Abstract
Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IValpha cytosolic PLA2 (cPLA2alpha) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i). MC+/+, which lack group IIa and V PLA2s, and (ii). MC-/-, which lack groups IIa, V, and IValpha PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC-/-. It has been argued that cPLA2alpha plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IValpha PLA2s were expressed in MC-/- or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2alpha in H2O2-treated MC-/- increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC-/-. When sPLA2 and cPLA2alpha are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N',N'-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2alpha and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2alpha is responsible for AA release.
Highlights
Introduction ofphospholipase A2 (PLA2) Enzymes into Primary Murine Mesangial Cells—Subconfluent mesangial cell (MC) were infected with adenoviral vectors at varying levels of infection, as reflected by plaque forming units/cell, for 48 h in RPMI 1640 with 2% (v/v) fetal bovine serum
To study the effect of cPLA2␣ on H2O2-induced arachidonic acid (AA) release from MC, MCϪ/Ϫ infected with varying m.o.i. of Ad-cPLA2␣, were stimulated with 75 M H2O2 for periods of 3 and 6 h and AA released into media was measured (Fig. 1B)
We avoided some of the ambiguities associated with nonspecific inhibitors and incomplete protein suppression by using MC derived from mice with genetic defects, either spontaneous or engineered, leading to the absence of two forms of PLA2
Summary
Materials—[5,6,8,9,11,12,4,15-3H]Arachidonic acid ([3H] AA, 98.6 Ci/ mmol; 1 Ci ϭ 37 GBq) and [1-14C]oleic acid ([14C]OA, 50 mCi/mmol; 1 Ci ϭ 38 GBq) were from PerkinElmer Life Sciences. 1-Palmitoyl-2[14C]linoleoyl-sn-glycero-3-phosphoethanolamine ([14C]PE, 56 mCi/ mmol; 1 Ci ϭ 37 GBq) was from Amersham Biosciences. The medium was removed, and cells were washed three times with RPMI containing 0.2% bovine serum albumin. Cells grown on coverslips coated with bovine collagen type I were rinsed with phosphate-buffered saline and loaded with 3 M Fura-2AM in Earle’s balanced salt solution. Cells were loaded with Fura-2AM for 1 h at 37 °C and washed 2–3 times with Earle’s balanced salt solution containing probenecid. Introduction of PLA2 Enzymes into Primary Murine Mesangial Cells—Subconfluent MC were infected with adenoviral vectors at varying levels of infection, as reflected by plaque forming units/cell, for 48 h in RPMI 1640 with 2% (v/v) fetal bovine serum. The adenovirus-mediated gene transfers were followed by the expression of GFP under UV light for Ad-IIa and VPLA2s and Ad-GFP/LacZ, and by Western blot analysis of cell lysate for Ad-cPLA2␣ and Ad-MEK1-DD, respectively. Each experiment was performed in triplicate and independently three to five times
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