(C) Lysates from mEFs transfected with control and VAMP2 siRNA analyzed as in A. and VAMP2 at secretory vesicle membranes of bronchial epithelial cells, and that absence of ANXA2 prospects to retention of COL6 in a late-Golgi, VAMP2-positive compartment. These results define a new role for ANXA2 in the COL6 secretion pathway, and further show that this pathway establishes cellmatrix interactions that underlie normal pulmonary function and epithelial cell survival. KEY WORDS:Annexin A2, Collagen VI, Secretion == INTRODUCTION == The basement membrane is an electron-dense, 50- to 100-nm-thick, sheet-like structure composed of intricately put together extracellular matrix proteins that individual epithelial cell monolayers from their underlying connective tissue (LeBleu et al., 2007). Not only do basement membranes provide structural support and serve as barriers to toxins and pathogens, they also provide a conduit for signaling and mechanotransduction (Bateman et al., 2009;Lu et al., 2011). Polarized epithelial cells, such as those in the lung, constantly remodel the basement membrane by secreting and proteolyzing Rabbit Polyclonal to OR2T2 its component proteins; in turn, basement membrane proteins support epithelial cell-adhesion, survival and differentiation (Pozzi and Zent, 2011). Alterations in basement membrane composition accompany an array of human lung disorders, including asthma, chronic obstructive pulmonary disease and interstitial fibrosis (Cox and Erler, 2011;Kranenburg et al., 2006;Lambrecht and Hammad, 2012;Proud and Leigh, 2011). Most basement membranes consist of a collagen IV and laminin superstructure, whose stability and compliance are processed by expression of additional proteins (LeBleu et al., 2007). In the lung, the basement membrane scaffolding is usually buttressed by agrin, fibulin, netrin, nidogen, perlecan, tropoelastin (Manabe et al., 2008) and collagen VI (COL6), which together impart elasticity, FRAX486 mechanical support and signaling capability to the bronchial wall (Bober et al., 2010;Gelse et al., 2003;Specks et al., 1995). COL6, a microfibrillar collagen, forms filaments that anchor basement membranes FRAX486 to other collagen fibers (Bonaldo et al., 1990;Engvall et al., 1986;Keene et al., 1988). Abnormalities in COL6 lead to myopathy in the mouse (Bonaldo et al., 1998;Irwin et al., 2003) and zebrafish (Telfer et al., 2010), and to Ullrich and Bethlem congenital muscular dystrophy in humans (Baker et al., 2005;Bnnemann, 2011). Many basement membrane polypeptides undergo intracellular synthesis and maturation, followed by extracellular assembly (LeBleu et al., 2007). Laminin, for example, consists FRAX486 of polypeptide chains that form intracellular trimers (800 kDa), which aggregate into a hexagonal polymer only after secretion (LeBleu et al., 2007). Nidogen and perlecan are secreted without prior complex formation (LeBleu et al., 2007). Collagen IV, which accounts for about 50% of most basement membrane protein mass, assembles into triple helical, kilodalton-scale protomers (500 kDa), which are also secreted before extracellular assembly into a stable network (Gelse et al., 2003). This is similar to many other collagens that form classical fibrils (Tooley et al., 2010). However, in sharp contrast, COL6 undergoes considerable intracellular assembly of highly crosslinked megadalton-scale 12-subunit complexes (2000 kDa), which then, upon secretion, associate into a microfibrillar network (Bateman et al., 2009;Colombatti et al., 1995;Engvall et al., 1986). Annexin A2 (ANXA2), a component of a cell surface fibrinolytic receptor complex (Dassah et al., 2009;Flood and Hajjar, 2011), belongs to a family of conserved, membrane-binding proteins that has been implicated in intracellular membrane-fusion events (Gerke et al., 2005). Intracellularly, ANXA2 and S100A10 form a heterotetramer, which appears to have a calcium-regulated membrane-bridging function. In cultured cells, ANXA2 regulates secretion of surfactant-containing lamellar body in the type II pneumocyte (Wang et al., 2007), release of catecholamines from dense core vesicles in adrenal chromaffin cells (Umbrecht-Jenck et al., 2010), and exocytosis of ultra-large von Willebrand factor multimers from endothelial cell WeibelPalade body (Knop et al., 2004). In these processes, ANXA2 appearsin vitroto crosslink secretory granules to the plasma membrane by interacting with soluble N-ethylmaleimide-sensitive factor acceptor proteins 23 and 25 (SNAP-23 and SNAP-25), as well as vesicle associated membrane protein 2 (VAMP2), which are both components of the SNAP receptor (SNARE) complex (Nakata et al., 1990;Umbrecht-Jenck et al., 2010;Wang et al., 2007). On the basis of previous findings that implicate ANXA2 in membrane-fusion eventsin vitro, we sought to define the role of ANXA2 in basement membrane secretionin vivo, and its physiological relevance. We noted thatAnxA2/mice exhibit reduced exercise tolerance and abnormal pulmonary stiffness. Examination of lung tissue from resting mice revealed dysmorphic bronchial epithelial cells, high-level apoptosis and cell dropout. Specific absence of COL6 within theAnxA2/basement membrane correlated with.
