AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling

Abstract

Background: Gonadotropin-releasing hormone (GnRH) receptors are essential for reproduction and are expressed in numerous urogenital, reproductive, and non-reproductive cancers. In addition to canonical G protein-coupled receptor signaling, GnRH receptors functionally interact with several receptor tyrosine kinases. AXL is a receptor tyrosine kinase expressed in numerous tissues as well as multiple tumors. Here we tested the hypothesis that AXL, along with its endogenous ligand Gas6, impacts GnRH receptor signaling.

Methods: We used clonal murine pituitary αT3-1 and LβT2 gonadotrope cell lines to examine the effect of AXL activation on GnRH receptor-dependent signaling outcomes. ELISA and immunofluorescence were used to observe AXL and GnRH receptor expression in αT3-1 and LβT2 cells, as well as in murine and human pituitary sections. We also used ELISA to measure changes in ERK phosphorylation, pro-MMP9 production, and release of LHβ. Digital droplet PCR was used to measure the abundance of Egr-1 transcripts. A transwell migration assay was used to measure αT3-1 and LβT2 migration responses to GnRH and AXL.

Results: We observed AXL, along with the GnRH receptor, expression in αT3-1 and LβT2 gonadotrope cell lines, as well as in murine and human pituitary sections. Consistent with a potentiating role of AXL, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LβT2 cells. Further, and consistent with enhanced post-transcriptional GnRH receptor responses, we found that Gas6 increased the abundance of Egr-1 transcripts. Suggesting functional significance, in LβT2 cells, Gas6/AXL signaling stimulated LHβ production and enhanced GnRH receptor-dependent generation of pro-MMP9 protein and promoted cell migration.

Conclusions: Altogether, these data describe a novel role for AXL as a modulator of GnRH receptor signaling. Video Abstract.

Keywords: Gas6; Matrix metalloproteinase 9; TAM receptor tyrosine kinase.

Fig. 1

AXL and GnRH receptor-like immunoreactivity in murine and human pituitaries. A Representative images showing AXL-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. B Frequency of overlapping AXL- and GnRH receptor-like immunoreactivity in a human pituitary; > 100 cells were analyzed from 7 different images for both AXL- and GnRH receptor-like immunoreactivity. C Representative images showing Gas6-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. D Representative images showing AXL-like (red) and Gas6-like (green) immunostaining in a human pituitary section. Cell nuclei (blue) blue are shown in the merged images. Images are representative of ≥ 3 paraffin sections; scale bars = 20 μm

Fig. 2

AXL receptor expression in murine gonadotrope cell lines. AXL-like immunoreactivity (A) and protein abundance (B) in αT3-1 and LβT2 murine gonadotrope cell lines and AXL protein abundance in control cells and cells incubated with GnRH (10 nM for 24 h; n = 3 independent cultures). Images representative of ≥ 3 αT3-1 and LβT2 cell cultures; n = number of independent experiments; numerical data presented as mean ± SEM; * P < 0.05

Fig. 3

Gas6-dependent AXL receptor activation enhances GnRH receptor signaling processes. A Proportion of phosphorylated ERK relative to total ERK (pERK/ERK) in αT3-1 and LβT2 cells under control conditions and after incubation with Gas6 (100 nM for 3 h); n = 3 for each condition. B Time course of Gas6-dependent changes (100 nM) in pERK/ERK in LβT2 cells (from left); effect of incubating LβT2 cells with GnRH (10 nM) and GnRH plus Gas6 (100 nM) for 5 min on pERK/ERK (at right); n = 3 for each condition. C Time course of Gas6-dependent (100 nM) changes on Egr-1 transcript abundance in αT3-1 and LβT2 cells; n = 3. D LHβ protein abundance in the culture media and lysates of LβT2 cells under control conditions and after 2 h incubations with Gas6 (100 nM) and GnRH (10 nM); n = 3. € LHβ protein abundance in LβT2 culture media under control conditions and after 2 h incubations with Gas6 (100 nM), GnRH (10 nM), the GnRH receptor antagonist cetrorelix (2 nM), the AXL receptor antagonist R428 (50 µM), and the MEK inhibitor U0126 (10 µM); n = 3 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; * P < 0.05

Fig. 4

AXL and GnRH receptor stimulation promotes pro-MMP9 release. Pro-MMP9 abundance in the culture media and lysates of αT3-1 (A) and LβT2 (B) cells following incubation with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, and the AXL receptor antagonist R428 (50 µM), the MEK inhibitor U0126 (10 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05

Fig. 5

AXL and GnRH receptor activation promotes migration of clonal gonadotropes. Transwell migratory responses (% cells) of αT3-1 (A) and LβT2 (B) cells following 48 h incubations with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, the MEK inhibitor U0126 (10 µM), and the MEK activator PAF C-16 (100 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05. C Working model of AXL and GnRH receptor signaling integration in gonadotropes