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, Gpr116Δexon17, were generated. Gpr116Δexon17 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 Gpr116Δexon17 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 Gpr116Δexon17 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.
The molecular pathways that sense and regulate surfactant levels in the alveolar saccules are incompletely understood. In this work we provide evidence that the G protein–coupled receptor GPR116 is a master regulator of surfactant homeostasis. The long-term goal of this work is to identify potential therapeutic targets, including GPR116, for modulating endogenous surfactant levels in the treatment of lung disease.
Pulmonary surfactant is synthesized by alveolar type II cells and is primarily composed of phospholipids, which constitute 80% of the total mass. The remaining components include neutral lipids, the lipid-associated surfactant proteins SFTPB and SFTPC, and the hydrophilic surfactant proteins SFTPA and SFTPD (1, 2). Saturated phosphatidylcholine (SatPC) is uniquely enriched in surfactant and, in concert with SFTPB and SFTPC, is fundamentally required to reduce surface tension at the air/liquid interface within the alveolus (3, 4). After synthesis, the lipid and lipid-associated proteins SFTPB and SFTPC are routed to and stored in membrane-enclosed secretory organelles called lamellar bodies. Through constitutive pathways or upon stimulation by secretagogues (including purines [5, 6], β-agonists [7–9], and adenosine [10, 11]) or mechanical stretch (12), lamellar bodies fuse with the plasma membrane and are exocytosed from type II cells into the alveolar space.
The quantity of surfactant in the mammalian lung, referred to as surfactant pool size, increases dramatically during late gestation to facilitate the transition to air breathing at birth. In the fetal lung, the majority of surfactant is stored within type II cells, with minimal amounts of secreted surfactant in the airspace. During the fetal-to-neonatal transition, stored surfactant is rapidly secreted into the alveoli; tissue and alveolar pool levels subsequently decline within 2 to 5 days due to decreased secretory rates and increased catabolic rates (13–15). Alveolar surfactant pool sizes are tightly regulated and maintained at a steady-state level in the adult lung through a net balance of synthesis, secretion, and recycling by alveolar type II cells and by catabolism by alveolar macrophages and type II cells (16). Alterations in surfactant pool size or composition significantly affect lung function and have been associated with severe lung diseases in children and adults, including respiratory distress syndrome, acute lung injury, chronic lung disease, and pulmonary alveolar proteinosis. The ability to pharmacologically manipulate pathways that modulate endogenous surfactant pool sizes, both positively and negatively, may provide therapeutic benefit for diseases associated with dysregulation of the surfactant system.
Secretion of surfactant-containing lamellar bodies from type II cells occurs by two pathways: constitutive and regulated secretion. Three G protein–coupled receptor (GPCR)-mediated pathways have been implicated in controlling regulated secretion from isolated type II cells: the P2RY2 purinoreceptor pathway, the β2 adrenergic receptor (β2AR) pathway, and the adenosine A2B pathway (5–10). Activation of these G protein–coupled pathways by their cognate agonists results in increased cytosolic levels of the second messengers Ca2+ and/or cAMP, culminating in the activation of one or more of three downstream protein kinases, protein kinase (PK)A, PKC, and Ca2+/calmodulin-dependent protein kinase, that lead to surfactant release (17–20). Likewise, compounds that bypass the receptors to activate PKA or PKC directly or that increase cytosolic Ca2+ levels are sufficient to stimulate surfactant secretion in vitro (7, 9, 21). Mechanical stretch of isolated type II cells in vitro or by hyperventilation ex vivo is the most physiologically relevant stimulus identified to date and induces surfactant secretion via ATP- and Ca2+–dependent pathways (12, 22). Although many exogenous extracellular and intracellular mediators are capable of stimulating surfactant secretion in vitro, the endogenous, physiologically dominant in vivo agonists and receptors that control surfactant pool sizes have not been established.
In the present study, we report that an orphan member of the adhesion GPCR family, GPR116, is highly expressed on alveolar epithelial cells and that mice with a genetic loss of GPR116 function, Gpr116Δexon17, have a marked accumulation of pulmonary surfactant that is progressive in nature. We further demonstrate that incorporation of surfactant precursors into SatPC was increased in Gpr116Δexon17 mice and that surfactant accumulation was associated with increased expression of P2RY2, a GPCR known to mediate surfactant secretion, in alveolar type II cells. Collectively, these data demonstrate that GPR116 is a major regulator of surfactant homeostasis.
Details regarding the generation of Gpr116Δexon17 animals and GPR116 antisera are listed in the online data supplement and Figure E1 in the online supplement.
Bronchoalveolar lavage fluid (BALF) was collected by intratracheal intubation followed by serial lavaging with 0.9% saline (5 × 1 ml total, pooled and placed on ice). BAL cells were isolated from the lavage via centrifugation at 900 × g for 10 minutes at 4°C. After lavage, lungs were homogenized in 0.9% saline. Lipids were extracted from the lavage and postlavaged lung homogenate by the Bligh and Dyer method (23). SatPC was isolated using the osmium tetroxide–based method of Mason and colleagues (24) and quantitated by phosphorous measurement. SatPC levels in BALF reported in this manuscript represent the total BALF, including the BAL cell pellet, due to our inability to reliably and accurately separate the BAL cells from the abnormally aggregated surfactant in the BALF of Gpr116Δexon17 animals; SatPC levels reported in the lung homogenate are from postlavaged lung homogenate.
Phospholipid composition was performed on chloroform-methanol extracts of BALF by two-dimensional thin-layer chromatography as previously described (25). SatPC synthesis was determined in vivo as previously described using [3H]-palmitic acid and [methyl-3H]-choline chloride (25). Briefly, 0.5 μCi [3H]-palmitic acid or [3H]-choline chloride/g body weight was injected intraperitoneally into 4-week-old wild-type (WT) and Gpr116Δexon17 mice. Eight hours after injection, BALF and lavaged lung tissue were harvested, SatPC was isolated, and radioactivity was determined in the SatPC fraction. The 8-hour time point was chosen for this analysis because we have previously shown this time point to best reflect the net incorporation of precursor into SatPC and secretion of labeled SatPC into the alveoli with minimal recycling and catabolism of labeled SatPC by type II cells and alveolar macrophages (26).
Total RNA was purified from adult murine organs, whole lung homogenates, or isolated type II cell preparations with the RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed into cDNA with the iScript kit (BioRad, Hercules, CA). Real-time qPCR was performed on a 7300 real-time PCR system (Applied Biosystems, Foster City, CA) with TaqMan gene expression assays ( Table E1) and normalized to endogenous ActinB.
Details are provided in the online supplement.
Lungs from 20-week-old animals were fixed, processed, and analyzed as previously described (27).
Details are provided in the online supplement.
Details are provided in the online supplement.
All data are presented as means ± SEM, with P ≤ 0.05 considered significant. Multiple group comparisons were made by one-way ANOVA with a Tukey post hoc analysis. Two-way comparisons were performed by two-tailed, unpaired Student’s t test. All analyses were performed using GraphPad Prism software version 5.0.
We first sought to define the expression pattern of murine Gpr116 by analyzing several tissues via real-time qPCR. Gpr116 mRNA was detectable in multiple organs of the adult mouse and was highly expressed in the lung (Figure 1A). These data are consistent with the expression profile of GPR116 in the adult rat (28). To define the developmental expression pattern of GPR116 in the lung, an ontogenic study of Gpr116 mRNA was performed using lung tissue from E11.5 embryos through 8-week-old adult mice. Gpr116 mRNA was developmentally regulated in the murine lung, reaching peak expression at postnatal Day 1 with maintenance of high expression levels throughout adulthood (Figure 1B). The sharp increase in Gpr116 expression in late gestation is temporally consistent with maturation of the surfactant system in alveolar type II cells. To assess the relative degree to which Gpr116 is expressed in distal epithelial cells, Gpr116 mRNA expression in primary adult type II cells was quantitated and compared with Sftpc, a gene that is highly expressed in type II cells. Gpr116 mRNA expression is comparable with Actin and 7.9-fold lower than Sftpc (Figure 1C).
To determine which cell types within the lung express GPR116 protein, immunohistochemistry was performed on adult lung sections with polyclonal antisera directed against the C-terminus of GPR116. In the distal airspaces, GPR116 protein is expressed on the apical surface of type I and type II epithelial cells (Figure 1D, inset), consistent with data reported in adult rat lung (28). In the proximal airways, GPR116 is present on ciliated cells (Figure 1D, arrowheads) but is not detected on other epithelial cells, including club cells (Clara cells) and neuroendocrine cells. Although Gpr116 mRNA is expressed in transformed endothelial cell lines (29) and isolated mouse lung endothelial cells (30), GPR116 protein is undetectable in pulmonary endothelial cells analyzed by flow cytometry (Figure E2), and Gpr116 mRNA and protein are not expressed in alveolar macrophages (data not shown).
To define the role of GPR116 in vivo, we generated mice with a conditional allele of Gpr116 by inserting loxP sites into the intronic sequences flanking exon17, which encodes all seven transmembrane domains of GPR116 (Figure E1A). Successful generation of the Gpr116 floxed allele was confirmed by Southern blot and PCR analyses (Figures E1B and E1C). To ablate GPR116 function in all tissues, Gpr116f/f mice were crossed to CMV-Cre transgenic mice and backcrossed to BALB/c mice to remove the CMV-Cre transgene. Homozygous germline GPR116 loss-of-function mice, resultant from heterozygous parents backcrossed to BALB/c mice for more than 12 generations, were used exclusively in this study and are herein referred to as Gpr116Δexon17 mice.
Homozygous Gpr116Δexon17 mice were born at normal Mendelian ratios and were viable, indicating that GPR116 is dispensable for normal embryonic development. Perinatal development of Gpr116Δexon17 mice was normal, as demonstrated by body weights that were comparable to WT control mice from E16.5 through 12 weeks of age (data not shown). To confirm the loss of Gpr116 mRNA in Gpr116Δexon17 mice, qPCR analyses was performed on Gpr116Δexon17 lung tissue using a TaqMangene expression assay targeting the exon16/17 boundary of Gpr116. Gpr116 mRNA was undetectable in Gpr116Δexon17 lung tissue with this assay, indicating complete ablation of exon17 (Figure E3A). However, using a distinct gene expression assay specific for the exon4/5 boundary, we were able to detect Gpr116 mRNA in Gpr116Δexon17 lung tissue (Figure E3B). These data demonstrated that the Gpr116Δexon17 allele generated mRNA that was devoid of exon17. Because exon18 is in frame with exon16, it was possible that the Gpr116Δexon17 mRNA was capable of generating a protein that contained exon sequences downstream of exon17. To test this possibility, Gpr116Δexon17 lung sections were stained with GPR116 polyclonal antisera. GPR116 immunoreactivity was indeed observed in distal alveolar epithelial cells of Gpr116Δexon17 mice, albeit at reduced levels and in a discontinuous staining pattern compared with WT control mice (Figure E3C). Consistent with these data, transient expression of human GPR116 cDNA in which exon 17 was deleted in HEK293 cells resulted in diffuse, cytoplasmic staining compared with the uniform localization of the WT protein to the plasma membrane (Figure E3D). Expression and mislocalization of GPR116Δexon17 protein did not invoke the unfolded protein response, as demonstrated by similar levels of Hsp15 (BiP), Ddit3 (CHOP), and spliced Xbp-1 in isolated WT and Gpr116Δexon17 type II cells (Figure E3E). Collectively, these results demonstrate that targeting of exon17 in Gpr116Δexon17 mice was successful and that the Gpr116Δexon17 allele generates a protein product devoid of the transmembrane domains encoded by exon17 that is low in abundance, fails to traffic to the plasma membrane, and does not elicit an unfolded protein response in type II cells.
Young Gpr116Δexon17 mice appeared healthy and were phenotypically indistinguishable from heterozygous and WT littermate control mice. However, the majority of Gpr116Δexon17 mice exhibited signs of severe respiratory distress, including profound tachypnea and chest retractions, between 20 and 32 weeks of age. Consistent with these observations, BALF from 20-week-old animals in respiratory distress was extremely turbid and viscous compared with BALF from WT control mice, indicative of increased lipid and/or protein content (Figure E4A).
To determine if surfactant phospholipids were increased in Gpr116Δexon17 mice, SatPC levels in BALF and lung homogenate were quantified at three ages: E18.5, 4 weeks, and 20 weeks. SatPC levels in lung homogenate were comparable in Gpr116Δexon17 and WT control mice at E18.5. However, alveolar and tissue SatPC levels were significantly increased in Gpr116Δexon17 mice at 4 weeks of age (BALF increase, 13.9-fold; lavaged lung homogenate increase, 3.7-fold) and further increased at 20 weeks of age (BALF increase, 33.3-fold; lavaged lung homogenate incease, 29.8-fold) (Figure 2A). Furthermore, the amount of SatPC in the alveoli compared with the amount of SatPC in the total lung, referred to as the SatPC alveolar–tissue index, was increased 2.2-fold in Gpr116Δexon17 mice over WT control mice at 4 weeks of age (Figure 2B). Next, we quantitated SatPC levels in media and the lysates of primary WT and Gpr116Δexon17 type II cells isolated from 4-week-old animals and cultured for 7 days. SatPC levels in WT and Gpr116Δexon17 type II cells were comparable after 7 days of culture, whereas SatPC levels were increased 1.9-fold in the media of Gpr116Δexon17 cells (Figure 2C). Mice heterozygous for the Gpr116Δexon17 allele had SatPC levels comparable to WT mice at 6 months of age (data not shown).
SatPC synthesis rates were evaluated in Gpr116Δexon17 mice by independently measuring the incorporation of two radiolabeled phospholipid precursors, palmitic acid or choline, into SatPC in vivo. Incorporation of palmitic acid and choline into SatPC was increased over 2-fold in BALF and lung homogenate of Gpr116Δexon17 mice at 4 weeks of age (Figures 3A and 3B). To determine if SatPC was uniquely enriched in Gpr116Δexon17 mice or if all surfactant phospholipid species were increased proportionately with SatPC, the composition of surfactant phospholipid species in the lavage fluid was quantified by two-dimensional thin-layer chromatography. All of the major phospholipid species examined, including phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, phosphatidylserine, and lysobisphosphatidic acid, were present at comparable levels in the lavage of Gpr116Δexon17 and WT mice (Figure 3C). Taken together, these data demonstrate that Gpr116Δexon17 mice exhibited profound increases of alveolar and tissue surfactant phospholipid pool sizes that initiated in the postnatal period and progressed throughout adulthood. The accumulation of SatPC was associated with an increased SatPC alveolar–tissue index and synthesis in vivo and with SatPC accumulation in the media of isolated Gpr116Δexon17 type II cells in vitro. Furthermore, all of the major surfactant phospholipid species examined increased proportionately with SatPC in Gpr116Δexon17 animals at 4 weeks of age.
To determine if surfactant protein production and alveolar levels were influenced by GPR116, surfactant protein levels were quantitated from the lavage and lung homogenate of Gpr116Δexon17 and WT animals. Due to the large increase of alveolar SatPC pools in Gpr116Δexon17 mice, inputs for the BALF samples used in these analyses were normalized to recovered BALF volume, and inputs for postlavaged lung homogenates were normalized to total protein. Alveolar levels of surfactant protein A (SFTPA) and SFTPD were unchanged in the BALF from Gpr116Δexon17 mice relative to WT mice, whereas mature SFTPB and SFTPC proteins were increased 2.5- and 1.7-fold, respectively (Figures 4A and 4B). In contrast, tissue-associated levels of SFTPB and SFTPC were comparable, whereas SFTPA was decreased 1.8-fold and SFTPD was increased 3.7-fold (Figure 4B). To determine the amount of SFTPB protein that was associated with lamellar bodies (LBs), LBs were isolated from the lung tissue of Gpr116Δexon17 and WT mice, and mature SFTPB protein was quantitated. When normalized to total protein content in the isolated LBs, mature SFTPB protein was increased 1.6-fold in Gpr116Δexon17 LBs compared with WT LBs (Figure 4C). Taken together, these data demonstrate that the hydrophobic surfactant proteins SFTPB and SFTPC were increased in the BALF of Gpr116Δexon17 mice, although not to the same extent as SatPC levels, and that SFTPA and SFTPD levels were selectively altered in the lung tissue of Gpr116Δexon17 mice.
Histopathology was performed on lung tissue of 4- and 20-week-old Gpr116Δexon17 mice to determine the effects of accumulated surfactant on lung structure. The distal airspaces of Gpr116Δexon17 lungs were significantly enlarged at 4 and 20 weeks of age compared with WT control mice (Figures 5A and 5B). The airspace enlargement was associated with the presence of lipid-laden macrophages in 20-week-old Gpr116Δexon17 lungs, consistent with the large increase of surfactant phospholipids observed at this age (Figure 5A, asterisk; Figure E4B). BAL cell counts and differentials on 4- and 20-week-old animals demonstrated a progressive increase in the total number of macrophages and neutrophils in Gpr116Δexon17 lungs (Figure E4C). In addition, a proportion of 20-week-old Gpr116Δexon17 animals demonstrated focal clusters of lymphoid cells localized primarily to subpleural regions of the lung (Figure 5B, arrow), the majority of which were identified as B220/CD45R+ B cells (Figure E4D). Total protein content, as measured by bicinchoninic acid assay, was increased 2.6-fold in Gpr116Δexon17 BALF compared with WT control mice at 4 weeks of age (53.4 ± 1.7 versus 20.9 ± 3.9 μg total protein/body weight, respectively); total protein levels of postlavaged lung homogenate were not statistically different between the two genotypes (data not shown). BALF and lung homogenates from 20-week-old Gpr116Δexon17 mice failed to grow bacterial colonies on blood agar plates, indicating the absence of bacterial infection (data not shown). Ultrastructural analysis of secreted surfactant in the alveoli of Gpr116Δexon17 mice showed an abundance of aggregated lipid whorls compared with the prototypical “loose” ultrastructure of surfactant seen in the WT lung (Figure 6, middle panel versus left panel). In addition, Gpr116Δexon17 airspaces contained compact, electron-dense surfactant particles that contained tubular myelin structures (Figure 6, far right panel). Taken together, these data demonstrate an association between surfactant phospholipid accumulation, abnormal ultrastructure of alveolar surfactant, lymphocytic and neutrophilic inflammation, and altered alveolar architecture in Gpr116Δexon17 mice at 20 weeks of age, suggesting that chronic surfactant overload in these animals induces pathophysiological alterations in lung structure and function.
Due to the marked accumulation of surfactant in Gpr116Δexon17 mice associated with increased SatPC synthesis, we hypothesized that GPR116 primarily regulates surfactant phospholipid synthesis genes at the transcriptional level. To test this hypothesis using an unbiased approach, we compared the transcriptomes of purified type II cells from 4-week-old Gpr116Δexon17 and WT control mice by RNA microarray analysis (n = 3 mice per genotype). Data from this experiment has been submitted to Gene Expression Omnibus (accession number GSE41417). Very few transcriptional changes were detected in Gpr116Δexon17 type II cells, including differences in genes required for surfactant phospholipid synthesis (Figure E5). In fact, several phospholipid synthesis genes were significantly decreased in Gpr116Δexon17 type II cells, including Acox2, Chpt1, and Slc2a1 (Figure E4). Levels of Il-4 and Csf2 (also known as Gmcsf), genes that are known to alter surfactant homeostasis (26, 31), were comparable in Gpr116Δexon17 and control mice, as were mRNA levels of all of the surfactant proteins (Figure E5 and data not shown). However, while analyzing the data for genes related to surfactant secretion, we found that the purinergic receptor, P2RY2, was increased 2.1-fold in Gpr116Δexon17 type II cells. Induction of P2RY2 mRNA was confirmed by qPCR in purified type II cells and in the lung homogenate of Gpr116Δexon17 mice (Figures 7A and 7B). P2RY2 was significantly increased in Gpr116Δexon17 lung tissue at E18.5, a time point at which SatPC levels were normal (Figure 2A). Increased P2RY2 mRNA expression correlated with a 1.9-fold increase in P2RY2 protein in whole lung homogenate of 4-week-old Gpr116Δexon17 mice (Figures 7C and 7D).
The goal of this study was to define the role of GPR116 in pulmonary surfactant homeostasis. Our data show that expression of GPR116 in the murine lung was developmentally regulated, reaching maximal levels 1 day after birth, with sustained expression through adulthood. We further demonstrated that GPR116 protein was highly expressed on the apical membrane of alveolar type I and type II epithelial cells and on cilia of proximal epithelia but is not expressed on alveolar macrophages or pulmonary endothelial cells. GPR116 loss-of-function mice developed marked increases in alveolar surfactant phospholipids and proteins that began postnatally and progressed with age. Increased surfactant in Gpr116Δexon17 animals was associated with augmented SatPC synthesis and an increased SatPC alveolar-tissue index in juvenile animals, the presence of inflammatory infiltrates (including lipid-laden macrophages), and alveolar enlargement. Microarray analyses demonstrated that surfactant phospholipid synthesis genes were not induced in purified Gpr116Δexon17 type II cells, suggesting that the principle mechanism by which GPR116 regulates surfactant pool size is not at the level of transcription of surfactant synthesis genes. However, we found that P2RY2, a GPCR known to mediate surfactant secretion (5, 6), was significantly increased at the mRNA and protein level in 4-week-old Gpr116Δexon17 type II cells associated with increased SatPC levels in the media of cultured primary Gpr116Δexon17 type II cells. Collectively, these data demonstrate that GPR116 is a major regulator of surfactant homeostasis and are consistent with the hypothesis that GPR116 maintains alveolar surfactant pool sizes via control of the regulated secretory pathway in alveolar type II cells.
The targeting strategy used to engineer the Gpr116f/f allele was intended to fully ablate the function of GPR116 by eliminating all seven transmembrane domains of the protein upon Cre-mediated excision. Through initial characterization, we demonstrated that the Gpr116Δexon17 allele produces a protein that is low in abundance and mislocalized compared with WT GPR116 (Figure E3). Based on these data, the increased surfactant pool sizes in Gpr116Δexon17 mice could be due to loss-of-function of the WT protein or to gain-of-function of the GPR116Δexon17 protein. Two key pieces of data argue against a gain-of-function explanation for the observed phenotype. First, mice heterozygous for the Gpr116Δexon17 allele were healthy and fertile, had SatPC levels comparable to WT mice at 6 months of age, and lacked histological pulmonary abnormalities (data not shown). Second, the surfactant accumulation phenotype in Gpr116Δexon17 mice was identical to that reported (in abstract form) for a distinct GPR116 loss-of-function mouse in which exon1 was replaced with a lacZ reporter (32). These data strongly support the concept that the surfactant accumulation in Gpr116Δexon17 mice was due to a loss-of-function and demonstrate that the transmembrane domains of GPR116 are critically required for its function in vivo.
Similar to Gpr116Δexon17 mice, increased alveolar surfactant pool sizes were observed in Sftpd−/− and Csf2−/− mice (33–35). In addition, mutations in the α chain of the CSF2 receptor, CSF2R (36, 37), or autoantibodies against CSF2 itself (38) cause primary pulmonary alveolar lipoproteinosis in humans. Detailed analyses of surfactant metabolism in vivo in Sftpd−/− and Csf2−/− mice have shown that neither surfactant synthesis nor secretion was increased in these models (13, 26, 39). Rather, the defect was attributed to defective uptake and/or catabolism of surfactant by type II cells and alveolar macrophages due to altered surfactant ultrastructure (Sftpd−/− ) or impaired macrophage differentiation (Csf2−/− ). In contrast, SatPC synthesis and the SatPC alveolar-tissue SatPC index were significantly increased in 4-week-old Gpr116Δexon17 mice. These increases occurred before the abundant accumulation of lipid-laden macrophages observed in older animals, suggesting that the primary defect is due to surfactant hypersecretion rather than to defective surfactant catabolism. Furthermore, Gpr116 is not detectable on alveolar macrophages, and P2RY2, a receptor known to stimulate surfactant secretion in an ATP/UTP-dependent manner (6, 10), was increased in Gpr116Δexon17 type II cells associated with increased SatPC levels in the media of cultured Gpr116Δexon17 type II cells. Mouse models of the human disease Hermansky-Pudlak syndrome also demonstrate altered surfactant homeostasis. However, unlike Gpr116Δexon17, Sftpd−/−, and Csf2−/− mice, mice with mutations in Hermansky-Pudlak syndrome–associated genes exhibit decreased alveolar pools of surfactant phospholipids and increased tissue phospholipid levels due to defects in surfactant trafficking and decreased secretion in type II cells (42–44). Taken together, our data support the hypothesis that GPR116 controls alveolar surfactant pool sizes by negatively regulating surfactant secretion from type II cells.
Although GPR116 expression is highest in the lung, GPR116 mRNA is detected in multiple organs, including adipose tissue (Figure 1A). Recent data reported by Nie and colleagues implicate a role for GPR116 in adipocyte differentiation and energy homeostasis. In this study, knockdown of GPR116 in 3T3-L1 cells resulted in impaired differentiation into adipocytes in vitro, and adipocyte-specific ablation of GPR116 function in vivo resulted in increased susceptibility to high-fat diet–induced glucose intolerance and insulin resistance (45). It is unknown if GPR116 ablation in adipocytes or other GPR116-expressing tissues contributed to altered surfactant homeostasis in Gpr116Δexon17 mice.
The endogenous ligand and intracellular pathways by which GPR116 signals to control surfactant homeostasis are unknown. Expression of GPR116 on the apical surface of type I and type II epithelial cells (Figure 1D and Ref. 28) suggests that the ligand resides in the alveolar subphase fluid and/or is a component of pulmonary surfactant. Our microarray analysis demonstrated that P2RY2 expression is increased in the absence of GPR116 function. Therefore, one possible mechanism by which GPR116 functions is by regulating P2RY2 expression to control surfactant secretion. Alternatively, because GPCRs of the melatonin receptor subfamily (46) and human cytomegalovirus encoded GPCRs (47) are known to functionally inhibit each other via heterodimerization, GPR116 may also negatively control P2RY2 activity or the activity of other GPCRs involved in surfactant secretion, including β2AR or A2B, via heterodimerization to effect secretion. P2ry2−/− mice do not display an overt pulmonary phenotype in the absence of challenge, indicating that P2RY2 is dispensable for baseline pulmonary function (48). However, these data do not preclude the possibility that increased P2RY2 expression and/or activity is driving surfactant accumulation in Gpr116Δexon17 mice. It is also possible that GPR116 controls surfactant homeostasis independent of surfactant secretion by positively regulating surfactant uptake and catabolism after ligand engagement directly within type II cells or indirectly through alveolar macrophages.
It is unclear whether type I– or type II–specific expression of GPR116, or expression on both cell types, is required for surfactant homeostasis. Experimental evidence supports a role for type I/type II cell crosstalk in regulating surfactant secretion. Calcium imaging studies in an isolated rat lung model demonstrated that alveolar expansion rapidly increased intracellular calcium levels in type I and type II cells, triggering surfactant secretion (49). Inhalation-induced surfactant exocytosis in this model was inhibited by intracellular calcium chelation and by functional blockade of intracellular gap junctions, demonstrating a role for type I/type II cell communication in regulated secretion. Furthermore, mechanical stretch of cocultured rat “type I–like” cells (transdifferentiated from type II cells in culture) and type II cells resulted in type I–mediated ATP release that stimulated surfactant secretion from type II cells (50). Recent data using similar coculture techniques demonstrated that activation of a type I–specific purinergic receptor, P2RX7, was sufficient to release ATP from type I cells, triggering P2RY2-dependent surfactant release from type II cells (51).
Collectively, our data support a working model in which GPR116 is highly induced during the later stages of lung development and is poised for activation in the perinatal lung. After the burst of surfactant secretion from type II cells that promotes the transition to air breathing at birth, a newly presented ligand in the alveolar space binds to the extracellular domain of GPR116 on alveolar epithelial cells, activating downstream signaling pathways to regulate surfactant secretion via suppression of P2RY2 expression and/or activity or to regulate surfactant uptake and degradation by type II cells and/or macrophages to maintain alveolar surfactant pool sizes at homeostatic levels. The long-term goal of this work is to identify potential therapeutic targets, including GPR116 itself, for modulating endogenous surfactant levels in the treatment of lung diseases associated with surfactant dysfunction.
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This work was supported by divisional start-up funds, a Perinatal Institute Pilot and Feasibility grant, and a Trustee grant from Cincinnati Children’s Hospital Medical Center (J.P.B.) and by Mational Institutes of Health grants HL110964 (J.A.W.) and HL105433 (Y.X.).
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2012-0439OC on April 5, 2013