p44/42 MAP Kinase ELISA Kit

Acute H2O2 exposure to placental artery endothelial cells induced an array of tyrosine-phosphorylated proteins, including caveolin 1 (CAV1) rapid and transient tyr(14) phosphorylated in a time- and concentration-dependent manner. Basal tyr(14) phosphorylated CAV1 was primarily located at the edges of cells and associated with actin filaments. Phosphorylated CAVI was markedly increased and diffused with the disorganization of actin filaments at 20 min, disappeared at 120 min treatment with 0.2 mM H2O2. Treatment with H2O2 also disorganized actin filaments and changed cell shape in a time-dependent manner. Pretreatment with antioxidants catalase completely, whereas the other tested superoxide dismutase, N-acetyl-L-cysteine and sodium formate partially attenuated H2O2-induced CAV1 phosphorylation in a concentration-dependent manner. Acute treatment with H2O2-activated multiple signaling pathways, including the mitogen-activated protein kinases (MAPK) members (MAPK3/1-ERK2/1, MAPK8/9-JNK1/2, and MAPK11-p38(mapk)) and the c-src tyrosine kinase (CSK). Pharmacological studies demonstrated that, among these pathways, only the blockade of CSK activation abolished H2O2-induced CAV1 phosphorylation. Additionally, H2O2-induced CAV1 phosphorylation was reversible rapidly (< 10 min) upon H2O2 withdrawal. Because maternal and fetal endothelia must make dynamic adaptations to oxidative stress resulting from enhanced pregnancy-specific oxygen metabolism favoring prooxidant production, which is emerging as one of the leading causes of the dysfunctional activated endothelium during pregnancy, these unique features of CAV1 phosphorylation on oxidative stress observed implicate an important role of CAV1 in placental endothelial cell biology during pregnancy.

Aims/hypothesis Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1(-/-) mice) and pharmacological approaches. Methods NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. Results Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. Bycontrast, ERK1 was required forglucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1(-/-) mouse beta cells (p < 0.01) compared with Erk1(+/+) mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1(-/-) mice was slightly increased in young animals (4-month-oldmice) vs Erk1(+/+) mice (section occupied by islets [mean +/- SEM]: 0.74% +/- 0.03% vs 0.62% +/- 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic periinsulitis (infiltrated islets [mean +/- SEM]: 7.51% +/- 1.34% vs 2.03% +/- 0.51%; p < 0.001). Conclusions/interpretation ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1(-/-) mice shows that ERK1 is dispensable in normal conditions.