Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

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.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets 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 attenuates neuronal dysfunction induced by amyloid-ß oligomers through endocytic internalization of NMDA receptors.

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aß) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aß (Aß42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aß toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aß42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aß42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aß42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aß42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.

Highly Charged Ru(II) Polypyridyl Complexes as Photosensitizer Agents in Photodynamic Therapy of Epithelial Ovarian Cancer Cells.

Ovarian cancer recurrence is frequent and associated with chemoresistance, leading to extremely poor prognosis. Herein, we explored the potential anti-cancer effect of a series of highly charged Ru(II)-polypyridyl complexes as photosensitizers in photodynamic therapy (PDT), which were able to efficiently sensitize the formation of singlet oxygen upon irradiation (Ru12+ and Ru22+) and to produce reactive oxygen species (ROS) in their corresponding dinuclear metal complexes with the Fenton active Cu(II) ion/s ([CuRu1]4+ and [Cu2Ru2]6+). Their cytotoxic and anti-tumor effects were evaluated on human ovarian cancer A2780 cells both in the absence or presence of photoirradiation, respectively. All the compounds tested were well tolerated under dark conditions, whereas they switched to exert anti-tumor activity following photoirradiation. The specific effect was mediated by the onset of programed cell death, but only in the case of Ru12+ and Ru22+ was preceded by the loss of mitochondrial membrane potential soon after photoactivation and ROS production, thus supporting the occurrence of apoptosis via type II photochemical reactions. Thus, Ru(II)-polypyridyl-based photosensitizers represent challenging tools to be further investigated in the identification of new therapeutic approaches to overcome the innate chemoresistance to platinum derivatives of some ovarian epithelial cancers and to find innovative drugs for recurrent ovarian cancer.

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

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.

S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes.

BACKGROUND: Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle. METHODS AND RESULTS: Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited. CONCLUSION: Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.

A2 B Adenosine Receptors and Sphingosine 1-Phosphate Signaling Cross-Talk in Oligodendrogliogenesis.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain. Impairments in the process of myelination, or demyelinating insults, might cause chronic diseases such as multiple sclerosis (MS). Under physiological conditions, remyelination is an ongoing process throughout adult life consisting in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs). During pathological events, this process fails due to unfavorable environment. Adenosine and sphingosine kinase/sphingosine 1-phosphate signaling axes (SphK/S1P) play important roles in remyelination processes. Remarkably, fingolimod (FTY720), a sphingosine analog recently approved for MS treatment, plays important roles in OPC maturation. We recently demonstrated that the selective stimulation of A2 B adenosine receptors (A2 B Rs) inhibit OPC differentiation in vitro and reduce voltage-dependent outward K+ currents (I K ) necessary to OPC maturation, whereas specific SphK1 or SphK2 inhibition exerts the opposite effect. During OPC differentiation A2 B R expression increases, this effect being prevented by SphK1/2 blockade. Furthermore, selective silencing of A2 B R in OPC cultures prompts maturation and, intriguingly, enhances the expression of S1P lyase, the enzyme responsible for irreversible S1P catabolism. Finally, the existence of an interplay between SphK1/S1P pathway and A2 B Rs in OPCs was confirmed since acute stimulation of A2 B Rs activates SphK1 by increasing its phosphorylation. Here the role of A2 B R and SphK/S1P signaling during oligodendrogenesis is reviewed in detail, with the purpose to shed new light on the interaction between A2 B Rs and S1P signaling, as eventual innovative targets for the treatment of demyelinating disorders.

Phosphatidic Acid Stimulates Myoblast Proliferation through Interaction with LPA1 and LPA2 Receptors.

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [3H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.

Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes.

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P1-5. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P3 prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.

Genome-wide analysis of copy number alterations led to the characterisation of PDCD10 as oncogene in ovarian cancer.

Copy Number Alterations (CNAs) represent the most common genetic alterations identified in ovarian cancer cells, being responsible for the extensive genomic instability observed in this cancer. Here we report the identification of CNAs in a cohort of Italian patients affected by ovarian cancer performed by SNP-based array. Our analysis allowed the identification of 201 significantly altered chromosomal bands (70 copy number gains; 131 copy number losses). The 3300 genes subjected to CNA identified here were compared to those present in the TCGA dataset. The analysis allowed the identification of 11 genes with increased CN and mRNA expression (PDCD10, EBAG9, NUDCD1, ENY2, CSNK2A1, TBC1D20, ZCCHC3, STARD3, C19orf12, POP4, UQCRFS1). PDCD10 was selected for further studies because of the highest frequency of CNA. PDCD10 was found, by immunostaining of three different Tissue Micro Arrays, to be over-expressed in the majority of ovarian primary cancer samples and in metastatic lesions. Moreover, significant correlations were found in specific subsets of patients, between increased PDCD10 expression and grade (p < 0.005), nodal involvement (p < 0.05) or advanced FIGO stage (p < 0.01). Finally, manipulation of PDCD10 expression by shRNA in ovarian cancer cells (OVCAR-5 and OVCA429) demonstrated a positive role for PDCD10 in the control of cell growth and motility in vitro and tumorigenicity in vivo. In conclusion, this study allowed the identification of novel genes subjected to copy number alterations in ovarian cancer. In particular, the results reported here point to a prominent role of PDCD10 as a bona fide oncogene.

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".

Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P2 receptor axis.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P2 receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.

Glycans Meet Sphingolipids: Structure-Based Design of Glycan Containing Analogues of a Sphingosine Kinase Inhibitor.

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator associated with diverse homeostatic and signaling roles. Enhanced biosynthesis of S1P, mediated by the sphingosine kinase isozymes (SK1 and SK2), is implicated in several pathophysiological conditions and diseases, including skeletal muscle fibrosis, inflammation, multiple sclerosis, and cancer. Therefore, therapeutic approaches that control S1P production have focused on the development of SK1/2 inhibitors. In this framework, we designed a series of natural monosaccharide-based compounds to enhance anchoring of the known SK1 inhibitor PF-543 at the polar head of the J-shaped substrate-binding channel. Herein, we describe the structure-based design and synthesis of new glycan-containing PF-543 analogues and we demonstrate their efficiency in a TGFß1-induced pro-fibrotic assay.

The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-ß activation by Gq-coupled receptors.

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-ß and the thromboxane A2 receptor-ß (TPß receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPß receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCß, WDR36 augmented TPß receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPß receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPß towards PLC and SphK1, most likely by scavenging Gαq and PLCß. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.