Lucía Fuentes

Bromoenol Lactone Promotes Cell Death by a Mechanism Involving Phosphatidate Phosphohydrolase-1 Rather than Calcium-independent Phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

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.

Cooperation Between Secretory Phospholipase A2 and TNF-Receptor Superfamily Signaling Implications for the Inflammatory Response in Atherogenesis

Abstract— Atherogenesis is the consequence of a variety of effector mechanisms rather than the result of a single functional molecule. In this connection, type IIA secretory phospholipase A2 (sPLA2) is an acute-phase reactant, which accumulates in atherosclerotic arterial walls, elicits several effects on monocytes, and has been related to the development of atherosclerosis. CD40/CD40 ligand pair is also a strong proatherogenic system. sPLA2 produced an increase of the surface expression of CD40 in THP-1 monocytes and enhanced the effect of CD40 ligation on the expression of both Fas and FasL, thus indicating the existence of a positive cooperation between sPLA2 and different elements of the TNF-receptor superfamily. Activation of the CD40/CD40L dyad with anti-CD40 antibody produced a small release of arachidonic acid and lacked any significant effect on the induction of cyclooxygenase-2, whereas the secretion of the chemokine MCP-1 and the surface display of CD11b, the &agr; chain of the integrin Mac-1, were upregulated. Engagement of CD40 did not influence the survival of THP-1 monocytes, but coincubation of THP-1 monocytes pretreated with anti-CD40 antibody and Jurkat cells induced a significant increase of the number of Jurkat cells showing binding of annexin-V, and nuclear condensation and fragmentation, thus indicating that this treatment might trigger a juxtacrine/paracrine mechanism of apoptotic death in sensitive cell types. This data indicates the existence of overlapping routes for the response to CD40, TNF-&agr;, and sPLA2, thus allowing the development of distinct patterns of response in monocytic cells.

Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis

AIMS Human atherosclerotic plaques express markers of macrophage/dendritic cells as well as high levels of inflammatory proteins such as secreted phospholipase A(2) type IIA (sPLA(2)-IIA). To understand the cellular changes associated with the progress of atherosclerosis, we evaluated the role of sPLA(2)-IIA in mediating monocyte recruitment and differentiation into antigen-presenting cells. METHODS AND RESULTS The effect of sPLA(2)-IIA on monocyte differentiation was evaluated in human THP-1 cells, a cellular line widely used as a model for monocyte-macrophage differentiation. Changes in functional processes, morphology and expression of antigens, characteristic of differentiated cells, were monitored over a 1-3 day period. sPLA(2)-IIA inhibited CD14 expression in a time- and concentration-dependent manner and upregulated dendritic cell-specific ICAM-3 grabbing non-integrin levels at the cell surface, findings that were the same for human monocytes. In addition, sPLA(2)-IIA-differentiated cells showed a dendritic cell phenotype characterized by the generation of fine dendritic protrusions and an increase in surface markers such as CD40, CD83, CD54, CD61, and CD62L. Furthermore, cell adhesion, migration, endocytic activity, and allogeneic T cell proliferation capacity were markedly increased after sPLA(2)-IIA treatment. CONCLUSION sPLA(2)-IIA induces the differentiation of mononuclear cells and increases their adhesive and migratory capabilities, which suggests a novel function for sPLA(2)-IIA as a mediator connecting innate and adaptive immunity. These findings may provide insight into the immuno-inflammatory processes occurring in atherosclerosis, helping us to understand the cellular changes associated with the development of atherosclerosis.

Effect of 4‐trifluoromethyl derivatives of salicylate on nuclear factor κB‐dependent transcription in human astrocytoma cells

The effect of two derivatives of salicylate, 2‐hydroxy‐4‐trifluoromethylbenzoic acid (HTB) and 2‐acetoxy‐4‐trifluoromethylbenzoic acid (triflusal), on the expression of several proteins displaying pro‐inflammatory activities the regulation of which is associated to the transcription factor NF‐κB, was assayed in the human astrocytoma cell line 1321N1. Tumour necrosis factor‐α (TNF‐α) activated NF‐κB as judged from both the appearance of κB‐binding activity in the nuclear extracts, the degradation of IκB proteins in the cell lysates, and the activation of IκB kinases using an immunocomplex kinase assay with glutathione S‐transferase (GST)‐IκB proteins as substrates. HTB up to 3 mM did not inhibit the nuclear translocation of NK‐κB/Rel proteins as judged from electrophoretic mobility‐shift assays; however, HTB inhibited the degradation of IκBβ without significantly affecting the degradation of both IκBα and IκBε. In keeping with their inhibitory effect on IκBβ degradation in the cell lysates, both HTB and triflusal inhibited the phosphorylation of GST‐IκBβ elicited by TNF‐α, without affecting the phosphorylation of GST‐IκBα. The effect of both HTB and triflusal on κB‐dependent trans‐activation was studied by assaying the expression of both cyclo‐oxygenase‐2 (COX‐2) and vascular cell adhesion molecule‐1 (VCAM‐1). HTB and triflusal inhibited in a dose‐dependent manner the expression of COX‐2 and VCAM‐1 mRNA and the induction of COX‐2 protein at therapeutically relevant concentrations. These findings show the complexity of the biochemical mechanisms underlying the activation of NF‐κB in the different cell types and extend the anti‐inflammatory effects of HTB and triflusal to neural cells.

Secreted phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells

Human group IIA secreted phospholipase A2 (sPLA2‐IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA2‐IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA2‐IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen‐activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA2‐IIA‐induced cell proliferation. We found that sPLA2‐IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA2‐IIA triggered Ras and Raf‐1 activation, with kinetics that paralleled ERK phosphorylation, and co‐immunoprecipitation assays indicated an association between Ras, Raf‐1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA2‐IIA treatment, effect that was abrogated by N‐acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N‐acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA2‐IIA‐induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.

Role of atrial tissue remodeling on rotor dynamics an in vitro study

The objective of this article is to present an in vitro model of atrial cardiac tissue that could serve to study the mechanisms of remodeling related to atrial fibrillation (AF). We analyze the modification on gene expression and modifications on rotor dynamics following tissue remodeling. Atrial murine cells (HL-1 myocytes) were maintained in culture after the spontaneous initiation of AF and analyzed at two time points: 3.1 ± 1.3 and 9.7 ± 0.5 days after AF initiation. The degree of electrophysiological remodeling (i.e., relative gene expression of key ion channels) and structural inhomogeneity was compared between early and late cell culture times both in nonfibrillating and fibrillating cell cultures. In addition, the electrophysiological characteristics of in vitro fibrillation [e.g., density of phase singularities (PS/cm(2)), dominant frequency, and rotor meandering] analyzed by means of optical mapping were compared with the degree of electrophysiological remodeling. Fibrillating cell cultures showed a differential ion channel gene expression associated with atrial tissue remodeling (i.e., decreased SCN5A, CACN1C, KCND3, and GJA1 and increased KCNJ2) not present in nonfibrillating cell cultures. Also, fibrillatory complexity was increased in late- vs. early stage cultures (1.12 ± 0.14 vs. 0.43 ± 0.19 PS/cm(2), P < 0.01), which was associated with changes in the electrical reentrant patterns (i.e., decrease in rotor tip meandering and increase in wavefront curvature). HL-1 cells can reproduce AF features such as electrophysiological remodeling and an increased complexity of the electrophysiological behavior associated with the fibrillation time that resembles those occurring in patients with chronic AF.

Inflammatory protein sPLA2-IIA abrogates TNFα-induced apoptosis in human astroglioma cells: Crucial role of ERK

Brain injury induces the expression of well-known cytokines, such as tumor necrosis factor-alpha (TNFalpha), and other, which functions are less understood, as secreted phospholipase A(2) group IIA (sPLA(2)-IIA). Since in pathological processes, cytokines function coordinately in networks, to further explore the actions of sPLA(2)-IIA in tumorigenesis, we investigated the effect of sPLA(2)-IIA in the presence of TNFalpha in human 1321N1 astrocytoma cells. In these cells, TNFalpha activates the apoptotic programme that is accompanied of cytoskeleton changes; however, simultaneous treatment with sPLA(2)-IIA prevents TNFalpha-mediated apoptosis and reverses the modification of the markers associated to this response. In fact, the mitogenic activity elicited by the phospholipase alone is preserved. This inhibitory effect is not found in other TNFalpha-mediated responses, even a functional cooperation is observed on COX-2 protein induction. The cross-talk between TNFalpha and sPLA(2)-IIA is associated with ERK activity since its pharmacological inhibition attenuates both synergistic and inhibitory responses. We have also observed that upon sPLA(2)-IIA stimulation, endogenous ERK has the capacity to bind and phosphorylate sequences present within the cytoplasmic domain of TNFR1/CD120a. These findings thus indicate that sPLA(2)-IIA and TNFalpha transduction pathways interact to modulate inflammatory responses and provide additional insights about the capacity of sPLA(2)-IIA to promote apoptosis resistance in astrocytoma cells.