Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells

Abstract

Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

“…The molecular mechanism underlying exocytosis is a very specialized process that involves cytoskeletal machinery (F-Actin) [12] and particular proteins like Annexin 2 and 7 [13,14], SNAP-23 [15] and ABCA-3 [16]. As with many other secretory pathways, the LB secretory machinery uses Ca 2+ as a second messenger [17].…”

Annexin A7 and SNAP23 interactions in alveolar type II cells and in vitro: A role for Ca2+ and PKC

“…We have previously demonstrated that our purified antibody to recombinant annexin A7 recognizes a single band at ~47 kDa in lung, 24 h-cultured type II cells or in isolated lung lamellar bodies [23]. The specificity of antibody was demonstrated by loss of reactivity following pre-incubation with recombinant A7.…”

“…In vitro phosphorylation of recombinant annexin A7 with commercially available PKC (EMD Biosciences, Philadelphia, PA) was carried out as described previously [23]. Briefly, the phosphorylation mixture was incubated for 2h at 30° C (1 μg recombinant A7 in 10 μl reaction volume) in presence of 0.05 mUnits PKC, 4 μg phosphatidylserine, 100 nM PMA, 1 mM Na orthovanadate, 1 mM Ca 2+ and 0.1 mM ATP.MgCl 2 .…”

“…The binding to plasma membrane or lamellar bodies from secretagogue-stimulated cells were higher in comparison to the fractions from untreated cells [22]. In our recent studies, we demonstrated that surfactant secretagogues promoted A7 relocation to the lamellar bodies in alveolar type II cells and that such relocation was regulated by protein kinase activation [23]. In the current study, we investigated if two of the surfactant secretagogues (phorbol myristate acetate, PMA, and calcium ionophore A23187) also increased relocation of A7 to the t-SNARE containing domains in type II cells.…”

Molecular Machinery Regulating Exocytosis

“…This has facilitated the functional study of individual proteins as targets of particular protein kinases, whilst uncovering the significance of phosphorylation/dephosphorylation events in the regulation of exocytosis, such as in: synaptic transmission and cell plasticity (Amin, et al, 2008;Barclay, et al, 2003;Boczan, et al, 2004), neuronal morphogenesis (Chernyshova, et al, 2011), insulin secretion (Butelman, 1990;Sugawara, et al, 2009;Wang & Thurmond, 2010), insulin stimulated GLUT4 transport (Aran, et al, 2011;X. W. Chen, et al, 2011a;Sano, et al, 2011), mast cell and platelet degranulation (Fitzgerald & Reed, 1999;Foger, et al, 2011), exocytosis of factors required for neutrophil adhesion (Fu, et al, 2005), acrosomal exocytosis in sperm (Castillo Bennett, et al, 2010;Zarelli, et al, 2009) and lung surfactant exocytosis (Gerelsaikhan, et al, 2011). It has now become obvious that phospho-regulation of exocytosis is a complex and dynamic process implicated at almost all points along the exocytic route, from recruitment and transport of vesicles to their ultimate fusion at the plasma membrane ( Figure 1&2).…”