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

Biochem J. 2006 Apr 1;395(1):21-30. doi: 10.1042/BJ20051459.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Antigens, CD / metabolism
  • Antigens, Differentiation, Myelomonocytic / metabolism
  • Antigens, Surface / genetics*
  • Antigens, Surface / metabolism*
  • Apoptosis
  • Epithelial Cells / cytology
  • Epithelium / metabolism
  • Female
  • Gene Expression Regulation*
  • Immunohistochemistry
  • Lactation / metabolism
  • Macrophages / metabolism
  • Mammary Glands, Animal / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • Milk / metabolism
  • Milk Proteins / genetics*
  • Milk Proteins / metabolism*
  • Phosphatidylserines / metabolism*
  • Protein Binding
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Secretory Vesicles / metabolism
  • Subcellular Fractions
  • Weaning*

Substances

  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Antigens, Surface
  • CD68 protein, mouse
  • Mfge8 protein, mouse
  • Milk Proteins
  • Phosphatidylserines
  • RNA, Messenger