Weaning-induced expression of a milk-fat globule protein, MFG-E8, in mouse mammary glands, as demonstrated by the analyses of its mRNA, protein and phosphatidylserine-binding activity

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Biochemical Journal (Impact Factor: 4.4). 05/2006; 395(1):21-30. DOI: 10.1042/BJ20051459
Source: PubMed


A milk membrane glycoprotein, MFG-E8 [milk fat globule-EGF (epidermal growth factor) factor 8], is expressed abundantly in lactating mammary glands in stage- and tissue-specific manners, and has been believed to be secreted in association with milk fat globules. In the present paper, we describe further up-regulation of MFG-E8 in involuting mammary glands, where the glands undergo a substantial increase in the rate of epithelial cell apoptosis, and a possible role of MFG-E8 in mediating recognition and engulfment of apoptotic cells through its specific binding to PS (phosphatidylserine). Immunoblotting and RNA blotting analyses revealed that both MFG-E8 protein and MFG-E8 mRNA were markedly increased in mammary tissue within 3 days of either natural or forced weaning (pup withdrawal) of lactating mice. Using immunohistochemical analysis of the mammary tissue cryosections, the MFG-E8 signal was detected around the epithelium of such involuting mammary glands, but was almost undetectable at early- and mid-lactation stages, although strong signals were obtained for milk fat globules stored in the alveolar lumen. Some signals double positive to a macrophage differentiation marker, CD68, and MFG-E8 were detected in the post-weaning mammary tissue, although such double-positive signals were much smaller in number than the MFG-E8 single-positive ones. Total MFG-E8 in milk was also increased in the post-weaning mammary glands and, furthermore, the free MFG-E8 content in the post-weaning milk, as measured by in vitro PS-binding and apoptotic HC11 cell-binding activities, was much higher than that of lactation. In addition, the post-weaning milk enhanced the binding of apoptotic HC11 cells to J774 macrophages. Sucrose density-gradient ultracentrifugation analyses revealed that such enhanced PS-binding activity of MFG-E8 was present in membrane vesicle fractions (density 1.05-1.13 g/ml), rather than milk fat globule fractions. The weaning-induced MFG-E8 might play an important role in the recognition and engulfment of apoptotic epithelial cells by the neighbouring phagocytic epithelial cells in involuting mammary glands.

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    • "This contradiction may explain re-uptake of milk fat globules (MFGs) by AECs after weaning. Milk fat globule EGF factor VIII (MFG-E8) is a secreted glycoprotein that is up-regulated after weaning (Nakatani et al. 2006). MFG-E8 binds to MFGs in the alveolar lumen and facilitates the uptake of MFGs into HC11 mammary epithelial cells (Nakatani et al. 2013). "
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    ABSTRACT: The mammary gland is a highly specialized organ that is able to repeat development and regression (involution) of alveolar structures for milk production. Mammary involution consists in two phases. The first phase is reversible and lasts until approximately 48 h after weaning in mice. Interestingly, an extended milking interval can change the milk-secretory activity of alveolar epithelial cells (AECs) before the first phase of involution begins. In this study, we investigate the changes in the ability of AECs to secrete milk during the involution progression. Careful observation of the number and locations of cleaved caspase-3 positive AECs revealed that the first phase of involution occurred approximately 24 h after weaning and the second phase began between 48 and 72 h after weaning. However, initial changes in the milk production ability of AECs began just 1 h after weaning and milk production gradually ceased within 24 h. In addition, activation of STAT3 and inactivation of STAT5 had occurred in some AECs by 6 h after weaning and more broadly by 24 h. In addition, milk production processes such as nutrient uptake, synthesis, and secretion ceased by 24 h post-weaning. Interestingly, enlarged cytoplasmic lipid droplets were observed in AECs 12 h after weaning even though the expression levels of genes relevant to triglyceride production (Srebp1 and AQP3) were down-regulated. These results indicate that several changes in the milk production ability of AECs occur during expanded suckling intervals and prior to involution.
    Cell and Tissue Research 10/2014; 359(2). DOI:10.1007/s00441-014-2013-7 · 3.57 Impact Factor
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    • "Finally, in contrast to Beclin 1 ECD, the C2 domain of MFG-E8 exhibited a strong preference for PS-enriched liposome (Supplementary information, Figure S5B). The result of this control experiment is consistent with previous reports 26, 27 and supports the validity of results derived from our liposome binding assays. "
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    Cell Research 03/2012; 22(3):473-89. DOI:10.1038/cr.2012.24 · 12.41 Impact Factor
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    ABSTRACT: Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.
    Bioscience Biotechnology and Biochemistry 10/2006; 70(9):2019-27. DOI:10.1271/bbb.60142 · 1.06 Impact Factor
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