GnRH-(1–5) was purchased from Bachem (Torrance, CA, USA) and reconstituted to 10 mM in distilled water and stored in 12 µL aliquots at −80°C. GN11 cells (18) generously donated by Dr. Sally Radovick (Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech Inc., Herndon, VA, USA) without antibiotics and supplemented with 7% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 3% newborn calf serum (Hyclone), 25 mM glucose, and 5 mM l-glutamine (7, 11, 19). Cells were maintained at 37°C in an atmosphere with 5% CO₂. In addition, all experiments were conducted at 37°C in an atmosphere with 5% CO₂. All GN11 cells used for experimentation were below passage 25. GN11 cells originated from an olfactory tumor extracted from a male mouse (18). The antibodies used in this study were specific for the TGF-βRI (1:1,000; Cat. No. SC-9048, Santa Cruz Biotechnology, Dallas, TX, USA), TGF-βRII (1:1,000; Cat. No. SC-400, Santa Cruz Biotechnology), GAPDH (1:2,000; Cat. No. SC-25778, Santa Cruz Biotechnology), and anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000; Cat. No. 170-6515, Bio-Rad, Hercules, CA, USA).

RNA extracted from a mouse nasal region was obtained at embryonic day (ED) 12.5. This sample was generously donated by Dr. Susan Wray (National Institutes of Health, Bethesda, MD, USA) and described previously by us for detection of GPR173 (7) and by Wray and colleagues (20). All experimental procedures were conducted in accordance with the National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, revised 1996) and were approved by the Uniformed Services University of the Health Sciences Institutional Animal Care and Use Committee.

GN11 cells were plated on six-well plates and allowed to grow until 80% confluency. Subsequently, the media were changed to serum-free media and starved for at least 24 h prior to treatments. Cells were treated with or without 100 nM GnRH-(1–5) in 10% charcoal-stripped FBS (volume of 700 μL/well) for 5 min or 24 h. At the end of each experiment, the cells were placed on ice and the conditioned media collected and immediately frozen at −80°C for future analysis of both chemokine and TGF-β content. Cell lysates were also collected to determine protein concentration using a Pierce protein BCA assay (Thermo Fisher; Waltham, MA, USA). The chemokines CXCL1, CXCL2, CXCL9, CXCL10, CCL3, CCL4, CCL5, and MCSF were measured by the multiplex assay Milliplex Mouse Chemokine Panel (Cat. # MPXMCYTO-70K; EMD Millipore, Billerica, MA, USA) according to manufacturer’s instructions. The levels of TGF-β1, 2, and 3 were measured by the multiplex assay Bio-Rad Pro TGF-β Assay 3-plex (Cat. # 171W4001M; Hercules, CA, USA) according to the provided protocol. All multiplexing assays were performed using the Bio-Plex 200 Luminex assay system (Bio-Rad; Hercules, CA, USA).

The conditioned media of GN11 cells treated with ±100 nM GnRH-(1–5) for 5 min or 24 h were used to measure bioactive TGF-β using a mink lung epithelial cell (MLEC) line stably expressing the TGF-β-responsive plasminogen activator-1 luciferase reporter (21, 22). Briefly, MLECs were plated on 24-well plates and allowed to grow until 80% confluent in DMEM supplemented with 10% FBS and 80 µg/mL G418 (Sigma; St. Louis, MO, USA). Subsequently, the media were changed to serum-free DMEM supplemented with 0.1% insulin/transferrin/sodium selenite (Cat. # 354352; Corning, Corning, NY, USA), and the MLECs were allowed to incubate overnight. The following day MLECs were treated with 20 µL of conditioned media harvested from treated GN11 cells. After 16 h, MLECs were lysed with Promega Passive Lysis Buffer (Madison, WI, USA) followed by the addition of the luciferase substrate. Samples were subsequently analyzed using a Synergy H1 Hybrid Plate Reader (BioTek; Winooski, VT, USA), and values were expressed as a percentage change relative to VEH-treated GN11 cells. Each treatment condition was assayed in three replicates, and the experiments were performed at least five times.

Analysis of genes involved in ECM remodeling in GN11 cells was performed using a pre-designed PrimePCR Pathway Plate (Cat. No. 10029332; Bio-Rad). Total RNA was extracted using TRIzol reagent (Invitrogen) 2 h post treatment with GnRH-(1–5) and purified using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. A total of 2 µg of RNA were reverse transcribed using the iScript Advanced cDNA Synthesis kit (Bio-Rad). SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and cDNA were added to each well up to a total 20 µL according to manufacturer’s instructions. All samples were assayed using the CFX Connect Real-time System (Bio-Rad). The cycling conditions were performed as follows: initial denaturation and enzyme activation at 95°C for 2 min followed by 40 cycles of denaturation (95°C, 5 s), annealing, and reading (60°C, 30 s). Melt curve analysis was performed to ensure the presence of a single amplicon.

Droplet digital PCR relies on water-emulsion technology to form ~20,000 nL-sized droplets for absolute quantitation of gene expression. The generation of droplets for our experiments was performed using the Automated Droplet Generator (AutoDG; Bio-Rad). Each sample was prepared using 2x QX200 ddPCR EvaGreen Supermix (Bio-Rad), forward and reverse primers (Table 1; final concentration of 200 nM), template (variable volume; 200 pg cDNA), and ultra-pure H₂O for a final volume of 22 µL. Samples were loaded onto a 96-well PCR plate (Eppendorf, Hamburg, Germany), which was then loaded into the AutoDG instrument for droplet generation. Generated droplets were transferred by the AutoDG to a new 96-well PCR plate which was then heat-sealed with a pierceable foil using a PX1 PCR Plate Sealer (Bio-Rad). The plate was loaded into a conventional thermal cycler (C1000 Touch, Bio-Rad), and the following thermal cycling conditions were used: initial denaturation and enzyme activation at 95°C for 5 min, 40 cycles of denaturation (95°C, 30 s) and annealing/extension (55°C, 1 min), and final signal stabilization steps at 4°C (5 min) and then 90°C (5 min). After thermal cycling, the 96-well plate was loaded into the QX200 Droplet Reader (Bio-Rad), and the droplets were automatically aspirated and read. All data were analyzed using the provided analysis software (QuantaSoft, Bio-Rad).

Total protein lysates were harvested from GN11 cells with O’dell’s lysis buffer [10 mM EGTA, 10 mM EDTA, 80 µM Na₂MoO₄, 5 mM NaPO₄, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 4 mM p-nitrophenyl phosphate, 1% Triton X-100, and Sigma Inhibitor Cocktail I, II, and Roche Complete Protease Inhibitor (Roche; Indianapolis, IN, USA)]. Cell lysates were briefly sonicated, and the supernatant (30 µg) was subjected to SDS-PAGE (4–12%, Lonza) after centrifugation (20,000 × g for 20 min at 4°C). After electrophoresis, the gel was transferred onto a polyvinyl difluoride membrane using a Trans-Blot Turbo Transfer System (BioRad) and incubated in 5% non-fat dry milk at room temperature for 1 h to block non-specific antibody binding. The membrane was then rinsed with TBS-Tween (TBST) and incubated with the primary antibody overnight at 4°C. The membrane was subsequently rinsed 3 × 10 min with TBST, incubated with the secondary antibody at an appropriate dilution conjugated to horseradish peroxidase (Bio-Rad) for 1 h at room temperature, then rinsed 5 × 15 min with TBST. The blots were visualized by chemiluminescence (Millipore, Billerica, MA, USA) using a Fujifilm LAS-3000 imager (Fujifilm; Stamford, CT, USA).

The effect of GnRH-(1–5) on matrix metalloproteinase (MMP)-2 and 9 activity in conditioned medium was determined by zymography (23–27). Serum-starved GN11 cells in six-well plates (at 80% confluency) were treated with 700 µL of ±100 nM GnRH-(1–5) in 10% charcoal-stripped FBS. After 1 h, 25 µL of the conditioned media was diluted 1:1 with sample buffer (Bio-Rad) and subjected to electrophoresis in 10% gelatin polyacrylamide gels (Bio-Rad) under non-reducing conditions. Proteins within the gel were renatured by removing the SDS via washing for 30 min in 2.5% Triton X-100. Gels were developed in collagenase buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl₂, and 0.02% Brij-35] for 72 h and stained with 0.5% Coomassie blue R-250 (Bio-Rad). After destaining gels in 30% methanol and 10% acetic acid, areas of digestion were digitized using the Fujifilm LAS-3000 imager (Fujifilm; Stamford, CT, USA). MMP activity is represented by clear bands and quantified with Fujifilm Image Gauge Software (Valhalla, NY, USA).

The invasion assay was conducted using transwell inserts with 8 µm pores coated with a reduced growth factor matrigel (BD Biosciences; Palo Alto, CA, USA). The matrigel is a proteinaceous mixture composed of collagen IV, laminin, entactin, and proteoglycan to resemble the constituents of the ECM. Cells exhibiting a more invasive and migratory phenotype will be better able to digest the matrigel to traverse insert. In our model, GN11 cells were grown in six-well plates until 80% confluent and allowed to serum-starve for at least 24 h. Subsequently, the cells were harvested with dissociation buffer (2 mM EDTA diluted in 0.1 M phosphate-buffered saline). The cells were briefly centrifuged (5 min at 500 g) to remove the dissociation buffer and resuspended in 2 mL of DMEM per well of a six-well plate. A total of 250 µL of cell suspension was added to the upper chamber of the transwell assay followed by 250 µL of DMEM with or without 200 nM GnRH-(1–5) (final concentration of 100 nM). The lower chamber contained 750 µL of 10% charcoal-stripped FBS as a chemoattractant. Chambers were subsequently maintained at 37°C in an atmosphere with 5% CO₂. After 24 h, cells that had not migrated through the matrigel-coated inserts were gently removed using cotton swabs, and the invading cells on the underside of the membrane were fixed and stained with 0.5% crystal violet in 10% buffered paraformaldehyde. Stained GN11 cells were imaged at 20× with the Leica AF6000 microscope and counted using Image J (NIH, Bethesda, MD, USA). Multiple images were taken of each insert and averaged. Values were expressed as a percentage change relative to VEH (24, 28, 29). Each condition was assayed in duplicate and represents at least three independent experiments.

GN11 cells were plated at 20% confluency and transfected with a cocktail of siRNA (25 nM) specific to GPR173 (SMARTpool; Cat. No. L-059639-00-0005, Dharmacon, Lafayette, CO, USA). Control cells were transfected with a non-targeting siRNA mixture (25 nM) (SMARTpool; Cat. No. D-001810-10-05, Dharmacon). Dharmafect4 (Dharmacon) was used as the transfection reagent. All treatments were done in 10% charcoal-stripped dextran-treated FBS (Atlanta Biologicals; Lawrenceville, GA, USA). Cells were incubated with the siRNA treatments for 48 h and then serum-starved for at least 24 h prior to experimentation.

The efficiency of GPR173 silencing by siRNA was measured in GN11 cells by qPCR. Total RNA was harvested 48 h post-transfection with siRNA as described above. A total of 2 µg of RNA was reverse transcribed using the Maxima First Strand cDNA synthesis kit (Fermentas; Glen Burnie, MD, USA), and the cDNA was analyzed by qPCR using the FAST SYBR Green Mastermix (Cat. No. 330609, Qiagen, Valencia, CA, USA) using 200 nM of the appropriate primer pair. All samples were assayed in duplicate using the CFX Connect Real-time System (Bio-Rad; Hercules, CA, USA). The qPCR conditions were performed as follows: initial denaturation and enzyme activation at 95°C for 10 min followed by 40 cycles of denaturation (95°C, 15 s), annealing, and reading (60°C, 30 s). Melt curve analyses was conducted after each qPCR reaction to demonstrate the presence of a single amplicon. Additionally, qPCR products were visualized by agarose gel electrophoresis (1%) using the GelStar Nucleic Acid Gel Stain (Lonza; Rockland, ME, USA) under UV light and verified by sequencing. Primers specific to GPR173 and the reference gene Cyclophilin A are shown in Table 1. qPCR conditions were optimized to produce a greater than 95% efficiency for both primer pairs as determined by a 10-fold serial dilution of cDNA (29). The delta delta Ct method was used to measure fold change in GPR173 expression relative to levels obtained from VEH-treated cells. This experiment was repeated over three consecutive passages.

Statistics were conducted using the analysis software GraphPad Prism 6 (GraphPad Software, Inc.; La Jolla, CA, USA). For most experiments, normalized data were analyzed using a paired t-test to determine significance. Studies using siRNA, data were analyzed by a one-way ANOVA followed by Fisher’s least significant difference post hoc test. ddPCR data were presented as target copy number/TATA box binding protein copy number and analyzed by a non-parametric paired-sample Wilcoxon test. A value of p < 0.05 was considered significant.

To determine the effect of GnRH-(1–5) on GN11 cellular invasion, a transwell migratory assay was implemented where the inserts were coated with and without a matrigel. The matrigel is a proteinaceous mixture of ECM proteins that mimics the extracellular environment. GN11 cells treated with 100 nM GnRH-(1–5) had no effect in empty inserts but significantly inhibited the ability of GN11 cells to traverse the matrigel environment (Figures 1A,B). Subsequently, we determined whether the effect of GnRH-(1–5) on invasion was dependent on GPR173 using a siRNA approach. The expression of GPR173 was decreased by GPR173-specific siRNA, and the effect of GnRH-(1–5) on GN11 cellular invasion was assessed. GnRH-(1–5) treatment significantly inhibited the cellular invasion of GN11 cells, which was reversed in cells pre-treated with GPR173-specific siRNA (Figures 2A,B). GPR173 downregulation was confirmed by qPCR demonstrating greater than 60% knockdown (Figure 2C).

Chemokines are secreted factors that are implicated in the regulation of cellular migration and invasion, including the migratory response of GN11 cells (30). Therefore, we measured chemokine release using a multiplex approach to quantify multiple chemokines from the same sample. GN11 cells were treated with 100 nM GnRH-(1–5) for 24 h, and the conditioned media were collected for analysis. GnRH-(1–5) treatment had no significant effect on the release of CXCL1, CXCL2, CXCL9, CXCL10, CCL3, CCL4, CCL5, and MCSF (Figure 3A).

Since there were no differences in chemokine release in response to GnRH-(1–5), we measured the levels of TGF-β release. TGF-β signaling has been implicated in the development of the CNS including the regulation of neuronal migration (31). To measure all TGF-β isoforms, we used a multiplex approach that can quantitate the levels of TGF-β1, 2, and 3. GN11 cells significantly produce more TGF-β1 than TGF-β2 and TGF-β3 (Figure 3B). Furthermore, GN11 cells treated with 100 nM GnRH-(1–5) for 24 h had significantly less total TGF-β1 compared to the other two isoforms (Figure 3C). To determine whether the modest but significant decrease in TGF-β1 release was biologically effective in decreasing TGF-β signaling, we used the MLEC line that stably expresses the TGF-β-responsive plasminogen activator-1 luciferase reporter and has been previously described to measure bioactive TGF-β1 in the pM range (21, 22). MLECs were treated with conditioned media of GN11 cells that were treated with or without 100 nM GnRH-(1–5) for 5 min or 24 h. GnRH-(1–5) treatment significantly decreased activation of the reporter construct at 5 min, indicating that the levels of bioactive TGF-β were diminished (Figure 3D). In addition, there was a decrease at the longer time point of 24 h; however, this effect was not significant (Figure 3D). Subsequently, we sought to determine whether GN11 cells express the TGF-β receptors and whether there were changes in total receptor expression, which may contribute to the decrease in TGF-β signaling. We conducted time course studies (5 min, 30 min, and 24 h) in GN11 cells treated with and without 100 nM GnRH-(1–5) and measured TGF-βRI and TGF-βRII by Western blot. In addition, time course studies were conducted to determine changes in TGF-βRI and TGF-βRII levels in response to GnRH-(1–5) treatment. GN11 cells express both TGF-βRI and TGF-βRII, but neither receptor is significantly regulated by GnRH-(1–5) treatment (Figure 4A).

Matrix metalloproteinases, especially MMP-2, have been implicated in the proteolytic activation of TGF-β (32, 33). Therefore, we sought to determine whether GnRH-(1–5) inhibits MMP-2 or MMP-9 activity to decrease the availability of bioactive TGF-β. We measured MMP-2 and MMP-9 release and activity by gel zymography from conditioned media of GN11 cells treated with or without 100 nM GnRH-(1–5) for 1 h. No significant changes were observed with GnRH-(1–5) treatment (Figure 4B).

Since we did not see changes in MMP-2 or MMP-9 activity, it is likely that GnRH-(1–5) may regulate ECM remodelers involved in the activation of TGF-β. Thus, we implemented a high-throughput focused array assay that measures the expression of 43 genes implicated in ECM remodeling. GN11 cells were treated with or without 100 nM GnRH-(1–5) for 2 h, and subsequently the RNA was isolated and analyzed using the focused array. GN11 cells did not express any of the kallikrein family members, which are a group of serine proteases (data not shown). GnRH-(1–5) treatment modestly increased the levels of tissue inhibitors of metalloproteinase (TIMP) 1 and TIMP3 but not TIMP2 (Figure 5A). TIMPs are inhibitors of proteinase activity, which may play a role in regulating proteinases involved in the proteolytic cleavage of latent TGF-β to active TGF-β. Therefore, we confirmed TIMP1, 2, and 3 expressions in GN11 cells and nasal tissue extracted from a mouse at ED 12.5 (Figure 5B). Furthermore, statistical analysis was performed on ddPCR measuring copy numbers of TIMP1, 2, and 3 in GN11 cells in response to GnRH-(1–5) treatment to confirm the PCR array data. GnRH-(1–5) treatment significantly increased TIMP1 levels but not TIMP2 or TIMP3 (Figure 5C). Likewise, ddPCR was performed on nasal tissue from a mouse (ED12.5) to quantitate the relative abundance of each TIMP in this region (Figure 5D).