GnRH-(1-5) Inhibits TGF-β Signaling to Regulate the Migration of Immortalized Gonadotropin-Releasing Hormone Neurons

Abstract

Gonadotropin-releasing hormone (GnRH) neurons originate outside the central nervous system (CNS) in the nasal placode where their migration to the basal forebrain is dependent on the integration of multiple signaling cues during development. The proper migration and establishment of the GnRH neuronal population within the CNS are critical for normal pubertal onset and reproductive function. The endopeptidase EP24.15 is expressed along the migratory path of GnRH neurons and cleaves the full-length GnRH to generate the metabolite GnRH-(1-5). Using the GN11 cell model, which is considered a pre-migratory GnRH neuronal cell line, we demonstrated that GnRH-(1-5) inhibits cellular migration in a wound closure assay by binding the orphan G protein-coupled receptor 173 (GPR173). In our current experiments, we sought to utilize an in vitro migration assay that better reflects the external environment that migrating GnRH neurons are exposed to during development. Therefore, we used a transwell assay where the inserts were coated with or without a matrigel, a gelatinous mixture containing extracellular matrix (ECM) proteins, to mimic the extracellular environment. Interestingly, GnRH-(1-5) inhibited the ability of GN11 cells to migrate only through ECM mimetic and was dependent on GPR173. Furthermore, we found that GN11 cells secrete TGF-β1, 2, and 3 but only TGF-β1 release and signaling were inhibited by GnRH-(1-5). To identify potential mechanisms involved in the proteolytic activation of TGF-β, we measured a panel of genes implicated in ECM remodeling. We found that GnRH-(1-5) consistently increased tissue inhibitors of metalloproteinase 1 expression, which is an inhibitor of proteinase activity, leading to a decrease in bioactive TGF-β and subsequent signaling. These results suggest that GnRH-(1-5) activating GPR173 may modulate the response of migrating GnRH neurons to external cues present in the ECM environment via an autocrine-dependent mechanism involving TGF-β.

Keywords: EP24.15; G protein-coupled receptor 173; G protein-coupled receptors; gonadotropin-releasing hormone; migration.

Figure 1

Effect of GnRH-(1–5) on GN11 cellular invasion. The migration of GN11 cells was measured in response to GnRH-(1–5) using a transwell assay with inserts either coated with matrigel or not. (A) The left panel indicates representative photomicrographs of migrated GN11 cells treated with VEH or 100 nM GnRH-(1–5) for 24 h through uncoated inserts. The right panel is a quantification of the results and demonstrated that GnRH-(1–5) treatment did not have an effect on the migration of GN11 cells exposed to empty inserts. (B) The lower panels indicate that cells exposed to a matrigel environment and treated with 100 nM GnRH-(1–5) for 24 h had decreased invasion relative to respective VEH-treated cells. The results are averages from four independent experiments. Data were normalized to VEH-treated cells and expressed as a percentage change. A paired t-test was conducted to determine significance. Scale bar indicates 100 µm. *p < 0.05 relative to VEH.

Figure 2

Effect of G protein-coupled receptor 173 (GPR173) silencing on the GnRH-(1–5)-mediated inhibition of invasion. GPR173 was downregulated by siRNA in GN11 cells, and the effect of GnRH-(1–5) to regulate invasion was investigated. (A) Upper panel shows representative photomicrographs of migrated GN11 cells treated with the indicated siRNA conditions with and without 100 nM GnRH-(1–5). (B) Lower panel indicates the quantification of the invasion assay results. The effect of GnRH-(1–5) to inhibit invasion was blocked in cells exposed only to siRNA targeting GPR173 levels. (C) GN11 cells exposed to GPR173 siRNA had a greater than 60% reduction in GPR173 mRNA levels (n = 3). Migration assay data are representative of three independent experiments and were analyzed by a one-way ANOVA followed by a least significant difference post hoc test. Scale bar indicates 100 µm. *p < 0.05 relative to VEH.

Figure 3

Effect of GnRH-(1–5) on chemokine release and TGF-β signaling. (A) GN11 cells were treated with 100 nM GnRH-(1–5) for 24 h, and the media were collected to measure chemokine release. GnRH-(1–5) had no effect on the release of CXCL1, CXCL2, CXCL9, CXCL10, CCL3, CCL4, CCL5, and MCSF (N = 3). (B) We also measured TGF-β1, 2, and 3 and found that GN11 cells significantly secrete more TGF-β1 than the other isoforms (N = 3). (C) Next, we determined whether GnRH-(1–5) regulates TGF-β release. GN11 cells were treated with 100 nM GnRH-(1–5) for 24 h, and the media were measured for total TGF-β1, TGF-β2, and TGF-β3 release. GnRH-(1–5) treatment inhibited TGF-β1 release but not TGF-β2 and TGF-β3 (N = 4). (D) Similarly, using an established TGF-β bioassay to measure bioactive TGF-β, conditioned media collected from GN11 cells treated with GnRH-(1–5) for 5 min and 24 h had decreased TGF-β activity (N = 5–6). A paired t-test was performed to determine significance. *p < 0.05 relative to VEH.

Figure 4

Effect of GnRH-(1–5) on TGF-βR levels and matrix metalloproteinase (MMP) activity. (A) Time course studies were conducted to confirm TGF-βRI and TGF-βRII expression and whether GnRH-(1–5) treatment regulates receptor levels. Western blot analysis revealed the expression of both TGF-βRI and TGF-βRII in GN11 cells, but neither was regulated by GnRH-(1–5) treatments. (B) Subsequently, we measured MMP-2 and MMP-9 activity by gel zymography to determine whether the decrease observed in bioactive TGF-β is mediated by these proteinases. Conditioned media isolated from GN11 cells treated with or without 100 nM GnRH-(1–5) did not change the activity of MMP-2 and MMP-9. Data are representative of three independent experiments and were analyzed by a paired t-test.

Figure 5

Effect of GnRH-(1–5) on extracellular matrix (ECM) remodelers. (A) A focused array was implemented to determine changes in ECM remodelers in response to GnRH-(1–5). GN11 cells were treated with 100 nM GnRH-(1–5) for 2 h and the RNA isolated for these studies. GnRH-(1–5) treatment modestly increased the levels of tissue inhibitors of tissue inhibitors of metalloproteinase (TIMP) 1 and TIMP3 but not TIMP2 (N = 2). (B) TIMP1, 2, and 3 expression was confirmed by gel electrophoresis in GN11 cells and nasal tissue extracted from a mouse at embryonic day (ED) 12.5. A reaction in which the reverse transcriptase is absent was used as a negative control (−RT). (C) To confirm the PCR array results, droplet digital PCR (ddPCR) was performed to measure copy numbers of TIMP1, 2, and 3 in GN11 cells in response to GnRH-(1–5) treatment. GnRH-(1–5) treatment significantly increased TIMP1 but not TIMP2 or TIMP3 levels (N = 6). A non-parametric paired-sample Wilcoxon test was used to determine significance. The medians and interquartile ranges (error bars) are indicated for each group. (D) Likewise, ddPCR was performed on nasal tissue from a mouse (ED12.5) to quantitate the relative abundance of each TIMP in this region (N = 1).