Sphingosine-1-phosphate receptor 3 is a non-hormonal target to counteract endometriosis-associated fibrosis.

OBJECTIVE: To study the molecular mechanisms responsible for fibrosis in endometriosis by investigating whether the protein expression levels of sphingosine-1-phosphate receptor 3 (S1PR3), one of the five specific receptors of the bioactive sphingolipid sphingosine-1-phosphate (S1P), correlate with fibrosis extent in endometriotic lesions and which are the cellular mechanisms involved in this process. DESIGN: Case-control laboratory study and cultured endometriotic cells. SETTING: University research institute and university hospital. PATIENT(S): A total of 33 women, with and without endometriosis, were included in the study. INTERVENTIONS(S): Endometriotic lesions were obtained from women with endometriosis (ovarian endometrioma, n = 8; deep infiltrating endometriosis, n = 15; [urological n = 5, gastrointestinal n = 6, and posterior n = 4]) and control endometrium from healthy women, n = 10, by means of laparoscopic and hysteroscopic surgery. The expression of S1PR3 was evaluated using immunohistochemistry and the extent of fibrosis was assessed using Masson's trichrome staining. Human-cultured epithelial endometriotic 12Z cells were used to evaluate the mechanisms involved in the profibrotic effect of S1PR3 activation. MAIN OUTCOME MEASURE(S): The expression of S1PR3 in endometriotic lesions is positively correlated with endometriosis-associated fibrosis. In addition, S1P induced epithelial-mesenchymal transition (EMT) and fibrosis in epithelial endometriotic cells. Using RNA interference and pharmacological approaches, the profibrotic effect of S1P was shown to rely on S1PR3, thus unveiling the molecular mechanism implicated in the profibrotic action of the bioactive sphingolipid. RESULT(S): The protein expression levels of S1PR3 were significantly augmented in the glandular sections of endometrioma and deep infiltrating endometriosis of different localizations with respect to the control endometrium and positively correlated with the extent of fibrosis. Sphingosine-1-phosphate was shown to have a crucial role in the onset of fibrosis in epithelial endometriotic cells, stimulating the expression of EMT and fibrotic markers. Genetic approaches have highlighted that S1PR3 mediates the fibrotic effect of S1P. Downstream of S1PR3, ezrin and extracellular-signal-regulated kinases 1 and 2 signaling were found to be critically implicated in the EMT and fibrosis elicited by S1P. CONCLUSION(S): Sphingosine-1-phosphate receptor 3 may represent a possible innovative pharmacological target for endometriosis.

Endometriosis: Cellular and Molecular Mechanisms Leading to Fibrosis.

Endometriosis is a chronic inflammatory condition affecting women of reproductive age. A relevant feature of endometriosis is the presence of fibrotic tissue inside and around the lesions, thus contributing to the classic endometriosis-related symptoms, pain, and infertility. The molecular mechanisms responsible for the development of fibrosis in endometriosis are not yet defined. The present review aimed to examine the biological mechanisms and signalling pathways involved in fibrogenesis of endometriotic lesions, highlighting the difference between deep infiltrating and ovarian endometriosis. The main cell types involved in the development of fibrosis are platelets, myofibroblasts, macrophages, and sensory nerve fibers. Members of the transforming growth factor (TGF) -ß family, as well as the receptor Notch, or the bioactive sphingolipid sphingosine 1-phosphate (S1P), play a role in the development of tissue fibrosis, resulting in their metabolism and/or their signalling pathways altered in endometriotic lesions. It is relevant the knowledge of the molecular mechanisms that guide and support fibrosis in endometriosis, to identify new drug targets and provide new therapeutic approaches to patients.

Sphingosine 1-phosphate pathway is dysregulated in adenomyosis.

RESEARCH QUESTION: Is sphingosine 1-phosphate (S1P) pathway involved in the process of fibrosis in adenomyosis? DESIGN: RNA was extracted from paraffin-embedded slices collected from the ectopic endometrium of patients with nodular adenomyosis (n = 27) and eutopic endometrium of healthy controls women (n = 29). Expression of genes involved in the metabolism and signalling of S1P, and actin-alpha-2 smooth muscle, encoded by ACTA2 gene, a gene involved in fibrogenesis, was evaluated by real-time polymerase chain reaction analysis. RESULTS: In adenomyotic samples, the expression of sphingosine kinase 1 (SPHK1), the enzyme responsible for the synthesis of S1P, and of S1P phosphatase 2 (SGPP2), the enzyme responsible for the conversion of S1P back to sphingosine, was lower (P = 0.0006; P = 0.0015), whereas that of calcium and integrin-binding protein 1, responsible for membrane translocation of SPHK1, was higher (P = 0.0001) compared with healthy controls. In S1P signalling, a higher expression of S1P receptor S1P3 (P = 0.001), and a lower expression of S1P2 (P = 0.0019) mRNA levels, were found compared with healthy endometrium. In adenomyotic nodules, a higher expression of ACTA2 mRNA levels were observed (P = 0.0001), which correlated with S1P3 levels (P = 0.0138). CONCLUSION: Present data show a profound dysregulation of the S1P signalling axis in adenomyosis. This study also highlights that the bioactive sphingolipid might be involved in the fibrotic tract of the disease, correlated with the expression of ACTA2, suggesting its role as novel potential biomarker of adenomyosis.

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect.

OBJECTIVE: To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A. DESIGN: Case-control laboratory study. SETTING: University institute and university hospital. PATIENT(S): Patients with uterine fibroids (n = 26). INTERVENTIONS(S): Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy. MAIN OUTCOME MEASURE(S): Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines. RESULT(S): The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P2, S1P3, and S1P5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P2. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells. CONCLUSION(S): This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.

Sphingosine 1-phosphate receptors are dysregulated in endometriosis: possible implication in transforming growth factor ß-induced fibrosis.

OBJECTIVE: To study the molecular mechanisms involved in the appearance of the fibrotic trait in endometriosis by investigating whether the signaling pathway of the bioactive sphingolipid sphingosine 1-phosphate (S1P) was altered in endometriotic lesions. DESIGN: Case-control laboratory study. SETTING: University research institute and university hospital. PATIENT(S): A total of 75 women, with and without endometriosis, were included in the study. INTERVENTIONS(S): Endometrial samples were obtained from women affected (n = 15 endometrioma [OMA]; n = 30 deep infiltrating endometriosis [DIE]) and not (n = 30) by endometriosis by means of laparoscopic surgery, followed by clinical and imaging investigation and checking for the expression of fibrosis markers and genes implicated in S1P metabolism and signaling by means of real-time polymerase chain reaction. MAIN OUTCOME MEASURE(S): The role of the S1P signaling axis in endometriosis-associated fibrosis was studied in vitro, where RNA interference approaches were used to investigate if S1P synthesis by sphingosine kinases (SKs) and specific S1P receptors (S1PRs) are implicated in the profibrotic effect of the cytokine transforming growth factor (TGF) ß1. RESULT(S): mRNA expression analysis of S1PR demonstrated a deep dysregulation of S1P signaling in endometriosis, characterized by increased expression of fibrosis markers: S1P1 was transcriptionally more expressed in OMA, and S1P3 and S1P5 mRNA levels were significantly augmented in both OMA and DIE. SK1 and its activating protein calcium- and integrin-binding protein 1 (CIB1) were significantly up-regulated in OMA and DIE. A crucial role for the SK/S1PR axis in the profibrotic effect elicited by TGFß1 was highlighted in vitro. CONCLUSION(S): The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis.

Role of sphingosine 1-phosphate signalling in tissue fibrosis.

Fibrosis is characterized by the excessive accumulation of extracellular matrix components, leading to loss of tissue function in affected organs. Although the majority of fibrotic diseases have different origins, they have in common a persistent inflammatory stimulus and lymphocyte-monocyte interactions that determine the production of numerous fibrogenic cytokines. Treatment to contrast fibrosis is urgently needed, since some fibrotic diseases lead to systemic fibrosis and represent a major cause of death. In this article, the role of the bioactive sphingolipid sphingosine 1-phosphate (S1P) and its signalling pathway in the fibrosis of different tissue contexts is extensively reviewed, highlighting that it may represent an innovative and promising pharmacological therapeutic target for treating this devastating multifaceted disease. In multiple tissues S1P influences different aspects of fibrosis modulating the recruitment of inflammatory cells, as well as cell proliferation, migration and transdifferentiation into myofibroblasts, the cell type mainly involved in fibrosis development. Moreover, at the level of fibrotic lesions, S1P metabolism is profoundly influenced by multiple cross-talk with profibrotic mediators, such as transforming growth factor ß, thus finely regulating the development of fibrosis. This article is part of a Special Issue entitled "Physiological and pathological roles of bioactive sphingolipids".