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

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.

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Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90233.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. L658-L669 ◽

Cited By ~ 20

Author(s):

Sandra R. Bates ◽

Altaf S. Kazi ◽

Jian-Qin Tao ◽

Kevin J. Yu ◽

Daniel S. Gonder ◽

...

Keyword(s):

Specific Binding ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Type Ii Cells ◽

Type Ii ◽

Calcium Dependent ◽

Type Ii Pneumocytes ◽

Surfactant Secretion

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of 125I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of 125I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of 125I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

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Permissive Effects of Oxygen on Cyclic AMP and Interleukin-1 Stimulation of Surfactant Protein A Gene Expression Are Mediated by Epigenetic Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.2901-2912.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 2901-2912 ◽

Cited By ~ 44

Author(s):

Kazi Nazrul Islam ◽

Carole R. Mendelson

Keyword(s):

Cyclic Amp ◽

Interleukin 1 ◽

Protein A ◽

Histone H3 ◽

Surfactant Protein ◽

Immune Defense ◽

Surfactant Protein A ◽

Type Ii Cells ◽

Type Ii ◽

Stimulation Of

ABSTRACT Surfactant protein A (SP-A) is important for immune defense within the alveolus. Cyclic AMP (cAMP) stimulation of SP-A expression in lung type II cells is O2 dependent and mediated by increased phosphorylation and binding of thyroid transcription factor 1 (TTF-1) to an upstream response element (TTF-1-binding element [TBE]). Interleukin-1 (IL-1) stimulation of SP-A expression is mediated by NF-κB (p65/p50) activation and increased binding to the TBE. In this study, we found that O2 also was permissive for IL-1 induction of SP-A expression and for cAMP and IL-1 stimulation of type II cell nuclear protein binding to the TBE. Using chromatin immunoprecipitation, we observed that when type II cells were cultured in 20% O2, cAMP and IL-1 stimulated the recruitment of TTF-1, p65, CBP, and steroid receptor coactivator 1 to the TBE region of the SP-A promoter and increased local acetylation of histone H3; these effects were prevented by hypoxia. Hypoxia markedly reduced global levels of CBP and acetylated histone H3 and increased the expression of histone deacetylases. Furthermore, hypoxia caused a global increase in histone H3 dimethylated on Lys9 and increased the association of dimethyl histone H3 with the SP-A promoter. These results, together with findings that the histone deacetylase inhibitor trichostatin A and the methyltransferase inhibitor 5′-deoxy(5′-methylthio)adenosine markedly enhanced SP-A expression in lung type II cells, suggest that increased O2 availability to type II cells late in gestation causes epigenetic changes that permit access of TTF-1 and NF-κB to the SP-A promoter. The binding of these transcription factors facilitates the recruitment of coactivators, resulting in the further opening of the chromatin structure and activation of SP-A transcription.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00415.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. L436-L446 ◽

Cited By ~ 40

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.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.2.l193 ◽

1993 ◽

Vol 265 (2) ◽

pp. L193-L199 ◽

Cited By ~ 12

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)

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Design-based stereological analysis of the lung parenchymal architecture and alveolar type II cells in surfactant protein A and D double deficient mice

The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology ◽

10.1002/ar.a.20225 ◽

2005 ◽

Vol 286A (2) ◽

pp. 885-890 ◽

Cited By ~ 25

Author(s):

Anja Jung ◽

Lennell Allen ◽

Jens R. Nyengaard ◽

Hans Jørgen G. Gundersen ◽

Joachim Richter ◽

...

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Stereological Analysis ◽

Deficient Mice

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Potential Role of Nuclear Factor B and Reactive Oxygen Species in cAMP and Cytokine Regulation of Surfactant Protein-A Gene Expression in Lung Type II Cells

Molecular Endocrinology ◽

10.1210/me.16.6.1428 ◽

2002 ◽

Vol 16 (6) ◽

pp. 1428-1440 ◽

Cited By ~ 9

Author(s):

K. N. Islam

Keyword(s):

Potential Role ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Type Ii Cells ◽

Nuclear Factor ◽

Oxygen Species ◽

Type Ii ◽

Factor B

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Pathways for clearance of surfactant protein A from the lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00250.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. L1011-L1018 ◽

Cited By ~ 15

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.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.2.l345 ◽

2001 ◽

Vol 281 (2) ◽

pp. L345-L360 ◽

Cited By ~ 24

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.

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