COX-2 and PGE2 signaling is essential for the regulation of IDO expression by curcumin in murine bone marrow-derived dendritic cells.

Indoleamine 2,3-dioxygenase (IDO), a key enzyme that catalyzes the initial, rate-limiting step in tryptophan degradation, is expressed in dendritic cells (DCs) which are stimulated by lipopolysaccharide (LPS) or interferons. In this study we show that curcumin inhibits IDO expression in vitro and in vivo in DCs, leading to the suppression of LPS-induced DC maturation. The effect of curcumin relative to LPS is not limited to the above, as it also enhances LPS-induced expression of cyclooxygenase (COX)-2 and production of prostaglandin E2 (PGE2). Additionally, PGE2 diminished the LPS-induced IDO expression in DCs, thereby contributing to the inhibition of expression of the surface molecules (CD80, CD86 and MHC class I) and the production of the proinflammatory cytokines (IL-12 p70 and TNF-alpha) by LPS stimulation. Under our experimental conditions, curcumin plays an immunomodulatory role by downregulating IDO expression via a COX-2/PGE2-dependant pathway, thus impacting DC maturation in vitro and in vivo.

FcepsilonRI-mediated mast cell migration: signaling pathways and dependence on cytosolic free Ca2+ concentration.

IgE-sensitized rat basophilic leukemia (RBL)-2H3 mast cells have been shown to migrate towards antigen. In the present study we tried to identify the mechanism by which antigen causes mast cell migration. Antigen caused migration of RBL-2H3 cells at the concentration ranges of 1000-fold lower than those required for degranulation and the dose response was biphasic. This suggests that mast cells can detect very low concentration gradients of antigen (pg/ml ranges), which initiate migration until they degranulate near the origin of antigen, of which concentration is in the ng/ml ranges. Similar phenomenon was observed in human mast cells (HMCs) derived from CD34(+) progenitors. As one mechanism of mast cell migration, we tested the involvement of sphingosine 1-phosphate (S1P). Fc epsilon RI-mediated cell migration was dependent on the production of S1P but independent of a S1P receptor or its signaling pathways as determined with S1P receptor antagonist VPC23019 and Gi protein inhibitor pertussis toxin (PTX). This indicated that the site of action of S1P produced by antigen stimulation was intracellular. However, S1P-induced mast cell migration was dependent on S1P receptor activation and inhibited by both VPC23019 and PTX. Cell migration towards antigen or extracellular S1P was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, while only migration towards antigen was inhibited by the inhibitors of sphingosine kinase and phospholipase C (PLC) and intracellular calcium chelator BAPTA. In summary, our data suggest that the high affinity receptor for IgE (Fc epsilon RI)-mediated mast cell migration is dependent on the production of S1P but independent of S1P receptors. Cell migration mediated by either Fc epsilon RI or S1P receptors involves activation of both PI3K and MAPK.

Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner.

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcepsilonRI-mediated Ca(2+) response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP(3)) production. This suggests that the Ca(2+) signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cgamma was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca(2+) pathway to be dependent on clathrin.

Agonists of the MAS-related gene (Mrgs) orphan receptors as novel mediators of mast cell-sensory nerve interactions.

IgE-dependent activation of mast cell activation is often associated with symptoms attributed to activation of sensory nerves. Depending on the tissues involved such symptoms include itching, sneezing, irritation, vasodilation, and reflex secretions. In the present study, we hypothesize that sensory neuroactive mediators released from mast cells may include agonists of recently discovered orphan receptors referred to as sensory nerve specific receptors or products of mas related genes. HEK-293 cells expressing MrgC11 receptors and wild-type HEK-293 cells were loaded with the calcium indicator Fura-2. A known stimulant of MrgC11 receptors the RF-amide, neuropeptide FF, evoked a rapid increase in cytosolic calcium in the MrgC11 expressing cells but not in the wild-type HEK-293 cells. IgE-dependent stimulation of either rat basophilic leukemia-2H3 cells (RBL-2H3 cells) or mouse bone marrow-derived mast cells, released a substance(s) that stimulated increases in cytosolic calcium in the MrgC11 expressing cells that far exceeded that seen in control cells. RT-PCR revealed that both mouse mast cells and RBL-2H3 cells express the RF-amide precursor gene proneuropeptide FF (A). Immunohistochemical analysis demonstrated RF-amide immunoreactivity in mouse skin mast cells in situ and in mast cells isolated from mouse skin. These data support the hypothesis that agonists of certain sensory nerve specific receptors or mas related genes may participate in mast cell sensory nerve interactions.

Cloning and characterization of rat sphingosine kinase 1 with an N-terminal extension.

Sphingosine kinase (SK) is an enzyme that converts sphingosine to sphingosine 1-phosphate, a lysophospholipid with various cellular functions. Here, we report cloning and characterization of a novel isoform of rat SK1 (rSK1) with an N-terminal extension (long-rSK1). Long-rSK1 is 458 amino acid-long and has a calculated molecular weight of 49.8kDa. Deduced amino acid sequences of rat and human long-SK1 have high homology (71.3% identity and 78.9% similarity). Long-rSK1 mRNA is widely expressed throughout the tissues including brain, heart, lung, liver, kidney, and spleen, whereas mRNA encoding rSK1 has been shown not to be expressed in heart or liver. When the long-rSK1 was transfected in COS-7 cells, SK activity was 53-fold increased. Substrate specificity and dependency on divalent cations of long-rSK1 were similar to those of rSK1. Taken together, the fact that long-rSK1 with similar enzymatic characteristics to rSK1 displays differential tissue distribution may suggest an additional role of long-rSK1.

Antigen-induced Ca2+ mobilization in RBL-2H3 cells: role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production.

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.