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

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

De-phosphorylation of GR at Ser203 in nuclei associates with GR nuclear translocation and GLUT5 gene expression in Caco-2 cells

Glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) inactivation are considered to be important in small-intestinal differentiation/maturation. In this study, we found that co-treatment with glucocorticoid hormone agonist dexamethasone and p44/42 MAPK inhibitor PD98059 in intestinal cell line Caco-2 strongly induced GLUT5 gene expression. Glucocorticoid hormone receptor (GR) was translocated from the cytoplasm to the nucleus and de-phosphorylated at serine residue 203 in the nucleus, by combined treatment with dexamethasone and PD98059. The binding of GR, as well as acetylated histones H3 and H4, to the promoter/enhancer region of GLUT5 gene was enhanced by combined treatment with dexamethasone and PD98059. These results suggest that the inactivation of p44/42 MAP kinase enhances glucocorticoid hormone-induced GLUT5 gene expression, probably through controlling the phosphorylation at serine 203 and nuclear transport of GR, as well as histone acetylation on the promoter/enhancer region of GLUT5 gene.