Signalling mechanisms involved in TNF-α mediated cytoprotection during ischaemic injury in a C2C12 muscle cell line

[摘要] ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolicphospholipase A2 (cPLA2) are crucial driving forces in mediating the cellularinflammatory response and are involved in ischaemic injury. During an ischaemicinsult, TNF-α is endogenously generated. Apart from the recognized effects of TNF-α, such as the induction of apoptosis, proliferation and differentiation, if present inlow dosages, it also mediates cytoprotective effects. Upon activation, cPLA2contributes to the ischaemic challenge with the generation of mediators of cellularinjury and apoptosis. Upon stimulation, this calcium dependent enzyme translocatesto the phospholipid compartment of the cell membrane and induces the hydrolysis ofsn-2 ester bonds in phospholipids. It governs the release of free fatty acids andlysophospholipids and generates role players of inflammation. We suggest a role forcPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation andtranslocation pattern.AimsThe involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murineskeletal muscle cell line in tolerance to ischaemia was examined. To investigate thenature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases(MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2phosphorylation and activation, were examined at appropriate time points. To dissectout the cPLA2 interplay and dependencies with these MAPKs within the pathwaycontext, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3)was employed and its effect on cell viability was examined. Fluorescence microscopywas used to substantiate cPLA2 activation, by assessing its cellular distribution,translocation and cell organelle target preference, using co-localization and z-stacktechniques. In addition, the induction of the apoptotic pathway through analysis ofcaspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. Therole of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor,Z-DEVD-FMK. MethodsCells were grown in Dulbecco's Modified Eagles Medium (DMEM) with 10% fetalbovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70%confluent. Using DMEM supplemented with 1% horse serum, cell differentiation intomyotubes was induced. Differentiated cells were preconditioned for 30 minclassically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10μM) respectively. Followed by a 60 min washout period the cells were subjected to 8hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- andtranslocation events were assessed using Western blots and advancedimmunocytochemistry and imaging techniques.ResultsPreconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2inhibitor AACOCF3, attenuated the decrease is cell viability brough about byischaemia. Western blot analysis indicates the induction of the apoptotic pathway withcaspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to thenuclear region in the TNF-α preconditioned group compared to the ischaemic group,as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stackanalysis confirmed that the nuclear and endonuclear region was the target organellefor cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear markernucleoporin p62 mirrored these results.Discussion and conclusionOur results provide evidence that there is a role for cPLA2 in TNF-α mediatedcytoprotection. Although we do not observe a differential activation pattern in termsof cPLA2 phosphorylation at various time points within the ischaemic event, and nodifferential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describea differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition ofcPLA2 translocation, a functional cPLA2 inhibition might be achieved. This wouldimply inhibition of the inflammatory pathway and a subsequent reduction in thegeneration of inflammatory mediators. In addition we describe an effect of TNF-αpreconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cellin a setting of TNF-α mediated cytoprotection. This might lead to novel approaches inthe context of inflammation treatment, through agents that control differential cPLA2trafficking within the cell.

[发布机构] Stellenbosch University