Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells

1993 ◽  
Vol 106 (4) ◽  
pp. 1221-1226 ◽  
Author(s):  
M.D. Turner ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Differential Effect ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Brefeldin A ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Secretory Proteins ◽  
Mammary Cells ◽  
Regulated Secretory Pathway

The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.

Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein

1993 ◽  
Vol 106 (2) ◽  
pp. 641-648 ◽  
Author(s):  
M.E. Rennison ◽  
M. Kerr ◽  
C.V. Addey ◽  
S.E. Handel ◽  
M.D. Turner ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Regulatory Protein ◽  
Brefeldin A ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Feedback Inhibitor

The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulse-chase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.

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.

17-β-estradiol treatment maintains differentiative potential of virgin mouse mammary gland in culture and its responsiveness to insulin

1988 ◽  
Vol 119 (4) ◽  
pp. 543-548
Author(s):  
Hiroko Komura ◽  
Hideto Fukui ◽  
Hiroshi Wakimoto ◽  
Tetsuo Miyake ◽  
Naoki Terakawa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Thymidine Incorporation ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Insulin Binding ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Estradiol Treatment ◽  
Lactogenic Hormones

Abstract. Synthesis of the milk proteins, casein and α-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-β-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H] thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P< 0.001) of that obtained in intact mice, and 17-β-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P< 0.001) enhanced [3H] thymidine incorporation into DNA in cultured mammary explants from both intact and 17-β-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 ± 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P<0.05) lower than those from both intact (2870 ± 300 sites/cell) and 17-β-estradiol-treated castrated animals (2860 ± 190 sites/cell). The present findings suggest that 17-β-estradiol maintains growth and differentiative responses of mammary epithelial cells to insulin, which may be through preserving the number of insulin binding sites in the cells.

GENE ACTIVATION IN MAMMARY CELLS

1972 ◽  
Vol 71 (2_Suppla) ◽  
pp. S346-S368 ◽  
Author(s):  
Roger W. Turkington ◽  
Nobuyuki Kadohama
Keyword(s):  
Cell Differentiation ◽  
Gene Transcription ◽  
Hormonal Regulation ◽  
Gene Activation ◽  
Milk Proteins ◽  
Mouse Mammary Gland ◽  
Nuclear Proteins ◽  
Mammary Cells ◽  
Alveolar Cells ◽  
Mammary Cell

ABSTRACT Hormonal activation of gene transcription has been studied in a model system, the mouse mammary gland in organ culture. Transcriptive activity is stimulated in mammary stem cells by insulin, and in mammary alveolar cells by prolactin and insulin. Studies on the template requirement for expression of the genes for milk proteins demonstrate that DNA methylation has an obligatory dependence upon DNA synthesis, but is otherwise independent from hormonal regulation of mammary cell differentiation. Incorporation of 5-bromo-2′deoxyuridine into DNA selectively inhibits expression of the genes for specific milk proteins. Undifferentiated mammary cells activate the synthesis of specific acidic nuclear proteins when stimulated by insulin. Several of these induced acidic nuclear proteins are undetectable in unstimulated undifferentiated cells, but appear to be characteristic components of the nuclei of differentiated cells. These results indicate that mammary cell differentiation is associated with a change in acidic nuclear proteins, and they provide evidence to support the concept that acidic nuclear proteins may be involved in the regulation of gene transcription and of mammary cell differentiation.

scholarly journals Sorting and storage during secretory granule biogenesis: looking backward and looking forward

1998 ◽  
Vol 332 (3) ◽  
pp. 593-610 ◽  
Author(s):  
Peter ARVAN ◽  
David CASTLE
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Membrane Composition ◽  
Granule Formation ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

scholarly journals ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

1999 ◽  
Vol 147 (7) ◽  
pp. 1457-1472 ◽  
Author(s):  
Chung-Chih Lin ◽  
Harold D. Love ◽  
Jennifer N. Gushue ◽  
John J.M. Bergeron ◽  
Joachim Ostermann
Keyword(s):  
Secretory Pathway ◽  
Mutant Cell ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Intermediate Step ◽  
Golgi Stack ◽  
Transport Vesicles ◽  
Direct Fusion

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

scholarly journals The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins

Biomaterials ◽  
2019 ◽  
Vol 218 ◽  
pp. 119337 ◽  
Author(s):  
A.J. Kent ◽  
N. Mayer ◽  
J.L. Inman ◽  
C. Hochman-Mendez ◽  
M.J. Bissell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Mammary Epithelial

scholarly journals In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

1987 ◽  
Vol 105 (2) ◽  
pp. 659-668 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Signal Peptide ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
In Vitro Mutagenesis ◽  
Regulated Secretory Pathway

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

Roads taken by milk proteins in mammary epithelial cells

2001 ◽  
Vol 70 (1-2) ◽  
pp. 49-61 ◽  
Author(s):  
R. Boisgard ◽  
E. Chanat ◽  
F. Lavialle ◽  
A. Pauloin ◽  
M. Ollivier-Bousquet
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Mammary Epithelial
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