Role of sphingosine 1-phosphate signaling axis in endometriosis: insights into cellular invasion, fibrosis and inflammation

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid implicated in numerous biological processes, such as cell survival, proliferation, migration, cytoskeleton remodeling, calcium mobilization, vascular development, inflammation and immunity. S1P is synthetised intracellularly by two isoform of sphingosine kinase 1 and 2 (SK1 and SK2) and it can be dephosphorylated by S1P phosphatases (SPP1 and SPP2) or degraded by S1P lyase (SPL). After the export of S1P in the extracellular environment, several actions are mediated by its binding to five specific G protein-coupled receptors (GPCRs), defined S1P1-5. In this work, we explored the role of S1P in endometriosis pathogenesis, focusing on the invasive, fibrotic and inflammatory traits of the disease. Endometriosis is a benign gynaecological endocrine inflammatory disease characterized by chronic pelvic pain and infertility and about 10% of women of reproductive age are estimated to be affected. Despite this high prevalence, disease recognition is inadequate with high rate of diagnosis delay or misdiagnosis. The disease is defined by the presence of endometrium-like tissue outside the uterus even though the aetiology and physiopathology must be entirely defined yet. One of the most important pathological features of endometriosis is endometrial invasion. Despite endometriosis is considered a benign disorder, there are characteristics of malignancy in the way endometrial cells attach to and invade surfaces. Additionally, fibrosis is another fundamental trait of the disease. Many and various growth factors, cytokines, and chemokines are released by activated platelets, macrophages, ectopic endometrial cells and sensory nerve fibres within lesions establishing a microenvironment that may promote fibrogenesis directly. Along with these physio-pathological characteristics, inflammation encompasses them all, leading to systemic symptoms like debilitating chronic pain, dysmenorrhea, dyspareunia and dysuria. Since the lack of curative treatments and the incomplete understanding of the molecular mechanisms involved in the pathogenesis, pursuing the study of endometriosis pathogenesis is pivotal. Recently, S1P metabolism and signaling has been found to be deeply dysregulated in endometriosis. In this work we evaluated the crosstalk between S1P signaling axis and neuropeptide S (NPS) to promote biological actions in human endometriotic epithelial cells 12Z. We showed that NPS could enhance the invasive phenotype of 12Z cells, promoting the invasion and the remodeling of the F-actin cytoskeleton, after the activation of both SK1 and SK2 and the engagement of S1P1/S1P3-RhoA-RhoA associated kinase (ROCK) signaling axis. Moreover, we demonstrated that in human endometriotic 2 epithelial cells 12Z, a different number of GPCR ligands mediate [Ca2+]i increase, such as oxytocin (OXT), bradykinin (BK), histamine (HIS), lysophosphatidic acid (LPA), and S1P. We showed that pretreatment with pertussis toxin significantly reduced S1P-dependent [Ca2+]i increase in 12Z cells, highlighting the involvement of Gi-mediated signaling. Employing a pharmacological approach, we showed that S1P1, S1P3 and S1P5, but not S1P2 and S1P4 elicited the [Ca2+]i increase. Moreover, we assessed that while OXT, BK and HIS exerted a limited pro invasive effect, the bioactive lipids LPA, and particularly S1P, acting via S1P1, S1P4 and S1P5, strongly stimulated cell invasion. Finally, we demonstrated the pivotal role of ryanodine receptors in mediating the S1P-induced invasion of 12Z cells. Successively, we highlighted that the protein expression levels of S1P3 were significantly augmented in the glandular sections of endometriosis tissue compared to the control endometrium and positively correlated with the extent of fibrosis. In addition, the treatment with S1P was shown to have a fundamental role in the onset of fibrosis in human epithelial endometriotic cells, stimulating the EMT and the expression of fibrotic markers. Genetic approaches have highlighted that S1P3 mediates the fibrotic effect of S1P. Downstream of S1P3, Ezrin and ERK 1/2 signaling were found to be critically implicated in the EMT and fibrosis elicited by S1P. Finally, we investigated the role of S1P in the endometrium employing human endometrial stromal cells (HESC). We showed that S1P via S1P1/S1P3 serves as a pro-inflammatory stimulus in HESCs, increasing the production of reactive oxygen species (ROS) and the expression of pro-inflammatory cytokines, through the activation of the mitogen-activated protein kinase ERK5. In conclusion, in this work we investigated different aspects of S1P signaling in endometriosis, with a special focus on the pro-migratory, pro-fibrotic and pro-inflammatory effects of the sphingolipid, in order to deepen our comprehension of S1P pathophysiological role and provide support for using this pathway as a potential therapeutic target.