Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D

Abstract

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+) and Ig-Hepta(-/-) mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.

Figure 1. Targeted disruption of the mouse Ig-Hepta/GPR116 gene.

A, Schematic maps of the Ig-Hepta locus, the targeting vector, and the recombinant locus after targeting. Dark box denotes the exon containing the initiation codon. Dotted lines delineate the regions of homology between the wild-type allele and the vector. The nuclear localization signal-β-galactosidase gene (LacZ), the neomycin phosphotransferase gene (neo), and the diphtheria toxin-A gene (DT) are shown as an open box. The positions of the probes used for Southern blot analysis (closed bars) and primers for PCR analysis (closed triangles) are also shown. B, Southern blot analysis of ES cells. Genomic DNAs isolated from wild-type (+/+) and mutant (+/−) ES cell clones were digested with EcoRV and blotted. Fragments obtained from wild-type allele (21.0 kb) and targeted allele (5.5 kb) were detected by external probe (probe 1) and internal probe (probe 2). The data is representative of blotting with probe 1. C, Determination of mice genotype by PCR analysis of tail-derived DNA. The 500-bp fragment amplified with primers A and B shows the presence of the wild-type allele (+/+); the 300-bp fragment amplified with primers A and C indicates the mutant allele (−/−). Both alleles are detected in heterozygous mice (+/−). D, Northern blot analysis of Ig-Hepta-deficient mice. RNA samples from lung of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice were analyzed with probes for mouse Ig-Hepta and β-actin mRNAs. E, Western blot analysis of Ig-Hepta-deficient mice. Protein samples from lung (40 µg) and kidney (100 µg) were analyzed by Western blotting with anti-Ig-Hepta polyclonal antibody (upper panel) and anti-β-actin monoclonal antibody (Sigma) (lower panel). Molecular mass markers are indicated on the right.

Figure 2. Accumulation of surfactants in the lung of Ig-Hepta^−/− mice.

A, Lungs of Ig-Hepta^+/+ and Ig-Hepta^−/− mice. B, A picture of BALF from Ig-Hepta^+/+ and Ig-Hepta^−/− mice. C, Hematoxylin-eosin staining of lung sections of Ig-Hepta^+/+ and Ig-Hepta^−/− mice. Arrows point to surfactant aggregates and double arrowheads indicate macrophages whose number was increased in the lung of Ig-Hepta^−/− mice. The average diameter of the cross section of alveoli was 43±4 µm for Ig-Hepta^+/+ mice and 63±8 µm for Ig-Hepta^−/− mice (P = 0.03). D, Transmission electron microscopy of the lung of an Ig-Hepta^−/− mouse. Arrows and asterisks indicate tubular myelin in the alveolar lumen and lamellar bodies in an AT-II cell, respectively. E, Quantification of surfactant lipid (SatPC) and total proteins in BALF from Ig-Hepta^+/+ and Ig-Hepta^−/− mice. **P<0.001. F, Western blot analyses of surfactant proteins. Equal volumes of BALFs or tissue homogenates were loaded. G, Quantification of mRNA expressions of surfactant proteins in the lung of Ig-Hepta^+/+ and Ig-Hepta^−/− mice. Average ratios compared to GAPDH mRNA were as follows (mean ± SEM, n = 4). Sp-A: 0.64±0.06 (+/+), 0.49±0.07 (−/−), P = 0.16; Sp-B: 0.24±0.03 (+/+), 0.28±0.02 (−/−), P = 0.33; Sp-C: 9.2±0.7 (+/+), 7.3±0.5 (−/−), P = 0.06; and Sp-D: 0.16±0.02 (+/+), 0.11±0.03 (−/−), P = 0.28. ns, not significant. H, Western blot analyses of surfactant proteins. BALFs or tissue homogenates containing equal amounts of SatPC were loaded. In the protein basis, lanes 1–4 contain 10, 4, 40, and 7 µg proteins, respectively. I, Activity staining of β-galactosidase with its substrate X-gal for determining Ig-Hepta-expressing cells (green) in the mouse lung.

Figure 3. Ig-Hepta-expressing cells determined by in situ hybridization and immunohistochemical double-staining.

A and B, In situ hybridization with antisense and sense probes to Ig-Hepta mRNA. C and D, Differential interference contrast images of (A) and (B), respectively. Arrowheads indicate the cells that exhibit the appearance of type II cell. Ig-Hepta signal is detected in these cells. E and F, Double-staining pictures of Ig-Hepta^+/+ and Ig-Hepta^−/− mouse lung sections. G and H, Higher magnification images of type II cell. FITC fluorescence (green) represents β-galactosidase signal, which indicates altered Ig-Hepta expression in Ig-Hepta^−/− mice. Cy3 fluorescence (red) shows pro-Sp-C, an AT-II cell marker. Nuclei were stained by Hoechst 33342 (blue). Scale bars, 10 µm.

Figure 4. Ig-Hepta/GPR116-expressing cells determined by β-galactosidase assay.

Expression of Ig-Hepta was determined in various tissues, other than the lung (Fig. 2I), including renal cortex (A), post thalamic region of the brain (B), choroid plexus (C), heart (D), intestine (E), spleen (F), liver (G), stomach (H), and testis (I). In the kidney, intercalated cells, glomerular endothelial cells, and capillary endothelial cells around the renal tubules were stained. Concerning the other tissues examined, most capillary endothelial cells were stained. Arrows in (A) indicate intercalated cells of renal collecting duct. G, glomerulus. Asterisk in (B) shows hippocampus. Scale bars, 50 µm.

Figure 5. Age-dependent expression of Ig-Hepta mRNA and accumulation of surfactants and Mmp12.

A, Expression of Ig-Hepta mRNA was quantified by real-time PCR using mRNA preparations from fetal, neonatal, young, and adult lungs of Ig-Hepta^+/+ mice. dpc, days post coitum. B, Age-dependent accumulation of surfactant lipid (SatPC) in the lung of Ig-Hepta^+/+ and Ig-Hepta^−/− mice (n = 3, ***P<0.001). C and D, Induction of expression of Mmp12 mRNA and its protein product in the lung of Ig-Hepta^−/− mice revealed by real time PCR and Western blotting, respectively.

Figure 6. Hypertrophic alveolar macrophages in Ig-Hepta^−/− mice (D–F) compared to Ig-Hepta^+/+ mice (A–C).

A and D, Hematoxylin and eosin stains. B and E, Viability of macrophages confirmed by a Hoechst/propidium iodide double stain apoptosis detection kit. C and F, Transmission electron microscope images of an alveolar macrophage of Ig-Hepta^+/+ and Ig-Hepta^−/− mice. Scale bars, 10 µm.

Figure 7. Increased synthesis and reduced catabolism of DPPC in Ig-Hepta^−/− mouse lung monitored by radiotracer uptake.

A and B, Radioactivity of SatPC in BALF and lung tissues corrected by their lung weights at 8 and 48 h. [³H]Choline uptake was measured at 8 h to evaluate surfactant synthesis and secretion, and at 48 h to assess surfactant catabolism (n = 4 for each condition). C, Amounts of SatPC secreted relative to those synthesized, which were evaluated by the ratio of [³H]choline incorporation into SatPC in BALF at 8 h [marked “α” in (A)] to that in lung tissues at 8 h [marked “γ” in (B)]. D, Reduced SatPC catabolism in Ig-Hepta^−/− mice evaluated by the ratio of [³H]choline incorporation into SatPC in BALF at 48 h [marked “β” in (A)] to that in BALF at 8 h [marked “α” in (A)]. Values are means ± SE. *P<0.05, **P<0.001.

Figure 8. Interaction of Sp-D-Myc with the N-terminal extracellular domain (ECD) of mouse Ig-Hepta.

A, Confirmation of expression of Sp-D-Myc in 293T cells and its secretion into culture medium. 293T cells were transiently transfected with mock (lanes 1 and 3) or Sp-D-Myc (Lanes 2 and 4). At 48 h after transfection, culture medium was harvested and the cells were extracted with 1% Triton in PBS. The cell lysates (20 µg of proteins, lanes 1 and 2) and the medium (20 µl, lanes 3 and 4) were analyzed by Western blotting (WB) with anti-Myc antibody. Arrows and arrowhead indicate multimeric forms and a monomer of Sp-D-Myc, respectively. Double arrowhead indicates non-glycosylated Sp-D-Myc . B, 293T cells were transfected with mock (lane 1) or Sp-D-Myc alone (lane 2) or along with ECD-FLAG (lane 3). At 48 h after transfection, culture media were subjected to immunoprecipitation (IP) with anti-FLAG M2 beads followed by Western blotting (WB) with anti-Myc antibody (top panel). The blot was reprobed with anti-FLAG antibody (middle panel). The cell lysates (20 µg of proteins) used for the immunoprecipitation were analyzed by Western blotting with anti-Myc antibody (bottom panel). Asterisks indicate the bands corresponding to IgG.

Figure 9. Schematic illustration of roles of Ig-Hepta in maintaining pulmonary surfactant homeostasis.

A, Metabolic and catabolic pathways of pulmonary surfactants. B, Surfactant homeostasis in lungs of Ig-Hepta ^+/+ and Ig-Hepta ^−/− mice. According to our working model, the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of saturated phosphatidylcholine (SatPC) and surfactant proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) by monitoring the levels of Sp-D in alveolar space. Deletion of Ig-Hepta/GPR116 results in massive accumulation of surfactants in alveolar space, which in turn activates phagocytosis of macrophages as evidenced by appearance of enlarged foamy macrophages. The activated macrophages gradually release matrix metalloproteinase 12 (Mmp12) and inflammatory cytokines & chemokines, which attract white blood cells (an inflammatory response as a secondary effect; unpublished observation; Supplementary Fig. S1). Concerning the regulation of biosynthesis of the surfactant proteins, a translational regulation seems to be a key mechanism for coordinated production of surfactant lipids and proteins since no significant changes were seen in the mRNA levels of the surfactant proteins between Ig-Hepta ^+/+ and Ig-Hepta ^−/− mice.