Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

Reversal of an existing hearing loss by gene activation in Spns2 mutant mice.

Hearing loss is highly heterogeneous, but one common form involves a failure to maintain the local ionic environment of the sensory hair cells reflected in a reduced endocochlear potential. We used a genetic approach to ask whether this type of pathology can be reversed, using the Spns2tm1a mouse mutant known to show this defect. By activating Spns2 gene transcription at different ages after the onset of hearing loss, we found that an existing auditory impairment can be reversed to give close to normal thresholds for an auditory brainstem response (ABR), at least at low to mid stimulus frequencies. Delaying the activation of Spns2 led to less effective recovery of ABR thresholds, suggesting that there is a critical period for intervention. Early activation of Spns2 not only led to improvement in auditory function but also to protection of sensory hair cells from secondary degeneration. The genetic approach we have used to establish that this type of hearing loss is in principle reversible could be extended to many other diseases using available mouse resources.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Structural insights into sphingosine-1-phosphate receptor activation.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

Sphingosine-1-phosphate and its receptors in vascular endothelial and lymphatic barrier function.

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system.

Actin-binding protein filamin B regulates the cell-surface retention of endothelial sphingosine 1-phosphate receptor 1.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of ∼1 µM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed ß2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.

Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.

Pancreatic Acinar Cells-Derived Sphingosine-1-Phosphate Contributes to Fibrosis of Chronic Pancreatitis via Inducing Autophagy and Activation of Pancreatic Stellate Cells.

BACKGROUND & AIMS: Studies have demonstrated that activated pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis (CP); however, the precise mechanism for PSCs activation has not been fully elucidated. We analyzed the role of injured pancreatic acinar cells (iPACs) in the activation of PSCs of CP. METHODS: Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling was evaluated in experimental CP induced by cerulein injection or pancreatic duct ligation, as well as in PACs injured by cholecystokinin. The activation of PSCs and pancreatic fibrosis in CP samples was evaluated by immunohistochemical and immunofluorescence analyses. In vitro coculture assay of iPACs and PSCs was created to evaluate the effect of the SPHK1/S1P pathway and S1P receptor 2 (SIPR2) on autophagy and activation of PSCs. The pathogenesis of CP was assessed in SPHK1-/- mice or PACs-specific SPHK1-knockdown mice with recombinant adeno-associated virus serotypes 9-SPHK1-knockdown, as well as in mice treated with inhibitor of SPHK1 and S1P receptor 2 (S1PR2). RESULTS: SPHK1/S1P was remarkably increased in iPACs and acinar cells in pancreatic tissues of CP mice. Meanwhile, the pathogenesis, fibrosis, and PSCs activation of CP was significantly prevented in SPHK1-/- mice and recombinant adeno-associated virus serotypes 9-SPHK1-knockdown mice. Meanwhile, iPACs obviously activated PSCs, which was prevented by SPHK1 knockdown in iPACs. Moreover, iPACs-derived S1P specifically combined to S1PR2 of PSCs, by which modulated 5' adenosine monophosphate-activated protein kinase/mechanistic target of rapamycin pathway and consequently induced autophagy and activation of PSCs. Furthermore, hypoxia-inducible factor 1-α and -2α promoted SPHK1 transcription of PACs under hypoxia conditions, which is a distinct characteristic of the CP microenvironment. Coincidently, inhibition of SPHK1 and S1PR2 activity with inhibitor PF-543 and JTE-013 obviously impeded pancreatic fibrogenesis of CP mice. CONCLUSIONS: The activated SPHK1/S1P pathway in iPACs induces autophagy and activation of PSCs by regulating the S1PR2/5' adenosine monophosphate-activated protein kinase/mammalian target of rapamycin pathway, which promotes fibrogenesis of CP. The hypoxia microenvironment might contribute to the cross talk between PACs and PSCs in pathogenesis of CP.

Sphingosine-1-Phosphate Shapes Healthy Monocytes into An Immunosuppressive Phenotype.

BACKGROUND/AIMS: The physiological phenotype of individuals can influence and shape real-life phenomena in that it can contribute to the development of specific characteristics that can affect the immune response to specific stimuli. In this study we aimed to understand whether the sphingosine/sphingosine-1-phoshate (S1P) axis can modulate the immunotype of circulating cells. METHODS: To pursue this goal, we performed bioinformatic analyses of public datasets. RESULTS: The transcriptomic profile of healthy subjects of GSE192829 dataset identified two clusters with different transcriptional repertoire. Cluster 1 expressed higher levels of enzymes for S1P formation than cluster 0 which was characterized by enzymes that lead to ceramide formation, which represent the opposite metabolic direction. Inference analysis showed that cluster 1 was higher populated by monocytes, CD4+ T and B cells than cluster 0. Of particular interest was the phenotype of the monocytes in cluster 1 which showed an immunosuppressive nature compared to those in cluster 0. The role of S1P signature in healthy PBMCs was confirmed with other dataset analyses, supporting that circulating monocytes positive to the ceramidase, unlike the negative ones, had an immunosuppressive phenotype characterized by hub immunosuppressive markers (i.e. TYROBP, FCER1G, SYK, SIRPA, CSF1R, AIF1, FCGR2A, CLEC7A, LYN, PLCG2, LILRs, HCK, GAB2). This hub genes well discriminated the immunotype of healthy subjects. CONCLUSION: In conclusion this study highlights that S1P-associated hub markers can be useful to discriminate subjects with pronounced immunosuppression.

Dominant missense variants in SREBF2 are associated with complex dermatological, neurological, and skeletal abnormalities.

PURPOSE: We identified 2 individuals with de novo variants in SREBF2 that disrupt a conserved site 1 protease (S1P) cleavage motif required for processing SREBP2 into its mature transcription factor. These individuals exhibit complex phenotypic manifestations that partially overlap with sterol regulatory element binding proteins (SREBP) pathway-related disease phenotypes, but SREBF2-related disease has not been previously reported. Thus, we set out to assess the effects of SREBF2 variants on SREBP pathway activation. METHODS: We undertook ultrastructure and gene expression analyses using fibroblasts from an affected individual and utilized a fly model of lipid droplet (LD) formation to investigate the consequences of SREBF2 variants on SREBP pathway function. RESULTS: We observed reduced LD formation, endoplasmic reticulum expansion, accumulation of aberrant lysosomes, and deficits in SREBP2 target gene expression in fibroblasts from an affected individual, indicating that the SREBF2 variant inhibits SREBP pathway activation. Using our fly model, we discovered that SREBF2 variants fail to induce LD production and act in a dominant-negative manner, which can be rescued by overexpression of S1P. CONCLUSION: Taken together, these data reveal a mechanism by which SREBF2 pathogenic variants that disrupt the S1P cleavage motif cause disease via dominant-negative antagonism of S1P, limiting the cleavage of S1P targets, including SREBP1 and SREBP2.

Re-positioning of low dose paclitaxel against depressive-like behavior and neuroinflammation induced by lipopolysaccharide in rats: Crosstalk between NLRP3/caspase-1/IL-1ß and Sphk1/S1P/ NF-κB signaling pathways.

AIMS: Depression is a potentially fatal illness affecting millions of individuals worldwide, across all age groups. Neuroinflammation is a key factor in depression development. Paclitaxel (PXL), a well-known chemotherapeutic agent has been used as therapy for several types of cancer. This study aims to evaluate the ameliorative effect of low-dose PXL against lipopolysaccharide (LPS)-induced depression in rats. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were administrated a single dose of LPS (5 mg/kg, i.p.); 2 h later, rats received PXL (0.3 mg/kg, i.p. three times/week) for one week. KEY FINDINGS: Low-dose PXL alleviated LPS-induced depressive-like behavior in rats as evidenced by significantly improving behavioral changes in both forced swim test (FST) and open field test (OFT), successfully mitigated depletion of monoamines (serotonin, norepinephrine, and dopamine), in addition to markedly decreasing lipid peroxidation with antioxidant levels elevation in brain tissues. Low-dose PXL substantially decreased inflammation triggered by LPS in brain tissue via repressing the expression of NLRP3 and its downstream markers level, caspase-1 and IL-1ß jointly with a corresponding decrease in proinflammatory cytokine levels (TNF-α). Furthermore, low-dose PXL remarkably down-regulated Sphk1/S1P signaling pathway. Concurrent with these biochemical findings, there was a noticeable improvement in the brain tissue's histological changes. SIGNIFICANCE: These findings prove the role of low-dose PXL in treatment of LPS-induced neuroinflammation and depressive-like behavior through their anti-depressant, antioxidant and anti-inflammatory actions. The suggested molecular mechanism may entail focusing the interconnection among Sphk1/S1P, and NLRP3/caspase-1/IL-1ß signaling pathways. Hence PXL could be used as a novel treatment against LPS-induced depression.

The emerging roles of sphingosine 1-phosphate and SphK1 in cancer resistance: a promising therapeutic target.

Cancer chemoresistance is a problematic dilemma that significantly restrains numerous cancer management protocols. It can promote cancer recurrence, spreading of cancer, and finally, mortality. Accordingly, enhancing the responsiveness of cancer cells towards chemotherapies could be a vital approach to overcoming cancer chemoresistance. Tumour cells express a high level of sphingosine kinase-1 (SphK1), which acts as a protooncogenic factor and is responsible for the synthesis of sphingosine-1 phosphate (S1P). S1P is released through a Human ATP-binding cassette (ABC) transporter to interact with other phosphosphingolipids components in the interstitial fluid in the tumor microenvironment (TME), provoking communication, progression, invasion, and tumor metastasis. Also, S1P is associated with several impacts, including anti-apoptotic behavior, metastasis, mesenchymal transition (EMT), angiogenesis, and chemotherapy resistance. Recent reports addressed high levels of S1P in several carcinomas, including ovarian, prostate, colorectal, breast, and HCC. Therefore, targeting the S1P/SphK signaling pathway is an emerging therapeutic approach to efficiently attenuate chemoresistance. In this review, we comprehensively discussed S1P functions, metabolism, transport, and signaling. Also, through a bioinformatic framework, we pointed out the alterations of SphK1 gene expression within different cancers with their impact on patient survival, and we demonstrated the protein-protein network of SphK1, elaborating its sparse roles. Furthermore, we made emphasis on different machineries of cancer resistance and the tight link with S1P. We evaluated all publicly available SphK1 inhibitors and their inhibition activity using molecular docking and how SphK1 inhibitors reduce the production of S1P and might reduce chemoresistance, an approach that might be vital in the course of cancer treatment and prognosis.

Longitudinal increase of humoral responses after four SARS-CoV-2 vaccinations and infection in MS patients on fingolimod.

BACKGROUND: Humoral responses after SARS-CoV-2 vaccination are greatly impaired in multiple sclerosis (MS) patients on fingolimod. Effects of repeated vaccination and infections on long-term responses are unclear. METHODS: Prospective study in 60 MS patients on fingolimod measuring humoral responses after up to four vaccinations and 8 months after fourth vaccination. RESULTS: Anti-WH1 antibody titers increased with each additional vaccination. At long-term follow-up titers increased further and most patients developed new humoral responses against the BA.1 omicron variant. CONCLUSION: Repeated SARS-CoV-2 vaccinations boost humoral immunity and, probably together with SARS-CoV-2 infections, induce humoral responses on the long-term in almost all patients.

Status epilepticus: Is there a Stage 1 plus?

In status epilepticus (SE), "time is brain." Currently, first-line therapy consists of benzodiazepines (BDZs) and SE is classified by the response to treatment; stage 2 or established SE is defined as "BDZ-resistant SE." Nonetheless, this classification does not always work, especially in the case of prolonged convulsive SE, where many molecular changes occur and γ-aminobutyric acid signaling becomes excitatory. Under these circumstances, BDZ therapy might not be optimal, and might be possibly detrimental, if given alone; as the duration of SE increases, so too does BDZ resistance. Murine models of SE showed how these cases might benefit more from synergistic combined therapy from the start. The definition of Stage 1 plus is suggested, as a stage requiring combined therapy from the start, which includes prolonged SE with seizure activity going on for >10 min, the time that marks the disruption of receptor homeostasis, with increased internalization. This specific stage might require a synergistic approach from the start, with a combination of first- and second-line treatment.

The placenta in fetal death: molecular evidence of dysregulation of inflammatory, proliferative, and fetal protective pathways.

BACKGROUND: It is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. OBJECTIVE: The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. STUDY DESIGN: Cases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birthweight. Placental messenger RNA expression of inflammatory (interleukin 6), proliferative (activin A, transforming growth factor ß1) and regulatory (vascular endothelial growth factor, vascular endothelial growth factor receptor 2, ATP-binding cassette transporters (ABC) ABCB1 and ABCG2, sphingosine 1-phosphate signaling pathway) markers was conducted using real-time polymerase chain reaction. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student's t test was used, and P values<.05 were considered significant. RESULTS: Placental mRNA expression of interleukin 6 and vascular endothelial growth factor receptor 2 resulted significantly higher in the fetal death group compared to controls (P<.01), while activin A, ABCB1, and ABCG2 expression resulted significantly lower (P<.01). A significant alteration in the sphingosine 1-phosphate signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3, and 4 (sphingosine 1-phosphate1, sphingosine 1-phosphate3, sphingosine 1-phosphate4) and of sphingosine kinase 2, 1 of the enzyme isoforms responsible for sphingosine 1-phosphate synthesis (P<.01). CONCLUSION: The present study confirmed a significantly increased expression of placental interleukin 6 and vascular endothelial growth factor receptor 2 mRNA, and for the first time showed an increased expression of sphingosine 1-phosphate receptors and sphingosine kinase 2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells.

BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

Cystathionine γ-lyase-derived H2S negatively regulates thymic egress via allosteric inhibition of sphingosine-1-phosphate lyase.

Thymic egress is a crucial process for thymocyte maturation, strictly regulated by sphingosine-1-phosphate lyase (S1PL). Recently, cystathionine γ-lyase (CSE), one of the enzymes producing hydrogen sulfide (H2S), has emerged as a vital immune process regulator. However, the molecular connection between CSE, H2S and thymic egress remains largely unexplored. In this study, we investigated the regulatory function of CSE in the thymic egress of immune cells. We showed that genetic knockout of CSE or pharmacological inhibition by CSE enzyme inhibitor NSC4056 or D,L-propargylglycine (PAG) significantly enhanced the migration of mature lymphocytes and monocytes from the thymus to the peripheral blood, and this redistribution effect could be reversed by treatment with NaHS, an exogenous donor of H2S. In addition, the CSE-generated H2S significantly increased the levels of S1P in the peripheral blood, thymus and spleen of mice, suppressed the production of proinflammatory cytokines and rescued pathogen-induced sepsis in cells and in vivo. Notably, H2S or polysulfide inhibited S1PL activity in cells and an in vitro purified enzyme assay. We found that this inhibition relied on a newly identified C203XC205 redox motif adjacent to the enzyme's active site, shedding light on the biochemical mechanism of S1PL regulation. In conclusion, this study uncovers a new function and mechanism for CSE-derived H2S in thymic egress and provides a potential drug target for treating S1P-related immune diseases.

Effects and action mechanism of gonadotropins on ovarian follicular cells: A novel role of Sphingosine-1-Phosphate (S1P). A review.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective Gs protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting Gq proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells.

Reversible oxidation/reduction steps in the metabolic degradation of the glycerol side chain of the S1P1 modulator ponesimod.

1. Ponesimod is a selective modulator of the sphingosine 1-phosphate receptor 1 (S1P1) approved for the treatment of active relapsing forms of multiple sclerosis. The chemical structure of ponesimod contains a glycerol side chain which is the major target of drug metabolism in humans.2. The two major metabolic pathways give the acids M12 (-OCH2CH(OH)COOH) and M13 (-OCH2COOH). While the former results from oxidation of the terminal alcohol, the mechanism yielding the chain-shortened acid M13 is less obvious. A detailed mechanistic study with human liver microsomes and hepatocytes using ponesimod, M12 and some of the suspected intermediates revealed an unexpectedly complex pattern of enzyme-mediated and chemical reactions.3. Metabolic pathways for both acids were not independent and several of the transformations were reversible, depending on reaction conditions. Formation of M13 occurred either via initial oxidation of the secondary alcohol, or as a downstream process starting from M12.4. The phenol metabolite M32 was produced as part of several pathways. Control experiments at various pH values and in the absence of metabolising enzymes support the conclusion that its formation resulted from chemical degradation rather than from metabolic processes.