Danilo Guerini

Proton-sensing G-protein-coupled receptors

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8—site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors

Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a functional antagonist by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

Oxysterols direct immune cell migration via EBI2.

Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate

BAF312 is a next‐generation sphingosine 1‐phosphate (S1P) receptor modulator, selective for S1P1 and S1P5 receptors. S1P1 receptors are essential for lymphocyte egress from lymph nodes and a drug target in immune‐mediated diseases. Here, we have characterized the immunomodulatory potential of BAF312 and the S1P receptor‐mediated effects on heart rate using preclinical and human data.

Requirement for O-linked N-acetylglucosaminyltransferase in lymphocytes activation

The dynamic modification of nuclear and cytoplasmic proteins with O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) by the O‐linked N‐acetylglucosaminyltransferase (OGT) is a regulatory post‐translational modification that is responsive to various stimuli. Here, we demonstrate that OGT is a central factor for T‐ and B‐lymphocytes activation. SiRNA‐mediated knockdown of OGT in T cells leads to an impaired activation of the transcription factors NFAT and NFκB. This results in a reduction of IL‐2 production consistent with prevention of T‐cell activation. OGT is also required for the early activation of B cells mediated by stimulation of the B‐cell receptor. Mechanistically, we demonstrate that NFκB as well as NFAT are glycosylated with O‐GlcNAc after direct binding to OGT. Moreover, kinetic experiments show that O‐GlcNAc modification prominently increased shortly after activation of lymphoid cells and it might be required for nuclear translocation of the transcription factors NFκB and NFAT.

Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors

Sphingosine‐1‐phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein‐mediated signaling cascades. We also show that FTY720P as well as the S1P1‐selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.

Second generation S1P pathway modulators: research strategies and clinical developments.

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Expression, Purification, and Characterization of Isoform 1 of the Plasma Membrane Ca2+ Pump FOCUS ON CALPAIN SENSITIVITY

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.

A Potent and Selective S1P1 Antagonist with Efficacy in Experimental Autoimmune Encephalomyelitis

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.

The O-linked N-acetylglucosamine modification in cellular signalling and the immune system. 'Protein modifications: beyond the usual suspects' review series.

The intracellular modification of proteins by the addition of a single O‐linked N‐acetylglucosamine (O‐GlcNAc) molecule is a ubiquitous post‐translational modification in eukaryotic cells. It is catalysed by O‐linked N‐acetylglucosaminyltransferase, which attaches O‐GlcNAc to serine/threonine residues, and it is counter‐regulated by β‐N‐acetylglucosaminidase, which is the antagonistic glycosidase that removes the O‐GlcNAc group. O‐GlcNAc modification competes with phosphorylation by protein kinases at similar sites, thereby affecting important signalling nodes. Accumulating evidence supports a central role for O‐GlcNAc modifications and the corresponding enzymes in the regulation of immune cells, particularly in the activation processes of T and B lymphocytes. Here, we discuss recent advances in the field of O‐GlcNAc modifications, focusing on the cells of the immune system.