beta1-Integrins induce phosphorylation of Akt on serine 473 independently of focal adhesion kinase and Src family kinases

Adhesion by means of beta1-integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3-kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion-induced activation of class Ia PI3K. Here we show that PI3K-dependent phosphorylation of Akt in response to ligation of beta1-integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant beta1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion-dependent activation of PI3K/Akt is triggered by the membrane proximal part of the beta1 subunit in a FAK and Src-independent manner.     

N-cadherin mediates angiogenesis by regulating monocyte chemoattractant protein-1 expression via PI3K/Akt signaling in prostate cancer cells

Over the past decade, evidence continues to mount showing that N-cadherin is a critical protein in cancer progression and metastasis. In the present study, we evaluated the expression of N-cadherin in human prostate cancer tissue specimens and cell lines. Enhanced expression of N-cadherin was observed in both the malignant and bone-metastasized prostate tissue specimens compared to the healthy prostate tissues. Consistent with the tissue array data, N-cadherin was highly expressed in PC3, but not in Du145 and LNCaP human prostate cell lines. Based on cell to cell binding assay, we found that N-cadherin expression facilitates homotypic interaction between human prostate cancer cells and human microvascular endothelial cells (HMEC). Human angiogenesis antibody array and in vitro angiogenesis assay showed that siRNA-mediated knockdown of N-cadherin reduced the secretion of monocyte chemoattractant protein-1 (MCP-1), which played a potential role in stimulating capillary network formation of HMEC. Additionally, culture supernatant of Du145 cells transfected with full-length N-cadherin expressing plasmid showed increased MCP-1 expression and chemoattractant ability compared to normal Du145 cells. Further, we noticed that blocking PI3K activity inhibited N-cadherin mediated MCP-1 expression. Our data demonstrated that N-cadherin in prostate cancer cell mediates cell–cell adhesion and regulates MCP-1 expression via the PI3K/Akt signaling pathway.     

Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of alpha v beta 3 and alpha 5 beta 1 integrins

Protein kinase B (PKB)/Akt is known to promote cell migration, and this may contribute to the enhanced invasiveness of malignant cells. To elucidate potential mechanisms by which PKB/Akt promotes the migration phenotype, we have investigated its role in the endosomal transport and recycling of integrins. Whereas the internalization of alpha v beta 3 and alpha 5 beta 1 integrins and their transport to the recycling compartment were independent of PKB/Akt, the return of these integrins (but not internalized transferrin) to the plasma membrane was regulated by phosphatidylinositol 3-kinases and PKB/Akt. The blockade of integrin recycling and cell spreading on integrin ligands effected by inhibition of PKB/Akt was reversed by inhibition of glycogen synthase kinase 3 (GSK-3). Moreover, expression of nonphosphorylatable active GSK-3 beta mutant GSK-3 beta-A9 suppressed recycling of alpha 5 beta 1 and alpha v beta 3 and reduced cell spreading on ligands for these integrins, indicating that PKB/Akt promotes integrin recycling by phosphorylating and inactivating GSK-3. We propose that the ability of PKB/Akt to act via GSK-3 to promote the recycling of matrix receptors represents a key mechanism whereby integrin function and cell migration can be regulated by growth factors.     

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.     

Helicobacter pylori activate epidermal growth factor receptor- and phosphatidylinositol 3-OH kinase-dependent Akt and glycogen synthase kinase 3beta phosphorylation

The signalling pathways leading to the development of Helicobacter pylori -induced gastric cancer remain poorly understood. We tested the hypothesis that H. pylori infections involve the activation of Akt signalling in human gastric epithelial cancer cells. Immunoblot, immunofluorescence and kinase assays show that H. pylori infection of gastric epithelial cells induced phosphorylation of Akt at Ser 473 and Thr 308. Mutations in the H. pylori virulence factor OipA dramatically reduced phosphorylation of Ser 473, while the cag pathogenicity island mutants predominantly inhibited phosphorylation of Thr 308. As the downstream of Akt activation, H. pylori infection inactivated the inactivation of glycogen synthase kinase 3β at Ser 9 by its phosphorylation. As the upstream of Akt activation, H. pylori infection activated epidermal growth factor receptor (EGFR) at Tyr 992, phosphatidylinositol 3-OH kinase (PI3K) p85 subunit and PI3K-dependent kinase 1 at Ser 241. Pharmacologic inhibitors of PI3K or mitogen-activated protein kinase kinase (MEK), Akt knock-down and EGFR knock-down showed that H. pylori infection induced the activation of EGFR→PI3K→PI3K-dependent kinase 1→Akt→extracellular signal-regulated kinase signalling pathways, the inactivation of glycogen synthase kinase 3β and interleukin-8 production. The combined functions of cag pathogenicity island and OipA were necessary and sufficient for full activation of signalling at each level. We propose activation of these pathways as a novel mechanism for H. pylori -mediated carcinogenesis.     

Salvia miltiorrhiza inhibits intimal hyperplasia and monocyte chemotactic protein-1 expression after balloon injury in cholesterol-fed rabbits

Antioxidants that prevent low density lipoproteins (LDL) from oxidation may inhibit atherosclerosis and post-angioplasty restenosis. Salvia miltiorrhiza (SM) has been shown to inhibit LDL oxidation and reduce atherosclerosis in cholesterol-fed rabbits. The effects of SM on neointimal hyperplasia and monocyte chemotactic protein-1 (MCP-1) expression after balloon injury were studied. Male New Zealand white rabbits were fed a 2% cholesterol diet together with daily SM (4.8 gm/kg body wt.) treatment (SM; n=10) or without SM as a control (C; n=9) for 6 weeks. Probucol-treated (0.6 gm/kg body wt.) rabbits (P; n=9) were used as a positive control group. A balloon injury of the abdominal aorta was performed at the end of the third week. Aortas were harvested at the end of 6 weeks. The plasma cholesterol levels were lowered in SM group. The neointimal hyperplasia in abdominal aortas was significantly inhibited in SM group [neointima/media area ratio: 0.63+/-0.05 (SM) versus 0.78+/-0.05 (C); P < 0.05] and in P group [0.45+/-0.02 (P) versus 0.78+/-0.05 (C); P < 0.05] when compared with C group. SM treatment significantly reduced MCP-1 mRNA and protein expression in balloon-injured abdominal aorta. These inhibitory effects on intimal response after balloon injury might be attributed to antioxidant capacity and cholesterol lowering effect of SM. SM treatment may offer some protection against post-angioplasty restenosis.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by estrogen and progesterone.

There is a cyclicity in the number of endometrial macrophages that is most likely secondary to changes in steroid hormone levels. One cytokine that controls macrophage migration is monocyte chemotactic protein-1 (MCP-1). In the endometrium, highest levels of MCP-1 are detected perimenstrually, when estrogen levels are low; however, when estrogen levels are high (around the time of ovulation), MCP-1 levels are lowest. We hypothesized that sex steroids may be involved in the regulation of macrophage migration by regulating MCP-1 expression. We investigated the regulation of MCP-1 expression in human endometrial stromal cells by estradiol 17beta (E2) and progestins. We found that MCP-1 mRNA levels decreased in response to E2 (5 x 10(-8) M), with biphasic nadirs at 8 h and 24 h. MCP-1 protein production was also inhibited by E2 in a concentration-dependent manner. Tamoxifen, an anti-estrogen, alone (10(-7) M) did not affect MCP-1 expression, but it reversed the E2-induced inhibition up to 80%. Progesterone (10(-7) M) alone slightly decreased MCP-1 levels, and the combination of E2 and progesterone further decreased them, but that decrease was not different from that observed using E2 treatment alone. In summary, we found that E2 inhibits MCP-1 expression in endometrial stromal cells, and we speculate that E2 may control endometrial macrophage migration by regulating MCP-1 expression.     

Carbon monoxide differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phosphatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during anoxia-reoxygenation injury

Carbon monoxide (CO), previously considered a toxic waste product of heme catabolism, is emerging as an important gaseous molecule. In addition to its important role in neurotransmission, exogenous CO protects against vascular injury, transplant rejection, and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. We have recently shown that CO attenuates endothelial cell apoptosis during anoxia-reoxygenation injury by activating MKK3/p38alpha mitogen-activated protein kinase (MAPK) pathways. Our current study is the first to demonstrate that CO can differentially modulate STAT1 and STAT3 activation and, specifically, that STAT3 activation by CO is responsible for the anti-apoptotic effect in endothelial cells. In addition, we show that the anti-apoptotic effects of CO depend upon both phosphatidylinositol 3-kinase/Akt and p38 MAPK signaling pathways in endothelial cells, whereas previous reports have implicated only the MKK3/p38 MAPK pathway. Using chemical inhibitors and dominant negative constructs, we show that CO enhances STAT3 activation via phosphatidylinositol 3-kinase/Akt and p38 MAPK pathways with subsequent attenuation of Fas expression and caspase 3 activity. These data highlight the anti-apoptotic signaling mechanisms of CO and, importantly, delineate potential therapeutic strategies to prevent ischemia-reperfusion or anoxia-reoxygenation injury in the vasculature.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Epoxyisoprostane and epoxycyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin-8 synthesis. Formation of these oxidized phospholipids in response to interleukin-1beta.

Monocyte recruitment to the vessel wall, mediated by monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), plays an important role in atherogenesis. We have shown previously that minimally oxidized low density lipoprotein, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), activates endothelial cells to produce MCP-1 and IL-8. By using liquid chromatography/mass spectrometry methods coupled with bioassay, we report a family of epoxyisoprostane (PEIPC) and epoxycyclopentenone (PECPC) phospholipids that are the components of Ox-PAPC responsible for the majority of this activity. Ox-PAPC contains five chromatographically distinguishable active PEIPC components (m/z 825.5) and four PECPC components (m/z 810.5). All nine components induced endothelial cell synthesis of IL-8 and MCP-1 in a dose-dependent fashion between 0.1 and 5 microm concentrations. The five PEIPC components had identical functional groups and all underwent dehydration to produce m/z 810.5. We present evidence that these phospholipids are regioisomers with epoxide groups at the 5,6-, 8,9-, 11,12-, or 14,15-positions of the sn-2 fatty acid and their epoxide groups is important for biological activity. We have shown previously that peroxisome proliferator-activated receptor alpha is involved in MCP-1 synthesis in response to Ox-PAPC. We now show that PEIPC and PECPC isomers are potent activators of peroxisome proliferator-activated receptor alpha. PEIPC and PECPC isomers are strongly recognized by specific circulating murine natural autoantibodies (EO6) and accumulate in cells treated with IL-1beta. These studies demonstrate that PEIPC and PECPC isomers are potent activators of endothelial cells increasing synthesis of IL-8 and MCP-1. Their accumulation in cells exposed to cytokines and in atherosclerotic lesions suggests that these lipids may play a role in a number of chronic disease processes.     

Insulin and the beta3-adrenoceptor differentially regulate uncoupling protein-1 expression

Cross-talk between insulin and the adrenergic system is important in the regulation of energy homeostasis. In cultured, differentiated mouse brown adipocytes, beta3-adrenergic stimulation induced a 4.5-fold increase in uncoupling protein-1 (UCP-1) expression, which was diminished by 25% in the presence of insulin. Beta3-adrenergic stimulation also activated mitogen-activated protein (MAP) kinase by 3.5-fold and caused a decrease in basal phosphoinositide (PI) 3-kinase activity detected in p110gamma- and Gbeta-subunit-immunoprecipitates in a time-dependent manner, whereas insulin stimulated p110alpha- and phosphotyrosine-associated PI 3-kinase activity. Inhibition of MAP kinase or PI 3-kinase potentiated the beta3-adrenergic effect on UCP-1 expression, both alone and in the presence of insulin. Thus, insulin inhibits beta3-adrenergic stimulation of UCP-1, and both MAP kinase and PI 3-kinase are negative regulatory elements in the beta3-adrenergic control of UCP-1 expression. Cross-talk between the adrenergic and insulin signaling systems and impaired regulation of UCP-1 might contribute to the development of a reduced energy balance, resulting in obesity and insulin resistance.     

Induction of monocyte chemotactic protein-1 (MCP-1) and TNF alpha by Trichinella spiralis in serum of mice in vivo

Effects of selenium deficiency, induced by thioacetamide, were investigated in rats. Thioacetamide (0.3 g/L) given in drinking water, as expected, caused a significant loss of selenium from the liver. It was accompanied by liver cirrhosis and a significant increase in the liver weight as well as liver to body weight ratio. A significant loss of selenium from spleen was also accompanied by an increase in its weight. Weights of lungs, testis and kidney, however, were not affected by thioacetamide and there was no change in their selenium content. Plasma levels of selenium were significantly reduced in the thioacetamide treated group. All these changes were confirmed to be due to selenium deficiency caused by thioacetamide, as supplementation with selenium reversed these changes. The mode of action of selenium is unknown but may involve anti-oxidant defense mechanisms.     

Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance.