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Journal of Lipid Research, Vol. 48, 782-793, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Endothelial cell-pericyte cocultures induce PLA₂ protein expression through activation of PKC and the MAPK/ERK cascade

Carmelina Daniela Anfuso^*, Gabriella Lupo^*, Loriana Romeo^*, Giovanni Giurdanella^*, Carla Motta^*, Alessia Pascale, Cataldo Tirolo, Bianca Marchetti and Mario Alberghina^1,*

^* Department of Biochemistry, University of Catania, 95126 Catania, Italy
Department of Experimental and Applied Pharmacology, University of Pavia, 27100 Pavia, Italy
OASI Institute for Research and Care on Mental Retardation and Brain Aging, Neuropharmacology Section, 94018 Troina, Italy

Published, JLR Papers in Press, January 31, 2007.

¹ To whom correspondence should be addressed. e-mail: malber{at}mbox.unict.it

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 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- (PKC) 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, PKC, and ERK1/2. By confocal microscopy, activation of PKC 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 PKC contributes to endothelial ERK1/2 and cPLA₂ phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKC-cPLA₂ pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation.

Supplementary key words blood-brain barrier • blood-retina barrier • phospholipase A₂ • protein kinase C • confocal microscopy • mRNA expression • mitogen-activated protein kinase/extracellular signal-regulated kinase cascade

Abbreviations: AA, arachidonic acid; AACOCF₃, arachidonoyl trifluoromethyl ketone; Akt, amino kinase terminal; BBB, blood-brain barrier; BEL, bromoenol lactone; cPLA₂, cytosolic phospholipase A₂; EC, endothelial cell; ERK, extracellular signal-regulated kinase; iPLA₂, calcium-independent intracellular phospholipase A₂; MAPK, mitogen-activated protein kinase; MEK-1, MAP/ERK kinase-1; PDK1, phosphoinositide-dependent protein kinase 1; PI3 kinase, phosphatidylinositol-3-kinase; PLA₂, phospholipase A₂; PKC, protein kinase C; TGF, tumor growth factor; vWF, von Willebrandt factor; WPB, Weibel-Palade body

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