MAPKs mediate the activation of cytosolic phospholipase A2 by amyloid β(25-35) peptide in bovine retina pericytes

We have previously shown that, in bovine retina pericytes, amyloid beta(1-42) and its truncated form containing amino acids 25-35, after 24 h treatment, stimulate arachidonic acid (AA) release and phosphatidylcholine hydrolysis, by activation of both cytosolic (cPLA(2)) and Ca2+- independent (iPLA,) phospholipase A,. A putative role for MAP kinases in this process emerged. Here we studied the role of the MAP-kinase family as well as both cPLA2 and iPLA, mRNA expression by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the same sublethal model of amyloid-beta (A beta) damage to pericytes in vitro. A beta(25-35) peptide evoked AA release as well as stimulated phosphorylation of ERK1/2, p38 MAPKs and cPLA(2), but not c-Jun N-terminal kinase (JNK/SAPK). PD98059, an inhibitor of ERK-activating kinase MEK-1, and SB203580, an inhibitor of p38 protein kinase, abolished the stimulation of AA release and MAPK activities. In cells stimulated by A beta(25-35) peptide, Western blotting and confocal microscopy analyses confirmed either an increase in the phosphorylated form of ERKs and p38 or their nuclear translocation. A complete inhibition of MAPK activation and AA release was also observed when pericytes were treated with GF109203X, a general PKC inhibitor, indicating the important role of both PKC and the two MAPKs in mediating the A beta peptide response. Compared with samples untreated or treated with reverse A beta(35-25) peptide, pretreatment with 50 mu M A beta(25-35) for 24 h significantly increased the level of constitutively expressed iPLA(2) mRNA by 25%, which seems to depend on the activation of kinases. By contrast, the level of cPLA, mRNA remained unchanged. Together, these data link either the stimulation of PKC-ERK-p38 cascades or PLA(2) activity by A beta peptide to prooxidant mechanism induced by amyloid, which may initially stimulate the cell reaction as well as metabolic repair, such as during inflammation. (c) 2005 Elsevier B.V. All rights reserved.