Introduction/Background
Variants of the neuropeptide S receptor 1 (NPSR1) gene like the G42C NPSR1 variant are linked to endometriosis, particularly advanced stages1. Its ligand NPS enhances actin cytoskeletal remodeling and cell invasion in endometrial cells via sphingosine signalling, indicating a role in disease progression2. Here, we investigated the effect of the G42C NPSR1 variant on NPS/NPSR1-mediated sphingosine signalling in endometriosis.
Materials and Methods
G42C-carrying SH-SY5Y cells and WT controls were stimulated with 100 nM NPS for 45 minutes alongside endometrial ECC-1 and 12Z cell lines. Expression of sphingosine signalling genes CIB1, SPHK1, SPHK2, and SPGL1 was measured by qRT-PCR (n=2 repeats). For cytoskeletal remodelling, cells were treated with 100, 300, and 900 nM NPS for 45 minutes and stained with TRITC-phalloidin/Hoechst 33258. Experiments were performed in duplicates and two repeats. Imaging was done by confocal microscope (Olympus FV1000), data were analysed using GraphPad Prism v10.
Results
The sphingosine signalling genes were significantly upregulated in ECC-1 (> 2 fold, p<0.05). SPHK1 and SPHK2 showed similarly high expression in ECC-1, with SPHK2 being particularly elevated in ECC-1 compared to the other lines. SPHK1 displayed moderate expression in both ECC-1 and 12Z, while SPGL1 was detectable only in ECC-1. Interestingly, SPHK2 was highly expressed. Surprisingly, SPHK1, SPHK2, or SPGL1 were not detected in the G42C-carrying SH-SY5Y clone following NPS stimulation (100 nM). Interestingly, cytoskeletal actin staining revealed no marked differences between NPS-treated and untreated cells.
Conclusion
qRT-PCR results highlight the importance of genetic background in cellular responses to NPS and suggest that NPSR1 variants, such as the G42C-carrying SH-SY5Y, may significantly influence gene regulation preventing activation of downstream transcriptional responses, which might explain their involvement in endometriosis pathophysiology.