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.
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.
Nakatani
Hajime
H
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Aoki
Naohito
N
Nakagawa
Yoshimi
Y
Jin-No
Shinji
S
Aoyama
Koji
K
Oshima
Kenji
K
Ohira
Sachiyo
S
Sato
Chihiro
C
Nadano
Daita
D
Matsuda
Tsukasa
T
D000818:Animals; D000831:Animals, Newborn; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics* / Q000378:metabolism*; D017209:Apoptosis; D004847:Epithelial Cells / Q000166:cytology; D004848:Epithelium / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D007774:Lactation / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008892:Milk / Q000378:metabolism; D008894:Milk Proteins / Q000235:genetics* / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D013347:Subcellular Fractions; D014886:Weaning; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000954:Antigens, Surface; D008894:Milk Proteins; D010718:Phosphatidylserines; D012333:RNA, Messenger; C081026:CD68 protein, mouse; D000097382:CD68 Molecule; C490919:Mfge8 protein, mouse