IL-1ß is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells.

In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1ß, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1ß, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1ß caused rapid disruption of TJs at tricellular contact points. IL-1ß treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1ß on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1ß or anisomycin. These results indicate that IL-1ß is a key cytokine that adversely affects the TJs between MECs by activating p38.

IL-1ß directly inhibits milk lipid production in lactating mammary epithelial cells concurrently with enlargement of cytoplasmic lipid droplets.

Mammary epithelial cells (MECs) in lactating mammary glands produce milk lipid, which provides a large percentage of calories and bioactive lipids for appropriate infant growth. However, secreted milk lipid is often reduced concurrently with increases in IL-1ß, IL-6, and TNF-α in mammary glands with mastitis. In this study, we investigated whether those cytokines directly influenced lipid production and secretion. A lactating MEC culture model with high lipid production ability was prepared by culture with oleic acid. TNF-α, IL-1ß, and IL-6 differentially affected lipid production and secretion in lactating MECs. In particular, IL-1ß treatment significantly reduced amounts of secreted triglycerides by 97% compared with the control concurrently with enlargement of cytoplasmic lipid droplets in MECs. IL-1ß also decreased mRNA expression of Fabp3 and Srebp1 and the amount of aquaporin 3, GLUT-1 and adipophilin in the milk lipid production pathway. Furthermore, IL-1ß inactivated STAT5 and glucocorticoid signaling to induce milk production in MECs, whereas STAT3 and NFκB signaling was activated. IL-1ß induced mRNA expression of IL-6 and TNF-α in MECs. Therefore, we suggest that IL-1ß is a key inhibitor of lipid production and secretion in lactating MECs.

Moderate High Temperature Condition Induces the Lactation Capacity of Mammary Epithelial Cells Through Control of STAT3 and STAT5 Signaling.

In lactating mammary glands, alveolar mammary epithelial cells (MECs) synthesize and secrete milk components. MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components. During lactation, MECs are exposed to temperature changes by metabolic heat production and air ambient temperature. In this study, we investigated whether temperature changes influence milk production ability and TJ barriers in MECs by using two lactating culture models. The results showed that 39 °C treatment activated milk production and enhanced the formation of less-permeable TJs. In contrast, 41 °C treatment caused adverse effects on the TJ barrier and cell viability, although the milk production ability of MECs was temporarily up-regulated. MECs cultured at 37 °C showed relatively low milk production ability and high proliferation activity. Furthermore, we investigated three kinds of transcription factors relating to lactogenesis, signal transducer and activator of transcription 5 (STAT5), STAT3 and glucocorticoid receptor (GR). STAT5 signaling was activated at 39 and 41 °C by an increase in total STAT5. However, long-term treatment led to a decrease in total STAT5. STAT3 signaling was inactivated by high temperature treatment through a decrease in total STAT3 and inhibited phosphorylation of STAT3. GR signaling was continuously activated regardless of temperature. These results indicate that a moderate high temperature condition at 39 °C induces a high lactation capacity of MECs through control of STAT5 and STAT3 signaling. In contrast, long-term exposure at 41 °C leads to a decline in milk production capacity by inactivation of STAT5 and a decrease in the total number of MECs.

Prolactin and glucocorticoid signaling induces lactation-specific tight junctions concurrent with ß-casein expression in mammary epithelial cells.

Alveolar mammary epithelial cells (MECs) in mammary glands are highly specialized cells that produce milk for suckling infants. Alveolar MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components after parturition. In the formation process of less permeable TJs, MECs show a selective downregulation of Cldn4 and a localization change of Cldn3. To investigate what induces less permeable TJs through these compositional changes in Cldns, we focused on two lactogenesis-related hormones: prolactin (Prl) and glucocorticoids. Prl caused a downregulation of Cldn3 and Cldn4 with the formation of leaky TJs in MECs in vitro. Prl-treated MECs also showed low ß-casein expression with the activation of STAT5 signaling. By contrast, dexamethasone (Dex), a glucocorticoid analogue, upregulated Cldn3 and Cldn4, concurrent with the formation of less permeable TJs and the activation of glucocorticoid signaling without the expression of ß-casein. Cotreatment with Prl and Dex induced the selective downregulation of Cldn4 and the concentration of Cldn3 in the region of TJs concurrent with less permeable TJ formation and high ß-casein expression. The inhibition of Prl secretion by bromocriptine in lactating mice induced the upregulation of Cldn3 and Cldn4 concurrent with the downregulation of milk production. These results indicate that the coactivation of Prl and glucocorticoid signaling induces lactation-specific less permeable TJs concurrent with lactogenesis.

Phytoestrogens Weaken the Blood-Milk Barrier in Lactating Mammary Epithelial Cells by Affecting Tight Junctions and Cell Viability.

During lactation, mammary epithelial cells (MECs) form the blood-milk barrier by less-permeable tight junctions (TJs) to prevent the leakage of milk components. Phytoestrogens affect the proliferation, differentiation, and apoptosis of MECs. However, it remains unclear whether phytoestrogens are involved in the blood-milk barrier. Therefore, we investigated the influence of phytoestrogens (coumestrol, genistein, and daidzein) by using an in vitro mouse-MEC-culture model. The results showed that coumestrol and genistein changed the expression of TJ proteins (claudins-3 and -4 and occludin), weakened barrier function, and reduced ß-casein production. Daidzein also weakened barrier function without inhibiting ß-casein production. Additionally, coumestrol and genistein induced apoptosis in MECs. These results indicate that phytoestrogens weaken the blood-milk barrier by directly affecting TJs and the cellular viability of lactating MECs in different ways.

Isoflavones and their metabolites influence the milk component synthesis ability of mammary epithelial cells through prolactin/STAT5 signaling.

SCOPE: Isoflavones are a class of polyphonic compounds present in legumes and are called phytoestrogens because of their estrogen-like activity. Estrogen influences the behavior of mammary epithelial cells (MECs) during pregnancy and lactation. In this study, we investigated the direct influences of isoflavones and their metabolites in milk production ability of MECs. METHODS AND RESULTS: Mouse MECs were cultured with prolactin and dexamethasone (glucocorticoid analog) to induce milk production ability. Subsequently, lactating MECs were treated with each isoflavone. Coumestrol, biochanin A, genistein, and formononetin decreased the intracellular and secreted ß-casein. On the other hand, p-ethylphenol, daidzein, and equol did not significantly influence ß-casein production at any concentration. Coumestrol, biochanin A and genistein down-regulated the mRNA expression of whey acidic protein (WAP), lactoferrin and α-lactalbumin. In contrast, p-ethylphenol, daidzein and equol up-regulated ß-casein and/or WAP with α-lactalbumin. Furthermore, coumestrol and genistein down-regulated the expression of prolactin receptor and signal transducer and activator of transcription 5 (STAT5) accompanied by a decrease in STAT5 phosphorylation. CONCLUSION: Isoflavones and their metabolites influence the milk production ability of MECs through different interactions with prolactin/STAT5 signaling. Simultaneous intake of multiple isoflavones by consumption of legumes may induce promotive or adverse effects on lactating MECs.

Distinct roles of prolactin, epidermal growth factor, and glucocorticoids in ß-casein secretion pathway in lactating mammary epithelial cells.

Beta-casein is a secretory protein contained in milk. Mammary epithelial cells (MECs) synthesize and secrete ß-casein during lactation. However, it remains unclear how the ß-casein secretion pathway is developed after parturition. In this study, we focused on prolactin (PRL), epidermal growth factor (EGF), and glucocorticoids, which increase in blood plasma and milk around parturition. MECs cultured with PRL, EGF and dexamethasone (DEX: glucocorticoid analog) developed the ß-casein secretion pathway. In the absence of PRL, MECs hardly expressed ß-casein. EGF enhanced the expression and secretion of ß-casein in the presence of PRL and DEX. DEX treatment rapidly increased secreted ß-casein concurrent with enhancing ß-casein expression. DEX also up-regulated the expression of SNARE proteins, such as SNAP-23, VAMP-8 and Syntaxin-12. Furthermore, PRL and DEX regulated the expression ratio of αs1-, ß- and κ-casein. These results indicate that PRL, EGF and glucocorticoids have distinct roles in the establishment of ß-casein secretion pathway.