Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.

Les eicosanoïdes sont des médiateurs importants qui encadrent et régulent les fonctions osseuses. Leur production est sous la tutelle des phospholipases A[indice inférieur 2] qui permettent la relâche d’acide arachidonique et de lysophospholipides puis de leur métabolisme subséquent des membranes cellulaires. Les PLA[indice inférieur 2] sécrétées ont également comme particularité de pouvoir exercer leurs effets directement via leurs récepteurs membranaires comme ligand. Malgré l’implication connue des prostaglandines sur les fonctions ostéoclastiques et dans plusieurs processus pathologiques résultants en érosion osseuse, les phospholipases A[indice inférieur 2] ostéoclastiques restent inconnues et leurs rôles, spéculatifs. Les études présentées démontrent la présence de la cPLA[indice inférieur 2]-α et de la sPLA[indice inférieur 2] IIA chez les ostéoclastes humains. Par contre, leur expression semble différer selon l’état de l’os. En effet, la cPLA[indice inférieur 2]-α semble exprimée ubiquitairement, mais la sPLA[indice inférieur 2] IIA n’est détectable que dans l’os fœtal ou atteint de la maladie de Paget et n’est pas exprimée dans l’os sain, ostéoporotique ou arthrosique. La sPLA[indice inférieur 2] IIA semble donc exprimée en condition de fort remodelage osseux. Dans notre modèle, la cPLA[indice inférieur 2]-α revêt un rôle anti-ostéoclastogénique. En effet, la cPLA[indice inférieur 2]- α produit des écosanoïdes qui inhibent l’ostéoclastogenèse. Ces derniers sensibilisent les ostéoclastes à l’apoptose. En revanche, un certain degré d’activation de la cPLA[indice inférieur 2]-α est requis pour la résorption osseuse, car son inhibition bloque la résorption osseuse en désorganisant les anneaux d’actine requis pour la résorption, et ce, de façon dépendante de la production d’acide arachidonique. En ce qui a trait à la sPLA[indice inférieur 2] IIA, elle stimule l’ostéoclastogenèse indépendamment de son activité catalytique, probablement via l’un de ses récepteurs membranaires. D’autre part, elle confère une résistance à l’apoptose autant chez les ostéoclastes matures que chez leurs précurseurs CD14+. Par contre, l’inhibition de la sPLA[indice inférieur 2] IIA bloque la résorption osseuse indépendamment du remodelage du cytosquelette d’actine, probablement via leur apoptose. Les résultats inclus dans cette thèse semblent donc démontrer la présence de deux PLA[indice inférieur 2]s chez les ostéoclastes humains ainsi que leur attribuer des rôles en physiologie et pathologie osseuse. Ces évidences pourraient faire des PLA[indice inférieur 2] de nouvelles cibles thérapeutiques pour le traitement de pathologies ostéo-articulaires, dont la maladie osseuse de Paget. // Abstract : Eicosanoïds are important mediators of bone biology. The first regulated enzymes in the biosynthetic pathway of eicosanoids are the PLA[subscript 2]s, which release arachidonic acid and lysophospholipids from membrane phospholipids. Further metabolism of arachidonic acid will lead, among other things, to the synthesis of prostaglandins, which deeply influence bone metabolism. Secreted PLA[subscript 2]s (sPL[subscript 2]) may also act independently of their catalytic activity, as a ligand to their membrane bound receptors. Even though PLA[subscript 2]s have been associated with joint and bone pathologies, their presence and functions were never investigated in osteoclasts (OCs), the principal cell responsible for bone destruction. Our study established the presence of cPLA[subscript 2] and sPLA[subscript 2] in human OCs, but demonstrated a contrast in their expression. cPLA[subscript 2] seems to be expressed ubiquitously, contrary to sPLA[subscript 2] whose expression is restricted to OCs from foetal bone and bone suffering from Paget disease. There is no trace of sPLA[subscript 2] in healthy bone or bone suffering from osteoarthrosis or osteoporosis, thus sPLA[subscript 2] seems tightly linked to high bone turnover. In our model, cPLA[subscript 2] exerted an anti-osteoclastogenic effect. Indeed, cPLA[subscript 2] produces eicosanoïds that inhibit osteoclastogenesis and sensitize OCs to apoptosis. Nevertheless, a minimum cPLA[subscript 2] activity is required since complete cPLA[subscript 2] inhibition blocks osteoclast mediated bone resorption. Its inhibition leads to disorganization of the OC cytoskeleton and inhibits the actin ring formation required for bone resorption in an arachidonic acid dependent fashion. On the other hand, sPLA[subscript 2] stimulates osteoclastogenesis independently of its catalytic activity; probably via interaction with one of its membrane bound receptors. sPLA[subscript 2] decreases OC and their CD14+ precursors’ sensibility to apoptosis. Moreover, sPLA[subscript 2] inhibition inhibits bone resorption independently of actin cytoskeleton remodeling, probably by inducing mature OC apoptosis. Together, these results demonstrate the presence of two PLA[subscript 2]s in human OCs and highlight their important roles in bone physiology and pathophysiology. Highlighting those functions could eventually lead to the elaboration of new strategies to control hyperosteoclastic states by targeting PL[subscript 2]s.

<p>When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of &#946; interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.</p> / Dissertation