Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Abstract

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling.

The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.

Introduction

The mammary gland is a complex organ which undergoes repeated cycles of growth, differentiation and regression concomitantly with variations in the reproductive status. From the onset of pregnancy, the mammary epithelium enters a growth and differentiation period leading to a highly branched tubuloalveolar structure. Mammary epithelial cells (MECs) reach a highly differentiated state at the end of gestation, and organise into numerous alveolar structures which are surrounded by contractile myoepithelial cells and embedded in a stroma composed of connective and adipose tissues, blood vessels and nerve terminals. MECs are in close contact with the basement membrane and interactions between these two entities are crucial for morphogenesis and differentiation of both the mammary gland and the MECs (Howlett and Bissell, 1993, Edwards and Streuli, 1995, Hennighausen and Robinson, 1998). After parturition, MECs produce large amounts of milk to feed the newborn offspring. Milk is essentially composed of water, proteins (mainly caseins assembled in micellar structures), fat globules and soluble components such as lactose and minerals. Milk protein genes are activated in a defined temporal time course from pregnancy to lactation (Robinson et al., 1995). During lactation, pituitary prolactin (PRL) is released in response to suckling and stimulates MECs to maintain the transcription of casein genes, but also acts post-transcriptionally by activating phospholipase A2 to produce arachidonic acid which was demonstrated to accelerate casein transport and/or secretion (Ollivier-Bousquet, 1978, Blachier et al., 1988, Pechoux et al., 2005). Thus, despite the fact that casein release appears to be mostly continuous, MECs may possess both constitutive and regulated secretory pathways (Ollivier-Bousquet, 1983, Turner et al., 1992).

Whatever their secretory mode, late trafficking steps within the secretory pathway and exocytosis, i.e. fusion of casein-containing vesicles with the plasma membrane might involve SNARE (Soluble N-ethylmaleimide-Sensitive Fusion (NSF) Attachment Protein (SNAP) Receptor) proteins, as already described in other cell types (Sollner et al., 1993b, Jahn and Scheller, 2006). The different SNAREs predominantly reside in distinct cellular compartments and are involved in specific trafficking pathways, with SNARE pairing contributing to the specificity of membrane fusion events (McNew et al., 2000, Scales et al., 2000). All SNARE proteins contain a characteristic “SNARE motif” of about 70 amino acids with a coiled-coil secondary structure. The association of R-SNAREs (formerly termed v-SNAREs) contained in the vesicle membrane with Q-SNAREs (formerly termed t-SNAREs) localised on target membranes leads to the formation of a highly stable four-helix bundle complex termed the SNARE complex. By zippering of the coil-coiled domains, SNARE complex formation brings the two lipid bilayers in close proximity and promotes their fusion (Sollner et al., 1993b, Sutton et al., 1998, Weber et al., 1998). After membrane fusion, SNARE complexes are dissociated by the NSF adenosine triphosphatase and its adaptor protein SNAP (Sollner et al., 1993a). Other proteins have been described to also interact with SNAREs to inhibit or promote the formation of SNARE complexes (Brunger, 2005, McNew, 2008). For example, proteins such as Munc-18 (Mammalian Uncoordinated-18) were first isolated bound to syntaxin-1 (Hata et al., 1993, Pevsner et al., 1994a, Pevsner et al., 1994b), maintaining it in a closed conformation, hence preventing its engagement in the formation of a SNARE complex (Dulubova et al., 1999). The interaction of SM (Sec1/Munc-18-like) proteins with SNAREs may therefore spatially and temporally regulate the formation of SNARE complexes (Dulubova et al., 2007, Shen et al., 2007).

To date, only a few studies have directly addressed the functions of SNAREs in MECs. One of these suggests that VAMP-8 (Vesicle-Associated Membrane Protein-8) may be involved in casein secretion (Wang et al., 2007). Moreover, the expression of the small GTPase Rab3A, as well as some SNAREs and regulatory proteins, has been observed in the mammary-derived MCF-7 and HC11 cell lines (Vadlamudi et al., 2000). On the other hand, SNAP-23 (Synaptosomal-Associated Protein 23), syntaxin-3 and -5, and Ykt6 have been described as being associated with lipid droplets (Boström et al., 2007, Reinhardt and Lippolis, 2008). SNAP-23 has been shown to be involved in the size increase of intracellular lipid droplets by homotypic fusion. Moreover, the large amount of membrane necessary for milk fat globule secretion by budding of the apical plasma membrane could be partly provided by casein-containing vesicles exocytosis (Mather and Keenan, 1998).

In the present study we asked which SNARE genes are expressed in MECs, and which of the corresponding proteins might play a role in milk secretion during lactation. With this aim, the endogenous expression levels of a large set of SNAREs were investigated in mouse mammary gland and purified mouse acini (formed by MECs) at different physiological stages. The intracellular localisation of those known to be involved in the late secretory pathway and exocytosis events was studied. Furthermore, the SNARE complexes present in membrane preparations from lactating acini were explored by mass spectrometry after co-immunoprecipitation. Since characterisation of SNARE expression, subcellular localisation and pairing are prerequisites to understand their function, our results therefore provide substantial data for further study of the functional role of SNAREs in milk product secretion.