Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells

1998 ◽  
Vol 275 (1) ◽  
pp. L38-L46 ◽  
Author(s):  
Qiping Chen ◽  
Aron B. Fisher ◽  
David S. Strayer ◽  
Sandra R. Bates
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Cell Membranes ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Cell

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of 125I-A2R and125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of ∼32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was ∼210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

scholarly journals Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes

2010 ◽  
Vol 299 (6) ◽  
pp. L794-L807 ◽  
Author(s):  
Altaf S. Kazi ◽  
Jian-Qin Tao ◽  
Sheldon I. Feinstein ◽  
Li Zhang ◽  
Aron B. Fisher ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Protein A ◽  
Surfactant Protein ◽  
Pi3 Kinase ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocytes ◽  
Kinase Signaling Pathway ◽  
Stimulation Of

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

2006 ◽  
Vol 291 (3) ◽  
pp. L436-L446 ◽  
Author(s):  
Nisha Gupta ◽  
Yefim Manevich ◽  
Altaf S. Kazi ◽  
Jian-Qin Tao ◽  
Aron B. Fisher ◽  
...  
Keyword(s):  
Binding Protein ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Receptor Complex ◽  
Type Ii ◽  
Surface Binding ◽  
Intact Mouse ◽  
The Cross

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

Pulmonary surfactant protein A-mediated uptake of phosphatidylcholine by alveolar type II cells

1993 ◽  
Vol 265 (2) ◽  
pp. L193-L199 ◽  
Author(s):  
A. Tsuzuki ◽  
Y. Kuroki ◽  
T. Akino
Keyword(s):  
Pulmonary Surfactant ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Pulmonary Surfactant Protein

Pulmonary surfactant protein A (SP-A)-mediated uptake of phosphatidylcholine (PC) by alveolar type II cells was investigated. SP-A enhanced the uptake of liposomes containing dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), or 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether), a diether analogue of DPPC, but about twice as much DPPC was taken up by type II cells as PLPC or DPPC-ether. When subcellular distribution was analyzed, 51.3 +/- 2.9% (mean +/- SD, n = 3) of cell-associated radiolabeled DPPC was recovered in the lamellar body-rich fraction in the presence of SP-A, whereas only 19.3 +/- 1.9% (mean +/- SD, n = 3) was found to this fraction in the absence of SP-A. When type II cells were incubated either with DPPC at 0 degree C or with DPPC-ether at 37 degrees C, or no cells were included, low proportions of the cell-associated lipids were present in the fractions corresponding to lamellar bodies even in the presence of SP-A. Anti-SP-A antibody significantly reduced the radioactivity incorporated into the lamellar body fraction. Phosphatidylcholine that had been incorporated into lamellar bodies remained largely intact when SP-A was present. Subcellular fractionations of type II cells with radiolabeled SP-A and DPPC revealed that the sedimentation characteristics of cell-associated SP-A are different from those of DPPC, although a small broad peak of radiolabeled SP-A was found in the lamellar body fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation and protein binding by two regions of the rat surfactant protein A promoter

1999 ◽  
Vol 277 (1) ◽  
pp. L134-L141
Author(s):  
Elizabeth Rosenberg ◽  
Feng Li ◽  
Candyce I. Smith ◽  
Samuel R. Reisher ◽  
Sheldon I. Feinstein
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Promoter Activity ◽  
Protein A ◽  
Surfactant Protein ◽  
Clara Cell ◽  
Surfactant Protein A ◽  
Type Ii ◽  
Recognition Element ◽  
Type Ii Cell

Surfactant protein A (SP-A) is expressed in lung alveolar type II cells and bronchiolar Clara cells. We have identified two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of transcription (−163 bp), linked to a reporter gene. Constructs were transfected into lung cell lines derived from each of the cell types that produces SP-A. We found a novel region of promoter activity at ∼90 bp before the transcriptional start (SP-A−90). Mutation of four nucleotides in SP-A−90 that are highly conserved among species (−92 to −89 bp) decreased expression of the SP-A construct by ∼50% in both cell lines. Electrophoretic mobility shift analysis showed specific binding to SP-A−90 by nuclear proteins from the cell lines, as well as from rat lung and liver. The electrophoretic mobility of the bands shifted by lung nuclear proteins changed late in fetal development. Although in the Clara cell line no reduction of promoter activity was seen on deletion of the region upstream of SP-A−90, in the type II cell line, deletion of residues −163 to −133 did reduce activity by ∼50%. This region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the SP-A construct better in the Clara cell line than in the type II cell line. These results suggest that the recognition element for TTF-1 has varying activity in the lung cell lines of different origin due to the availability of TTF-1.

Pathways for clearance of surfactant protein A from the lung

2005 ◽  
Vol 289 (6) ◽  
pp. L1011-L1018 ◽  
Author(s):  
Deepika Jain ◽  
Chandra Dodia ◽  
Aron B. Fisher ◽  
Sandra R. Bates
Keyword(s):  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Cytochalasin D ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Space ◽  
Alveolar Cells ◽  
Specific Inhibitors

Uptake and degradation of 125I-surfactant protein A (SP-A) over a 1-h period was studied in alveolar cells in culture and in isolated perfused lungs to elucidate the mechanism for clearance of the protein from the alveolar space. Specific inhibitors of clathrin- and actin-dependent endocytosis were utilized. In type II cells, uptake of SP-A, compared with controls, was decreased by 60% on incubation with clathrin inhibitors (amantadine and phenylarsine oxide) or with the actin inhibitor cytochalasin D. All agents reduced SP-A metabolism by alveolar macrophages. Untreated rat isolated perfused lungs internalized 36% of instilled SP-A, and 56% of the incorporated SP-A was degraded. Inhibitors of clathrin and actin significantly reduced SP-A uptake by ∼54%, whereas cytochalasin D inhibited SP-A degradation. Coincubation of agents did not produce an additive effect on uptake of SP-A by cultured pneumocytes or isolated perfused lungs, indicating that all agents affected the same pathway. Thus SP-A clears the lung via a clathrin-mediated pathway that requires the polymerization of actin.

Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes

2001 ◽  
Vol 281 (2) ◽  
pp. L345-L360 ◽  
Author(s):  
Heide Wissel ◽  
Andrea Lehfeldt ◽  
Petra Klein ◽  
Torsten Müller ◽  
Paul A. Stevens
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Type Ii Pneumocytes ◽  
Early Endosomes

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.

Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro

1995 ◽  
Vol 268 (5) ◽  
pp. L772-L780 ◽  
Author(s):  
J. R. Wright ◽  
D. C. Youmans
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Dependent Manner ◽  
Type Ii ◽  
Alveolar Type Ii Cell ◽  
Degradation Rates ◽  
Type Ii Cell

Pulmonary surfactant is synthesized and secreted into the airspaces by the alveolar type II cell. After it is secreted, surfactant undergoes a series of poorly understood transformations resulting in formation of a surface tension-reducing surface at the air-liquid interface. The by-products of the surface film and/or other products of surfactant metabolism are eventually cleared from the alveolar space. Both the alveolar type II cell and the macrophage are thought to be involved in surfactant clearance and have been shown to internalize surfactant lipid in vitro. The goal of the current investigation was to characterize further and to quantitate the role of the macrophage in surfactant clearance by investigating the uptake and metabolism of surfactant lipids and surfactant protein A (SP-A) by macrophages in vitro. SP-A enhanced the uptake of lipids by macrophages in a time-, temperature-, and concentration-dependent manner. In contrast, neither of the collagen-like proteins SP-D or C1q enhanced the uptake. Phosphatidylcholine was rapidly degraded by macrophages and the degradation occurred both in the presence and absence of SP-A. In addition, macrophages degrade SP-A by a process that is time- and temperature-dependent. These results and calculations of uptake and degradation rates suggest that macrophages may contribute significantly to the process of surfactant clearance.

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