Activation of phospholipase A2 and MAP kinases by oxidized low-density lipoproteins in immortalized GP8.39 endothelial cells

https://doi.org/10.1016/j.bbalip.2005.05.008Get rights and content

Abstract

In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A2 (cPLA2). A putative role for MAPKs in this process has emerged. Here, we studied the contribution of Ca2+-independent phospholipase A2 (iPLA2), and the role of the MAP kinase family as well as both cPLA2 and iPLA2 mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH2-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA2 activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 μM hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA2 protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA2 protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC–ERK–p38–JNK pathways and PLA2 activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.

Introduction

Oxidative modifications of circulating low-density lipoproteins (LDL) is a key step in the pathogenesis of atherosclerosis. Oxidized LDL (oxLDL) contains a complex, variable, incompletely characterized mixture of toxic oxidation products. Lipid hydroperoxides (Hpx) appear relatively early in the oxidation and aggressive aldehydes arise from their subsequent breakdown (for review, see Ref. [1]). Modified forms of LDL, including oxLDL, exert their pro-atherogenic effect on the walls of vasculature as well as an oxidative injury in both components of microvessels, endothelial cells (ECs) and pericytes [1], [2]. It is well recognized that oxidized lipoprotein interacts in a saturable manner with a number of multiligand scavenger receptors on the macrophage surface (SR-AI/II, SR-BI/II, CD36, LRP or LDL receptor-related protein), but the same knowledge does not arise from the less abundant studies on cells of the microvascular system. As expected, there are also receptors for modified LDLs on endothelial cells, but their activity is not mediated by type I and II macrophagic receptors [3]. In fact, human coronary and bovine, rabbit, rat aortic endothelial cells possess a scavenger receptor upregulated by oxLDL, named LOX-1 (lectin-like oxLDL-1) [4], which is endowed with an unusual broad binding specificity, and is distinct from the normal LDL receptor (ApoB/E receptor) and from the classical scavenger receptor SRs. In turn, the activation of LOX-1 induces the generation of reactive oxygen species (ROS) and decreases NO released from ECs [5].

Among its wide variety of biological properties, oxLDL exhibits a cytotoxic effect on the three cell types relevant to the development of atherosclerosis, namely macrophages, smooth muscle and ECs. Toxic doses of oxidized low-density lipoprotein or its lipid fractions induce apoptosis of both primary human or bovine ECs and immortalized ECs, as shown in numerous studies by characteristic morphological and biochemical changes [1], [6], [7].

We recently reported that oxLDL, in quiescent cells, triggers peroxidation processes and membrane phospholipid hydrolysis, activation of cytosolic phospholipase A2, without inducing significant cell death [8]. These culture conditions mimic a pre-apoptotic stage in which the endothelial cover of atherosclerotic areas may be found in the initial phase of the injury process or less advanced sclerotic lesions.

Several studies underscore the importance of the mitogen activated protein kinases (MAPK) cascade in regulating acute events in macro- and microvascular endothelium. In both systems, increasing evidence has been collected on the role of signal transduction pathways and on the protein tyrosine phosphorylation-dependent mechanism. Studies designed to determine the modulation of apoptosis by oxLDL in human coronary endothelial cells (HCAEC) after 24–27 h of treatment have shown increased Fas expression, decreased bcl-2 expression and an activation of PKCα and PTKs (protein tyrosine kinases). On the contrary, when the same quiescent cells were treated with 10 μM lysoPtdCho, this agent acutely activated ERK and JNK in a dose-dependent manner, but not conventional PKC [10]. Thus, the activation of PKC appears to vary in dependence on stimuli and cell types. More interestingly, mildly oxidized LDL induces apoptosis, mediated by Fas and TNF receptors, in primary cultures (24-h treatment) of HCAEC and smooth muscle cells [11]. This preparation also activates ERK1/2 and SAPK/JNK kinases, which are depressed by their respective inhibitors.

As mentioned above, the study of the effect of oxLDL on ECs has been previously approached in terms of MAPK signaling, particularly the short-term effects. We want to contribute to this subject, firstly presenting novel data concerning the involvement of Ca2+-independent PLA2 (iPLA2, type VI), an enzyme activity showing selectivity for hydrolysis of phospholipids bearing arachidonate, inhibition by bromoenol lactone (BEL), and activation during a variety of pathological processes [12], [13]. Among the growing number of PLA2s that have been isolated and characterized thus far, a calcium-dependent high molecular mass cytosolic PLA2 (cPLA2) and a calcium-independent PLA2 have been shown to play an important role in arachidonic acid release in response to a number of stimulants. In regard to iPLA2, the mechanism of regulation of its activity is unclear, for instance, whether or not its response to mitogenic signal events or to prooxidants is mediated by MAPKs. A second reason for turning our attention to the study of oxLDL action on ECs is that the rat brain GP8.39 cells we used are microvascular in origin, and their response to oxLDL may be distinct from that in large vessel ECs where the vast majority of previous studies were performed. Besides, our findings must be placed in the context of the long-term pre-apoptotic effects of oxLDL. Our results demonstrate that, in cerebral GP8.39 cells, ERK1/2, p38 and JNK MAPK phosphorylation status is the major regulatory mechanism leading to the cPLA2 activation (increase in cPLA2 enzyme activity and cPLA2 phosphorylation as shown in our previous study [8]) by oxLDL; in addition, cPLA2 and iPLA2 activations are sustained by the increase in protein and steady-state mRNA expression relative to both enzymes.

Section snippets

Materials and antibodies

Reagent grade chemicals were purchased from Sigma Chemicals Co. (St. Louis, MO) or E. Merck (Darmstadt, Germany). Collagenase-dispase, fatty acid-free BSA, arachidonic acid, lipid standards, NADH, and DTT were purchased from Sigma (St. Louis, MO). Bromoenol lactone (BEL), arachidonoyl trifluoromethyl ketone (AACOCF3), PD98059, SB203580, SP600125 and GF109203X were purchased from Calbiochem (La Jolla, CA). Rabbit polyclonal antibody against ERK1 or ERK2, mouse monoclonal anti-p38, mouse

Phospholipase A2 activity and the effect of inhibitors

iPLA2, in addition to cPLA2, might be involved in arachidonic acid (AA) liberation in stimulated endothelial cells. The participation of both phospholipases in stimulus-induced AA liberation remained to be elucidated. In the present study, to clarify the role of group VI iPLA2 upon oxLDL stimulation in ECs, we evaluated the effect of PLA2 inhibitors such as BEL, AACOCF3 and EDTA. The enzyme activity insensitive to BEL represents the Ca2+-dependent PLA2, whereas that insensitive to EDTA

Discussion

Expanding on our previous study [8], we examined the effect of oxLDL on the regulatory pathways of stimulated cPLA2 and iPLA2 activities. Our findings must be placed in the context of the long-term pre-apoptotic effects of oxLDL. We have previously reported that a significant increase in malondialdehyde levels and LDH release attested to the induction of oxidative changes in endothelial cell membranes due to free radical-mediated injury. In addition, prolonged exposure of quiescent ECs to oxLDL

Acknowledgements

The authors wish to thank Mr. Antonino Costa, Mr. Alessandro Giacchetto and Mr. Piero Rapisarda for their excellent technical assistance. This work was supported by grants from MIUR-Italy and Consorzio Interuniversitario Biotecnologie (CIB)-Italy.

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