Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

The modulation of apoptosis by cyclic AMP involves Akt and epidermal growth factor receptor

Adenosine 3',5'-cyclic monophosphate (cAMP) and transforming growth factor-beta are important regulators of many biological processes. In this study we investigated the effect and its potential mechanism of cAMP on transforming growth factor-beta1- and serum deprivation-induced apoptosis in Mv1Lu cells. Transforming growth factor-beta1 treatment or serum deprivation induces apoptotic response in Mv1Lu cells. Forskolin, a cAMP-elevating agent, or 8-Bromo-cAMP (8-B-cAMP), a cell permeable cAMP analogue, inhibited the cell proliferation and markedly enhanced apoptosis induced by transforming growth factor-beta1, but completely suppressed serum deprivation-induced apoptosis. Furthermore, forskolin decreased the Akt phosphorylation, and the inhibition of phosphatidylinositol-3 kinase by LY294002 sensitized Mv1Lu cells to transforming growth factor-beta1-induced apoptosis. In addition, forskolin treatment induced tyrosine phosphorylation of epidermal growth factor receptor. Inhibition of epidermal growth factor receptor by specific inhibitor PD153035 blocked the cAMP-mediated suppression of serum deprivation-induced apoptosis. The results indicate that cAMP exerts its opposite effects in transforming growth factor-beta1- and serum deprivation-induced apoptosis via a mechanism involving the modulation of signaling components of phosphatidylinositol-3-kinase/Akt and epidermal growth factor receptor in Mv1Lu cells.     

Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.     

Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-beta1 depends on the enhanced TGF-beta signaling and requires the decreased phosphorylation of PKB/Akt

We demonstrate how co-treatment of low-dose staurosporine (STS) and TGF-beta1, which alone have little effect on cell death, markedly induces apoptosis in Mv1Lu mink lung epithelial cells, but not in its clonal variant R1B cells lacking functional TGF-beta signaling. This process was associated with mitochondria-dependent apoptosis and the enhanced TGF-beta/Smad signaling in Mv1Lu cells. When R1B cells were infected with adenovirus carrying wild-type ALK5, a functional TGF-beta type I receptor gene, the apoptotic cell death was significantly restored in these cells following co-treatment of low-dose STS and TGF-beta1. Treatment of Mv1Lu cells with both low-dose STS and TGF-beta1 decreased the activity of phospho-Akt, which is involved in cell survival signal. In addition, pre-treatments of PI3 kinase inhibitors, LY294002 and wortmannin, further increased the apoptosis of MvlLu cells induced by co-treatment of low-dose STS and TGF-beta1. And overexpression of constitutively active Akt (myr-Akt) using adenoviral expression system inhibited the apoptotic cell death of Mv1Lu cells by about 50% upon co-treatment of low-dose STS and TGF-beta1. These results suggest that co-treatment of low-dose STS and TGF-beta1 induces apoptosis of mink lung epithelial cells by enhancing TGF-beta signaling and in part suppressing cytoprotective signaling.     

Amplification mechanisms of inflammation: paracrine stimulation of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid

In macrophages and other major immunoinflammatory cells, two phospholipase A(2) (PLA(2)) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A(2) (cPLA(2)) and a secreted phospholipase A(2) (sPLA(2)). In this study, the molecular mechanism underlying cross-talk between the two PLA(2)s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA(2)-dependent manner, and addition of exogenous group V sPLA(2) to the activated cells increases the release. This sPLA(2) effect is abolished if the cells are pretreated with cPLA(2) inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA(2). In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA(2) activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA(2) response to levels found in cells displaying normal cPLA(2) activity. Moreover, 12- and 15-HPETE are able to enhance sPLA(2) activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA(2)-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA(2) with cPLA(2) for a full AA mobilization response.     

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

Ceramide-induced enhancement of secretory phospholipase A2 expression via generation of reactive oxygen species in tumor necrosis factor-alpha-stimulated mesangial cells

Since prostanoids such as prostaglandin E2 play a pivotal role in modulating renal function, we investigated the involvement of ceramide in expression of secretory phospholipase A2 (sPLA2) and cyclooxygenase-2 (COX-2) in tumor necrosis factor-alpha (TNF-alpha)-stimulated mesangial cells. TNF-alpha stimulation increased ceramide generation in parallel with a decrease in sphingomyelin. Pretreatment with exogenous sphingomyelinase (SMase) dose-dependently enhanced TNF-alpha-stimulated increases in COX-2 protein and sPLA) activity. SMase also augmented TNF-alpha-mediated nuclear factor kappaB (NF-kappaB) activation. N-acetylcysteine (NAC), an antioxidant, completely inhibited the SMase-induced increase in sPLA2 activity, whereas NAC inhibited partially the activity stimulated with TNF-alpha alone. Under the conditions, NAC completely inhibited reactive oxygen species (ROS) production induced by SMase followed by TNF-alpha. These results suggest that ceramide elicits up-regulation of NF-kappaB through ROS production, which, in turn, leads to stimulation of COX-2 and sPLA2 expression. Therefore, ceramide may be implicated in the pathogenesis of renal abnormalities.     

The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609

The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1-5 min) and sustained eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2alpha phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin B1 or l-cycloserine. Interestingly, short-chain ceramide promoted eIF2alpha phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways. Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2alpha phosphorylation) or late (apoptotic) cytotoxic events.     

SPARC inhibits epithelial cell proliferation in part through stimulation of the transforming growth factor-beta-signaling system

Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell-cell and cell-matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-beta-sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-beta-insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-beta, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-beta and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-beta antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-beta-responsive reporter gene expression through a TGF-beta receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-beta signaling system, doing so through coupling of SPARC-EC to a TGF-beta receptor- and Smad2/3-dependent pathway.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Abstract

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.     

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.     

Daunorubicin- and Ara-C-induced interphasic apoptosis of human type II leukemia cells is caspase-8-independent

Drug-induced interphasic apoptosis in human leukemia cells is mediated through intracellular signaling pathways, of which the most proximal (initiating) event remains unclear. Indeed, both early ceramide generation and procaspase-8 cleavage have been individually identified as the initial apoptotic signaling events which precede the mitochondrial control of the apoptotic execution phase in Type II cells. In order to evaluate whether or not procaspase-8 cleavage is requisite for initial ceramide generation and rapid interphasic apoptosis, we investigated the chronological ordering of early ceramide generation and caspase-8 cleavage induced by daunorubicin (DNR) and 1-beta-D-arabinofuranosylcytosine (Ara-C) in U937 cells. We further evaluated the impact of these two drugs on initial ceramide generation and apoptosis in wild-type Jurkat cells and Jurkat clones mutated for caspase-8 and Fas-associated death domain. We show that while both DNR and Ara-C similarly induced early ceramide generation (within 5-20 min) and interphasic apoptosis in all cell models, caspase-8 cleavage was only observed farther downstream (4.5 h) and only in DNR-treated cells. Furthermore, neither DNR or Ara-C induced caspase-8 activation. These results demonstrate that caspase-8 cleavage is not requisite for the drug-induced activation of the ceramide-mediated interphasic apoptotic pathway in human Type II leukemic cells.     

Identification of a soluble form phospholipase A2 receptor as a circulating endogenous inhibitor for secretory phospholipase A2

Venomous snakes have various types of phospholipase A(2) inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A(2)s (PLA(2)s). Here we show the first evidence for the existence of circulating PLI against secretory PLA(2)s (sPLA(2)s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA(2)s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA(2) receptor (PLA(2)R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA(2) affinity column confirmed the identity of serum sPLA(2)-binding protein as a soluble form of PLA(2)R (sPLA(2)R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA(2)R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA(2), including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA(2)R-overexpressing Chinese hamster ovary cells suggested that sPLA(2)R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA(2)R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA(2)s in mice.     

Biological effects of group IIA secreted phosholipase A(2)

Group IIA secreted phospholipase A(2) (sPLA(2)-IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A(2), which shows properties different from those displayed by the cytosolic phospholipase A(2) involved in the release of arachidonic acid. sPLA(2)-IIA behaves as a ligand for a group of receptors inside the C-type multilectin mannose receptor family and also interacts with heparan sulfate proteoglycans such as glypican, the dermatan/chondroitin sulfate-rich decorin, and the chondroitin sulfate-rich versican, thus being able to internalize to specific compartments within the cell and producing biological responses. This review provides a short summary of the biological actions of sPLA(2)-IIA on intracellular signaling pathways.     

Saturated free fatty acid release and intracellular ceramide generation during apoptosis induction are closely related processes

Apoptosis induced by cells from the immune system is frequently associated with an increase in the ceramide content of target cells, due to the activation of sphingomyelinases (SMase). Some studies have also reported the release of saturated and monounsaturated free fatty acids (FFA) from apoptotic cells. However, the possible relationship between these lipid biochemistry events has not been characterized. We have analysed for the first time the release of FFA triggered by tumor necrosis factor-alpha (TNF-alpha), Fas/CD95 or the perforin/granzyme system of cytotoxic T lymphocytes (CTL) and their relationship to intracellular ceramide generation. TNF-alpha- and Fas-induced apoptosis are associated with both intracellular ceramide generation from sphingomyelin (SM) and release of palmitic-derived FFA, with similar kinetics. Intracellular SMase activation and FFA release from target cells during Fas-induced apoptosis are much more rapid and efficient if Fas-based cytotoxicity is exerted by alloantigenic CTL. In the case of perforin/granzyme-based cytotoxicity exerted by CTL, intracellular ceramide generation and FFA release from target cells seem to depend on the type of lysis induction used. Importantly, the correlation between intracellular SMase activation and the release of palmitic acid-derived FFA from target cells has been observed in all types of cytotoxicity assayed. In addition, exogenous natural ceramide induces the rapid release of the same FFA, well before any apoptotic sign is detected, and FFA release during Fas-induced apoptosis is inhibited in SM-depleted cells by chronic fumonisin-B(1) treatment. These results demonstrate a novel connection between the release of palmitic acid-derived FFA and intracellular ceramide accumulation during apoptosis induction.     

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.