Phosphatidylinositol 3-kinase Inhibitor LY294002 Research Articles

Emodin induces neurite outgrowth through PI3K/Akt/GSK-3β-mediated signaling pathways in Neuro2a cells

In this study, a neurite outgrowth-inducing substance was isolated from the ethylacetate extract of the Polygonum multiflorum roots and identified as emodin by gas–liquid chromatography–mass spectrometry and 1H NMR and 13C NMR. Emodin displayed remarkable neurite outgrowth-inducing activity in Neuro2a cells, as demonstrated by morphological changes and immunocytochemistry for class III β-tubulin. Emodin exhibited a stronger neutrophic activity than retinoic acid (RA) known as inducer of neurite outgrowth in Neuro2a cells. Emodin treatment resulted in marked increases in phosphorylation of Akt a direct downstream signaling molecule of phosphatidylinositol 3-kinase (PI3K), but upstream of glycogen synthase kinase-3β (GSK-3β) and cAMP response element-binding protein (CREB). These augmentations and neurite-bearing cells induced by emodin were remarkably reduced by the addition of PI3K inhibitor LY294002. These results demonstrate that emodin induces neuronal differentiation of Neuro2a cells via PI3K/Akt/GSK-3β pathway.

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ 25–35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ 25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ 25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease.

Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells.

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.

Both creatine and its product phosphocreatine reduce oxidative stress and afford neuroprotection in an in vitro Parkinson's model.

Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson’s model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK3β) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine473) and GSK3β (Serine9). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons.

Hypoxia-inducible factor 1α mediates neuroprotection of hypoxic postconditioning against global cerebral ischemia.

Hypoxia administered after transient global cerebral ischemia (tGCI) has been shown to induce neuroprotection in adult rats, but the underlying mechanisms for this protection are unclear. Here, we tested the hypothesis that hypoxic postconditioning (HPC) induces neuroprotection through upregulation of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), and that this involves phosphatidylinositol-3-kinase (PI3K), p38 mitogen-activated protein kinase (p38 MAPK), and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) pathways. The expression of HIF-1α, VEGF, and cleaved caspase-9 were determined by immunohistochemistry and Western blot. As pharmacologic interventions, the HIF-1α inhibitor 2-methoxyestradiol (2ME2), PI3K inhibitor LY294002, p38 MAPK inhibitor SB203580, and MEK inhibitor U0126 were administered before HPC or after tGCI. We found that HPC maintained the higher expression of HIF-1α and VEGF and decreased cleaved caspase-9 levels in CA1 after tGCI. These effects were reversed by 2ME2 administered before HPC, and the neuroprotection of HPC was abolished. LY294002 and SB203580 decreased the expression of HIF-1α and VEGF after HPC, whereas U0126 increased HIF-1α and VEGF after tGCI. These findings suggested that HIF-1α exerts neuroprotection induced by HPC against tGCI through VEGF upregulation and cleaved caspase-9 downregulation, and that the PI3K, p38 MAPK, and MEK pathways are involved in the regulation of HIF-1α and VEGF.

Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication

Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells

The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer cells and suggest that patients׳ hormonal status through this activity may play a significant role in some therapeutic outcomes.

Effect of a heat shock protein 90-specific inhibitor on the proliferation and apoptosis induced by VEGF-C in cervical cancer cells

The aim of the present study was to investigate the effect of heat shock protein 90 (Hsp90)-specific inhibitor geldanamycin (GA) on the proliferation and apoptosis induced by vascular endothelial growth factor-C (VEGF-C) in cervical cancer cells. HeLa cells (1×106/ml) in the logarithmic growth phase were incubated without serum for 24 h. The cells were pretreated with kinase insert domain receptor antibody (KDR)-Ab (20 μg/ml), phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (3 μmol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (30 μmol/l) or Hsp90-specific inhibitor GA (10 μmol/l) for 30 min, and then treated with VEGF-C (50 ng/μl) for a further 24 h. The cells were harvested for MTT analysis, annexin V-FITC/propidium iodide double staining for early apoptosis and SDS-PAGE and western blot analysis in order to determine Hsp90, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and cyclin D1 expression. Treatment with VEGF-C alone induced Hsp90 protein expression in HeLa cells at all time-points. Hsp90 expression was increased 3.31-fold in VEGF-C treated HeLa cells, and this increase was attenuated in the treatment groups (2.17-, 1.69-, 1.82-fold in VEGF-C + KDR-Ab, VEGF-C + PD98059 and VEGF-C + LY294002, respectively). The proliferation of the VEGF-C-treated HeLa cells was increased ~2.13-fold, while that of the VEGF-C + GA-treated HeLa cells decreased 0.87-fold (P<0.05). Even low concentrations of GA (0.02 μmol/l) were found to inhibit the Bcl-2 and cyclin D1 protein expression induced by VEGF-C. Therefore, the results indicate that the Hsp90-specific inhibitor GA has a critical role in the proliferation and apoptosis induced by VEGF-C in cervical cancer cells.

Involvement of two distinct signalling pathways in IGF-1-mediated central control of hypotensive effects in normotensive and hypertensive rats.

Insulin-like growth factor-1 (IGF-1) is abundantly expressed in the nucleus tractus solitarii (NTS). In a previous study, we revealed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP). It is well known that both acute administration and chronic administration of IGF-I reduce BP. The aim of this study was to evaluate the short-term hypotensive effect of IGF-1 in the NTS and to delineate the underlying molecular mechanisms of IGF-1 in the NTS of normotensive WKY rats and spontaneously hypertensive rats (SHRs). Microinjections of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the MAP kinase-ERK kinase (MEK) inhibitor PD98059 into the NTS in WKY rats and SHRs were used to study the involvement of IGF-1-induced depressor effects. An IGF-1 (7.7pmol) injection into the NTS resulted in a significant decrease in BP and HR in WKY rats and SHRs. Immunoblotting and immunohistochemical analysis showed that the microinjection of LY294002 (0.6pmol) or PD98059 (3.0pmol) into the NTS attenuated the IGF-1-induced depressor effects and Akt or ERK phosphorylation in WKY rats. An attenuation effect of LY294002, but not PD98059, was found in the SHRs. However, the mRNA and protein expression levels of the IGF-1R showed no significant differences in the NTS of the WKY rats and the SHRs. These results suggest that distinct Akt and ERK signalling pathways mediated the IGF-1 control of the central depressor effects in WKY rats and SHRs. ERK signalling defects may be associated with the development of hypertension.

Effect of CD95 on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes

ObjectiveMany CD95-expressing cells don’t always undergo apoptosis after stimulation with CD95 ligation. The purpose of this paper is to investigate the role of expression of CD95 (Fas/Apo1) on inflammatory response in fibroblast-like synoviocytes (FLS) obtained from rheumatoid arthritis (RA) and to evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathways within this process. MethodsThe expression levels of CD95 were monitored by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Apoptotic cells were detected by in situ apoptosis detection (TUNEL) assay. The RA-FLS were treated with agonistic anti-CD95 antibody or CD95 siRNA. Then the proliferation was detected by CCK-8, and mRNA level of inflammatory cytokines was detected by RT-PCR. After the RA-FLS were treated with agonistic anti-CD95 antibody, the total Akt and pAkt protein expression was analyzed by Western blot, and the changes mentioned above were observed while pre-incubated with the PI3K inhibitor LY294002. ResultsA significant increase of CD95 antigen was found in RA compared with osteoarthritis (OA) samples, while apoptosis in RA synovial tissue was not obvious. Low concentrations of agonistic anti-CD95 antibody could promote RA-FLS growth and interleukin-6 (IL-6) mRNA expression, while high concentrations could induce apoptosis. And both of these phenomena could be inhibited by CD95 siRNA. Agonistic anti-CD95 antibody could stimulate the expression of pAkt, and PI3K specific inhibitor LY294002 could induce opposite changes. ConclusionStimulation of CD95 could promote RA-FLS proliferation and inflammation, and activation of the PI3K/Akt signaling pathway might be the possible mechanism.

Decreased expression and prognostic role of cytoplasmic BRSK1 in human breast carcinoma: Correlation with Jab1 stability and PI3K/Akt pathway

ObjectiveJun activation domain-binding protein 1 (Jab1) was overexpressed in breast cancer, which was involved in degradation of the cyclin-dependent kinase inhibitor p27Kip1. The objective of this study was to examine the effect of brain specific kinase 1 (BRSK1) expression on Jab1 over-expression and related signaling pathway in breast cancer. MethodsImmunohistochemical analysis was performed in 95 human breast carcinoma samples and the data were correlated with clinicopathologic features. Furthermore, Western blot analysis was performed for BRSK1 and Jab1 in breast carcinoma samples and cell lines to evaluate their protein levels and molecular interaction. ResultsWe found that the cytoplasmic BRSK1 expression was inversely associated with Jab1 expression (P<0.01) and correlated significantly with histologic grade (P=0.006), however nuclear BRSK1 expression couldn't obtain similar results. Kaplan–Meier analysis revealed that survival curves of low versus high expressers of cytoplasmic BRSK1 and Jab1 showed a highly significant separation in breast cancer (P<0.01). While in vitro, following release of breast cancer cell lines from serum starvation, the expression of Jab1, phosphor-Akt (p-Akt) was up-regulated, whereas BRSK1 and p27Kip1 were decreased. Treatment of phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 could diminish Jab1 expression but increase BRSK1 expression. In addition, we employed siRNA technique to knock down Jab1 and/or BRSK1 expression and observed their effects on MDA-MB-231 cell growth. ConclusionsBRSK1 is a novel tumor suppressor in breast cancer which inversely correlated with Jab1 expression, may involve in the restoring Jab1-induced suppression of p27Kip1 and may regulate cell cycle through the PI3K/Akt pathway.

Expression and prognostic role of SGTA in human breast carcinoma correlates with tumor cell proliferation.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was reported to be implicated in various cellular processes and involved in control of cell cycle regulation and transcription. It may play a critical role in oncogenesis. In this study, to investigate the potential roles of SGTA in breast cancer, expression patterns, interaction and the correlation with clinical/prognostic factors of SGTA and Ki-67 were examined among patients with breast cancer. Immunohistochemistry and Western blot analysis were performed for SGTA in 100 breast carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine the prognostic significance. We found that SGTA was overexpressed in breast carcinoma compared with the adjacent normal tissues. High expression of SGTA was positively associated with histological grade (P=0.002) and Ki-67 (P=0.001). Univariate analysis showed that SGTA expression was associated with a poor prognosis (P=0.002). Kaplan-Meier survival curves of the study population showed that high expression level of SGTA significantly correlated with short-term survival. While in vitro, SGTA depletion by small interfering RNA inhibited cell proliferation and cell cycle in breast cancer cell lines. Western blot analyses showed that SGTA depletion decreased cyclin A, cyclin B and CDK2, whereas increased p27 levels. Additionally, treatment of phosphatidylinositol 3-kinase inhibitor LY294002 could arrest cells growth and diminish SGTA expression. These results suggested that SGTA overexpression was involved in the pathogenesis of breast cancer which might serve as a future target for novel treatment in breast cancer.

Lithiation of a silyl ether: formation of an ortho-Fries hydroxyketone.

A hydroxy-directed alkylation of an N,N-diethylarylamide using CIPE-assisted α-silyl carbanions (CIPE = complex-induced proximity effect) has been developed using a simple reagent combination of LDA (lithium diisopropylamide) and chlorosilane. A study of the mechanism, and the application of the procedure to an anionic Snieckus-Fries rearrangement for a highly efficient synthesis of the potent phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, are reported.

Cytokine-induced iNOS and ERK1/2 inhibit adenylyl cyclase type 5/6 activity and stimulate phosphodiesterase 4D5 activity in intestinal longitudinal smooth muscle.

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.

Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

Fertilization-induced autophagy in mouse embryos is independent of mTORC1.

Autophagy is a dynamically regulated intracellular degradation system that is important for cellular processes such as amino acid production during starvation and intracellular quality control. Previously, we reported that autophagy is suppressed in oocytes but is rapidly up-regulated after fertilization. During this period, autophagy is thought to be important for the generation of amino acids from the bulk degradation of maternal proteins that have accumulated during oogenesis. However, the mechanism of autophagy induction after fertilization is presently unknown. In most cell types, autophagy is negatively controlled by mammalian target of rapamycin complex 1 (mTORC1), which is typically regulated by amino acids and insulin or related growth factors. In this study, we determined the role of mTORC1 in fertilization-induced autophagy. On the basis of the phosphorylation status of mTORC1 substrates, we found that mTORC1 activity was relatively high in metaphase II (MII) oocytes but was rapidly decreased within 3 h of fertilization. However, chemical inhibition of mTORC1 by Torin1 or PP242 in MII oocytes or fertilized embryos did not induce autophagy. In addition, activation of mTORC1 by cycloheximide did not inhibit fertilization-induced autophagy in fertilized embryos. By contrast, the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 effectively suppressed autophagy in these embryos. These data suggest that, even though autophagy induction and postfertilization mTORC1 activity are inversely correlated with each other, as observed in other cell types, mTORC1 suppression is neither essential nor sufficient for fertilization-induced autophagy, highlighting a unique feature of the regulation mechanism of autophagy-mediated intracellular turnover in early embryos.

Role of PI3K/Akt signaling pathway in dexmedetomidine postconditioning-induced reduction of myocardial ischemia-reperfusion injury in rats

Objective To evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein-serinethreonine kinases (Akt) signaling pathway in dexmedetomidine postconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats.Methods Seventy-two healthy male Sprague-Dawley rats,aged 4-5 months,weighing 200-240 g,were heparinized and anesthetized with intraperitoneal pentobarbital sodium 40 mg/kg.Their hearts were rapidly excised and perfused in a Langendorff apparatus with oxygenated (95％ O2-5％ CO2) K-H solution at 0-4℃.The isolated hearts were randomly divided into 6 groups (n =12 each):control group (group C),group I/R,dexmedetomidine postconditioning group (gruop D),solvent group (group DMSO),PI3K inhibitor LY294002 group (group L) and LY294002 + dexmedetomidine postconditioning group (group L + D).After a 20 min stabilization period,the hearts were continuously perfused with K-H solution for 120 min in group C and were subjected to 30 min of global no-flow ischemia followed by 90 min of repeffusion in the other groups.Dexmedetomidine 100 nmo/L was added during the initial 30 min of reperfusion in D and L + D groups.LY294002 15 μmol/L was added in L group.0.02％ DMSO containing 15 μmol/L LY294002 was added in L + D group.0.02％ DMSO was added in DMSO group.HR,left ventricular end-diastolic pressure,left ventricular developed pressure and ± dp/dtmax were recorded at the end of 20 min stabilization and 15,30 and 90 min of reperfusion.The expression of phosphorylated Akt (p-Akt) and Akt was measured at 30 min of reperfusion.Myoeardial infarct size was determined using TTC staining at 90 min of reperfusion.Results Compared with group C,HR,left ventricular developed pressure and ± dp/dtmax were significantly decreased,and left ventricular enddiastolic pressure and myocardial infarct size were increased in the other 5 groups,and the expression of p-Akt was significantly up-regulated in group D (P ＜ 0.05).Compared with group I/R,HR was significantly decreased at 15 and 30 min of reperfusion,the expression of p-Akt was up-regulated,and myocardial infarct size was decreased in group D (P ＜ 0.05).Compared with group D,the expression of p-Akt was down-regulated,and myocardial infarct size and HR was increased in L + D group (P ＜ 0.05).Conclusion Dexmedetomidine postconditioning attenuates myocardial I/R injury throuth activating PI3K/Akt signaling pathway in rats. Key words: 1-PhosphatidyIinositol 3-kinase ; Protein-serine-threonine kinases ; Dexmedetomidine ; Myocardial reperfusion injury; Postconditioning

Apelin-13 protects the brain against ischemia/reperfusion injury through activating PI3K/Akt and ERK1/2 signaling pathways

Apelin has been proved to protect the heart against ischemia/reperfusion (I/R) injury via the activation of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways. Whether this protective effect applies to brain I/R injury needed to be explored. We therefore investigated the potential neuroprotective role of Apelin-13 and the underlying mechanisms. Focal transient cerebral I/R model in male ICR mice was induced by 60min of ischemia followed by reperfusion. Apelin-13 intracerebroventricular injection was performed 15min before reperfusion. Neurological function, infarct volume, brain edema and apoptosis were measured at 24h after stroke. To further test the mechanism of Apelin-13, PI3K inhibitor LY294002 and ERK1/2 inhibitor PD98059 were injected into the lateral cerebral ventricle 15min before ischemia. Compared with the Vehicle group, Apelin-13 significantly ameliorated neurological deficit, infarct volume, brain edema and reduced TUNEL-positive cells. Bax, caspase-3 and cleaved caspase-3 were down-regulated and Bcl-2 up-regulated. While, the effect of Apelin-13 on Bax, Bcl-2, caspase-3 and cleaved caspase-3 was attenuated by LY294002 and PD98059. Apelin protected the brain against I/R insult injury, and this effect may be through activation of PI3K/Akt and ERK1/2 signaling pathways.

Genistein suppresses tumor necrosis factor &amp;alpha;-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor &amp;Kappa;B and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

AimsGenistein, an isoflavone derivative found in soy, is known as a promising treatment for rheumatoid arthritis (RA). However, the detailed molecular mechanism of genistein in suppression of proinflammatory cytokine production remains ambiguous. The aim of this work was to evaluate the signal pathway by which genistein modulates inflammatory cytokine expression.Materials and methodsMH7A cells were stimulated with tumor necrosis factor (TNF)-α and incubated with genistein, and interleukin (IL)-1β, IL-6, and IL-8 production was measured by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor (NF)-κB was measured by a confocal fluorescence microscopy. The intracellular accumulation of reactive oxygen species (ROS) was monitored using the fluorescent probe 5-6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Signal-transduction protein expression was measured by Western blot.ResultsGenistein decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. Genistein prevented TNF-α-induced NF-κB translocation as well as phosphorylation of IκB kinase-α/β and IκBα, and also suppressed TNF-α-induced AMPK inhibition. The production of IL-1β, IL-6, and IL-8 induced by TNF-α was decreased by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting that inhibition of Akt activation might inhibit IL-1β, IL-6, and IL-8 production induced by TNF-α. In addition, we also found that pretreatment with the adenosine monophosphate-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside obviously inhibited TNF-α-induced proinflammatory cytokine production. These observations suggest that the inhibitory effect of genistein on TNF-α-induced proinflammatory cytokine production is dependent on AMPK activation.ConclusionThese findings indicate that genistein suppressed TNF-α-induced inflammation by inhibiting the ROS/Akt/NF-κB pathway and promoting AMPK activation in MH7A cells.

Phosphatidylinositol‐3,4,5‐triphosphate‐dependent Rac exchange factor 1 regulates epinephrine‐induced exocytosis of Weibel–Palade bodies

Weibel-Palade bodies (WPBs) function as storage vesicles for von Willebrand factor (VWF) and a number of other bioactive compounds, including angiopoietin-2 and insulin-like growth factor-binding protein 7. WPBs release their content following stimulation with agonists that increase the level of intracellular Ca²⁺, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory β-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Our findings show that PREX1 regulates epinephrine-induced release of WPBs.