animal
Animal (2009), 3:12, pp 1733–1742 & The Animal Consortium 2009
doi:10.1017/S1751731109990589
Mammary gland secretion: hormonal coordination of
endocytosis and exocytosis
S. Truchet and M. Ollivier-BousquetINRA, UR1196 Génomique et Physiologie de la Lactation, F78352 Jouy-en-Josas, France
(Received 26 January 2009; Accepted 24 June 2009; First published online 17 August 2009)
The mammary epithelium coordinates the uptake of milk precursors and the transport of milk components in order to produce
milk of relatively constant composition at a particular stage of lactation, as long as the mammary gland is healthy. The
mammary epithelial cell controls the uptake of blood-borne molecules at its basal side and the release of products into milk
at its apical side, through mechanisms of internalization (endocytosis) and mechanisms of release (exocytosis). These events
are strictly dependent on the physiological stage of the mammary gland. This review addresses the mechanisms responsible
for these processes and points out new questions that remain to be answered concerning possible interconnections between
them, for an optimal milk secretion.
Keywords: mammary epithelial cell, milk secretion, hormonal regulation
Implications
This paper is a review on the mechanisms of milk secretion.
The understanding of these mechanisms is fundamental to
the research on milk. Research on milk and its safety as a
source of substances that are beneficial to growth and
health in children and adults is a major concern in developed countries. The production of dairy products as basic
nutrients is a priority in the fight against under-nourishment.
Research on milk quality and milk composition is important
for a sustainable dairy production.
Introduction
The mammary epithelium finely orchestrates the uptake of
blood-borne molecules at its basal side (endocytosis),
intracellular trafficking across cells and the release of products into milk at its apical side (exocytosis). In this way,
the mammary gland has a remarkable capacity to control
the concentrations of milk constituents (nutrients, growth
factors, hormones, immunoglobulins, minerals and ions,
etc.) as well as total secretion. To achieve their ends,
cells must carry components to the correct place using
specific receptors and intracellular compartmentalization.
As described in a pioneering work, ‘The cell membranes
are making products and undergoing transformations
-
E-mail: [email protected]
simultaneously. More specifically, milk components packaged in membranes are moving through and out of the cell.
Thus, there is product flow and membrane flow’ (Patton,
1976a). An important goal for the physiologists is therefore
to understand the cellular mechanisms leading to optimum
milk secretion.
The mammary epithelial cell (MEC) is atypical among the
exocrine cells because of the huge quantity of plasma
membrane, enrobing milk fat droplets, which is pinched off
and expelled from the cell into milk. Thus, the mechanisms
required to maintain membrane homeostasis in mammary
cells differ from those governing membrane recycling in
other exocrine glands (exocrine pancreas, lacrimal and
parotid glands) (Meldolesi, 1974; Herzog and Farquhar,
1977), and from coupling of the fusion and recycling of
vesicles in the presynaptic nerve terminal (Gundelfinger
et al., 2003; Ryan, 2006). The highly polarized MEC
represents a cell type in which the transfer of material
between intracellular compartments occurs by means of
membrane-bound vesicles without compromising the identity of the compartments. Numerous questions, however,
remain concerning the process of membrane reconstitution
in the mammary cell. Does a process of internalization of a
portion of the basolateral plasma membrane correspond to
an equivalent incorporation of membranes of the secretory
vesicles at the apical membrane? How is the apical membrane reconstituted after release of the portion enveloping
the lipid droplet?
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Truchet and Ollivier-Bousquet
Another unique feature of MEC functioning is the hormonal control of milk secretion. The lactogenic hormones
released at each suckling or milking not only control the
gene expression of milk proteins but also maintain cellular
architecture and stimulate the mechanisms of MEC intracellular transport by secretagogue effects (Ollivier-Bousquet
and Devinoy, 2005 and references therein; Lollivier et al.,
2006). In addition, some blood-borne hormones are carried
to the milk across MECs (Ollivier-Bousquet, 1993). Moreover, in the case of prolactin (PRL), its intracellular transport
is required for its secretagogue effect.
How are events occurring at the basolateral side and
apical side related? Are endocytosis and intracellular
transport required for the triggering of different intracellular
signals? Is endocytosis itself regulated by hormonal effects?
We attempt here to review some of the old and new data
available on the relationship between endocytosis and
exocytosis in MECs. Because these events have often been
studied in mammary gland from lagomorphs and rodents,
we mainly refer to these species. However, an important
aim is to use the data obtained in these species to understand the functioning of the ruminant mammary gland. We
will first describe some of the routes used by different blood
constituents to enter cells. Some aspects of the exocytosis
of milk constituents and its regulation will also be presented. Finally, the possible crosstalks between the two
mechanisms will be discussed.
Endocytosis
The microvasculature constituted by a basket-like capillary
network surrounding the mammary acini ensures the supply of
nutrients to the mammary epithelium (Djonov et al., 2001)
(Figure 1a). After crossing the capillary endothelium, blood
nutrients and other components are sequestered or diffused
in the extracellular space by unknown mechanisms. They
have access to the basolateral membrane of the MEC up to
the tight junctions that separate the basal and apical sides
Figure 1 Organization of exocytosis and endocytosis sites in the mammary epithelium. (a) The microvasculature constitutes a basket-like capillary network
around the mammary acini. Capillaries were visualized with fluorescein isothiocyanate dextran injected in the vein of the tail of lactating rat and detected
by Fibered Confocal Fluorescence Microscopy (image kindly provided by R. Boisgard, CEA-Saclay, France). Bar 50 mm. (b) Section of lactating rat mammary
tissue. Actin was visualized with tetramethyl rhodamine isothiocyanate-phalloidin. Actin is abundant in myoepithelial cells (arrow) and appears
concentrated in the vicinity of both the basolateral (empty arrowheads) and the apical plasma membrane (arrowheads) in the MECs. L 5 lumen. Bar 20 mm.
(c) Membrane trafficking in the MECs. Endocytosis mainly occurs at the basolateral side of the MEC and materials are then transported to the recycling
endosomes. At this level, internalized components can be addressed to different cellular compartments. Proteins that are synthesized by the MEC follow the
classical secretory pathway and are targeted to the basolateral or the apical side of the MEC. E 5 endosome; rEb 5 recycling basal endosome;
rEa 5 recycling apical endosome; ER 5 endoplasmic reticulum; GA 5 Golgi apparatus; TGN 5 trans-Golgi network; SV 5 secretory vesicle. (d) Electron
micrograph of the apical region of a MEC from a lactating rabbit. Secretory vesicles containing casein micelles close to the apical membrane (empty
arrows). Tight junction (black arrows). L 5 lumen. (e) Electron micrograph of the basal region of a MEC from a lactating rabbit incubated in the presence of
cationized ferritin for 30 min at 48C. Cationized ferritin accumulated close to the basal membrane and was internalized in tubulo-vesicular endosomes
(arrow). A coated vesicle does not contain the marker (small arrow). IS 5 interstitial space. Bar 0.5 mm.
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Mammary gland secretion
Figure 2 Different endocytic internalization pathways. Clathrin-mediated endocytosis is involved in receptor-mediated internalization of different hormones
and biologically active molecules (transferrin, Immunoglobulin A, etc.). Pinocytic endocytosis relies on membranes microdomains and mediates the cellular
entry of dextran and GPI-AP (glycosylphospatidylinositol-anchored protein). Caveolin-dependent endocytosis takes place at some membrane microdomains
(caveolae) containing caveolin and promotes the internalization of the cholera toxin B subunit.
of the epithelium (Pitelka and Hamamoto, 1983). The general
molecular mechanisms underlying endocytosis are well
described in different cell types (Gundelfinger et al., 2003 and
references therein) (Figure 2). The basolateral membrane of
MECs from lactating animals offers a large surface for the
endocytosis of blood supplies. Besides convulated interdigitations of epithelial and myoepithelial surfaces, the basal
membrane contains clathrin-coated pits and vesicles as well as
non-coated vesicles. Caveolae are abundant in myoepithelial
cells and the capillary endothelium but very rare at the
basolateral membranes of MECs (Pitelka and Hamamoto,
1983; Hue-Beauvais et al., 2007; Figure 1). To understand how
the mammary epithelium regulates the internalization of milkconstituent precursors, it is necessary to characterize the
routes used by different blood supplies to enter the MECs.
Ions
The movement of sodium (Na1), potassium (K1) and
chloride (Cl2) across the basal membrane contributes to the
gradients between interstitial and intracellular fluids (Linzell
and Peaker, 1971; Shennan and Peaker, 2000). Based on
histochemical localization, the presence of Na1-K1 ATPases
on the basolateral membrane supports the scheme of a
functional Na1-K1 pump on the blood-facing side of the
mammary epithelium. A Na1-K1-2Cl2 co-transport system
has also been described. In mammary explants and cell
cultures, a long-term effect of PRL up-regulates K1 uptake
(Shennan and Peaker, 2000 and references therein). Moreover, in HC11 cells, PRL exerts an early effect (after 5 and
10 min of stimulation) on Cl2 transport (Selvaraj et al.,
2000) via phosphorylation of the Na1-K1-2Cl2 co-transporter.
Another example of a transport system localized on the
basolateral membrane of mammary cells is given by the
NA1/I2 symporter (Cho et al., 2000), the expression of
which is dependent on PRL and oxytocin.
During lactation, the mammary gland extracts large quantities of calcium (Ca21) from plasma (Neville and Peaker,
1979). The total calcium content of blood is about 3 mM.
Milk calcium is very highly concentrated (from approximately 8 mM in humans to about 100 mM in rabbits) and
is found associated with casein micelles (about 20%), free
ionized or non-ionized (about 32%) or complexed to inorganic anions such as phosphate and citrate (about 46%).
The mechanism for calcium entry into mammary cells may
be related to the presence of calcium channels in the
basolateral membrane. The intracellular compartmentalization of calcium is dependent on cytoplasmic binding proteins
(VanHouten and Wysolmerski, 2007).
Trace elements
Trace elements such as iron (Fe), copper (Cu) and zinc (Zn)
are essential nutrients in all mammalian tissues. Fe is
essential for the oxygen metabolism, mitochondrial function, normal growth and maturation of tissues. Cu is
necessary for cell metabolism as a cofactor of metabolic
enzymes, and Zn for both DNA and protein synthesis and
cell division (Kelleher and Lönnerdal, 2005; Lutsenko et al.,
2007). Although high fetal stores of trace elements are
mobilized during the early neonatal period, adequate supply
of trace elements from milk is crucial to ensure neonate
survival. At a given stage of lactation, trace element
concentrations remain remarkably stable, independently of
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Truchet and Ollivier-Bousquet
the mother’s diet (Lönnerdal, 2007 and references therein).
These stable levels are achieved by the mammary epithelium that tightly regulates both the uptake of elements from
the blood and their release into milk.
Fe concentrations in mouse milk are approximately three
times those found in serum (Zhang et al., 2000). Fe uptake
by the mammary cell mainly occurred by the transferrin–
transferrin receptor (Tf–TfR) pathway, as described in other
cell types. Plasmatic diferric Tf binds to the TfR at the basal
membrane of MEC and is internalized in clathrin-coated
vesicles (Ollivier-Bousquet, 1998 and references therein;
Zhang et al., 2000). Fe is released from Tf due to the acidic
environment of endosomes, Tf and TfR being recycled to the
plasma membrane. Fe is transferred to the cytoplasm by an
unknown transport mechanism where it is either bound to a
divalent ion transporter-1 (DMT1) or to a ferroportin, or
sequestered by ferritin for storage. The internalization of Fe
depends on the Tf–TfR complex, whereas, the quantity of Fe
internalized and secreted in milk is not correlated with TfR
expression, suggesting that receptor-dependent uptake is
not the only step regulating its concentration in milk
(Lönnerdal, 2007).
Cu intake is essential for the neonate and, despite high Cu
stores at birth, milk constitutes an important dietary supply.
Specialized transport mechanisms tightly regulate cellular Cu
levels in MECs (Lönnerdal, 2007 and references therein). A Cu
transporter-1 (Ctr1) is believed to form a membrane channel
by multimerization of several Ctr1 proteins. Such channels are
located in both plasma and intracellular membranes and are
likely involved in Cu import into the cell. The intracellular
trafficking of Cu is mediated by the metallochaperone Atox1
which interacts with the two Cu-ATPases: ATP7A and ATP7B.
ATP7B is localized in a perinuclear region associated with the
trans-Golgi network (TGN). ATP7B promotes the transport of
cytoplasmic Cu into this compartment, where it could bind to
ceruloplasmin, the major copper-carrying protein. ATP7A is a
cytosolic protein, which relocalized to the plasma membrane
during lactation. On the other hand, PRL stimulates the
transport of Ctr1 and ATP7A to the plasma membrane in
HC11 cells and may also stimulate copper transport (Kelleher
and Lönnerdal, 2006). Moreover, human ATP7A accumulates
at the basolateral side of MEC when over-expressed in the
mammary gland of transgenic mice. However, the concentrations of Cu in mammary cells and milk of these animals
are decreased, suggesting that the role of this transporter
may not be to carry Cu to the milk but rather to remove
excess Cu from mammary cells (Llanos et al., 2008).
Another example of the regulation of trace element
transport from the maternal circulation to milk is that of Zn
transport. In humans, 0.5–1 mg of Zn21/day is secreted in
milk. A Zn-specific transport protein (Zip3) has been characterized in the mammary gland (Kelleher and Lönnerdal,
2003). Based on the localization of Zip3 in HC11 cells after
in vitro differentiation and 2 h of PRL exposure, it has been
suggested that PRL may regulate the relocalization of Zip3
from the intracellular perinuclear region to the basal
membrane, thus facilitating Zn import into mammary cells.
Much is still unknown regarding the precise nature of
internalization compartments. However, taken together,
the results referred to above reveal that specific carriers
for trace elements, located in the basolateral region are
controlled by the hormonal status of MECs.
Glucose
Glucose is the obligate precursor for lactose synthesis in
MEC. In some species it is also used to synthesize lipids and
amino acids. Therefore the uptake of glucose and its
transport across the basolateral membrane are important
processes during milk synthesis. The glucose transporter
(GLUT1) is present at the basolateral membrane of rodent
MEC (Madon et al., 1990; Nemeth et al., 2000) and is the
predominant glucose transporter in the lactating bovine
mammary gland (Zhao and Keating, 2007). The expression
of genes encoding the glucose transporters is under the
control of lactogenic hormones in the mammary gland
during pregnancy and lactation (Shennan and Peaker, 2000
and references therein; Zhao and Keating, 2007). In addition, the compartmentalization of GLUT1 between the cell
surface and the intracellular site of lactose synthesis
has been described in CIT3, a mouse mammary cell line.
Following a short exposure to PRL (a few minutes to a
few hours), GLUT1 was targeted to a brefeldin-A sensitive
intracellular compartment via the endosomal pathway
(Haney, 2001; Riskin et al., 2008).
Thus, in addition to a long-term effect of lactogenic hormones on the expression of various transporters during
mammary gland differentiation, mention should also be made
of the early effects of PRL in highly differentiated mammary
cells. This hormone may control the localization of numerous
transporters between intracellular sites of synthesis and the
basolateral membrane via vesicular transport.
Hormones and growth factors
The basolateral membrane is the binding site of plasmaborne polypeptidic hormones and growth factors to their
receptors. For most polypeptidic hormones, internalization
of the hormone–receptor complex leads to sorting towards
the lysosomes, with recycling or degradation of the receptor
and ligand. In contrast, numerous hormones and growth
factors are carried from the plasma to the milk in MEC
during lactation. For example, after binding to its receptor,
PRL is internalized and transported in an intact and biologically active form to the milk (Ollivier-Bousquet, 1993). The
mechanisms governing the initial steps of internalization of
PRL in MECs have only been partly described. The involvement of clathrin-coated vesicles in PRL–PRL receptor
(PRLR) endocytosis has been shown in different cell lines
transfected with PRLR. However, PRL mainly binds to noncoated domains of the basal membrane in lactating MECs.
PRL is then targeted to endosomes, multivesicular bodies
and to a specific pathway that meets the exocytotic
pathway (Ollivier-Bousquet, 1998 and references therein;
Castino et al., 2008). The fate of PRLR is not known in MEC
secreting milk. In different cell types transfected with PRLR,
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Mammary gland secretion
the ubiquitination of PRLR may lead to its degradation in
the lysosomes (Varghese et al., 2008). In MECs from lactating animals incubated with various antibodies directed
against PRLR, the route followed by the complex PRLRantibody depends on the antibody used (Ollivier-Bousquet,
1998 and references therein).
Internalization by fluid-phase endocytosis or by
association with membranes
The albumin present in milk originates from plasma and is
carried to the milk by transcytosis. Whether albumin
transport across the mouse mammary epithelium is receptor-mediated or a function of fluid-phase endocytosis
remains unclear. In an interstitial space preparation of
mouse mammary gland, albumin and IgA (Immunoglobulin
A) were internalized via a clathrin-mediated endocytosis. In
endosomes, albumin colocalized with IgA, whereas Tf followed a different pathway (Monks and Neville, 2004).
The highly active membrane traffic of labeled basolateral
membrane is illustrated by the localization of some membrane markers. Concanavalin A, a classical mannose-binding
glycane, interacts with sugars on the cell membrane and is
subsequently internalized. Cationized ferritin binds to negative charges on the cell surface in mammary fragments
incubated in vitro. This marker is present on the basolateral
membrane and in tubulovesicular structures in the basal
region of cells as soon as 1 min (Figure 1), and in different
vesicles of the Golgi region and secretory pathway after 5 to
15 min. It should also be noted that an early effect of PRL
(1 min at 378C) is an accumulation of cationized ferritin in the
Golgi region (Ollivier-Bousquet, 1998; Castino et al., 2008).
Taken together, these examples show that in MEC from
lactating animals, endocytosis at the basolateral side is a
very strictly controlled process, which implies the sorting of
the different constituents and the hormonal regulation of
internalization.
Exocytosis
General features of exocytosis in mammary cells
Extensive overviews have summarized the major routes of
secretion across the mammary epithelium (Linzell and Peaker,
1971; Shennan and Peaker, 2000; Boisgard et al., 2001).
Transport of newly synthesized proteins proceeds in MECs
according to the general pathway described in exocrine
glands. Entry in the endoplasmic reticulum (ER) requires a
signal peptide. Once translocated into the lumen of the ER,
the polypeptide chains undergo various co-translational
modifications (formation of S–S bonds, N-glycosylation or
oligomerization). The proteins that are completely folded are
transported into and across the Golgi complex by a process
associating a vesicle shuttle mechanism and the maturation
of Golgi saccules. Then, proteins are carried to the TGN.
Enzymes, which reside in the Golgi saccules and in the TGN,
carry out functions including glycosylation, phosphorylation,
sulfation and proteolytic processings. The exact site where
casein micelle formation begins is not clear. Closely packed
casein micelles are clearly seen in the secretory vesicles
(Vilotte et al., 2003). The mechanism of lipid droplet growth
and translocation within the cell remain hypothetical. Droplets
are believed to form in the bilayer of the ER membrane, then
bud from the ER into the cytoplasm as droplets surrounded by
a phospholipids monolayer derived from the ER. Different
cytosolic proteins (adipophilin, TIP 47, etc.), known to be
related to membrane trafficking, have been detected in lipid
droplets and in the milk fat globule membrane. These proteins
may be involved in the growth and the translocation of lipid
droplets within the cell (Ollivier-Bousquet, 2002 and references therein). However, numerous questions remain
regarding the last step of secretion at the apical membrane.
The release of the milk constituents occurs at the apical
pole of MECs from lactating females, through two main
processes: (i) the envelopment of lipid droplets in the plasma
membrane followed by budding of the lipid globules secreted
in milk (Keenan, 2001); (ii) the exocytosis of secretory vesicles
content by fusion with the apical membrane (Figure 1).
General features of exocytosis are well described for the
release of neurotransmitters and may be used as a model for
the mammary cell (Figure 3). The secretory vesicles of the
MEC contain casein micelles, soluble proteins, lactose, ions,
minerals and numerous biologically active molecules (Vilotte
et al., 2003 and references therein). The morphological features of the association between secretory vesicles and the
plasma membrane of the MEC have been extensively
described (Franke et al., 1976). However, it is still not known
whether the secretory vesicles localized in the vicinity of the
apical membrane are docked and then readily releasable
under stimulation.
Directed movements in eukaryotic cells involve the
cytoskeleton. The disruption of microtubules, by colchicine
in vivo and in vitro, provokes an accumulation of secretory
vesicles in MECs and inhibits milk secretion (OllivierBousquet and Denamur, 1973; Patton, 1976b). Most
microtubules are found in the apical and medial cytoplasm
(Nickerson and Keenan, 1979), suggesting that they are
involved in the final steps of secretion. Moreover, a dense
web containing actin (Figure 1b) lines the apical side of the
mammary epithelium. Disruption of actin cytoskeleton by
cytochalasin B results in an increase of casein release
(Ollivier-Bousquet, 1979), thus demonstrating the barrier
role of this network.
Transcellular calcium transport in MEC is an important
process regarding both the transfer of calcium from mother
to neonate and its role as an intracellular second messenger
in many cellular processes and the exocrine function.
Calcium entry by channels, its storage in the endoplasmic
reticulum and its intracellular transport by specific calcium
transporters, have partly been described (VanHouten and
Wysolmerski, 2007). As for the role of calcium as a signaling molecule, previous and recent studies demonstrated
that PRL does not significantly modify the influx and efflux
of calcium in MECs (Ollivier-Bousquet, 1983; Anantamongkol et al., 2007). Activation of the calcium-sensing
receptor (CaR), a G-protein cell surface receptor located at
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Truchet and Ollivier-Bousquet
Figure 3 Coupling of exocytosis and endocytosis in the context of neurotransmitters release. Synaptic vesicles (SV) containing neurotransmitters are first
transported to the presynaptic active zone by the cytoskeleton elements (a). These vesicles then dock to the plasma membrane and are primed in an ATPdependent manner and become competent for fusion (b). Calcium (Ca21) increase (c) in response to a membrane depolarization induces the rapid fusion of
the vesicle with the plasma membrane that leads to the opening of the fusion pore and the release of the vesicle content (d). After exocytosis, membrane
retrieval occurs through clathrin-mediated endocytosis (e) that allows the recycling and the refilling of synaptic vesicles (f) but also eliminates the excess of
membrane created by the bulk of neurotransmitter secretion.
the basal side, increases Ca-ATPase activity in cultured
MECs. This activation implies the expression of PMCA2, an
ATPase located at the apical membrane (VanHouten et al.,
2007). Its activation also regulates the secretion of parathyroid hormone-related protein (PTHrP) by normal mammary cells (Mamillapalli et al., 2008 and references therein).
Thus the availability of calcium linked to activation of the
CaR may be coupled with a control of apical secretion (also
see section ‘Molecular mechanisms and regulation of exocytosis in MECs’). Taken together, these results suggest that
calcium concentration affects different types of signaling
through micro-localized modifications.
Hormonal control of exocytosis in MECs
Milk protein synthesis and secretion are dependent on the
action of lactogenic hormones (Ollivier-Bousquet, 1993). A
question arises as to whether lactogenic hormones play a role
in the specific mechanism of exocytosis. This seems to be the
case for oxytocin that, through its oxytocin receptors, acts
directly on MECs (Lollivier et al., 2006). Within one minute,
oxytocin was shown to induce an increase in the intracellular
transport of caseins to the lumen of acini. This effect was
accompanied by an increase in the number of secretory
vesicles containing casein micelles in the apical region and in
close contact with the apical membrane. We could then ask
whether the oxytocin play a role in the vesicular transport
and/or membrane fusion involved in exocytosis.
PRL exerts a secretagogue effect on MECs. The disappearance of secretory vesicles after bromocriptine treatment that markedly decreases the levels of plasma-borne
PRL, suggests a role of PRL in maintaining the complex
network of intracellular membranes (Ollivier-Bousquet,
1978; Akers et al., 1981). Interestingly, a very early effect of
PRL added in vitro to MECs of lactating rats and rabbits, is
to induce within a few minutes the release of caseins which
had been newly synthesized 45 min prior to addition of the
hormone (Lkhider et al., 2001). These results demonstrated
the rapid control, by PRL, of the last steps of apical transport and possibly the exocytosis of caseins. One important
actor in signaling of the secretagogue response to the
action of PRL is arachidonic acid, a polyunsaturated fatty
acid (PUFA) which is a component of membrane phospholipids, which is transiently released a few seconds after PRL
binds to its receptor. Arachidonic acid mimics the secretagogue effect of PRL, either directly or via its metabolites
(Ollivier-Bousquet, 1998).
It should be noted that heregulin b1, a combinatory
ligand for human epidermal growth factor receptors 3 and
4, has been proposed as a regulator of vesicles trafficking
in MEC (Vadlamudi et al., 2000). The physiological significance of these results obtained in cell culture remains to
be confirmed in vivo.
Molecular mechanisms and regulation of exocytosis
in MECs
The molecular machinery responsible for membrane fusion
has been particularly well described in the case of evoked
neurotransmitters release (Geppert and Südhof, 1998)
(Figure 4). Few data, however, are available concerning
the molecular control of exocytosis in MECs. Regulated
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Mammary gland secretion
Figure 4 Schematic representation of proteins involved in the release of neurotransmitters by synaptic vesicles. Guanine nucleotide-bound Rab3A binds to
vesicles that recruit rabphilin3A before docking on the presynaptic plasma membrane (active zone) through mechanisms involving GTP hydrolysis in a
calcium-dependent manner. Local increase of calcium concentration is sensed by synaptotagmin associated with the vesicular membrane. Once docked to
the target membrane, the vesicle is primed and the SNARE complex is formed. In the case of neurotransmitters release, the SNARE complex results of the
interaction of one v-SNARE (synaptobrevin or VAMP1/2) with t-SNAREs (syntaxin 1) and SNAP-25. Formation of the SNARE ternary complex is regulated by
cytosolic proteins such as Munc18. Polyunsaturated fatty acids, such as arachidonic acid, may also exert immediate effects on the fusion machinery by
direct interaction with SNARE proteins and with regulatory partners of the complex.
exocytosis essentially relies on the SNARE (Soluble NEthylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins that mediate specific fusion of transport vesicles
with target cellular membranes. In response to intracellular
calcium increase, the vesicular SNARE (v-SNARE) links cognate SNAREs located on the target membrane (t-SNAREs).
Thus, these proteins form a SNARE complex that promotes
the docking and the tethering of the vesicle to the membrane. The whole process of exocytosis is highly regulated by
numerous proteins working in close association with the
SNARE complex. In MECs, to date, only a v-SNARE (VAMP8/
endobrevin), also involved in the regulated exocytosis in
pancreatic, parotid and lacrimal acinar cells, has been
described (Wang et al., 2007). On the other hand, several
studies have shown that SNARE proteins are the targets of
arachidonic acid in different neuroendocrine cell types
(Darios and Davletov, 2006; Latham et al., 2007). An
intriguing possibility is that the SNARE proteins may be the
targets of arachidonic acid produced in response to PRL
thus providing a link between signal transduction, secretagogue effect and exocytosis in MECs (also see section
‘Coupling between endocytosis and exocytosis in the
MECs’). The expression of other proteins involved in the
docking and fusion of vesicles has also been described
in normal and cancerous mammary cells. For example, the
expression of the small GTPase Rab3A is increased during
pregnancy and post-weaning period. Heregulin b1 promotes the accumulation of Rab3A-associated vesicles in the
mammary cell lines MCF-7 and HC11, suggesting its role in
vesicle trafficking. These cells also express a number of
proteins involved in exocytosis (rabphilin 3A, Sec8, syntaxin 4,
SNAP 25, rabaptin and Doc2) (Vadlamudi et al., 2000). A
possible role for Annexin II in apical exocytosis has also
been suggested in MECs (Handel et al., 1991). Future work,
however, is needed to elucidate the molecular mechanisms
and regulations involved in caseins secretion during lactation. Differences may also exist in the regulation of secretion between species such as mouse, rabbit and cow, due to
differences in frequency of suckling. Regulations leading to
differences in the quality and quantity of milk, in relation
to the stage of lactation in dairy cows, may also be relevant
to these mechanisms.
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Truchet and Ollivier-Bousquet
Coupling between endocytosis and exocytosis in the
MECs. Questions and perspectives
In neurons and neuroendocrine cells, a compensatory process
has been described, corresponding to the tightly balanced
delivery of membranes by exocytosis and re-internalization by
endocytosis (Gundelfinger et al., 2003 and references therein).
In b-cells secreting insulin, a direct coupling between insulin
granule exocytosis and subsequent endocytosis occurs locally
at the site of the fusion pore (Min et al., 2007).
As reported above, the processes of endocytosis and
exocytosis in MECs are complex and a coupling of the two,
through a compensatory process, remains elusive.
Two examples of temporally coordinated events in MECs
raise more precise questions concerning a cross-connection
between endocytosis and exocytosis. The first example is
the regulation of these two events occurring on the same
side of the mammary epithelium. A release of different milk
constituents occurs at the basolateral side of the mammary
epithelium (Devinoy et al., 1995). Interestingly, endosomelysosome like vesicles localized at the basal side of MECs
release cathepsin D (CD) into the extracellular medium.
After binding to its receptor on the same side, PRL increases
the release of CD by these vesicles (Castino et al., 2008).
Questions then arise as to whether endocytosis of the
hormone may be compensated by a supply of membrane
due to the exocytosis of CD containing vesicles at the same
side of the mammary epithelium.
The second example is the different intracellular movements
described for molecules associated with the secretagogue effect
of PRL. After binding to its receptor, PRL is internalized but there
are no data available regarding the first steps of endocytosis
process. By contrast, the regulation of PRL transport in endosomes is well described and has been shown to be impaired
when the composition of membrane phospholipids is modified.
A marked decrease in n-6 family PUFAs induces an accumulation of PRL in early endosomes, whereas the addition of arachidonic acid restores PRL transport to the secretory pathway.
Furthermore, the inhibition of the intracellular transport of PRL
also leads to the disappearance of its secretagogue effect. In
parallel, it has been described that various phospholipases
A2 (PLA2) are involved in mediating the secretagogue effect of
PRL (Ollivier-Bousquet, 1998 and references therein; Lkhider
et al., 2001). It should be noted that the intracellular site where
PLA2 may be active is determinant for arachidonic acid release
and the subsequent PRL secretagogue effect (Ollivier-Bousquet
et al., 1991). Moreover, an early effect of PRL added to
mammospheres from a lactating rabbit, is associated with a
change, in a few seconds, in the localization of PLA2 (OllivierBousquet, unpublished results). Overall, these data strongly
suggest that the early effects of PRL linked to its internalization
have repercussions on its biological response.
These data raise questions regarding the precise links
between these effects and the structural components of MECs.
New perspectives have arisen with recent data on the role of
PUFAs in regulating the SNARE machinery in neuronal plasma
membranes (also see section ‘Molecular mechanisms and
regulation in MECs’). Arachidonic acid is involved in the
assembly of the SNARE complex in the presence of the cytoplasmic protein Munc18, intervening in functioning of the
complex (Rickman and Davietov, 2005; Latham et al., 2007).
Moreover, arachidonic acid has also been shown to be able to
directly interact with some SNARE proteins, thus promoting
the formation of SNARE complex (Latham et al., 2007). The
new concept of fatty acids ‘adding grease to exocytosis’
(Rohrbough and Broadie, 2005; Verhage, 2005) will now
make it possible to envisage a role for arachidonic acid in the
regulation of the exocytotic mechanism in MECs. Of interest is
the fact that MECs offer a model for the hormonal regulation
of PLA2-mediated phospholipid hydrolysis, leading to the
release of micromolar concentrations of arachidonic acid at
local sites, where the different elements of the SNARE
complex reside. A future challenge will be to understand
the physiological relevance of these molecular interactions
within the secretory machinery.
Conclusion
One key question that remains to be answered concerns the
extent to which endocytic proteins play roles that are
interconnected with the cellular processes leading to
secretion in MECs. At present, the experimental data
reported in this review underline the relationships between
these two processes, either in the field of receptor-mediated
signaling, or in that of membrane trafficking. Recent data
have shown that the strength of the signaling response
induced by transcription factors carried by the endocytic
pathway is related to endocytic membrane trafficking. Thus
interesting questions regarding endosomal proteins as
trafficking regulators are now clearly posed (McShane and
Zerial, 2008 and references therein).
Why are these questions now fundamental not only for
biologists but also in terms of dairy production? Research
on milk and its safety as a source of substances that are
beneficial to growth and health in children and adults is a
major concern in developed countries. On the other hand,
the production of dairy products as basic nutrients is a
priority in the fight against under-nourishment. In both
cases, research on milk and its safety may be useful in
optimization of sustainable dairy production. The control of
milk production requires an understanding of the molecules
involved in the cellular machinery (sorting of different
constituents in endosomes leading to different types of
intracellular signaling?), compartmentalization (regulation
of pathways leading to the synthesis of milk components?)
and membranes trafficking. It will be useful to explore
these points using model systems. Moreover, it is crucial to
validate the conclusions under physiological conditions, as a
function of environmental parameters.
Acknowledgements
We are grateful to Dr R. Boisgard and S. Delpal for the
micrographs, to M.-E. Marmillod for secretariat assistance and
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https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S1751731109990589
Mammary gland secretion
to B. Nicolas and M. Weber for their contribution to prepare
the digital files for the figures.
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