Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCα 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-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 cPLA 2 , PKCa, and ERK1/2. By confocal microscopy, activation of PKCa 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 PKCa contributes to endothelial ERK1/2 and cPLA 2 phosphorylation induced by either soluble factors or direct cell-tocell contact, and that the PKCa-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.-

Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly

“…An early overexpression of PLA2G4C would prematurely recruit HCV NS proteins to LDs. The mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) pathway has been found to be involved in cPLA2 protein expression in endothelial cells (4). Amphiregulin (AREG) and epidermal growth factor (EGF) are the activators of the MAPK-ERK pathway, but these proteins were not able to enhance PLA2G4C expression (data not shown).…”

“…The hydro- lyzation of phosphatidylcholine by PLA2G4C leads to the generation of lipid signaling molecules, such as arachidonic acid. Arachidonic acid is an inflammatory factor that is capable of activating inflammatory signaling (4,9,70). The addition of arachidonic acid did not reverse the inhibitory effects of siPLA2G4C or MAFP on HCV replication in Huh7.5.1 ( Fig.…”

Klebsiella pneumoniae Induces an Inflammatory Response in an In Vitro Model of Blood-Retinal Barrier

“…Controls were performed by incubation of cocultures with inhibitors for 120 min in the absence of bacteria. At the end of the incubations, the cells grown on both sides of the inserts were scraped with a rubber policeman and saved separately; BREC and BRPC were lysed as previously described (35), and equal amounts of cell lysates were incubated in a 96-well plate with the substrate arachidonoyl-thiophosphatidylcholine (ATPC), using a cPLA 2 assay kit (Cayman Chemicals Co., Ann Arbor, MI) and following the manufacturer's instructions. For sPLA 2 enzyme activity, an sPLA 2 enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals Co., Ann Arbor, MI) was used, following the manufacturer's instructions.…”

“…After incubation in coculture, BREC or BRPC grown on one side of the filter were removed by rubbing it on filter paper to leave only one cell type. The filters with BREC or BRPC were washed, fixed by adding 4% paraformaldehyde in PBS, and processed for immunocytochemistry as previously described (35), using the following antibodies to highlight cell architecture: mouse anti-␣-actin monoclonal antibody (BRPC marker), or rabbit anti-von Willebrand factor polyclonal antibody (BREC marker). All primary antibodies were used in a dilution of 1:100.…”

Melanoma-Induced Endothelial Cell Growth Involves Phospholipase A2 and COX2 Upregulation

“…Phospholipases A 2 are a diverse group of enzymes that catalyze the hydrolysis of the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid and 2-lysophospholipid acceptors (Balsinde et al, 2002). They are the rate limiting step for the AA production from membrane phospholipids, which in turn is the major precursor to prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids (via the cyclooxygenase, lipoxygenase, and epoxygenase pathways, respectively) that increase cell proliferation in response to various agonists in different cell types (Anfuso et al, 2007;Balsinde et al, 2002;Chakraborti, 2003;Lupo et al, 2002). Among the PLA 2 s is an 85 kDa cPLA 2 that requires Ca 2+ for catalysis, and a calcium independent PLA 2 , iPLA 2 β, for which several potential functions have been proposed, including a housekeeping role in phospholipid remodeling and a signaling role in cell growth, apoptosis, secretion, inflammation, and oxidant-induced cell injury (Chakraborti, 2003).…”