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

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

Caractérisation des protéines SNARE potentiellement impliquées dans la sécrétion des produits du lait

The mammary gland is a complex tissue which function is to ensure the offspring’s feeding by producing and secreting milk during lactation. Casein micelles and fat globules (MFGs) are essential constituents of milk and are both secreted at the apical side of mammary epithelial cells (MECs) during lactation. MFGs are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. The lactogenic hormone prolactin (PRL) not only induces the synthesis of caseins but also an increase of arachidonic acid (AA) in MECs, which may accelerate the apical transport and possibly the exocytosis of caseins (secretagogue effect of PRL). Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. By forming stable ternary complexes, SNARE (Soluble NSF Attachment Receptor) proteins constitute the core fusion machinery for cellular membrane trafficking and exocytosis events in many cell types. In addition, SNAREs have recently been described as target for AA, thus promoting exocytosis in neuroendocrine cells. We therefore attempted to identify the SNARE proteins involved in caseins secretion in the mouse mammary gland during lactation. An exciting possibility is that the SNAREs involved in caseins’ exocytosis can be the target of AA released in response to PRL

Caractérisation des protéines SNARE potentiellement impliquées dans la sécrétion des produits du lait

The mammary gland is a complex tissue which function is to ensure the offspring’s feeding by producing and secreting milk during lactation. Casein micelles and fat globules (MFGs) are essential constituents of milk and are both secreted at the apical side of mammary epithelial cells (MECs) during lactation. MFGs are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. The lactogenic hormone prolactin (PRL) not only induces the synthesis of caseins but also an increase of arachidonic acid (AA) in MECs, which may accelerate the apical transport and possibly the exocytosis of caseins (secretagogue effect of PRL). Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. By forming stable ternary complexes, SNARE (Soluble NSF Attachment Receptor) proteins constitute the core fusion machinery for cellular membrane trafficking and exocytosis events in many cell types. In addition, SNAREs have recently been described as target for AA, thus promoting exocytosis in neuroendocrine cells. We therefore attempted to identify the SNARE proteins involved in caseins secretion in the mouse mammary gland during lactation. An exciting possibility is that the SNAREs involved in caseins’ exocytosis can be the target of AA released in response to PRL

Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency

International audienceMutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-β signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-β binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency

Not All Floating-Harbor Syndrome Cases are Due to Mutations in Exon 34 of SRCAP

International audienceFloating-Harbor syndrome (FHS) is a rare disorder characterized by short stature, delayed bone age, speech delay, and dysmorphic facial features. We report here the molecular analysis of nine cases, fulfilling the diagnostic criteria for FHS. Using exome sequencing, we identified SRCAP as the disease gene in two cases and subsequently found SRCAP truncating mutations in 6/9 cases. All mutations occurred de novo and were located in exon 34, in accordance with the recent report of Hood et al. However, the absence of SRCAP mutations in 3/9 cases supported genetic heterogeneity of FH syndrome. Importantly, no major clinical differences were observed supporting clinical homogeneity in this series of FHS patients. Hum Mutat 34:88-92, 2013. (C) 2012 Wiley Periodicals, Inc