Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade
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Abstract
Little is known about the regulatorymechanisms of
endothelial cell (EC) proliferation by retinal pericytes and
vice versa. In a model of coculture with bovine retinal pericytes
lasting for 24 h, rat brain ECs showed an increase
in arachidonic acid (AA) release, whereas Western blot and
RT-PCR analyses revealed that ECs activated the protein
expression of cytosolic phospholipase A2 (cPLA2) and its phosphorylated
form and calcium-independent intracellular phospholipase
A2 (iPLA2). No activation of the same enzymes was
seen in companion pericytes. In ECs, the protein level of phosphorylated
extracellular signal-regulated kinase (ERK) 1/2 was
also enhanced significantly, a finding not observed in cocultured
pericytes. The expression of protein kinase C-a (PKCa)
and its phosphorylated form was also enhanced in ECs.
Wortmannin, LY294002, and PD98059, used as inhibitors of
upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway)
in cultures, markedly attenuated AA release and the expression
of phosphorylated forms of endothelial cPLA2,
PKCa, and ERK1/2. By confocal microscopy, activation of
PKCa in perinuclear regions of ECs grown in coculture as well
as strong activation of cPLA2 in ECs taken from a model of
mixed culture were clearly observed. However, no increased
expression of both enzymes was found in cocultured pericytes.
Our findings indicate that a sequential activation of
PKCa contributes to endothelial ERK1/2 and cPLA2 phosphorylation
induced by either soluble factors or direct cell-tocell
contact, and that the PKCa-cPLA2 pathway appears to play
a key role in the early phase of EC-pericyte interactions regulating
blood retina or blood-brain barrier maturation