Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.     

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.     

Integrin-mediated mechanotransduction processes in TGFbeta-stimulated monolayer-expanded chondrocytes

Previous studies have demonstrated that passage in monolayer detrimentally affects the response of articular chondrocytes to the application of dynamic compression. Transforming growth factor beta (TGFbeta) is known to regulate metabolic processes in articular cartilage and can enhance the re-expression of a chondrocytic phenotype following monolayer expansion. The current study tests the hypothesis that TGFbeta also modulates the response of monolayer-expanded human chondrocytes to the application of dynamic compression, via an integrin-mediated mechanotransduction process. The data presented demonstrate that TGFbeta3 enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release after 48 h of culture when compared to unsupplemented constructs. Dynamic compression also enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release in the presence of TGFbeta3. By contrast, dynamic compression did not alter these parameters in the absence of the growth factor. The addition of the peptide, GRGDSP, which acts as a competitive ligand for the alpha5beta1 integrin, reversed the compression-induced stimulation of 35SO4 incorporation, [3H]thymidine incorporation, and suppression of nitrite release. No effect was observed when the control peptide, GRADSP, was used. The current data clearly demonstrate that the dynamic compression-induced changes observed in cell metabolism for human monolayer-expanded chondrocytes were dependent on the presence of TGFbeta3 and are integrin-mediated.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Dynamic compression inhibits fibronectin fragment induced iNOS and COX-2 expression in chondrocyte/agarose constructs

Mechanical loading and the fibronectin fragments (FN-fs) are known to stimulate the anabolic and catabolic processes in articular cartilage, possible through pathways mediated by ^·NO. This study examined the combined effects of dynamic compression and the NH₂-hep I or COOH-hep II FN-fs on the expression levels of iNOS and COX-2 and production of ^·NO and PGE₂ release. Both types of fragments induced iNOS and COX-2 expression and stimulated the production of ^·NO release. This response was inhibited by dynamic compression. Inhibitor experiments indicated that both dynamic compression and the iNOS inhibitor were important in restoring cell proliferation and proteoglycan synthesis in the presence of the FN-fs. This is the first study which demonstrates a downregulation of the FN-f-induced iNOS and COX-2 expression by dynamic compression. The combination of mechanical and pharmacological interventions makes this study a powerful tool to examine further the interactions of biomechanics and cell signalling in osteoarthritis.     

Interleukin-1β Stimulates Mitogen-Activated Protein Kinase in U373 Astrocytoma Cells without the Production of Lipid Second Messengers

IL-1 is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 activated only MAPK. In cultured rat astrocytes, IL-1 caused activation of MAPK without inducing proliferation.     

The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.     

Prostaglandin E2 production is synergistically increased in cultured human glomerular mesangial cells by combinations of IL-1 and tumor necrosis factor-alpha 1

Both IL-1 alpha and IL-1 beta and TNF-alpha induced a time- and dose-dependent release of authentic PGE2 from cultured human glomerular mesangial cells (HMC). This release became significant only after a 4- to 6-h lag phase, and was abolished by inhibition of protein synthesis, and was not related to cell proliferation. Combinations of IL-1 and TNF-alpha when added simultaneously to HMC resulted in a dose-dependent synergistic increase in PGE2 production. These stimulatory effects were specifically inhibited by anticytokine antibodies and the synergistic effect required the simultaneous presence of both IL-1 and TNF-alpha. Arachidonic acid (AA) release experiments and measurement of cyclooxygenase activity, revealed that while both were increased by IL-1 beta and TNF-alpha alone (IL-1 beta greater than TNF-alpha), combinations of IL-1 beta and TNF-alpha resulted in only additive increases in AA release and cyclooxygenase activity. Taken together, these data suggest that stimulation of PGE2 in HMC, by combinations of these cytokines, is not rate limited by AA release or cyclooxygenase activation, but may be related to the induction of the distal enzymes controlling specific PG synthesis.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of TGF-beta, TNF-alpha, IL-beta and IL-6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, prostaglandin E2 production and bone resorption in mouse calvarial bone cells

Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E(2) (PGE(2)) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and, to a lesser extent, TNF-alpha, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1beta and TNF-alpha resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE(2) biosynthesis compared to cells treated with IL-1beta or TNF-alpha or IL-6 alone. In contrast, the presence of TGF-beta reduced COX-2 mRNA level, PGE(2) biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or a combination thereof. However, neither IL-1beta, TNF-alpha, IL-6 nor a combination of IL-1beta, TNF-alpha, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE(2) production induced by IL-1beta, TNF-alpha and IL-6 or a combination of IL-1beta, TNF-alpha, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-beta, TNF-alpha, IL-6 on PGE(2) production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss.     

Expression of the VEGF receptor-3 in osteoarthritic chondrocytes: stimulation by interleukin-1β and association with β<Subscript>1</Subscript>-integrins

Recent studies have demonstrated enhanced expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGFR)-1 and -2 in chondrocytes of rheumatoid and osteoarthritic cartilage. Since expression of VEGFR-3 (Flt-4) in chondrocytes has not yet been investigated, we studied the distribution of VEGFR-3 in osteoarthritic cartilage samples by immunohistochemistry and immunoelectron microscopy. Furthermore, we looked for functional colocalization of VEGFR-3 with the signal transduction receptor ₁-integrin. Superficial osteoarthritic chondrocytes exhibited VEGFR-3 expression in the cytoplasm and on the cell membrane. Using western blotting we could demonstrate that interleukin-1 (IL-1) stimulates the expression of VEGFR-3 in chondrocytes in vitro in a dose-dependent manner. By coimmunoprecipitation assay we found a functional complex between the ₁-integrin and VEGFR-3 in IL-1-stimulated chondrocytes indicating that activated VEGFR-3 may interact with ₁-integrin and associated subcellular pathways in osteoarthritic chondrocytes. Taken together with results of previous studies showing that ₁-integrins were also associated with other surface receptors and proteins in chondrocytes that cause cartilage destruction in arthritis (for example, urokinase-type plasminogen activator receptor and matrix metalloproteinases), we can hypothesize that signal transduction by these receptor complexes via ₁-integrins may play a crucial role in pathogenesis of osteoarticular disorders. Further work needs to be done to elucidate downstream signaling events activated by these receptors.     

Phagocytosis of N-acetyl-D-glucosamine particles, a Th1 adjuvant, by RAW 264.7 cells results in MAPK activation and TNF-alpha, but not IL-10, production

A practical and highly effective Th1 adjuvant should induce Th1 cytokines (IL-12, IL-18, and TNF-alpha) but not the Th2 cytokine IL-10, an inhibitor of Th1 responses. In this study, phagocytosis of N-acetyl-d-glucosamine polymer (chitin) particles by RAW 264.7 cells, a murine macrophage-like cell line, resulted in phosphorylation of MAPK (p38, Erk 1/2, and JNK), and production of relatively high levels of TNF-alpha and COX-2 with increased PGE(2) release. Similar results were observed in response to oligonucleotides with CpG motifs, mycobacterial components and endotoxin. However, these bacterial components also induced a large amount of IL-10. Chitin particles, in contrast, failed to induce detectable levels of IL-10, although the production of high levels of PGE(2) and TNF-alpha and the activation of MAPK's are potentially positive signals for IL-10 production. Thus, our results indicate that chitin particles act as a unique Th1 adjuvant for macrophages without inducing increased production of IL-10.     

Effects of IL-1β on Receptor-Mediated Poly-Phosphoinositide Signaling Pathway in Immortalized Astrocytes (DITNC)

Astrocytes are known to play multi-functional roles in support of many homeostatic mechanisms in the central nervous system including host defense mechanisms. Despite the ability of cytokines to alter gene expression and cellular activity, their effect on receptor-mediated poly-phosphoinositide (poly-PI) signaling pathway has not been examined in detail. In this study, an immortalized astrocyte cell line (DITNC) was used to test the effect of IL-1 exposure on the poly-PI signaling pathway. Similar to primary astrocytes, DITNC cells exhibit P₂-purinergic receptor response to ATP and UTP leading to transient increases in inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular calcium concentration, [Ca²⁺]i. Upon exposure of DITNC cells to IL-1 (100U/ml) for 24 hrs, an increased response to the poly-PI agonists was observed. The increase in ATP-mediated Ins(1,4,5)P₃ release could not be attributed to a shift in the ATP dose or an alteration of the time profile for the release of Ins(1,4,5)P₃. Since the increase in response required a lag time of 4 hr after IL-1 exposure, it is unlikely that this effect was due to a direct interaction of IL-1 with the purinergic receptor. On the other hand, an increase in ATP response could be observed in DITNC cells exposed to conditioned medium obtained after IL-1 treatment. It can be concluded that exposure of astrocytes to cytokines may lead to an increase in receptor-mediated poly-PI signaling activity and this may involve compounds secreted into the culture medium, e.g., the secretory phospholipase A₂.     

Thrombospondin-1 differentially regulates release of IL-6 and IL-10 by human monocytic cell line U937

We investigated possible regulatory effects of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, on cytokine release from macrophages. Immobilized TSP-1 enhanced IL-6 release from the human monocytic U937 cells stimulated with phorbol myristate acetate and LPS, whereas it inhibited IL-10 release. The 70-kDa fragment of TSP-1 containing the type 1 repeats showed the same regulatory effects. The enhanced IL-6 release by TSP-1 was inhibited by anti-CD36 antibody or antibody against the sequence of the binding site to CD36 in the type 1 repeats of TSP-1. Conversely, the decrease in IL-10 release by TSP-1 was strengthened by the blocking of the interaction between CD36 and TSP-1. Furthermore, the involvement of TGF-beta1 in the inhibition of IL-10 release by TSP-1 was indicated by the facts that (i) TSP-1 induced activation of TGF-beta1 produced by the U937 cells, (ii) exogenously added TGF-beta1 inhibited IL-10 release, and (iii) antibody against TGF-beta1 blocked the inhibition of IL-10 release by TSP-1. Together, the present findings suggest that TSP-1 enhances IL-6 release from macrophages by interaction with CD36, whereas IL-10 release is regulated by the balance between the enhancing effect of TSP-1 via CD36 and the suppressive effect by TSP-1-activated TGF-beta1.     

Antigen-induced Ca 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.