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.

Figure 1. Surfactant secretagogues cause increased membrane-association of endogenous annexin A7

A. Western blot demonstrates the specificity of antibodies to recombinant rat annexin A7. Proteins (10μg) in isolated lung lamellar bodies, lysates of isolated type II cells cultured for 20–22h, and 1μg of recombinant rat annexin A7 (rA7) were probed for annexin A7 using antibodies preincubated for overnight without (panel containing lanes 4–6 and M) or with (panel with lanes 1–3 and M) 10μg of recombinant rat annexin A7 (rA7). Chemiluminiscence was recorded by autoradiography on an X-ray film with both panels exposed simultaneously. The blot demonstrates the presence of annexin A7 in lamellar bodies and isolated type II cells. Preincubation of antibodies with excess antigen decreased the signal intensity in each lane. M – Molecular weight markers with each position highlighted with a marker pen. B. Isolated rat alveolar type II cells were incubated for 30min without or with ATP (1mM), A23187 (250nM) or PMA (80nM). All of the recovered cytosol (c) and the membrane (M) fractions were probed for annexin A7. The membranes were stripped and probed for actin. C. Densitometry results of western blots for the membranes and cytosol fractions from several experiments showing increased membrane-association of annexin A7 in cells treated for 30min with all three agents used, in comparison to the controls. For these experiments, equal proportions of the cytosol and membrane fractions were probed for annexin A7 levels by western blot analysis followed by densitometry analysis to determine relative distribution of annexin A7. Only membrane-associated A7 levels are shown in each case for better clarity. Each secretagogue increased membrane-association of annexin A7. Results are expressed as mean ± SE of number of cell preparations shown in parentheses. * P < 0.05 – % distribution in control membranes was different in comparison to all others.

Figure 2. Immuno-staining for annexin A7 and ABCA3 shows co-localization with surfactant secretagogues

Adherent type II cells were incubated for 30min in the absence (Control) or presence of 250nM A23187, 1mM ATP, 10μM Isoproterenol or 80nM PMA. Cells were then fixed and stained for annexin A7 and ABCA3. Confocal fluorescence microscopic images are shown after adjusting for fluorescence intensities to the same range. The DAPI-stained nuclei are shown only in the last panels to clearly show the fluorescence due to annexin A7 and ABCA3 reactivity. All agents increased co-localization of annexin A7 with ABCA3. Representative images are shown from experiments that were repeated at least two times.

Figure 3. Membrane-association of annexin A7 is inhibited by protein kinase inhibitors

Alveolar type II cells were incubated for 30min without or with protein kinase inhibitors, bisindolylmaleimideI (BisI) or H-89 before treatment for 30min without or with indicated secretagogues. Equal proportions of the membrane (M) and cytosol (c) fractions were probed for the annexin A7 levels by western blot analysis. Western blot for fractions from cells treated with A23187 (A) shows increased membrane-association of A7, which was inhibited in the presence of BisI. The distribution of actin was not affected by either A23187 or BisI. Similarly, PMA (B), ATP (C) and isoproterenol (D) increased membrane-association of A7, which was inhibited by respective protein kinase inhibitor. E. Results of densitometry analysis of western blot images from all experiments. Results are relative distributions in the membrane fraction as a percent of the total and are mean ± SE of 3 –12 cell preparations. * P < 0.05 in comparison to all other conditions in the absence of protein kinase (PK) inhibitor, ** P < 0.05 in comparison to respective condition in the absence of PK inhibitor (BisI or H-89, as appropriate).

Figure 4. Protein kinase inhibitors diminish co-localization of annexin A7 and ABCA3 in secretagogue-stimulated cells

Isolated rat type II cells were pre-incubated for 30min without or with PKC inihibitor, BisI (for ATP, A23187 or PMA), or PKA inhibitor, H89 (for isoproterenol), before incubation for 30min without or with indicated secretagogue. Confocal microscope fluorescence images are shown for each agent. The adjacent image for each condition shows co-localization pattern as determined using the Zeiss LSM co-localization software. The analysis showed that prior treatment with protein kinase inhibitor prevented the increase in co-localization of annexin A7 and ABCA3. Representative images from several experiments are shown. Isoprel – isoproterenol.

Figure 5. Annexin A7 phosphorylation in vitro and in vivo in alveolar type II cells

A. Phosphorylation of purified recombinant rat annexin A7 (rA7) by commercially available catalytic subunit of PKA and by purified rat brain PKC. Purified A7 (1μg) was incubated with ATP-γ-³²P in presence of required co-factors for 1h. The proteins were separated by SDS-PAGE, stained to localize A7, and imaged with a phosphor-imager to determine phosphorylation. Both the kinase enzyme and rA7 were phosphorylated. B. Phosphorylation of purified A7 in the absence or presence of PKC. Purified A7 was incubated in the described assay mixture in the absence or presence of PKC and in the presence of ATP-γ-³²P. Left panel shows phosphorylation of A7 as determined by imaging of radioactivity using a phosphorimager. The phosphorylation of PKC is indicated by double arrowhead. A7 position is just below the 50kDa maker. Right panel shows Coomassie Blue-stained gels showing comparable amounts of A7 in two lanes. Results show no authophosphorylation of A7 under these conditions. C. Type II cells were incubated for indicated periods without or with 100nM PMA and the immuno-precipitates (IPs) were obtained with A7 antibodies. Western blots of IPs with indicated antibodies showed a time-dependent increase in A7 phosphorylation at the serine and threonine residues. D. Type II cells were incubated for 30min without or with indicated agents and harvested in lysis buffer. Equal amounts of cell lysate proteins were immunoprecipitated using A7 antibodies. Western blots of IPs with A7, p-thr and p-ser antibodies showed equal levels of A7 but increased reactivity with p-ser and p-thr antibodies in stimulated cells in comparison to the controls.