Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling

Abstract

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

Keywords: Cell Biology; Pulmonology.

Figure 1. Epithelial deletion of GPR116 alters surfactant homeostasis in mice.

(A) Saturated phosphatidylcholine (SatPC) levels in bronchoalveolar lavage fluid from 4-week-old Gpr116^–/– mice, epithelial-specific GPR116 loss-of-function mice (ShhCre:Gpr116^f/f), AT2 cell–specific GPR116 loss-of-function mice (SftpcCreER:Gpr116^f/f), endothelial-specific GPR116 loss-of-function mice (Tie2Cre:Gpr116^f/f), and control mice (CON, littermate controls for each genotype). SftpcCreER:Gpr116^f/f mice were placed on tamoxifen chow for 7 days. Data represent 3 independent experiments, with n = 3–4 mice per group. (B) In vitro basal phospholipid secretion assays of primary WT and Gpr116^–/– AT2 cells. Secretion was measured as SatPC content in media after a 3-hour incubation time divided by SatPC content in cell lysate (n = 2 individual experiments, 2–3 biological replicates per group). (C) In vitro phospholipid secretion assays in the absence (unstim) and presence (stim) of ATP stimulation (n = 2 individual experiments, 2–3 biological replicates per group). (D) Surfactant uptake assays in primary CON and Gpr116^–/– type II cells (n = 2 individual experiments, 2–3 biological replicates per group). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA for A, C, and D; unpaired t test for B).

Figure 2. GPR116 CTF activates G_q/11 signaling.

(A) Model of FLAG-tagged GPR116 CTF protein in plasma membrane. (B) Confocal image of GPR116 CTF-FLAG protein in HEK cells 12 hours after transfection. Original magnification, ×100. (C–E) Inositol phosphate (IP) conversion assays of HEK cells transiently transfected with empty vector (EV), GPR116 WT, or CTF plasmids. (C) CTF expression resulted in dose-dependent IP conversion (100 ng, 250 ng, 750 ng GPR116 CTF-FLAG plasmid/12-well plate; n = 3 independent experiments, 2 biological replicates per group). (D) Pretreatment with U73122 (10 μM) or YM-254890 (10 nM) 1 hour prior to assay attenuated or completely inhibited CTF-induced IP conversion, respectively; pretreatment with pertussis toxin (PTX, 100 ng/ml) 18 hours prior to assay had no effect on CTF-induced IP conversion (n = 3 independent experiments, 2 biological replicates per group). (E) Coexpression of EV or GPR116 CTF with WT Gnaq (Gq wt) or dominant-negative Gnaq (dn Gq; G209L,D277N) potentiated or inhibited CTF-induced IP conversion, respectively (n = 3 independent experiments, 2 biological replicates per group). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA for C–E).

Figure 3. Peptide-induced activation of GPR116.

(A) Model of the full-length GPR116 protein with the GAP16 peptide corresponding to the amino acids of the CTF ectodomain and scrambled (SCR) peptide sequence. (B) Membrane localization of transfected V5-tagged GPR116 in HEK cells (inset, green = V5-tagged GPR116, blue = DAPI-stained nuclei; original magnification, ×60) and IP conversion assays. GAP16 treatment resulted in dose-dependent IP conversion that was inhibited by cotreatment with the Gq inhibitor YM-254890 (100 nM) (n = 3 independent experiments, 2 biological replicates per group). (C) Representative calcium transient traces of GAP16-stimulated GPR116-V5 stably transfected HEK cells. Ionomycin was added as a positive control; overlaying control lines in black include SCR peptide (50 μM, 100 μM, and 250 μM) and vehicle (n = 3 independent experiments, 3 biological replicates per group). (D) Quantification of calcium transient data (n = 4 independent experiments, 3 biological replicates per group). (E) Calcium transients of GAP16-stimulated (250 μM), GPR116-V5 stably transfected HEK cells pretreated with the G_q inhibitor YM-254890 (YM) 2 hours prior to stimulation (n = 3 independent experiments, 2 biological replicates per group). Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA for B and D).

Figure 4. GAP16 activation of GPR116 in isolated mouse primary alveolar type II cells.

Fluo-4 imaging–based calcium transient assays in adult WT and Gpr116^–/– primary mouse alveolar type II (AT2) cells. (A) GAP16 or SCR (250 μM) were added approximately 120 seconds after start of imaging. Representative images for WT and Gpr116^–/– cells at start of imaging and 170 seconds after peptide addition (scale bar: 10 μm for all images). Representative traces (B) and quantitation of peak calcium responses (C) from imaging data in WT or Gpr116^–/– adult primary mouse AT2 epithelial cells. Data represent 5 independent experiments for WT cells, 3 independent experiments for Gpr116^–/– cells, and 3 biological replicates per group. (D) Peak calcium responses in WT or Gpr116^–/– cells stimulated with ATP (50 μM). Data represent 1 experiment, 3–4 biological replicates per group. Data are expressed as mean ± SD. ****P < 0.0001 (1-way ANOVA for C, unpaired t test for D).

Figure 5. Characterization of GPR116 CTF ectodomain amino acids involved in receptor activation.

(A) Diagram of amino acid residues in the ectodomain of GPR116 CTF and alanine mutant constructs generated to identify residues required for IP conversion activity. (B) Representative Western analysis of GPR116 CTF alanine mutants compared with WT GPR116-FLAG. The graph represents quantitative data of Western blot analyses for n = 3 experiments. (C) IP conversion assays in transiently transfected HEK cells. Data represent 4 independent experiments, 2 biological replicates per group. (D) Activity of truncated activating peptides. HEK cells stably expressing GPR116 (cell line 3C) were stimulated with GAP16 as a reference and C-terminally truncated peptides (GAP15-GAP7). A representative graph from 3 independent calcium transient experiments is shown; mean EC₅₀ and control data are shown in Supplemental Figure 2. Data are expressed as mean ± SD. **P < 0.01, ****P < 0.0001 (1-way ANOVA for B and C).

Figure 6. GAP16-induced suppression of surfactant phospholipid secretion and alveolar pool sizes in vivo.

(A) Basal phospholipid secretion assays in GAP16-treated primary rat AT2 epithelial cells. (B–D) In vivo secretion assays. Representative in vivo fluorescent images (B) of lysotracker green–loaded AT2 cells in intact WT mouse alveoli injected with SCR16 or GAP16 peptide (100 μM). Images represent preinflation and 5 and 10 minutes after 30-second hyperinflation. Scale bar: 5 μm for all images. (C) Traces of lysotracker fluorescence as a function of time, before and after hyperinflation. (D) Quantitated data from in vivo secretion assays, as performed in C. Data represent n = 3 lungs per group, a total of 9 type AT2 cells analyzed per group. (E) SatPC levels in bronchoalveolar lavage fluid from 4-week-old SftpcCreER:Gnaq^f/f:Gna11^–/– mice. Mice were placed on tamoxifen chow for 4 weeks prior to harvest (n = 5 mice per group, n = 1 experiment). CON, littermate controls. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 (1-way ANOVA for C–E).

Figure 7. Expression and activation of human GPR116 in transfected and primary human alveolar epithelial cells.

(A) RT-PCR of full-length GPR116 mRNA from two primary human lung tissue samples. (B) Western analysis of HEK293 cell lysates stably transfected with V5-tagged human GPR116 cDNA (A6) versus nontransfected cells. (C) Confocal image of A6 cells stained with anti-V5 antibody (green) and DAPI (blue). Original magnification, ×60. (D) Representative traces of FLIPR-based calcium mobilization assay in the A6 hGPR116-V5 cell line. Cells were stimulated with 250 μM or 500 μM GAP16 (top and bottom red trace, respectively), 250 μM or 500 μM SCR control, or 1 μM ionomycin. The arrow indicates time of compound addition. (E and F) Calcium transient tracings (E) and quantitation of Fluo-4–based imaging–based calcium transient assays (F) in primary human AT2 epithelial cells. Cells were stimulated with 500 μM GAP10 or SCR10 peptide as indicated. Data are expressed as mean ± SD (1-way ANOVA for C–E).

Figure 8. GPR116 regulates cortical actin assembly and barrier function.

(A) Increased cortical F-actin staining in HEK293 cells transiently transfected with GPR116 CTF/mCherry for 48 hours compared with mCherry-negative control cells (red = GPR116 CTF/mCherry, green = phalloidin, blue = DAPI). Original magnification, ×60. (B) Increased cortical actin staining in GAP14-treated HEK293 cells stably expressing GPR116 (cell line 3C) compared with cells treated with SCR14 peptide (green = phalloidin, blue = DAPI). Scale bar: 50 μm for large images and insets; insets are magnified images of regions denoted by dashed boxes). (C) Representative cell impedance traces as a function of time for HEK cells stably expressing mGPR116 treated with mGAP14, scrambled peptide (SCR14), or thrombin as a positive control for Gq signaling. (D) Cell impedance measurements in HEK cells stably expressing mGPR116 following stimulation with activating peptides (GAP10, -12, -14, and -16) compared with cells treated with SCR16. Data are expressed as mean ± SD (1-way ANOVA for C and D).

Figure 9. Model of GPR116-mediated modulation of surfactant homeostasis in alveolar type II cells.

Present data support a model in which full-length GPR116 is held in an inactive state in the absence of ligand binding to the NTF. Subsequent ligand binding to the NTF fragment, perhaps present in the alveolar lumen, or cell stretch induced by the ventilator cycle induces a conformational change, or complete disengagement of the NTF from the CTF, that permits interaction of the activating CTF ectodomain with the 7TM domains, resulting in receptor activation, G_q/11 coupling, actin rearrangement, and decreased surfactant secretion/increased surfactant uptake. Activating peptides, such as GAP16, bypass the inhibitory effect of the NTF, resulting in receptor activation.