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

1992 ◽  
Vol 117 (2) ◽  
pp. 269-278 ◽  
Author(s):  
MD Turner ◽  
ME Rennison ◽  
SE Handel ◽  
CJ Wilde ◽  
RD Burgoyne
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Isolated Cells ◽  
Pulse Label ◽  
Constitutive Secretion

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.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments

1995 ◽  
Vol 108 (2) ◽  
pp. 519-527 ◽  
Author(s):  
P.L. Jones ◽  
N. Boudreau ◽  
C.A. Myers ◽  
H.P. Erickson ◽  
M.J. Bissell
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Protein Synthesis ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Active Regions ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Beta Casein ◽  
Casein Protein

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

scholarly journals Caffeic Acid Prevented LPS-Induced Injury of Primary Bovine Mammary Epithelial Cells through Inhibiting NF-κB and MAPK Activation

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Mingjiang Liu ◽  
Guoqing Fang ◽  
Shaojie Yin ◽  
Xin Zhao ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Caffeic Acid ◽  
Proinflammatory Cytokines ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Signal Pathways ◽  
Dependent Manner ◽  
Bovine Mammary Epithelial Cells

In our previous study, lipopolysaccharide (LPS) significantly reduced the cell viability of primary bovine mammary epithelial cells (bMEC) leading to cell apoptosis, which were prevented by caffeic acid (CA) through inhibiting NF-κB activation and reducing proinflammatory cytokine expression. While the underlying mechanism remains unclear, here, we determined that LPS induced the extensive microstructural damage of bMEC, especially the mitochondria and endoplasmic reticulum. Then, the obvious reduction of mitochondrial membrane potential and expression changes of apoptosis-associated proteins (Bcl-2, Bax, and casepase-3) indicated that apoptosis signaling through the mitochondria should be responsible for the cell viability decrease. Next, the high-throughput cDNA sequencing (RNA-Seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were employed to verify that the MAPK and JAK-STAT signaling pathways also were the principal targets of LPS. Following, the critical proteins (ERK, JNK, p38, and c-jun) of the MAPK signaling pathways were activated, and the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) regulated by NF-κB and MAPKs was significantly increased, which can promote a cascade of inflammation that induces cell injury and apoptosis. Meanwhile, CA significantly inhibited the activation of MAPKs and the release of proinflammatory cytokines in a dose-dependent manner, which were similar to its effects on the NF-κB activation that we previously published. So we concluded that CA regulates the proteins located in the upstream of multiple cell signal pathways which can reduce the LPS-induced activation of NF-κB and MAPKs, thus weakening the inflammatory response and maintaining cell structure and function, which accordingly inhibit apoptosis.

scholarly journals Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

2012 ◽  
Vol 79 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Damien S. Bouchard ◽  
Lucie Rault ◽  
Nadia Berkova ◽  
Yves Le Loir ◽  
Sergine Even
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Lactobacillus Casei ◽  
Mammary Epithelial Cells ◽  
Control Strategies ◽  
Mammary Epithelial ◽  
Dairy Herds ◽  
Dependent Manner ◽  
Content Type ◽  
Bovine Mammary Epithelial Cells

ABSTRACTStaphylococcus aureusis a major pathogen that is responsible for mastitis in dairy herds.S. aureusmastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability ofS. aureusto invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability ofLactobacillus caseistrains to prevent invasion of bMEC by twoS. aureusbovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively.L. caseistrains affected adhesion and/or internalization ofS. aureusin a strain-dependent manner. Interestingly,L. caseiCIRM-BIA 667 reducedS. aureusNewbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two otherL. caseistrains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate ofS. aureuswas not affected byL. casei. It should be noted thatL. caseiwas internalized at a low rate but survived in bMEC cells with a better efficiency than that ofS. aureusRF122. Inhibition ofS. aureusadhesion was maintained with heat-killedL. casei, whereas contact between liveL. caseiandS. aureusor bMEC was required to preventS. aureusinternalization. This first study of the antagonism of LAB towardS. aureusin a mammary context opens avenues for the development of novel control strategies against this major pathogen.

scholarly journals Exocytosis from permeabilized lactating mouse mammary epithelial cells. Stimulation by Ca2+ and phorbol ester, but inhibition of regulated exocytosis by guanosine 5′-[γ-thio]triphosphate

1992 ◽  
Vol 286 (1) ◽  
pp. 13-15 ◽  
Author(s):  
M D Turner ◽  
C J Wilde ◽  
R D Burgoyne
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Phorbol Ester ◽  
Mammary Epithelial Cells ◽  
Post Partum ◽  
Mammary Epithelial ◽  
Independent Manner ◽  
Permeabilized Cells ◽  
A Site ◽  
Golgi Network

Lactating mouse mammary epithelial cells secrete large amounts of milk protein via constitutive or regulated exocytotic pathways. Secretion through both pathways was quantified by assaying the release of [35S]methionine-labelled trichloroacetic acid-precipitable proteins from digitonin-permeabilized secretory acini isolated from mammary glands of 10-day-post-partum lactating mice. Protein secretion from the isolated permeabilized cells was either Ca(2+)-dependent (regulated) or Ca(2+)-independent (constitutive). In both cases there was a requirement for ATP. Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) caused a marked increase in the percentage protein secretion from the cells in a Ca(2+)-independent manner. However, the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) caused a partial inhibition of Ca(2+)-dependent exocytosis, while having no significant effect on Ca(2+)-independent exocytosis. Thus the GTP[S] is exerting its effect on the regulated pathway at a site subsequent to protein sorting and packaging into secretory vesicles at the trans-Golgi network.

Autocrine Regulation of Protein Secretion in Mouse Mammary Epithelial Cells

1998 ◽  
Vol 248 (3) ◽  
pp. 761-766 ◽  
Author(s):  
David R. Blatchford ◽  
Kay A.K. Hendry ◽  
Colin J. Wilde
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Autocrine Regulation

scholarly journals Indirubin Treatment of Lipopolysaccharide-Induced Mastitis in a Mouse Model and Activity in Mouse Mammary Epithelial Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Jin-lun Lai ◽  
Yu-hui Liu ◽  
Yong-chong Peng ◽  
Pan Ge ◽  
Chen-fei He ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Mammary Epithelial Cells ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Mammary Epithelial ◽  
Myelogenous Leukemia ◽  
Myeloperoxidase Activity ◽  
Dependent Manner

Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor-α, interleukin-1β(IL-1β), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein and inhibitor of kappa B. In addition to its effect on the NF-κB signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF-κB and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

The majority of clathrin coated vesicles from lactating rabbit mammary gland arises from the secretory pathway

1999 ◽  
Vol 112 (22) ◽  
pp. 4089-4100 ◽  
Author(s):  
A. Pauloin ◽  
S.A. Tooze ◽  
I. Michelutti ◽  
S. Delpal ◽  
M. Ollivier-Bousquet
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Bovine Brain ◽  
Mammary Epithelial ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Secretory Vesicles ◽  
Mannose 6 Phosphate

Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on (2)H2O-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9+/-10.5 nm and a density of 1.23 g × ml(−1). Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only approximately 5–10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents approximately 70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway. Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of alpha(s1)-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.

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