J Mammary Gland Biol Neoplasia DOI 10.1007/s10911-013-9309-1

The Role of Tight Junctions in Mammary Gland Function Kerst Stelwagen & Kuljeet Singh

Received: 11 August 2013 / Accepted: 29 October 2013 # Springer Science+Business Media New York 2013

Abstract Tight junctions (TJ) are cellular structures that facilitate cell-cell communication and are important in maintaining the three-dimensional structure of epithelia. It is only during the last two decades that the molecular make-up of TJ is becoming unravelled, with two major transmembranespanning structural protein families, called occludin and claudins, being the true constituents of the TJ. These TJ proteins are linked via specific scaffolding proteins to the cell’s cytoskeleton. In the mammary gland TJ between adjacent secretory epithelial cells are formed during lactogenesis and are instrumental in establishing and maintaining milk synthesis and secretion, whereas TJ integrity is compromised during mammary involution and also as result of mastitis and periods of mammary inflamation (including mastitis). They prevent the paracellular transport of ions and small molecules between the blood and milk compartments. Formation of intact TJ at the start of lactation is important for the establishment of the lactation. Conversely, loss of TJ integrity has been linked to reduced milk secretion and mammary function and increased paracellular transport of blood components into the milk and vice versa. In addition to acting as a paracellular barrier, the TJ is increasingly linked to playing an active role in intracellular signalling. This review focusses on the role of TJ in mammary function of the normal, non-malignant mam-

K. Stelwagen (*) SciLactis Ltd, Waikato Innovation Park, Hamilton 3240, New Zealand e-mail: [email protected] K. Singh AgResearch Ltd, Ruakura Research Centre, Hamilton 3240, New Zealand

mary gland, predominantly in ruminants, the major dairy producing species. Keywords Bovine . Mammary gland . Tight junction . Milk production Abbreviations ECM EGF IGF LPS LTA MEC ODM TER TGF TJ

Extracellular matrix Epidermal growth factor Insulin-like growth factor Lipopolysaccharide Lipoteichoic acid Mammary epithelial cell(s) Once-daily milking Transepithelial electrical resistance Transforming growth factor Tight junction(s)

Introduction The tight junction (TJ), or zonula occludens, is an important member of the junctional complex, which also consists of desmosomes (macula adherens) and intermediate junctions (zonula adherens). The junctional complex plays a key role in cell-cell interaction between adjacent epithelial or endothelial cells in all vertebrates. The TJ is a cytoskeletal- and membrane-linked structure that surrounds both epithelia and endothelial cells toward the apical proximity of the cell. The basic function of the TJ is two-fold, as it acts both as a barrier and a fence. The TJ prevents the vectoral paracellular transport

J Mammary Gland Biol Neoplasia

of ions and small molecules across the epithelial or endothelial cell bed (i.e. barrier function), and in addition, the TJ separates the plasma membrane into apical and basolateral domains of distinct protein and lipid composition (i.e. fence function) [1, 2]. Whilst the existence of TJ has been known for some time, their molecular composition was an enigma until the mid-1990s and continues to be unravelled to date. The TJ is made up of two major transmembrane spanning structural proteins, called occludin and claudin [3, 4]. These are linked intracellularly to the actin cytoskeleton via scaffolding proteins, such as ZO-1 [5]. The mammary epithelium is unique in that it is a secretory epithelium that synthesizes and secretes milk components apically into an alveolar lumen. A prerequisite for this secretory process is the existence and maintenance of a small transepithelial potential difference, in the order of 30 to 35 mV, between the basolateral or “blood” side and the apical or “milk” side of the mammary epithelial cell (MEC) [6, 7]. The TJ is instrumental in maintaining this polarized state across the mammary epithelium. Loss of TJ integrity during established lactation, experimentally induced or caused by mammary inflammation, has been linked to reduced milk secretion and mammary function and increased paracellular transport of blood components into the milk and vice versa [8]. Increasingly, the importance of TJ integrity is recognized in the prevention and progression of mammary cancer, as recently reviewed by Brennan et al. [9]. However, the current review addresses the role that TJ play in normal, non-malignant, mammary tissue and function, in particular focusing on the main dairy species, cows and goats. Where relevant, information obtained in other species, predominantly from rodents, will be referred to.

TJ Structure and Composition Linzell and Peaker [10] studied the movement of small solutes and ions in milk and colostrum around parturition in goats and suggested a selective paracellular route of transport across the mammary epithelium. Using the freeze-fracture technique on tissue sections from the lactating mouse mammary gland, Pitelka et al. [11] showed nicely that mammary TJ consisted of irregular strands (“ridges”) that run parallel to the alveolar luminal surface and suggested these may help regulate the movement of milk precursors and solutes and/or cell shape. It was these studies in the early 1970s that high-lighted the presence of mammary TJ and suggested a role for TJ in mammary function. Since these early studies it took at least another two decades until the discovery of the true and distinct tight junction proteins, occludin and claudin [3, 4]. Both occludin and claudin are transmembrane proteins, with each having two extracellular loops and one intracellular loop. Whilst both play

a role in maintaining the TJ barrier function, occludin may also have a protective function and disruption of occludin may play a key role in initiating apoptosis and cell death signalling [12, 13], whereas claudin’s role seems to be predominantly one of maintaining TJ barrier function [14]. Claudins comprises a large family of proteins; to date more than 25 members have been identified, however their expression is celland tissue-specific and different claudins selectively regulate the paracellular flux of specific ions [14, 15]. Occludin and claudins are also integral parts of the mammary TJ, but not all members of the claudin family appear to be expressed in the mammary gland [16]. In addition, the expression of different claudins may depend on the physiological state of the mammary gland. In mice claudin-3 appears to play a role in maintaining TJ integrity during lactation, whereas the expression of claudin-4 was almost undetectable during lactation [17]. Further, claudin-1 and 3 were upregulated 20 h after weaning and expression of claudin-2 and 16 were decreased at that time [16]. Initially, TJ were considered to be static cell structures that simply provided a physical barrier to the movement of small solutes and ions between adjacent cells. However, it is now clear that TJ not only serve as a barrier but are actively involved in cell function through various cell signalling pathways [18]. It has been shown that the TJ is linked to the actin cytoskeleton of the cell via TJassociated or scaffolding proteins [5, 19]. This means that the TJ is also indirectly linked to the basolaterally located extracellular matrix (ECM) domain of the cell; both the cell-ECM and the cell-cell (i.e. TJ) interactions are crucial to maintaining the three-dimensional cell structure, and as such, to cell function. Furthermore, there are a number of cell signalling proteins that can be recruited to the TJ and some may localize not only at the TJ but also at the cell nucleus [18]. One of these signalling components, the Rho effector protein PKN1, has been shown to play a role in TJ sealing, using a transgenic mouse model [20]. Moreover, disruption of occludin may play a key role in initiating mammary apoptosis and cell death signalling [12, 13]. The exact role of TJ, however, in cell signalling remains to be elucidated, but it is clear that TJ play an active role in cell function.

Endocrine Control of Mammary TJ Most biological processes within the mammalian body are subject to some form of, often complex, endocrine control. The mammary gland is no exception and mammary development, involution and functioning involve many systemic and locally operating and interacting endocrine pathways [21]. It is therefore not surprising that endocrine factors affect TJ functionality. Although by no means is our understanding of how hormones and growth factors affect TJ complete.

J Mammary Gland Biol Neoplasia

Glucocorticoids, prolactin and steroids are important hormones for mammary gland development and functioning. There is solid evidence from both in vitro [22–24] and in vivo experiments with goats and cows [25, 26] that glucocorticoids are required for the formation and maintenance of mammary TJ. How glucocorticoids stimulate TJ formation and maintenance is not fully understood, but suppression of parts of the Rho signalling pathway may be part of the role of glucocorticoids in stimulating TJ formation and maintenance [20, 27]. Studies in goats, receiving exogenous prolactin [28] or in which the release of pituitary prolactin was blocked using bromocriptine [29] demonstrated changes in milk composition that were indicative of loss of TJ integrity. In cows, treated with the prolactin inhibitor quinagolide, the effect of prolactin on mammary TJ appeared to be more variable, with one indicator showing suggesting a positive effect, whereas others suggested no effect of prolactin on TJ [30]. In an in vitro study the addition of prolactin to a murine mammary cell line, stimulated TJ formation, but the effect was much stronger in combination with glucocorticoids, indicating a synergistic effect between prolactin and glucocorticoids [22]. The effect of prolactin and/or glucocorticoid appears to be mediated through an up-regulation of the TJ protein occludin and the scaffolding protein ZO-1 [22]. Progesterone appears to play an important role in preventing mammary TJ formation. Mammary TJ are formed around parturition, as a prerequisite to milk secretion, and it appears that the normal immediate prepartum drop in the systemic progesterone level triggers the formation of TJ [31]. In a series of experiments involving ovariectomy and the administration of exogenous progesterone and/or of a progesterone antagonist these authors clearly demonstrated a role, albeit a negative one, for progesterone in the formation of mammary TJ. Unlike progesterone, estrogen, another steroid hormone that plays a role in mammogenesis, does not appear to have an effect on mammary TJ [32]. Oxytocin is the hormone that is necessary to induce milk ejection. It induces contraction of the myoepithelial cells surrounding mammary alveoli which leads to the milk being “squeezed” out of the alveoli and into the cisternal lumen. Whilst there is no evidence of a direct action of oxytocin on TJ, the TJ integrity may be compromised following administration of exogenous oxytocin [33, 34]; although it must be noted that high, non-physiological, doses of oxytocin were administered in these experiments. Given that the permeability is associated with higher than physiological doses, it is likely the increased permeability is due to a disruption of the cell-cell contact as a result of the mechanical forces caused by the sudden alveolar contraction. Growth factors, such as transforming growth factor (TGF), insulin-like growth factors (IGF) and epidermal growth factor (EGF), are of critical importance to proper mammogenesis and mammary functioning [21], however, much less is known

about their role in the formation and maintenance of TJ in the normal, non-malignant mammary gland. Using a murine cell line, Woo and co-workers [35] showed that TGF-β inhibited glucocorticoid-induced formation of TJ, by down regulating ZO-1, a protein that links TJ proteins to the cell’s cytoskeleton. Similarly, there is limited evidence that IGF-1, when constitutively expressed in a transfected bovine MEC cell line, induced TJ permeability, whereas no such effect was seen in the non-transfected parent cells that exhibited sealed TJ [36]. No data are available on the possible effect of EGF on mammary TJ. Given that growth factors, as their name implies, play a role in the growing and developing mammary gland [21], it is perhaps not surprising that they have an inhibitory or no effect on mammary TJ, which are the hallmark of a differentiated and secretory mammary state. Pitelka and co-workers [37] demonstrated that maintaining the extracellular level of calcium is necessary to maintain mammary TJ integrity. This would implicate parathyroid hormone-related peptide (PTHrP), which is involved in, amongst other roles, cellular calcium transport and homeostasis [38], as an ideal candidate to regulate mammary TJ [8]. Indeed, it was shown in a murine cell line that PTHrP stimulated the formation of mammary TJ, but only under conditions where extracellular calcium was limiting [39]. It appears that when there is insufficient calcium in the extracellular environment, initiating loss of TJ integrity, PTHrP increases calciumchannel activity in the apical membrane in an attempt to maintain intracellular calcium and ultimately resulting in upregulation of the TJ protein occludin [39]. Consistent with these in vitro results, in goats, milked unilaterally once- or four times a day, PTHrP production and secretion into milk were reduced in the once-daily milked (ODM) glands [40] and it is well known that ODM reduces milk secretion and is associated with a loss of TJ integrity [8]. The neurotransmitter serotonin is produced within MEC and has recently been implicated in regulating milk secretion [41]. It appears to conduct its regulatory role, at least in part, by inducing loss of mammary TJ integrity, as demonstrated both in vitro and in vivo [42, 43]. Interestingly preliminary evidence indicates that serotonin up-regulates gene expression of PTHrP [41]. This seems contradictory to the fact that PTHrP stimulates TJ formation, albeit when extracellular calcium is limiting [39]. Therefore, it appears that the effect of serotonin on PTHrP is one of general regulation of calcium homeostasis and that the adverse TJ effect of serotonin is mediated through a different mechanism.

TJ and Mammary Function Following development, the mammary gland undergoes successive phases of milk secretion (i.e. galactopoiesis), involution and regeneration, which are repeated each time the female

J Mammary Gland Biol Neoplasia

produces offspring. During this lactation cycle, mammary TJ play an important role but also continuously undergo changes, depending on the physiological state of the mammary gland [44]. Lactogenesis Lactogenesis is the period prior to parturition, when the mammary gland changes from a developing, non-secretory state to a secretory state. As early as 10 weeks before parturition, changes in the intramammary fluid can be detected [45]. Although milk components are likely to be present in mammary fluid during this time, this fluid is a mixture of milk and blood-derived components and cannot be classified as milk; this period is often referred to as lactogenesis stage I. In contrast, stage II lactogenesis comprises the immediate prepartum (i.e., -3 to 0 days) period, and is characterized by the onset of copious milk secretion. Tight junctions are leaky during stage I lactogenesis, but become tight at parturition during stage II lactogenesis [46]. As such, they are instrumental in the mammary epithelium becoming polarized [6, 7, 47] and for the onset of colostrogenesis [10]. Colostrum is the “first milk”, which has quite a distinct composition from normal milk, with colostrum being particularly rich in immune factors, especially immunoglobulins [48, 49]. Whilst some of these immune factors, may be synthesized by the mammary gland itself, most of them are derived from the blood and either enter via a transcellular route or a paracellular route [50, 51]. Thus mammary TJ play an important role in establishing and maintaining colostrum composition. Interestingly, due to the lack of in utero transfer of immune factors, the ruminant neonate relies exclusively on passive immunity during its initial lifespan, and it is the delay in intestinal TJ formation that allows for the transient uptake of the large intact immunoglobulin molecules and other immune factors across the gut wall during the first 48 h of life [52]. The rapid sealing of mammary TJ at parturition suggests a tightly controlled series of events. Nguyen et al. [31], using the mouse as a model, showed that the parturition-induced fall in progesterone was associated with a rapid closure of mammary TJ. Moreover, these authors showed in the same study that injection of progesterone in ovariectomized mice resulted in delayed TJ closure, whereas the use of a progesterone antagonist in late pregnant mice resulted in reduced TJ permeability. However, despite this clear systemic level of regulation by progesterone, it would appear that ultimately TJ formation at the onset of lactation is locally regulated within the mammary gland. Because when milking was initiated unilaterally (i.e. one udder half was milked, whereas the opposite half was not yet milked) during stage II lactogenesis at 9 and up to 19 days prepartum, the prematurely milked gland started to produce normal milk and of a composition

that was indicative of intact mammary TJ, in both goats [10] and cows [53]; yet, progesterone levels would have still been elevated as the animals were still pregnant at that point in time. The detailed regulation of mammary TJ formation at the establishment of lactation remains to be elucidated, but it is clear that intact mammary TJ are required for the initiation of full colostrum and subsequent milk production. Established Lactation Not only are intact mammary TJ required for the onset of lactation, maintenance of TJ integrity is also necessary to maintain milk synthesis and secretion during established lactation. Direct evidence to support this notion stems from experiments where mammary TJ were disrupted in lactating animals. Such an in vivo approach is made possible by the fact that maintenance of the extra-cellular calcium level is necessary to maintain TJ integrity [37]. Intramammary administration of the calcium chelator EGTA caused an immediate increase in TJ permeability and a resulting drop in milk secretion in lactating goats [47, 54]. This effect occurred only in the EGTA-treated gland, with milk secretion remaining unchanged in the contralateral gland receiving an isosmotic sucrose solution without EGTA [54]. In both studies it took about two days for TJ to reseal and milk production to return to pre-treatment levels. Changes in mammary TJ integrity can also be observed as milk accumulates in the mammary gland. Milk secretion is a continuous process and during normal lactation milk is periodically removed from the mammary gland through suckling or milking. However, if milk is not removed and is allowed to accumulate within the gland, mammary TJ in both cows and goats become leaky after only 18 to 21 h of milk accumulation [7, 55]. Moreover, these changes in TJ integrity are positively correlated with declines in mammary blood flow and milk synthesis and occur before the capacity of the gland to hold milk is exceeded and are reversible [7]. It is therefore important emphasize that these early changes in TJ permeability are not to be confused with those associated with mammary involution as a result of cessation of milking as these occur much later when mammary capacity has been reached and are associated with shut-down of lactation [56, 57]. These early changes in TJ integrity appear to be related to the ability to store milk in the gland’s cisternal compartment. It is hypothesized that once, at approximately 18 h, the cisternal compartment of the gland is filled and free movement of milk from the alveolar to the cisternal compartment is no longer possible, continuing milk secretion results in loss of TJ permeability in the alveolar compartment [8, 58]. Experiments with cows where the cisternal compartment was either “enlarged” or “reduced”, causing the time at which TJ become leaky to be delayed or shortened (Fig. 1) support this hypothesis. This has practical implications for milking management. Cows are

J Mammary Gland Biol Neoplasia

Cessation of Lactation Cessation of milk removal from the mammary gland through either ceasing suckling or milking leads to milk accumulation and mammary engorgement, resulting in a rapid decrease in milk synthesis and secretion and ultimately in mammary involution and apoptosis [57, 62]. In both rodents and cows it has been shown that cessation of lactation down-regulates cell survival signalling through β1-integrin and focal adhesion kinase at the MEC-ECM interface [57, 63]. Similarly, cell-cell communication also appears to become compromised during mammary involution as paracellular permeability increases [62]. In both cows and rats this appears to be associated with a decrease in the protein expression of TJ proteins occludin and ZO-1 and an initial increase and subsequent decrease in expression of claudin-1 [64]. Markov et al. [16] also showed that 20 h following cessation of milk removal protein expression of claudin-1 and -3 were upregulated in the rat mammary gland. Given the role of claudin in maintaining TJ barrier function [14] the initial increase in claudin expression may signify an initial TJ-rescue attempt, but as the process of mammary involution continues, eventually the expression becomes down-regulated. Intriguingly, in the same experiment the expression of claudin-2 and -16 were shown to be down-regulated from the start [16] demonstrating again the differential roles of the various members of the claudin family [14, 15]. The down-regulation of occludin during mammary involution appears to be consistent with its purported role in the initiation of mammary apoptosis and cell death [12, 13]. Further, down-regulation of the cytoskeletal-linking protein ZO-1 suggests a disruption of the communication of TJ with the rest of the cell. Although changes in TJ expression appear to be an early event during mammary involution, it is unclear if these changes in TJ protein expression are causal to the onset of mammary involution.

Plasma Lactose (µM)

a

200 Control Drained 150

100 Cistern drained 50

0

0

2

4

6

8

10

12

14

16

18

20

22

24

16

18

20

22

24

Time (h)

b Plasma Lactose (µM)

normally milked twice daily at regular intervals, although ODM or thrice-daily milking is not uncommon. The effects of milking frequency on milk yield are well-established, with, compared to twice-daily milking, ODM resulting in a decrease in milk yield and thrice-daily milking increasing milk yield [59, 60]. Once-daily milking is characterized by a rapid loss of TJ integrity during the first 24 h, after which the mammary gland appears to “reset” itself to a state of lower milk production, associated with a reduction in TJ permeability [55]. In contrast, thrice-daily milking appears to result in tighter TJ [61]. These changes in TJ permeability may, at least partly, explain the changes in milk yield associated with different milking frequencies. Interestingly, the approximate 15 to 30 % decrease in milk yield as a result of EGTA-induced disruption of mammary TJ [47, 54] is of a similar order as that observed with yield losses associated with ODM [59].

1000 Control Infused

800

600

400

Infusion

200

0

0

2

4

6

8

10

12

14

Time (h)

Fig. 1 Effect of cisternal filling on TJ permeability in the bovine mammary gland. When capacity of the mammary cistern is “enlarged”, though drainage of milk from the mammary cistern (a) or “reduced”, by infusing an isosmotic milk-like fluid into the mammary cistern (b), TJ permeability is delayed (a) or happens much sooner (b). In experiment A, lactating cows (n =5; cross-over design) were subjected to 24-h milk accumulation or the same, but with drainage of only cisternal milk through previously inserted teat cannulas. Cows received adrenalin (i.v. 3 ml of 1 mg/ml) prior to drainage to prevent alveolar contraction. In experiment B, lactating cows (n =6; cross-over design) received an intramammary infusion of the equivalent volume of five hours-worth of milk secretion in each gland, of an isosmotic lactose/sucrose solution (300 mOsm; pH 6.7). Jugular vein blood samples were taken hourly via an indwelling catheter and plasma lactose [55] was analysed as a marker of TJ permeability [Stelwagen et al. unpublished data]

Milk Factors and TJ It is clear that formation and maintenance of mammary TJ is important to initiate and maintain milk synthesis and secretion. However, once the lactation process is established it appears that the mammary gland may secrete components in milk that in turn help to maintain mammary TJ. A fraction derived from hyper-immune milk [65] and also normal milk [Unpublished] exhibited anti-inflammatory activity and consistent with such activity stimulated both the formation and maintenance of TJ in mammary and kidney cells. Moreover, Silanikove and colleagues have demonstrated the presence of

J Mammary Gland Biol Neoplasia

a milk peptide, derived from the milk protein β-casein, that disrupts mammary TJ and as a result rapidly decreases milk secretion in goats and cows [66, 67]. Further, serotonin, produced by MEC, is present in milk and has been shown to compromise mammary TJ integrity in vitro and in vivo [42, 43]. In fact, the presence of these milk-borne factors has been implicated in the regulation of milk secretion [41]. Not only do such milk-derived factors affect mammary TJ, colostrum and milk also appear to contain factors that affect intestinal permeability. Bovine colostrum prevented intestinal permeability following an indomethacin (i.e., nonsteroidal anti-inflammatory drug) or heat challenge in humans and rats [68, 69] and goat milk was even more effective with heat stress [69]. In contrast, in humans subjected to endurance exercise both bovine whey powder and colostrum increased intestinal permeability [70]. Overall, however bovine whey and colostrum are associated with an improved intestinal barrier function [71]. Although it must be cautioned that under these in vivo conditions it is not possible to ascertain if the effect was solely due to changes in TJ permeability or perhaps due to a combination of changes in TJ permeability and cell loss. The positive effects on intestinal permeability have been attributed to TGF-β, present in the milk and colostrum powder, up regulating the expression of claudins [71, 72].

Mastitis and TJ Mastitis is a commonly occurring and usually transient inflammation of the normal, non-malignant mammary gland and is predominantly caused by a bacterial infection. The mammary gland has a highly developed innate immune system, with both MEC and macrophages having the ability to sense pathogenic bacteria and subsequently mobilize an extensive host-immune response, initially involving the innate immune system and later the acquired immune system [50, 51]. Although some of the antimicrobial factors are MEC-derived and others may enter the alveolar lumen via a transcellular route, mammary TJ play an important role in the immune response by allowing various blood-derived cytokines and neutrophils to enter the alveolar lumen [50]. Initially it was thought that MEC were sacrificed in order to allow the massive influx of neutrophils during mastitis, however it is now evident that mammary TJ facilitate the diapedesis of neutrophils into the alveolar lumen [73]. Gram- positive and -negative mastitis pathogens give rise to endotoxins, respectively lipoteichoic acid (LTA) and lipopolysaccharide (LPS), which are part of the bacterial surface [74]. Both LTA and LPS are pro-inflammatory and help stimulate the immune response in the mammary gland, partly through increasing TJ permeability and allowing blood-borne immune factors to enter the alveolar lumen [75]. However, it appears that LTA and LPS affect TJ permeability to a different

extent, with LPS inducing a more efficient transfer of immune factors into milk compared to LTA [76]. How these changes in mammary TJ integrity are mediated at the molecular level remains to be elucidated, but a recent study in mice showed that the protein expression of different claudins was differentially up- or down-regulated in response to an LPS challenge [17]. Claudin-7 was up-regulated 3 h following LPS administration, whereas claudin-1 and -4 were significantly upregulated 6 h post-challenge. In contrast claudin-3 was down-regulated at 6 h postchallenge. Moreover it was shown that Toll-receptor-4, which can bind LPS, is located at the apical surface of MEC and it was suggested that LPS binding activates the NFkβ pathway and that this in turn disrupts mammary TJ through localization and/or up- or downregulation of claudins [17].

Conclusions Mammary TJ play a key role in maintaining a small potential difference across the mammary epithelium and as such are instrumental in establishing and maintaining milk synthesis and secretion. In addition, they play a role in the transport of certain immune factors into colostrum and milk. From what initially were assumed to be static structures, simply preventing paracellular transport of ions and small molecules across the epithelial cell bed, TJ are increasingly implicated in normal cell functioning through active involvement in intracellular signalling. At present little is known about the signalling roles of mammary TJ and future research will be aimed at elucidating more precisely how and to what extend mammary TJ play a role in intramammary signalling events underlying milk synthesis and secretion.

References 1. Schneeberger EE, Lynch RD. Structure, function, and regulation of cellular tight junctions. Am J Physiol. 1992;262:L647–61. 2. Stevenson BR, Keon BH. The tight junction: morphology to molecules. Annu Rev Cell Dev Biol. 1998;14:89–109. 3. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, et al. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123:1777–88. 4. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and 2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141:1539–50. 5. Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem. 1998;273:29745–53. 6. Berga SE. Electrical potentials and cell-to-cell dye movement in mouse mammary gland during lactation. Am J Physiol Cell Physiol. 1984;247:C20–5. 7. Stelwagen K, Davis SR, Farr VC, Prosser CG, Sherlock RA. Epithelial cell tight junction integrity and mammary blood flow

J Mammary Gland Biol Neoplasia

8. 9.

10.

11.

12.

13.

14. 15.

16.

17.

18. 19.

20.

21.

22.

23.

24.

25. 26.

during an extended milking interval in goats. J Dairy Sci. 1994;77: 426–32. Stelwagen K. Effect of milking frequency on mammary functioning and shape of the lactation curve. J Dairy Sci. 2001;84:E204–11. Brennan K, Offiah G, McSherry EA, Hopkins AM. Tight junctions: a barrier to the initiation and progression of breast cancer? J. Biomed Biotechnol. 2010;ID 460607 - doi:10.1155/2010/460607. Linzell JL, Peaker M. Changes in colostrum composition and in the permeability of the mammary epithelium at about the time of parturition in the goat. J Physiol. 1974;243:129–51. Pitelka DR, Hamamoto ST, Duafala JG, Nemanic K. Cell contacts in the mouse mammary gland. 1. Normal gland in postnatal development and the secretory cycle. J Cell Biol. 1973;56:797–818. Beeman NE, Baumgartner HK, Webb PG, Schack JB, Neville MC. Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance. BMC Cell Biol. 2009;10:85. Beeman N, Webb PG, Baumgartner HK. Occludin is required for apoptosis when claudin-claudin interactions are disrupted. Cell Death Dis. 2012;3:e273. Van Itallie CM, Anderson JM. The role of claudins in determining paracellular charge selectivity. Proc Am Thorac Soc. 2004;1:38–41. Schneeberger EE. Claudins from ion-selective channels in the paracellular pathway. Focus on “Claundin extracellular domains determine paracellular charge selectively and resistance but not tight junction fibril architecture”. Am J Physiol Cell Physiol. 2003;284: 1331–3. Markov AG, Kruglova NM, Fomina YA, Fromm M, Amasheh S. Altered expression of tight junction proteins in mammary epithelium after discontinued suckling in mice. Pflugers Arch - Eur J Physiol. 2012;463:391–8. Kobayashi K, Oyama S, Numata A, Rahman MM, Kumura H. Lipopolysaccharide disrupts the milk-blood barrier by modulating claudins in mammary alveolar tight junctions. PloS One. 2013;8: e62187. Matter K, Balda MS. Signalling to and from tight junctions. Nat Rev. 2003;4:225–36. Gonzalez-Mariscal L, Bautista P, Lechuga S, Quiros M. ZO-2, a tight junction scaffold protein involved in the regulation of cell proliferation and apoptosis. Ann NY Acad Sci. 2012;1257:133–41. Fischer A, Stuckas H, Gluth M, Russell TD, Rudolph MC, Beeman NE, et al. Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice. J Cell Sci. 2007;120: 2272–83. Akers RM. Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. J Dairy Sci. 2006;89:1222–34. Stelwagen K, McFadden HA, Demmer J. Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells. Mol Cell Endocrinol. 1999;156:55–61. Zettl KS, Sjaastad MD, Riskin PM, Parry G, Machen TE, Firestone GL. Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro. Proc Natl Acad Sci U S A. 1992;89:9069–73. Singer KL, Stevenson BR, Woo PL, Firestone GL. Relationship of serine/threonine phosphorylation/dephosphorylation signaling to glucocorticoid regulation of tight junction permeability and ZO-1 distribution in nontransformed mammary epithelial cells. J Biol Chem. 1994;269:16108–15. Thompson GE. Cortisol and regulation of tight junctions in the mammary gland of the late-pregnant goat. J Dairy Res. 1996;63:305–8. Stelwagen K, Van Espen DC, Verkerk GA, McFadden H, Farr VC. Elevated plasma cortisol reduces permeability of mammary tight junctions in the lactating bovine mammary epithelium. J Endocrinol. 1998;159:173–8.

27. Rubenstein NM, Guan Y, Woo PL, Firestone GL. Glucocorticoid down-regulation of RhoA is required for the steroid-induced organization of the junctional complex and the tight junction formation in rat mammary epithelial tumor cells. J Biol Chem. 2003;278: 10353–60. 28. Linzell JL, Peaker M, Tayler JC. The effects of prolactin and oxytocin on milk secretion and on the permeability of the mammary epithelium in the rabbit. J Physiol. 1975;253:547–63. 29. Flint DJ, Gardner M. Evidence that growth hormone stimulates milk synthesis by direct action on the mammary gland and that prolactin exerts effects on milk secretion by maintenance of mammary deoxyribonucleic acid content and tight junction status. Endocrinology. 1994;135:1119–24. 30. Ollier S, Zhao X, Lacasse P. Effect of prolactin-release inhibition on milk production and mammary gland involution at drying-off in cows. J Dairy Sci. 2013;96:335–43. 31. Nguyen D-A, Parlow AF, Neville MC. Hormonal regulation of tight junction closure in the mouse mammary epithelium during the transition from pregnancy to lactation. J Endocrinol. 2001;170:347–56. 32. Gulay MS, Hayen MJ, Head HH, Bachman KC. Effect of estrogen supplemented at dry-off on temporal changes in concentrations of lactose in blood plasma of Holstein cows. J Dairy Sci. 2009;92: 3815–8. 33. Linzell JL, Peaker M. The effects of oxytocin and milk removal on milk secretion in the goat. J Physiol. 1971;216:717–34. 34. Allen JC. Milk synthesis and secretion rates in cows with milk composition changed by oxytocin. J Dairy Sci. 1990;73:975–84. 35. Woo PL, Vha HH, Singer KL, Firestone GL. Antagonistic regulation of tight junction dynamics by glucocorticoids and transforming growth factor-β in mouse mammary epithelial cells. J Biol Chem. 1996;271:404–12. 36. Paye JM, Akers RMHWR, Forsten-Williams K. Autocrine production of insulin-like growth factor-I (IGF-I) affects paracellular transport across epithelial cells in vitro. Cell Commun Adhes. 2007;14: 85–98. 37. Pitelka DR, Taggart BN, Hamamoto ST. Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelium cells in culture. J Cell Biol. 1983;96: 613–24. 38. Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol R. 1996;76:127–73. 39. Stelwagen K, Callaghan MR. Regulation of mammary tight junctions through parathyroid hormone-related peptide-induced activation of apical calcium channels. J Endocrinol. 2003;178:257–64. 40. Thompson GE, Ratcliffe WA, Hughes S, Abbas SK, Care AD. Local control of parathyroid hormone-related protein secretion by the mammary gland of the goat. Comp Biochem Physiol. 1994;108A:485–90. 41. Collier RJ, Hernandez LL, Horseman ND. Serotonin as a homeostatic regulator of lactation. Dom Anim Endocrinol. 2012;43:161–70. 42. Stull MA, Pai V, Vomachka AJ, Marshall AM, Jacob GE, Horseman ND. Mammary gland homeostasis employs serotonergic regulation of epithelial tight junctions. PNAS. 2007;104:16708–13. 43. Hernandez LL, Collier JL, Vomachka AJ, Collier RJ, Horseman ND. Suppression of lactation and acceleration of involution in the bovine mammary gland by a selective serotonin reuptake inhibitor. J Endocrinol. 2011;209:45–54. 44. Kobayashi K, Kumura H. Distinct behaviour of claudin-3 and -4 around lactation period in mammary alveolus in mice. Histochem Cell Biol. 2011;136:587–94. 45. Fleet IR, Goode JA, Hamon MH, Laurie MS, Linzell JL, Peaker M. Secretory activity of goat mammary glands during pregnancy and the onset of lactation. J Physiol. 1975;251:763–73. 46. Morgan G, Wooding FB. A freeze-fracture study of tight junction structure in sheep mammary gland epithelium during pregnancy and lactation. J Dairy Res. 1982;49:1–11.

J Mammary Gland Biol Neoplasia 47. Neville MC, Peaker M. Ionized calcium in milk and the integrity of the mammary epithelium in the goat. J Physiol. 1981;313:561–70. 48. Butler JE. Synthesis and distribution of immunoglobulins. J Am Vet Med Assoc. 1973;163:795–8. 49. Smolenski G, Haines S, Kwan FY, Bond J, Farr VDSR, Stelwagen K, et al. Characterisation of host defence proteins in milk using a proteomics approach. J Proteome Res. 2007;6:207–15. 50. Wheeler TT, Hodgkinson AJ, Prosser CG, Davis SR. Immune components of colostrum and milk - a historical perspective. J Mammary Gland Biol Neoplasia. 2007;12:237–47. 51. Stelwagen K, Carpenter E, Haigh B, Hodgkinson A, Wheeler T. Immune components of bovine milk and colostrum. J Anim Sci. 2009;87 Suppl 1:3–9. 52. Bush LJ, Staley TE. Absoption of colostral immunuglobulins in new born calves. J Dairy Sci. 1980;63:672–80. 53. Guy MA, McFadden TB, Cockrell DCBTE. Effects of unilateral prepartum milking on concentrations of immunoglobulin G1 and prolactin in colostrum. J Dairy Sci. 1994;77:3584–91. 54. Stelwagen K, Farr VC, Davis SR, Prosser CG. EGTA-induced disruption of epithelial cell tight junctions in the lactating caprine mammary gland. Am J Physiol. 1995;269:R848–55. 55. Stelwagen K, Farr VC, McFadden HA, Prosser CG, Davis SR. Time course of milk accumulation-induced opening of mammary tight junctions and blood clearance of milk components. Am J Physiol. 1997;273:R379–86. 56. Peaker M. The effect of raised intramammary pressure on mammary function in the goat in relation to the cessation of lactation. J Physiol. 1980;301:415–28. 57. Singh K, Dobson J, Phyn CVC, Davis SR, Farr VC, Molenaar AJ, et al. Milk accumulation decreases expression of genes involved in cell-extracellular matrix communication and is associated with induction of apoptosis in the bovine mammary gland. Livest Prod Sci. 2005;98:67–78. 58. Stelwagen K, Knight CH, Farr VC, Davis SR, Prosser CG, McFadden TB. Continuous versus single drainage of milk from the bovine mammary gland during a 24-hour period. Exp Physiol. 1996;81:141–9. 59. Stelwagen K, Phyn CVC, Davis SR, Guinard-Flament J, Pomiés D, Roche JR, et al. Invited review: reduced milking frequency: milk production and management. J Dairy Sci. 2013;96:3401–13. 60. Erdman RA, Varner M. Fixed yield responses to increased milking frequency. J Dairy Sci. 1995;78:1199–203. 61. Sorensen BA, Muir DD, Knight CH. Thrice-daily milking throughout lactation maintains epithelial integrity and thereby improves milk protein quality. J Dairy Res. 2001;68:15–25. 62. Fleet IR, Peaker M. Mammary function and its control at the cessation of lactation in the goat. J Physiol Lond. 1978;279:491–507.

63. McMahon CD, Farr VC, Singh K, Wheeler TT, Davis SR. Decreased expression of ß1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands. J Cell Physiol. 2004;200:318–25. 64. Phyn CVC. Regulation of tight junction proteins during engorgement of the mammary gland. 1996. PhD Thesis. Massey University, Palmerston North, New Zealand. 65. Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyper-immunised cows reduces tight junction permeability in vitro. Inflam Res. 1998;47:384–8. 66. Shamay A, Shapiro F, Mabjeesh SJ, Silanikove N. Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats. Life Sci. 2002;70:2707–19. 67. Shamay A, Shapiro F, Leitner G, Silanikove N. Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows. J Dairy Sci. 2003;86:1250–8. 68. Playford RJ, MacDonald CE, Calnan DP, Floyd DN, Podas T, Johnson W, et al. Co-administration of the health food supplement, bovine colostrum, reduces the acute non-steroidal anti-inflammatory drug-induced increase in intestinal permeability. Clin Sci (Lond). 2001;100:627–33. 69. Prosser CG, Stelwagen K, Cummins R, Guerin P, Gill N, Milne C. Reduction in heat-induced gastrointestinal hyperpermeability in rats by bovine colostrum and goat milk powders. J Appl Physiol. 2004;96:650–4. 70. Buckley JD, Buler RN, Southcott E, Brinkworth GD. Bovine colostrum supplementation during running training increases intestinal permeability. Nutrients. 2009;1:224–34. 71. Kotler BM, Kerstetter JE, Ansogna KL. Claudins, dietary milk proteins, and intestinal barrier regulation. Nutr Rev. 2013;71:60–5. 72. Bodammer P, Kerkhoff C, Maletzki C, Lamprecht G. Bovine colostrum increases pore-forming claudin-2 protein expression but paradoxically not ion permeability possibly by a change of the intestinal cytokine milieu. PloS One. 2013;8:e64210. 73. Lin Y, Xia L, Turner JD, Zhao X. Morphologic observation of neutrophil diapedesis across bovine mammary gland epithelium in vitro. Am J Vet Res. 1995;56:203–7. 74. Warshakoon HJ, Burns MR, David SA. Structure-activity relationships of antimicrobial and lipoteichoic acid-sequestering properties in polyamine sulfonamides. Antimicrob Agents Chemother. 2009;53: 57–62. 75. Lehmann M, Wellnitz O, Bruckmaier RM. Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk. J Dairy Sci. 2013;96:889–96. 76. Wellnitz O, Arnold ET, Lehmann M, Bruckmaier RM. Differential immunoglobulin transfer during mastitis challenge by pathogenspecific components. J Dairy Sci. 2013;96:1681–4.