期刊
JOURNAL OF LIPID RESEARCH
卷 48, 期 4, 页码 782-793出版社
ELSEVIER
DOI: 10.1194/jlr.M600489-JLR200
关键词
blood-brain barrier; blood-retina barrier; phospholipase A(2); protein kinase C; confocal microscopy; mRNA expression; mitogen-activated protein kinase/extracellular signal-regulated; kinase cascade
Little is known about the regulatory mechanisms 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 add (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form and calcium-independent intracellular phospholipase A(2) (iPLA(2)). 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-alpha (PKC alpha) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (die PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA(2), PKC alpha, and ERK1/2. By confocal microscopy, activation of PKC alpha in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA(2) 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 PKC alpha contributes to endothelial ERK1/2 and cPLA(2) phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKC alpha-cPLA(2) pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation.
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