Stage specific expression of milk fat globule membrane glycoproteins in mouse mammary gland: comparison of MFG-E8, butyrophilin, and CD36 with a major milk protein, β-casein

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Abstract

The expression of mouse milk fat globule membrane (MFGM) glycoproteins, MFG-E8, butyrophilin, CD36 was analyzed by Northern blot analyses. MFG-E8 and butyrophilin mRNAs were specifically detected in the mammary gland of lactating mice, whereas CD36 mRNA was detected in the heart and lung as well as in the mammary gland of lactating mice. The mRNAs of the three MFGM glycoproteins accumulated at mid-lactation were about 2–10-times as much as those of the early and late gestation stages, whereas β-casein mRNA accumulation was dramatically increased; the mRNA at mid-lactation was no less than 40-times as much as that before lactation. In mouse mammary epithelial cell lines, HC11 and COMMA-1D, only a slight or almost no enhancement for the expression of MFG-E8, butyrophilin and CD36 mRNAs was induced simply by the treatment with the lactogenic hormones such as prolactin, insulin and dexamethasone, whereas the β-casein mRNA expression was remarkably enhanced only by that treatment. Furthermore, while the β-casein protein was constantly detected in milk throughout the lactation stage, the content of MFG-E8 and butyrophilin proteins increased during the lactation with an increase in the milk fat content. These results suggest that the stage-specific expression of milk fat globule membrane glycoproteins in mammary epithelial cells is regulated in a similar but not necessarily identical mechanism to that of a major milk protein, β-casein.

Introduction

Drastic changes occur in mammary glands during gestation and lactation periods for structural and functional development, and milk production. In lactating mammary glands, a great deal of milk proteins, fat and lactose are synthesized and secreted into milk fluid. Proteins such as casein and whey proteins are packaged into secretory vesicles followed by exocytosis from mammary epithelial cells, whereas milk fat, which is primarily synthesized as a small droplet in the mammary epithelial cells, gathers together and large droplets fuse to the apical plasma membrane. The milk fats are then secreted as droplets after being surrounded with milk fat globule membrane (MFGM), principally originated from the plasma membrane of the epithelial cells 1, 2.

MFGM glycoproteins are a diverse group of molecules, and some of them share one common characteristic that they are derived from the plasma membrane proteins of mammary epithelial cells in lactating mammary gland. The availability of MFGM proteins provides investigators with a unique source of epithelial plasma membrane proteins. Several major MFGM glycoproteins such as xanthine oxidase [3], butyrophilin 4, 5, 6, the components 15/16 (PAS-6 and -7) 7, 8, 9, MFG-E8 [10], MGP57/53 11, 12and CD36 (=PAS-4) 13, 14have been isolated from various species and characterized, though their biological and physiological functions in mammary gland or milk are not known. CD36 has been reported to possess some functions relating to cell adhesion 15, 16, 17and fatty acid transport [18], but the role of CD36 in MFGM or mammary gland is still uncertain.

Both cDNA and genomic clones specifying major milk proteins such as casein and whey proteins have been isolated from many species [19]. On the other hand, only limited numbers of cDNAs encoding bovine [5]and mouse [20]butyrophilins, mouse MFG-E8 [10], human BA46-1 [21]and bovine MGP57/53 [12]have been cloned. Butyrophilin was shown to express only in the mammary gland of lactating cows [5]and the maximum accumulation of MFG-E8 mRNA was observed in the lactating mammary gland and in the mammary epithelial cells on floating collagen gels [10]. However, the gene expression of major MFGM glycoproteins including MFG-E8, butyrophilin and CD36 in lactating mammary gland remains to be investigated.

To explore the possible biological functions of these MFGM proteins in the mammary gland and other tissues, we need to know how their expression in mammary epithelial cells is regulated during gestation and lactation periods. To achieve this, we amplified the cDNA fragments encoding mouse MFGM glycoproteins such as MFG-E8, butyrophilin and CD36 by a reverse transcriptase coupled-polymerase chain reaction (RT-PCR) method using RNA from mammary gland of pregnant and lactating mice, and investigated the accumulation of the glycoprotein mRNAs at gestation and lactation stages using the cDNA fragments as probes. The hormone-dependent expression was also investigated in the mouse mammary epithelial cell lines, HC11 and COMMA-1D. Moreover, we showed that the content of MFG-E8 and butyrophilin in mouse milk increased in proportion to triacylglycerol content of the milk.

Section snippets

Animals

Female pregnant ddY mice, 10 weeks of age, were purchased from Japan SLC (Hamamatsu, Japan) and the mice were fed laboratory chow (Japan SLC) ad libitum. Litter size was standardized to 8 pups within 24 h postpartum.

RNA preparation

Total RNA was prepared from the mammary tissues of lactating mice or cultured cells by the method using guanidine thiocyanate [22]. The RNA samples were stored at −85°C until use.

cDNA cloning by RT-PCR

cDNA fragments of MFGM glycoproteins were obtained by RT-PCR as described previously [20]. Briefly,

Northern blot analyses of mouse MFG-E8, butyrophilin and CD36 mRNAs

To investigate how the expression of MFGM glycoproteins is regulated in mammary gland and other tissues, cDNAs for MFGM glycoproteins have been cloned from one species, mouse, and used as probes for Northern blotting. The cDNA fragment encoding mouse MFG-E8 (about 340 bp) was cloned by RT-PCR based on the reported cDNA sequence [10]. Although CD36 (also referred to PAS-V) is known to constitute bovine MFGM [13], mouse CD36 of MFGM has not been well defined. The cDNA encoding mouse CD36 has

Discussion

In general, functions and properties of a protein can be speculated based on its amino acid sequence, its cDNA and genomic DNA sequences and the expression pattern depending on the tissues and developmental stages. This is also the case for the proteins constituting MFGM. The cDNA clones encoding bovine [5]and mouse [20]butyrophilins and mouse MFG-E8 [10]have been isolated and sequenced, and limited sequence similarities have been found between butyrophilins and rat myelin/oligodendrocyte

Acknowledgements

We acknowledge Drs. K. Maeda and H. Tsukamura (Department of Animal Sciences, Nagoya University) for immunizing animals and Dr. P. Neilsen (Max-Plank Institute) for the gift of S12 cDNA. This work was partially supported by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture of Japan.

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