, into secretory granules. We found that the effect of DTT on chromogranin B, which was already known to prevent disulfide bond formation in the endoplasmic reticulum, occurred in the TGN. We concluded that the sorting, in the TGN, of chromogranin B to secretory granules is dependent upon the integrity of its disulfide bond and that DTT treatment in vivo is as valuable tool to study the post-endoplasmic reticulum traffic of disulfide bond containing proteins.

Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.

Peptides co-released with luteinizing hormone by perifused pituitary cell aggregates.

The proteins co-released with gonadotropins were analyzed using perifusion of pituitary cell aggregates from 14-day-old female rats, after a pre-labeling period with [35S]methionine. Radioimmunoassays of hormones and electrophoretic analysis were performed on each 4 min effluent. Gonadotropin-releasing hormone (GnRH) pulses increased significantly (P less than 0.01) the release of several proteins (Mr range from 140,000 to 28,000). The main stimulation appeared for -1, a 87 kDa species, previously characterized as gonadotrope polypeptide 87 (GP87) in monolayer cultures and identified as a secretogranin II (SgII) form; -2, a second species of 80 kDa designated as B2. Secretory patterns of radiolabeled GP87 and B2 paralleled the luteinizing hormone (LH) ones. The release of these species was -1, GnRH dose dependent; -2, monophasic for short pulses but complex when the duration of GnRH pulses increased to 16 min, suggesting different pools of GP87 and B2 as for LH; -3, induced by thyrotropin-releasing hormone (TRH). A slight output was also elicited by corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), but this release was partly impaired in the presence of a potent anti-GnRH ([Ac-D-(2)-NAL1,pF-D-Phe2,D-Trp3,D-Arg6]-LAF) suggesting a non-specific effect of these two factors. GP87/SgII thus appeared mainly associated with the release of hormonal glycoproteins. In conclusion, perifusion of pituitary cell aggregates allows a precise minute-to-minute kinetic analysis of the various proteins co-released with hormones. The similar timing in output of LH, GP87 and B2 suggests that these three proteins co-exist in the same secretory granules inside gonadotropes.

The gonadotrope polypeptide (GP 87) released from pituitary cells under luteinizing hormone-releasing hormone stimulation is a secretogranin II form.

Cultured gonadotrope cells from 14 day old female rat pituitaries have been shown to release a highly acidic protein when incubated with LHRH: the gonadotrope polypeptide (GP 87). Moreover, a new tyrosine-sulfated acidic protein, secretogranin II (Sg II), clearly distinct from the chromogranin species, was described in the secretory granule matrix of endocrine cells secreting peptide hormones by the regulated pathway. Recently, the release of Sg II from female rat pituitary stimulated by LHRH was demonstrated in vitro. Several physicochemical (Mr; pI) and biological (cellular localization in the pituitary; LHRH-stimulated release) properties are common to Sg II and GP 87. To verify if these 2 polypeptides are similar or distinct components, other physicochemical characteristics (heat-stability, sulfation, phosphorylation) were compared using isotope incorporation followed by either 1- or 2-dimensional polyacrylamide gel electrophoresis and autoradiography. Furthermore, the similarity of GP 87 to Sg II was studied by immunoblotting on nitrocellulose sheets following electrophoresis of intracellular and secreted proteins. Antisera raised against bovine Sg II (extracted from whole pituitaries) and against rat GP 87 (released into the medium of cultured pituitary cells stimulated by LHRH) were used. The overall data presented here suggest that GP 87 is the Sg II form contained in, and released by, gonadotrope cells.

LHRH-stimulated release of stored and newly synthesized gonadotrop polypeptide (GP-87) by cultured rat gonadotrophs.

Enriched gonadotrophs from rat pituitaries were used to analyze the kinetics of release in vitro of the Gonadotrop Polypeptide (GP-87) under LHRH stimulation. Proteins in cultured cells were labeled with [35S]methionine. Labeling of the intracellular GP-87 pool was effected during 16 hours prior to LHRH (10(-7) M) stimulation. Proteins were analyzed either by one-dimensional SDS-PAGE (Medium content) or by two-dimensional PAGE (Cell content). An apparent half-life (intracellular catabolism + release) of 31 h for GP-87 was estimated from control cells; it dropped to 2.5 h in stimulated cells due to intense release (26% after 1 h and about 80% after 8 h of stimulation). When cells were simultaneously labeled and stimulated, the newly synthesized species appeared in the medium after a lag phase of 30 minutes, time required for synthesis and full subsequent processing. From both series of experiments, it is concluded that the hypothalamic decapeptide promotes exocytosis of the newly synthesized GP-87 well before the endogenous GP-87 pool is exhausted. Furthermore, the release of another discrete protein (B2, Mr: 81 kDa) is also stimulated by LHRH. These proteins being co-released with LH could be part of the sorting and/or routing process of hormones towards exocytosis.

LHRH promotes the synthesis and release of a 87,000 Da protein (GP-87) by enriched gonadotrophs.

Protein secretion by cultured pituitary cells from 14-day-old female rats was estimated using [35S]methionine incorporation followed by either one- or two-dimensional electrophoresis and autoradiography. Stimulation of total cells or gonadotrophs by LHRH promoted the synthesis and release of a specific polypeptide (apparent molecular weight 87,000, pI = 4.6). Silver staining of cellular proteins from both gonadotroph-enriched and gonadotroph-depleted populations prepared by centrifugal elutriation revealed a high concentration of this polypeptide in the gonadotrophs and a very low level in the other cell population. This species was thus called Gonadotrope Polypeptide GP-87. Release of labeled GP-87 by gonadotrophs was both time dependent (up to 4 h) and LHRH dose dependent (from 10(-9) M to 10(-7) M) as was the release of LH. Attempts to precipitate GP-87 from the incubation medium with anti-LH antiserum were unsuccessful suggesting that GP-87 is not a 'big' form of LH. TRH neither stimulated the release of GP-87 from gonadotrophs nor from lactotrophs though it did stimulated PRL release. From these data, we conclude that gonadotrophs in culture synthesize a specific polypeptide (GP-87), LHRH stimulates both the synthesis and release of GP-87, and the pituitary cell response is peptide specific. The LHRH-induced synthesis and release of GP-87 could be an important step in the molecular processes that regulate gonadotrophin secretion.

[Effect of LHRH on the synthesis and phosphorylation of proteins in gonadotropic cells]. / Action de la LHRH sur la synthèse et la phosphorylation de protéines dans les cellules gonadotropes.

Protein secretion by cultured pituitary cells from 14 day-old female rats was estimated using [35S]-méthionine incorporation, electrophoresis and autoradiography. Stimulation by LHRH promoted biosynthesis and fast release of a protein of apparent molecular weight 87,000 daltons and pI 4.6. Gonadotrophs enriched by centrifugal elutriation were particularly rich in this polypeptide which was thus called GP-87 (Gonadotrope polypeptide). Cells were then cultured with [32P]-orthophosphate and proteins were analyzed. Our first results tend to show that GP-87 is phosphorylated, at least in the cells. We suggest that this specific protein, the secretion of which is correlated with LH release, participates to the response mechanisms of gonadotrope cells stimulated by LHRH.