title: Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade.
abstract: 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 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 (PKCalpha) 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 cPLA(2), PKCalpha, and ERK1/2. By confocal microscopy, activation of PKCalpha 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 PKCalpha contributes to endothelial ERK1/2 and cPLA(2) phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCalpha-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.
authors.affiliation: Department of Biochemistry, University of Catania, 95126 Catania, Italy.
authors.forename: Carmelina Daniela
authors.initials: CD
authors.lastname: Anfuso
authors.forename: Gabriella
authors.initials: G
authors.lastname: Lupo
authors.forename: Loriana
authors.initials: L
authors.lastname: Romeo
authors.forename: Giovanni
authors.initials: G
authors.lastname: Giurdanella
authors.forename: Carla
authors.initials: C
authors.lastname: Motta
authors.forename: Alessia
authors.initials: A
authors.lastname: Pascale
authors.forename: Cataldo
authors.initials: C
authors.lastname: Tirolo
authors.forename: Bianca
authors.initials: B
authors.lastname: Marchetti
authors.forename: Mario
authors.initials: M
authors.lastname: Alberghina
mesh_terms: D000818:Animals; D002417:Cattle; D002450:Cell Communication / Q000502:physiology; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology; D005786:Gene Expression Regulation; D020935:MAP Kinase Signaling System; D020286:Pericytes / Q000166:cytology / Q000502:physiology*; D010741:Phospholipases A / Q000235:genetics*; D054467:Phospholipases A2; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051381:Rats
chemical_list: D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D054467:Phospholipases A2