Mariano Sánchez Crespo

Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1

The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such asp-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptorp-aminophenyl-α-d-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation.

Activation of monocytic cells through Fcγ receptors induces the expression of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and RANTES

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcγRI, FcγRIIA, and FcγRIII, LPS, TNF-α, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcγR with mAb reacting with FcγRI and FcγRIIA, but not with a mAb reacting with FcγRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcγRI and FcγRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcγR cross-linking, although expression of IFN-γ-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1α, MIP-1β, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1α using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein β sites and ruled out the functional significance of both NF-AT and AP-1 sites.

Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by Tumor necrosis factor-α : Phosphorylation of cytosolic phospholipase A2 and transactivation of cyclooxygenase-2

Abstract : Tumor necrosis factor‐α (TNF‐α) is a cytokine that elicits cell responses by activating the mitogen‐activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor‐kB (NF‐kB). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase‐2 (COX‐2), the effect of TNF‐α on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF‐α produced a phosphorylation of cPLA2, which was preceded by an activation of both c‐Jun N‐terminal kinase (JNK) and p38‐MAP kinase, and this was associated with the release of [3H]AA. In contrast, TNF‐α did not activate the extracellular signal‐regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic response. Analysis of [3H]AA metabolites by reverse‐phase HPLC showed that all of the [3H]AA released during the first hour after TNF‐α addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF‐α, 25% of the [3H]AA had been converted into COX products as compared with only 9% in control cells. In keeping with these findings, TNF‐α produced an increase of COX‐2 expression, as judged from both RT‐PCR studies and immunoblot of COX‐2 protein, and a long‐lasting activation of NF‐kB. These data show that TNF‐α produces in astrocytoma cells an early activation of both p38‐MAP kinase and JNK, which is followed by the phosphorylation of cPLA2 and the release of AA. On the other hand, the activation of NF‐kB may explain the induction of the expression of COX‐2 and the delayed generation of prostanoids.

4-trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid, are potent inhibitors of nuclear factor κB activation

The effect of two derivatives of salicylate, 2‐hydroxy‐4‐trifluoromethylbenzoic acid (HTB) and 2‐acetoxy‐4‐trifluoromethylbenzoic acid (triflusal), on the activation of NF‐κB elicited by tumour necrosis factor‐α (TNF‐α) on human umbilical vein endothelial cells (HUVEC) was tested. The expression of the mRNA of vascular cell adhesion molecule‐1 (VCAM‐1) was studied as an example of a gene the expression of which is regulated by NF‐κB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF‐κB. The calculation of the IC50 values showed ∼amp;2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. Comparison of the potency of these compounds on VCAM‐1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36–43% inhibition at the same concentration. Inhibition of NF‐κB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose‐dependent inhibition of nitrite production by the L‐arginine pathway via iNOS. IC50 values for this effect were 1.13±0.12 mM (triflusal), 1.84±0.34 (HTB), 6.08±1.53 mM (aspirin) and 9.16±1.9 mM (salicylate). These data indicate that the incorporation of a 4‐trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF‐κB activation, VCAM‐1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo‐oxygenase.

The Induction of IL-10 by Zymosan in Dendritic Cells Depends on CREB Activation by the Coactivators CREB-Binding Protein and TORC2 and Autocrine PGE2

Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated κB-binding activity, but inhibition of NF-κB was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE2, and inhibitors of PKA, COX, and glycogen-synthase kinase-3β showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE2 and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3β, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.

Costimulation of dectin-1 and DC-SIGN triggers the arachidonic acid cascade in human monocyte-derived dendritic cells.

Inflammatory mediators derived from arachidonic acid (AA) alter the function of dendritic cells (DC), but data regarding their biosynthesis resulting from stimulation of opsonic and nonopsonic receptors are scarce. To address this issue, the production of eicosanoids by human monocyte-derived DC stimulated via receptors involved in Ag recognition was assessed. Activation of FcγR induced AA release, short-term, low-grade PG biosynthesis, and IL-10 production, whereas zymosan, which contains ligands of both the mannose receptor and the human β-glucan receptor dectin-1, induced a wider set of responses including cyclooxygenase 2 induction and biosynthesis of leukotriene C4 and IL-12p70. The cytosolic phospholipase A2 inhibitor pyrrolidine 1 completely inhibited AA release stimulated via all receptors, whereas the spleen tyrosine kinase (Syk) inhibitors piceatannol and R406 fully blocked AA release in response to immune complexes, but only partially blocked the effect of zymosan. Furthermore, anti-dectin-1 mAb partially inhibited the response to zymosan, and this inhibition was enhanced by mAb against DC-specific ICAM-3-grabbing nonintegrin (SIGN). Immunoprecipitation of DC lysates showed coimmunoprecipitation of DC-SIGN and dectin-1, which was confirmed using Myc-dectin-1 and DC-SIGN constructs in HEK293 cells. These data reveal a robust metabolism of AA in human DC stimulated through both opsonic and nonopsonic receptors. The FcγR route depends on the ITAM/Syk/cytosolic phospholipase A2 axis, whereas the response to zymosan involves the interaction with the C-type lectin receptors dectin-1 and DC-SIGN. These findings help explain the distinct functional properties of DC matured by immune complexes vs those matured by β-glucans.

KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases

Potassium channel tetramerization domain (KCTD) proteins contain a bric‐a‐brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage‐gated potassium channels. Some BTB‐domain‐containing proteins have been shown recently to participate as substrate‐specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty‐two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post‐transcriptionally in peripheral blood lymphocytes stimulated through the T‐cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N‐terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate‐specific adaptor for cullin3‐based E3 ligases.

Biological effects of group IIA secreted phosholipase A2

Group IIA secreted phospholipase A2 (sPLA2‐IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A2, which shows properties different from those displayed by the cytosolic phospholipase A2 involved in the release of arachidonic acid. sPLA2‐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 sPLA2‐IIA on intracellular signaling pathways.

Polarization of the innate immune response by prostaglandin E2: A puzzle of receptors and signals

Eicosanoids tailor the innate immune response by supporting local inflammation and exhibiting immunomodulatory properties. Prostaglandin (PG) E2 is the most abundant eicosanoid in the inflammatory milieu due to the robust production elicited by pathogen-associated molecular patterns on cells of the innate immune system. The different functions and cell distribution of E prostanoid receptors explain the difficulty encountered thus far to delineate the actual role of PGE2 in the immune response. The biosynthesis of eicosanoids includes as the first step the Ca2+- and kinase-dependent activation of the cytosolic phospholipase A2, which releases arachidonic acid from membrane phospholipids, and later events depending on the transcriptional regulation of the enzymes of the cyclooxygenase routes, where PGE2 is the most relevant product. Acting in an autocrine/paracrine manner in macrophages, PGE2 induces a regulatory phenotype including the expression of interleukin (IL)-10, sphingosine kinase 1, and the tumor necrosis factor family molecule LIGHT. PGE2 also stabilizes the suppressive function of myeloid-derived suppressor cells, inhibits the release of IL-12 p70 by macrophages and dendritic cells, and may enhance the production of IL-23. PGE2 is a central component of the inflammasome-dependent induction of the eicosanoid storm that leads to massive loss of intravascular fluid, increases the mortality rate associated with coinfection by Candida ssp. and bacteria, and inhibits fungal phagocytosis. These effects have important consequences for the outcome of infections and the polarization of the immune response into the T helper cell types 2 and 17 and can be a clue to develop pharmacological tools to address infectious, autoimmune, and autoinflammatory diseases.

Lipopolysaccharides of Brucella abortus and Brucella melitensis Induce Nitric Oxide Synthesis in Rat Peritoneal Macrophages

ABSTRACT Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucellain humans.