At low levels, inorganic mercury interference with antigen signaling is associated with modifications to a panel of novel phosphoserine sites in B cell receptor pathway proteins.

Epidemiological studies indicate that human and animal exposure to environmental mercury (Hg) disrupts normal immune system function, but the molecular mechanism responsible for this is still unresolved. We have previously utilized phospho-proteomic mass spectrometry to demonstrate that in the absence of B Cell Receptor (BCR) stimulation, exposure of B cells to Hg induces significant changes to a great many elements of the BCR signaling pathway in a concentration dependent manner. In this report, we have extended those initial findings by utilizing mass spectrometry to evaluate in detail the effect of low-level Hg exposure on BCR induced phospho-proteomic changes. Specifically, murine WEHI-231 B lymphoma cells were exposed to environmentally relevant levels of Hg with or without concomitant BCR stimulation. The cellular phospho-proteomes were then profiled by LC-MS/MS. We found that for low-level exposures, Hg interference with signal transduction across the BCR pathway was predominantly associated with modification of phosphorylation of 12 phosphosites located on seven different proteins. Nine sites were serine, two sites tyrosine and one site threonine. Most of these sites are novel, in the sense that only the two tyrosine and one of the serine sites have previously been reported to be associated with BCR signaling.

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis.

BACKGROUND: Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co-localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved. METHODS: Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography-mass spectrometry. Phosphatidylserine-containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real-time PCR, immunohistochemistry enzyme-linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies. RESULTS: The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2-like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4 (TIM4) and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression. CONCLUSIONS: This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.

Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system.

Calcium phosphate-based bone cements have been widely adopted in both orthopedic and dental applications. Phosphoserine (pSer), which has a natural role in biomineralization, has been identified to possess the functionality to react with calcium phosphate phases, such as tetracalcium phosphate (TTCP) and α-tricalcium phosphate (α-TCP), and form a uniquely adhesive cement. This study investigated the chemical composition and phase evolution of a heterogeneous calcium phosphate (56% TTCP and 15% α-TCP) and pSer cement system with respect to pH. The coordination network of calcium phosphoserine monohydrate was discovered as the predominant crystalline phase of this adhesive apatitic cement system. Furthermore, it was determined that pH has a significant effect on the reaction kinetics of the system, whereby a lower pH tends to accelerate the reaction rate and favor products with lower Ca/P ratios. These findings provide a better understanding of the reaction and products of this adhesive organo-ceramic cement, which can be compositionally tuned for broad applications in the orthopedic and dental spaces. STATEMENT OF SIGNIFICANCE: The application of self-setting calcium phosphate cements (CPCs) in hard tissue regeneration has been a topic of significant research since their introduction to the field 30 years ago. Traditional CPCs, however, are limited by their suboptimal mechanical properties due to their solely inorganic composition. Recently, it was discovered that monomeric phosphoserine (pSer) is capable of serving as a setting reagent for a subset of CPC systems, resulting in an adhesive organo-ceramic composite. Despite its adhesive functionality and biomedical potential, its reaction chemistry and product composition were not well characterized. The present study identifies a calcium phosphoserine coordination network as the primary crystalline phase of this apatitic cement system and further characterizes compositional tunability of the products with respect to pH.

Resolvin D1 activates the sphingosine-1-phosphate signaling pathway in murine livers with ischemia/reperfusion injury.

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 µg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 µL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 µg/kg, i.p.), an antagonist for S1P1/S1P3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1, SK2, S1P1, and S1P3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling.

Elfn1-Induced Constitutive Activation of mGluR7 Determines Frequency-Dependent Recruitment of Somatostatin Interneurons.

Excitatory synapses onto somatostatin (SOM) interneurons show robust short-term facilitation. This hallmark feature of SOM interneurons arises from a low initial release probability that regulates the recruitment of interneurons in response to trains of action potentials. Previous work has shown that Elfn1 (extracellular leucine rich repeat and fibronectin Type III domain containing 1) is necessary to generate facilitating synapses onto SOM neurons by recruitment of two separate presynaptic components: mGluR7 (metabotropic glutamate receptor 7) and GluK2-KARs (kainate receptors containing glutamate receptor, ionotropic, kainate 2). Here, we identify how a transsynaptic interaction between Elfn1 and mGluR7 constitutively reduces initial release probability onto mouse cortical SOM neurons. Elfn1 produces glutamate-independent activation of mGluR7 via presynaptic clustering, resulting in a divergence from the canonical "autoreceptor" role of Type III mGluRs, and substantially altering synaptic pharmacology. This structurally induced determination of initial release probability is present at both layer 2/3 and layer 5 synapses. In layer 2/3 SOM neurons, synaptic facilitation in response to spike trains is also dependent on presynaptic GluK2-KARs. In contrast, layer 5 SOM neurons do not exhibit presynaptic GluK2-KAR activity at baseline and show reduced facilitation. GluK2-KAR engagement at synapses onto layer 5 SOM neurons can be induced by calmodulin activation, suggesting that synaptic function can be dynamically regulated. Thus, synaptic facilitation onto SOM interneurons is mediated both by constitutive mGluR7 recruitment by Elfn1 and regulated GluK2-KAR recruitment, which determines the extent of interneuron recruitment in different cortical layers.SIGNIFICANCE STATEMENT This study identifies a novel mechanism for generating constitutive GPCR activity through a transsynaptic Elfn1/mGluR7 structural interaction. The resulting tonic suppression of synaptic release probability deviates from canonical autoreceptor function. Constitutive suppression delays the activation of somatostatin interneurons in circuits, necessitating high-frequency activity for somatostatin interneuron recruitment. Furthermore, variations in the synaptic proteome generate layer-specific differences in facilitation at pyr → SOM synapses. The presence of GluK2 kainate receptors in L2/3 enhances synaptic transmission during prolonged activity. Thus, layer-specific synaptic properties onto somatostatin interneurons are mediated by both constitutive mGluR7 recruitment and regulated GluK2 kainate receptor recruitment, revealing a mechanism that generates diversity in physiological responses of interneurons.

Sphingosine 1-phosphate induces epicardial progenitor cell differentiation into smooth muscle-like cells.

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, induces the differentiation of EpiCs is unknown. In the present study, we demonstrated that S1P significantly induced the expression of smooth muscle cell specific markers α-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1P1/S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1P1 agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, increases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a secondary role.

Role of S1P/S1PR3 axis in release of CCL20 from human bronchial epithelial cells.

BACKGROUND: Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1 phosphate (S1P) following stimulation of the five plasma membrane G-protein-coupled receptors. The objective of this study is to clarify the role of S1P and its receptors (S1PRs), especially S1PR3 in airway epithelial cells. METHODS: The effects of S1P on asthma-related genes expression were examined with the human bronchial epithelial cells BEAS-2B and Calu-3 using a transcriptome analysis and siRNA of S1PRs. To clarify the role of CCL20 in the airway inflammation, BALB/c mice were immunized with ovalbumin (OVA) and subsequently challenged with an OVA-containing aerosol to induce asthma with or without intraperitoneal administration of anti-CCL20. Finally, the anti-inflammatory effect of VPC 23019, S1PR1/3 antagonist, in the OVA-induced asthma was examined. RESULTS: S1P induced the expression of some asthma-related genes, such as ADRB2, PTGER4, and CCL20, in the bronchial epithelial cells. The knock-down of SIPR3 suppressed the expression of S1P-inducing CCL20. Anti-CCL20 antibody significantly attenuated the eosinophil numbers in the bronchoalveolar lavage fluid (P<0.01). Upon OVA challenge, VPC23019 exhibited substantially attenuated eosinophilic inflammation. CONCLUSIONS: S1P/S1PR3 pathways have a role in release of proinflammatory cytokines from bronchial epithelial cells. Our results suggest that S1P/S1PR3 may be a possible candidate for the treatment of bronchial asthma.

Apolipoprotein M Protects Lipopolysaccharide-Treated Mice from Death and Organ Injury.

OBJECTIVE: High-density lipoprotein (HDL) has been epidemiologically shown to be associated with the outcome of sepsis. One potential mechanism is that HDL possesses pleiotropic effects, such as anti-apoptosis, some of which can be ascribed to sphingosine 1-phosphate (S1P) carried on HDL via apolipoprotein M (apoM). Therefore, the aim of this study was to elucidate the roles of apoM/S1P in the consequent lethal conditions of sepsis, such as multiple organ failure caused by severe inflammation and/or disseminated intravascular coagulation. METHODS AND RESULTS: In mice treated with lipopolysaccharide (LPS), both plasma apoM levels and the expression of apoM in the liver and kidney were suppressed. The overexpression of apoM improved the survival rate and ameliorated the elevated plasma alanine aminotransferase (ALT) and creatinine levels, while the knockout or knockdown of apoM deteriorated these parameters in mice treated with LPS. Treatment with VPC23019, an antagonist against S1P receptor 1 and 3, or LY294002, a PI3K inhibitor, partially reversed these protective properties arising from the overexpression of apoM. The overexpression of apoM inhibited the elevation of plasma plasminogen activator inhibitor-1, restored the phosphorylation of Akt, and induced anti-apoptotic changes in the liver, kidney and heart. CONCLUSION: These results suggest that apoM possesses protective properties against LPS-induced organ injuries and could potentially be introduced as a novel therapy for the severe conditions that are consequent to sepsis.

Probing the Hydrophobic Binding Pocket of G-Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS1 by Docking-Aided Structure-Activity Analysis.

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Fingolimod confers neuroprotection through activation of Rac1 after experimental germinal matrix hemorrhage in rat pups.

Fingolimod, a sphingosine-1-phosphate receptor (S1PR) agonist, is clinically available to treat multiple sclerosis and is showing promise in treating stroke. We investigated if fingolimod provides long-term protection from experimental neonatal germinal matrix hemorrhage (GMH), aiming to support a potential mechanism of acute fingolimod-induced protection. GMH was induced in P7 rats by infusion of collagenase (0.3 U) into the right ganglionic eminence. Animals killed at 4 weeks post-GMH received low- or high-dose fingolimod (0.25 or 1.0 mg/kg) or vehicle, and underwent neurocognitive testing before histopathological evaluation. Subsequently, a cohort of animals killed at 72 h post-GMH received 1.0 mg/kg fingolimod; the specific S1PR1 agonist, SEW2871; or fingolimod co-administered with the S1PR1/3/4 inhibitor, VPC23019, or the Rac1 inhibitor, EHT1864. All drugs were injected intraperitoneally 1, 24, and 48 h post-surgery. At 72 h post-GMH, brain water content, extravasated Evans blue dye, and hemoglobin were measured as well as the expression levels of phospho-Akt, Akt, GTP-Rac1, Total-Rac1, ZO1, occludin, and claudin-3 determined. Fingolimod significantly improved long-term neurocognitive performance and ameliorated brain tissue loss. At 72 h post-GMH, fingolimod reduced brain water content and Evans blue dye extravasation as well as reversed GMH-induced loss of tight junctional proteins. S1PR1 agonism showed similar protection, whereas S1PR or Rac1 inhibition abolished the protective effect of fingolimod. Fingolimod treatment improved functional and morphological outcomes after GMH, in part, by tempering acute post-hemorrhagic blood-brain barrier disruption via the activation of the S1PR1/Akt/Rac1 pathway.

Blockage of Central Sphingosine-1-phosphate Receptor does not Abolish the Protective Effect of FTY720 in Early Brain Injury after Experimental Subarachnoid Hemorrhage.

BACKGROUND: Although sphingosine 1-phosphate (S1P) receptor activation by FTY720 (fingolimod) has been suggested to improve the prognosis of experimental stroke, the effect of the drug in early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the precise mechanism of the effect are undetermined. In this study, we investigated the protective effect of systemic administration of FTY720 in EBI after SAH and assessed the mechanism using intracerebroventricular infusion of VPC23019 which is the S1P receptor antagonist. METHOD: SAH rats were produced by the endovascular perforation model and injected saline or 1mg/kg FTY720 intraperitoneally at 30 minutes after SAH induction. Neurological function, cerebral blood flow, amount of subarachnoid blood, and brain edema were evaluated to confirm the protective effect of systemic administration of FTY720. SAH rats also received VPC23019 intraventricularly before SAH induction to abolish the central S1P receptor activation. RESULTS: Systemic administration of FTY720 significantly ameliorated SAH-induced neurological deficits and brain edema without modulation of CBF and the amount of subarachnoid blood. Blockage of central S1P receptor with VPC23019 did not abolish the protective effects of FTY720. CONCLUSION: The present study suggests that systemic administration of FTY720 reduces EBI after SAH and that the effect might not come from central S1P activation but be associated with pleiotropic actions of the drug.

Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway.

Among sphingosine 1-phosphate receptors (S1PRs) family, S1PR1 has been shown to be the most highly expressed subtype in neural stem cells (NSCs) and plays a crucial role in the migratory property of NSCs. Recent studies suggested that S1PR1 was expressed abundantly in the hippocampus, a specific neurogenic region in rodent brain for endogenous neurogenesis throughout life. However, the potential association between S1PR1 and neurogenesis in hippocampus following traumatic brain injury (TBI) remains unknown. In this study, the changes of hippocampal S1PR1 expression after TBI and their effects on neurogenesis and neurocognitive function were investigated, focusing on particularly the extracellular signal-regulated kinase (Erk) signaling pathway which had been found to regulate multiple properties of NSCs. The results showed that a marked upregulation of S1PR1 occurred with a peak at 7 days after trauma, revealing an enhancement of proliferation and neuronal differentiation of NSCs in hippocampus due to S1PR1 activation. More importantly, it was suggested that mitogen-activated protein kinase-Erk kinase (MEK)/Erk cascade was required for S1PR1-meidated neurogenesis and neurocognitive recovery following TBI. This study lays a preliminary foundation for future research on promoting hippocampal neurogenesis and improving TBI outcome.

Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in HUVEC in the same experimental setting. Inhibition or knockdown of one of these receptor subtypes did not abolish the eNOS activation and subsequent NO production.

Sphingosine-1-phosphate receptor inhibition prevents denervation-induced dendritic atrophy.

A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways.

Blockade of Sphingosine 1-Phosphate Receptor 2 Signaling Attenuates High-Fat Diet-Induced Adipocyte Hypertrophy and Systemic Glucose Intolerance in Mice.

Sphingosine 1-phosphate (S1P) is known to regulate insulin resistance in hepatocytes, skeletal muscle cells, and pancreatic ß-cells. Among its 5 cognate receptors (S1pr1-S1pr5), S1P seems to counteract insulin signaling and confer insulin resistance via S1pr2 in these cells. S1P may also regulate insulin resistance in adipocytes, but the S1pr subtype(s) involved remains unknown. Here, we investigated systemic glucose/insulin tolerance and phenotypes of epididymal adipocytes in high-fat diet (HFD)-fed wild-type and S1pr2-deficient (S1pr2(-/-)) mice. Adult S1pr2(-/-) mice displayed smaller body/epididymal fat tissue weights, but the differences became negligible after 4 weeks with HFD. However, HFD-fed S1pr2(-/-) mice displayed better scores in glucose/insulin tolerance tests and had smaller epididymal adipocytes that expressed higher levels of proliferating cell nuclear antigen than wild-type mice. Next, proliferation/differentiation of 3T3-L1 and 3T3-F442A preadipocytes were examined in the presence of various S1pr antagonists: JTE-013 (S1pr2 antagonist), VPC-23019 (S1pr1/S1pr3 antagonist), and CYM-50358 (S1pr4 antagonist). S1P or JTE-013 treatment of 3T3-L1 preadipocytes potently activated their proliferation and Erk phosphorylation, whereas VPC-23019 inhibited both of these processes, and CYM-50358 had no effects. In contrast, S1P or JTE-013 treatment inhibited adipogenic differentiation of 3T3-F442A preadipocytes, whereas VPC-23019 activated it. The small interfering RNA knockdown of S1pr2 promoted proliferation and inhibited differentiation of 3T3-F442A preadipocytes, whereas that of S1pr1 acted oppositely. Moreover, oral JTE-013 administration improved glucose tolerance/insulin sensitivity in ob/ob mice. Taken together, S1pr2 blockade induced proliferation but suppressed differentiation of (pre)adipocytes both in vivo and in vitro, highlighting a novel therapeutic approach for obesity/type 2 diabetes.

Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

Intravenous administration of Factor VIII-O-Phospho-L-Serine (OPLS) complex reduces immunogenicity and preserves pharmacokinetics of the therapeutic protein.

Hemophilia A is a bleeding disorder caused by the deficiency of an important coagulation factor; Factor VIII (FVIII). Replacement therapy using exogenously administered recombinant FVIII is the most commonly used method of treatment. However, approximately 30% of Hemophilia A patients develop neutralizing antibodies (Nabs) against the recombinant protein. Nabs abolish FVIII activity and drastically influence efficacy of the protein. The immunogenic epitopes of FVIII reside predominantly in the C2 domain of FVIII. However, the C2 domain also contains a lipid binding region. O-Phospho-L-Serine (OPLS) which is the head-group moiety of phosphatidylserine, interacts with the lipid binding region of FVIII. Previous studies have shown that FVIII complexed with OPLS lowered Nab development against FVIII following subcutaneous administration. In dendritic cell-T-cell co-culture studies, OPLS treatment increased the secretion of immunosuppressive cytokines (Transforming Growth Factor-ß and Interleukin-10), and simultaneously decreased pro-inflammatory IL-17 cytokine. Here, we investigated FVIII immune response and pharmacokinetics upon intravenous administration of FVIII-OPLS complex. We studied the effect of FVIII-OPLS complex on the interaction between a professional antigen presenting cell; dendritic cell and T-cell, and T-cell clonal expansion. Pharmacokinetics parameters were estimated following intravenous administration of FVIII and FVIII-OPLS. The results suggest that OPLS lowers FVIII immune response following intravenous administration. OPLS also hinders FVIII-specific T-cell clonal proliferation and preserves FVIII PK profile. Thus, the ease of protein-lipid complexation, preservation of FVIII activity and in vivo behavior, and improved in vitro FVIII stability, makes OPLS an attractive excipient in the preparation of next generation or biosimilar FVIII products with improved safety profile.

Antioxidative stress effect of phosphoserine dimers is mediated via activation of the Nrf2 signaling pathway.

SCOPE: Phosphoserine-containing peptides have been shown to exert antioxidative stress effects, by lowering lipid peroxidation, increasing intracellular glutathione, and increasing the expression of antioxidant enzymes in human intestinal epithelial cells. However, the role of phosphoserine residues in antioxidative stress activity, and their mechanism of action, remains unknown. METHODS AND RESULTS: The antioxidative stress activity of phosphoserine and phosphoserine peptides was examined using an in vitro model of hydrogen peroxide (H2 O2 )-induced oxidative stress in Caco-2 cells. Phosphoserine dimers (2PS) reduced IL-8 secretion in H2 O2 -treated Caco-2 cells, and reduced H2 O2 -induced expression of genes involved in inflammation and generation of reactive oxygen species (ROS), including chemokine (C-C motif) ligand 5 (CCL5), lactoperoxidase (LPO), myeloperoxidase (MPO), neutrophil cytosolic factor 1/2 (NCF1/2), and nitric oxide synthase 2A (NOS2), and upregulated metallothionein 3 (MT3), peroxiredoxin 3 (PRDX3), and surfactant, pulmonary-associated protein D (SFTPD), which are involved in protection against oxidative stress and activation of the Nrf2 signaling pathway. At the protein level, 2PS reduced H2 O2 -induced phosphorylation of the ERK1/2 and JNK MAPKs, and increased Nrf2 expression. Moreover, the ability of 2PS to reduce H2 O2 -induced IL-8 secretion, a marker of inflammation and oxidative stress, was abrogated in Nrf2 knockdown cells. CONCLUSION: These results suggest that 2PS reduce H2 O2 -induced oxidative stress via the Nrf2 signaling pathway, and reveal a potential mechanism for the antioxidative stress activity of phosphoserine-containing peptides.

O-phospho-l-serine mediates hyporesponsiveness toward FVIII in hemophilia A-murine model by inducing tolerogenic properties in dendritic cells.

The clinical use of therapeutic proteins can be complicated by the development of anti-product antibodies. We have previously observed that O-phospho-l-serine (OPLS) reduced antibody response to FVIII in Hemophilia-A (HA) mice. However, the mechanism underlying this observation is not clear. We hypothesize that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells (DCs). We tested this hypothesis using in vivo, in vitro, and ex vivo methods. Naive HA mice that were pre-exposed to FVIII in the presence of OPLS showed substantially lower antibody response following rechallenge with OPLS free FVIII as compared with dexamethasone-pretreated mice. Exposure of OPLS to bone-marrow-derived dendritic cells (BMDCs) in culturing conditions resulted in an increase in the regulatory cytokine TGF-ß and a decrease in proinflammatory cytokines TNF-α and IL12p70. This was accompanied by a significant reduction in upregulation of costimulatory marker CD40, as measured by flow cytometry. Furthermore, ex vivo matured BMDCs in the presence of FVIII and OPLS failed to elicit a robust immune response in HA mice compared with FVIII-treated BMDCs. Our data suggest that OPLS modulates the immune response by altering the function and maturation of DCs, resulting in the induction of tolerogenic properties. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3457-3463, 2014.

Selectivity and evolutionary divergence of metabotropic glutamate receptors for endogenous ligands and G proteins coupled to phospholipase C or TRP channels.

To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G(i/o) proteins coupled to the ion channel TRPC4ß. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is L-serine-O-phosphate (L-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas L-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of L-glutamate and L-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both L-glutamate and L-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of L-SOP. Experiments with pertussis toxin and dominant-negative Gα(i/o) proteins revealed that mGluR1 couples strongly to TRPC4ß through Gα(i/o), in addition to coupling to PLC through Gα(q/11).