Orphan G protein-coupled receptor GPR116 regulates pulmonary surfactant pool size

Abstract

Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116(Δexon17), were generated. Gpr116(Δexon17) mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116(Δexon17) mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116(Δexon17) type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.

Figure 1.

GPR116 expression is enriched in type I and type II alveolar epithelial cells. (A) GPR116 mRNA expression in adult mouse tissues measured by qPCR analysis. Note the high expression in lung tissue (n = 4 samples per tissue). (B) Ontogeny of GPR116 mRNA expression in developing mouse lung. Note the dramatic increase just before birth at E18.5 (n = 3 lungs per gestational age). *P < 0.05 versus E11.5. (C) Quantitation of Gpr116, Sftpc, and Actin mRNA expression by qPCR analysis in type II cells isolated from wild-type (WT) mice (n = 3 individual isolations). *P < 0.05 versus Actin. (D) GPR116 immunostaining of adult WT lung with an antibody directed against C-terminus. Note staining of cilia on proximal airway cells (arrowheads) and apical staining pattern on type I and type II epithelial cells (inset). Scale bar = 50 μm in the large panel and 10 μm in inset.

Figure 2.

Progressive pulmonary surfactant accumulation in Gpr116^Δexon17 mice. (A) Saturated phosphatidylcholine (SatPC) levels in bronchoalveolar lavage fluid (BALF) and lung homogenate after BAL isolated by lipid extraction and osmium tetroxide chromatography followed by phosphorous analysis. SatPC levels were similar at E18.5 and significantly increased in 4- and 20-week-old Gpr116^Δexon17 mice (n = 4–6 lungs per group). BW = body weight; ND = not determined due to inability of lavaging E18.5 animals. (B) SatPC alveolar–tissue index, calculated from 4-week data in panel A [(SatPC in BALF)/(SatPC in BALF + SatPC in lung tissue)], was increased 2.2-fold in Gpr116^Δexon17 mice. (C) Cumulative SatPC levels in cell lysates and media from isolated type II cells cultured for 7 days. SatPC in the media of Gpr116^Δexon17 type II cells was increased 2.0-fold compared with WT. Data are pooled from four individual isolations (n = 2 mice/genotype/isolation). *P < 0.05 versus WT.

Figure 3.

Increased SatPC synthesis and normal surfactant phospholipid composition in Gpr116^Δexon17 lung tissue. (A and B) SatPC synthesis, determined by measuring the incorporation of ³H-palmitic acid into SatPC 8 hours after [3H]-palmitic acid injection (A) or the incorporation of ³H-choline into SatPC 8 hours after [methyl-3H]-choline chloride injection (B), was increased in the BALF and lung homogenate of 4-week-old Gpr116^Δexon17 mice. *P < 0.05 versus WT (n = 4–5 mice per genotype). (C) The composition of pulmonary surfactant phospholipids in the BALF of Gpr116^Δexon17 and WT mice are comparable at 4 weeks of age (n = 4 mice per genotype). LBPA = lysobisphosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PI = phosphatidylinositol; PS = phosphatidylserine; SM = sphingomyelin.

Figure 4.

Surfactant protein expression and secretion in GPR116^Δexon17 mice. (A) Representative Western blot analyses of surfactant proteins A (SFTPA), B (SFTPB), C (SFTPC), and D (SFTPD) in BALF of Gpr116^Δexon17 and WT control mice at 4 weeks of age. Input was normalized to recovered volume of unfractionated BALF; relative molecular weights = 26 to 38 kD (SFTPA), 16 kD (SFTPB), 4 kD (SFTPC), 43 kD (SFTPD), and 44 kD (ACTIN). The graph represents n = 7 mice per genotype. (B) Representative Western blot analyses of surfactant proteins in postlavaged lung homogenate of Gpr116^Δexon17 and WT control mice at 4 weeks of age. Input was normalized to total protein levels, and data were normalized to ACTIN; the graph represents n = 4 mice per genotype. (C) Quantitation of mature SFTPB protein levels in lamellar bodies isolated from 4-week-old Gpr116^Δexon17 and WT mice. Input was normalized to total protein. *P < 0.05 versus WT.

Figure 5.

Alveolar simplification and inflammation in Gpr116^Δexon17 mice. (A) Representative hematoxylin and eosin–stained lung sections from 4- and 20-week-old animals. Note enlarged alveoli at 4 and 20 weeks and the presence of lipid-laden macrophages (asterisk) in Gpr116^Δexon17 lungs at 20 weeks of age. (B) Mean linear intercept quantitation demonstrates enlarged airspaces in Gpr116^Δexon17 mice at 4 weeks (1.8-fold) and 20 weeks (1.7-fold) (n = 3 mice per genotype). *P < 0.05 versus WT. (C) Subpleural accumulation of lymphoid cells (arrow), neutrophilia (arrowheads), and foamy alveolar macrophages (asterisks) in 20-week-old Gpr116^Δexon17 animals. Scale bars = 50 μm.

Figure 6.

Ultrastructural analysis of secreted surfactant in Gpr116^Δexon17 mice. (A) Representative electron micrographs of WT and Gpr116^Δexon17 lung sections at 20 weeks showing morphology of secreted surfactant within an alveolus. Note the abundance and dense, aggregated nature of surfactant in the Gpr116^Δexon17 lung compared with prototypical ultrastructure seen in the WT lung. Scale bar in large panels = 2 mm; scale bar in inset = 500 nm.

Figure 7.

The purinergic receptor P2RY2 is increased in Gpr116^Δexon17 lungs. (A and B) qPCR analysis demonstrating increased expression of P2RY2 in type II cells isolated from GPR116^Δexon17 mice at 4 weeks of age (A) and in whole lung tissue at E18.5 and 4 weeks of age (B). *P < 0.05 versus WT at respective time points (n = 4 lungs per group). (C) P2RY2 protein expression by Western blot analysis of whole lung homogenates from 4-week-old animals. (D) Densitometry analysis of the data in C demonstrates a 1.9-fold increase in Gpr116^Δexon17 lungs. *P < 0.05 versus WT.