Phosphatidylinositol 3-kinase Akt Research Articles

Positive Feedback Regulation of Proliferation in Vascular Smooth Muscle Cells Stimulated by Lipopolysaccharide Is Mediated through the TLR 4/Rac1/Akt Pathway

Toll-like receptor 4 (TLR4) are important in inflammation and regulating vascular smooth muscle cells (VSMCs) proliferation, which are related to atherosclerosis and restenosis. We have investigated the mechanisms involved in Lipopolysaccharide (LPS)-induced proliferation of VSMCs. Stimulation of rat aortic VSMCs with LPS significantly increases the proliferation of VSMCs. This effect is regulated by Rac1 (Ras-related C3 botulinum toxin substrate l), which mediates the activation of phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathways. Inhibition of Rac1 activity by NSC23766 is associated with inhibition of Akt activity. Treatment with NSC23766 or LY294002 significantly decreases LPS-induced TLR4 protein and mRNA expression. The data show that positive feedback regulation of proliferation in VSMCs is mediated through the TLR4/Rac1/Akt pathway.

Human biliverdin reductase‐based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase-AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

AimsFibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia.Methods and ResultsFGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased.ConclusionIn an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia.

Herbal medicines for cardiovascular diseases.

Herbal medicines for cardiovascular diseases.

IN6 ADVANCES IN CYTOTOXIC AND OTHER TARGETED THERAPY OUT OF HER2, ER AND TRIPLE NEGATIVE METASTATIC BREAST CANCERS

transduction pathways. Particularly, in the past year, encouraging results have emerged from studies testing mammalian target of rapamycin (mTOR) inhibitors, such as the BOLERO-2 that showed an increase in the median progression-free survival from 4.1 to 10.6 months with the addition of everolimus to exemestane (central assessment), in the interim analysis (hazard ratio for progression/ death 0.36, p<0.001). Several other clinical phase II and phase III trials are underway combining anti-mTOR agents, such has BELLE-3 (BKM120 + fulvestrant), PrE0102 (fulvestrant ± everolimus) or TAMRAD (tamoxifen ± everolimus); insulin-like growth factor receptor-1 (IGFR1) inhibitors (AMG 479 + exemestane or fulvestrant); histone deacetylase (HDAC) inhibitors (vorinostat + tamoxifen) and phosphatidylinositol3-kinase/Akt (PI3K/AKT) inhibitors. So far, the most promising results come from the combination of everolimus and endocrine agents, as has been shown in the TAMRAD and BOLERO-2 studies. Because these new therapies carry a cost in terms of side effects and finances, it will be important to identify biomarkers of enhanced benefit: preliminary results in this field will be presented. Besides addressing the identification of the best partner for combination with an endocrine agent, trials examining the intermittent administration of endocrine therapy, as in prostate cancer, could also be appealing, in an attempt to obviate the menopausal symptoms experienced by patients, which are known to compromise treatment adherence, and, as a result, can “fuel” tumour resistance. Finding new targets is also sought. The androgen receptor (AR) as a target in breast cancer seems to have regained interest recently and research is underway to clarify its role both as a prognostic and predictive biomarker. The cross-talk between the AR and HER2 pathways and the relationship between AR levels and mutations in PI3K are being examined and constitute a possible new hope in the treatment of advanced breast cancer.

Decidual Natural Killer Cell Interactions with Trophoblasts Are Impaired in Pregnancies at Increased Risk of Preeclampsia

Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase-Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia.

Apoptosis induced by Staphylococcus aureus in human monocytic U937 cells involves Akt and mitogen-activated protein (MAPK) phosphorylation

Staphylococcus aureus (S. aureus) is a leading etiologic agent of nosocomial and community-acquired infectious diseases. Numerous studies have shown that, S. aureus could promote apoptosis in host cells. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. The present study aims to elucidate the signaling mechanisms involved in S. aureus-induced U937 cells apoptosis by investigating the role of phosphatidylinositol 3-kinase/Akt (PI3K/Akt), mitogen-activated protein (MAPK) and caspases. Our results showed that, S. aureus decreased the expression of phosphorylation-Akt. In contrast, S. aureus increased phosphorylation-JNK1/2, phosphorylation-ERK1/2 and phosphorylation-p38 MAPK. Treatment of U937 cells with S. aureus resulted in the activation of caspase-3 and -9. Furthermore, caspases inhibitors, SP600125 (JNK inhibitor), SB203580 (p38MAPK inhibitor) and PD98059 (ERK inhibitor) decreased apoptosis in U937 cells. However, LY294002 (Akt inhibitor) increased U937 cells apoptosis. Taken together, our study for the first time suggest that S. aureus is able to enhance apoptosis of U937 cells through inhibition of PI3K/Akt and activation of MAPK signaling pathways.

Nasal Administration of Recombinant Osteopontin Attenuates Early Brain Injury After Subarachnoid Hemorrhage

Neuronal apoptosis is a key pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Given that recombinant osteopontin (rOPN), a promising neuroprotectant, cannot pass through the blood-brain barrier, we aimed to examine whether nasal administration of rOPN prevents neuronal apoptosis after experimental SAH. Male Sprague-Dawley rats (n=144) were subjected to the endovascular perforation SAH model. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 and 72 hours after SAH induction. The expressions of cleaved caspase-3, phosphorylated focal adhesion kinase (FAK), and phosphorylated Akt were examined using Western blot analysis. Neuronal cell death was demonstrated with terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling. We also administered FAK inhibitor 14 and phosphatidylinositol 3-kinase inhibitor, Wortmannin, prior to rOPN to establish its neuroprotective mechanism. ELISA was used to measure rOPN delivery into the cerebrospinal fluid. Cerebrospinal fluid level of rOPN increased after its nasal administration. This was associated with improved neurological scores and reduced brain edema at 24 hours after SAH. rOPN increased phosphorylated FAK and phosphorylated Akt expressions and decreased caspase-3 cleavage, resulting in attenuation of neuronal cell death within the cerebral cortex. These effects were abolished by FAK inhibitor 14 and Wortmannin. Nasal administration of rOPN decreased neuronal cell death and brain edema and improved the neurological status in SAH rats, possibly through FAK-phosphatidylinositol 3-kinase-Akt-induced inhibition of capase-3 cleavage.

Insulin suppresses ischemic preconditioning-mediated cardioprotection through Akt-dependent mechanisms

It is believed that the diabetic myocardium is refractory to cardioprotection by ischemic preconditioning (IPC) mainly because of impaired insulin signaling to phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB or Akt). However, human as well as animal studies have clearly showed that the hearts of type 2 diabetic humans and animals may exhibit increased signaling through PI3K–Akt but yet are resistant to cardioprotection by IPC or ischemic post-conditioning. Therefore, this study was designed to determine whether activation of insulin signaling prior to IPC is detrimental for cardioprotection and to assess the role of insulin receptors (IRs) and Akt in mediating this effect. Wild-type (WT) hearts, hearts lacking IRs or hearts expressing an active form of Akt (myrAkt1) were perfused ex vivo using a Langendorff preparation and were subjected to IPC (3cycles of 5min ischemia followed by 5min reflow before 30min no flow ischemia and then by 45min reperfusion) in the presence or absence of 1nmol/L insulin. Interestingly, whereas insulin was protective against I/R (30min no flow ischemia and 45min reperfusion), it completely abolished cardioprotection by IPC in WT hearts but not in mice lacking insulin receptors (IRs) in cardiomyocytes (CIRKO) or in all cardiac cells (TIRKO). The suppression of IPC-mediated cardioprotection was mediated through downstream signaling to Akt and Gsk3β. In addition, transgenic induction of Akt in the heart was sufficient to abrogate IPC even when insulin was absent, further confirming the involvement of Akt in insulin's suppression of cardioprotection by IPC. These data provide evidence that excessive insulin signaling to Akt is detrimental for cardioprotection by IPC and could explain the failure of the diabetic myocardium to precondition.

Thymoquinone-induced reactive oxygen species causes apoptosis of chondrocytes via PI3K/Akt and p38kinase pathway

Thymoquinone (TQ), a bioactive ingredient of the volatile oil of black seed (Nigella sativa), has been shown to possess anti-neoplastic and anti-inflammatory effects on a variety of tumours. However, the precise mechanism of action is not clear in normal cells such as primary chondrocytes. So, we have investigated the effects of TQ on the apoptosis of chondrocytes with a focus on reactive oxygen species (ROS) production. In in vitro experiments, chondrocytes were cultured with increasing concentrations of TQ for 24 h or with 20 µmol/L TQ for the indicated time periods, and various experiments were performed to detect the apoptotic effects caused by TQ. The results showed that TQ significantly increases apoptosis. Apoptosis was dose- and time-dependently expressed, and the generation of ROS also dramatically increased in a dose-dependent manner. Pretreatment of N-acetyl-L-cysteine (NAC), an inhibitor of ROS, inhibited both TQ-induced apoptosis and ROS generation. Also, TQ up-regulated phosphorylation of phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinases ([MAPKs] p38kinase, ERK-1/-2, and JNKinase), and these effects were prevented by pretreatment of NAC. However, pretreatment with inhibitors of PI3K/Akt and MAPKs did not inhibit TQ-caused ROS generation. Among the inhibitors of PI3K/Akt, p38kinase, ERK-1/-2, and JNKinase, pretreatment with LY294002 and SB203580 abolished TQ-induced apoptosis, but PD98059 and SP600125 did not have any effect on TQ-caused apoptosis. These findings suggest that TQ-induced ROS generation regulates apoptosis by modulating PI3K/Akt and p38kinase pathways.

N-Acetylcysteine and allopurinol up-regulated the Jak/STAT3 and PI3K/Akt pathways via adiponectin and attenuated myocardial postischemic injury in diabetes

N-Acetylcysteine (NAC) and allopurinol (ALP) synergistically reduce myocardial ischemia reperfusion (MI/R) injury in diabetes. However, the mechanism is unclear. We postulated that NAC and ALP attenuated diabetic MI/R injury by up-regulating phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathways subsequent to adiponectin (APN) activation. Control (C) or streptozotocin-induced diabetic rats (D) were untreated or treated with NAC and ALP followed by MI/R. D rats displayed larger infarct size accompanied by decreased phosphorylation of Akt, STAT3 and decreased cardiac nitric oxide (NO) and APN levels. NAC and ALP decreased MI/R injury in D rats, enhanced phosphorylation of Akt and STAT3, and increased NO and APN. High glucose and hypoxia/reoxygenation exposure induced cell death and Akt and STAT3 inactivation in cultured cardiomyocytes, which were prevented by NAC and ALP. The PI3K inhibitor wortmannin and Jak2 inhibitor AG490 abolished the protection of NAC and ALP. Similarly, APN restored posthypoxic Akt and STAT3 activation and decreased cell death in cardiomyocytes. Gene silencing with AdipoR2 siRNA or STAT3 siRNA but not AdipoR1 siRNA abolished the protection of NAC and ALP. In conclusion, NAC and ALP prevented diabetic MI/R injury through PI3K/Akt and Jak2/STAT3 and cardiac APN may serve as a mediator via AdipoR2 in this process.

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Atrial natriuretic peptide suppresses Th17 development through regulation of cGMP-dependent protein kinase and PI3K–Akt signaling pathways

In recent years, accumulating evidence suggests that atrial natriuretic peptide (ANP), a hormone widely known as a result of its significant effects on the cardiovascular system mediated by natriuretic peptide receptor A (NPRA), may play a nonnegligible role in the regulation of immune responses. In this study, we firstly investigated whether ANP signaling could regulate the differentiation and capacity of Th17 cells and discovered ANP-dose (10−8–10−6M) dependently indeed suppressed the differentiation of Th17 cells along with the reduced IL-17 production by polarizing naïve CD4+ T cells isolated from splenocytes to Th17 phenotype in vitro. Moreover, ANP primarily signals through NPRA and cGMP-dependent protein kinase (PKG) which could be antagonized when pretreated with either ANP/NPRA signaling antagonist or PKG inhibitor. In addition, we also found that ANP signaling could upregulate the levels of phosphorylation of Akt which was hypothesized to be implicated in ANP-induced inhibition of Th17 development in our studies, and the effect of ANP on the development of murine Th17 cells seemed to be partially reversed when an inhibitor of phosphatidylinositol 3′-kinase (PI3K)/Akt had been performed in advance. Briefly, we showed for the first time that ANP signaling could suppress murine Th17 cell development from naïve CD4+ T cells in vitro through NPRA/PKG pathway and the PI3K–Akt signal was implicated in the ANP-mediated suppression of Th17 development.

Expression of p110, phosphorylation-protein kinase B and protein kinase B in hemangioma vascular endothelial cell in vitro

Objective To study the expression of phosphatidy linositol-3-kinase(PI3K) p110 catalytic subunit and phosphorylation-protein kinase B(p-Akt), protein kinase B(Akt) in cultured vascular endothelial cells, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Methods The hemangioma vascular endothelial cells which came from the sterile hemangioma were cultured by using tissue culture method and then synchronized cultured endothelial cells, PI3K p110, p-Akt and Akt of them were detected by way of Western blot, meanwhile cell cycle and cell apoptosis were examined by using flow cytometry technique, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Results The percentage of vascular endothelial cell was (94.00±3.00)% in the G0/G1 phase and the apoptosis rate was (11.24±2.34)% when p110 and p-Akt protein expression level were lower (0.19±0.01, 0.09±0.02), and they were (61.00±6.00)% and (0.51±0.03)% when p110 and p-Akt protein expression level were higher (0.37±0.04, 0.22±0.01). The percentage of vascular endothelial cell in the G0/G1 phase and the apoptosis rate were different in 2 groups(t=-8.42, -35.48, all P<0.01). But Akt protein expression level was not changed(0.72±0.00). Conclusions p110, p-Akt and Akt protein were expressed in the cultured hemangioma vascular endothelial cells in vitro.PI3K p110, p-Akt may be involved in cultured hemangioma vascular endothelial cells apoptosis in vitro. Key words: Hemangioma; Vascular endothelial cell; Cell culture; p110; Phosphorylation-protein kinase B; Protein kinase B; Cell apoptosis

Protective Effects of Shen-Yuan-Dan, a Traditional Chinese Medicine, against Myocardial Ischemia/Reperfusion InjuryIn VivoandIn Vitro

Objectives. The study was to investigate the effects and mechanisms of Shen-Yuan-Dan (SYD) pharmacological postconditioning on myocardial ischemia/reperfusion (I/R) injury. Methods. In the in vivo experiment, myocardial injury markers and histopathology staining were examined. In the in vitro experiment, cell viability and cell apoptosis were, respectively, detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and Hoechst 33342 fluorochrome staining. The protein expressions of Bcl-2 and Bax were determined by immunocytochemistry assay. Results. Both low and high doses of SYD protected myocardium against I/R injury in rat model by reducing lactic dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity and malondialdehyde (MDA) content, increasing superoxide dismutase (SOD) activity and attenuating histopathology injury. Meanwhile, in the in vitro experiment, SYD promoted cell viability and inhibited the cardiomyocyte apoptosis. The level of Bcl-2 protein was restored to the normal level by SYD pharmacological postconditioning. In contrast, the Bax protein level was markedly reduced by SYD pharmacological postconditioning. These effects of SYD were inhibited by LY294002. Conclusions. The results of this study suggested that SYD pharmacological postconditioning has protective effects against myocardial I/R injury in both in vivo and in vitro models, which are related to activating the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway.

Migration of retinal pigment epithelial cells is EGFR PI3K AKT dependent

Abnormal migration of retinal pigment epithelium (RPE) contributes to a variety of disorders such as proliferative vitreoretinopathy. Here, the effect of epidermal growth factor (EGF), and signaling by its receptor (ERGR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) on RPE cell migration was studied. The in vitro wound healing and migration of the human RPE cell line, ARPE19 cell was accelerated, in a dose dependent manner, in response to EGF stimulation, while pretreatment with EGFR, PI3K or AKT inhibitor, inhibited both events. Exposure of cells to EGF activated the AKT phosphorylation, whereas EGFR and PI3K inhibitors blocked EGF-induced AKT phosphorylation in a dose-dependent manner. These data suggest that EGF mediate ARPE-19 cell migration through EGFR/PI3K/AKT signaling pathway.

Malvidin, a red wine polyphenol, modulates mammalian myocardial and coronary performance and protects the heart against ischemia/reperfusion injury

A moderate red wine consumption and a colored fruit-rich diet protect the cardiovascular system, thanks to the presence of several polyphenols. Malvidin-3-0-glucoside (malvidin), an anthocyanidine belonging to polyphenols, is highly present in red grape skin and red wine. Its biological activity is poorly characterized, although a role in tumor cell inhibition has been found. To analyze whether and to which extent, like other food-derived polyphenols, malvidin affects the cardiovascular function, in this study, we have performed a quantitative analysis by high-performance liquid chromatography of polyphenolic content of red grape skins extract, showing that it contains a high malvidin amount (63.93±12.50 mg/g of fresh grape skin). By using the isolated and Langendorff perfused rat heart, we found that the increasing doses (1–1000 ng/ml) of the extract induced positive inotropic and negative lusitropic effects associated with coronary dilation. On the same cardiac preparations, we observed that malvidin (10−10–10−6 mol/L) elicited negative inotropism and lusitropism and coronary dilation. Analysis of mechanism of action revealed that malvidin-dependent cardiac effects require the activation of the phosphatidylinositol 3-kinase (PI3K)/nitric oxide (NO)/cGMP/PKG pathway and are associated with increased intracellular cGMP and the phosphorylation of endothelial NO synthase (eNOS), PI3K–AKT, ERK1/2, and GSK-3β. AKT and eNOS phosphorylation was confirmed in human umbilical vein endothelial cell. We also found that malvidin act as a postconditioning agent, being able to elicit cardioprotection against ischemia/reperfusion damages. Our results show the cardioactivity of polyphenols-rich red grape extracts and indicate malvidin as a new cardioprotective principle. This is of relevance not only for a better clarification of the beneficial cardiovascular effects of food-derived polyphenols but also for nutraceutical research.

Astaxanthin prevents loss of insulin signaling and improves glucose metabolism in liver of insulin resistant mice

This study investigates the effects of astaxanthin (ASX) on insulin signaling and glucose metabolism in the liver of mice fed a high fat and high fructose diet (HFFD). Adult male Mus musculus mice of body mass 25-30 g were fed either normal chow or the HFFD. After 15 days, mice in each group were subdivided among 2 smaller groups and treated with ASX (2 mg·(kg body mass)⁻¹) in olive oil for 45 days. At the end of 60 days, HFFD-fed mice displayed insulin resistance while ASX-treated HFFD animals showed marked improvement in insulin sensitivity parameters. ASX treatment normalized the activities of hexokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glycogen phosphorylase, and increased glycogen reserves in the liver. Liver tissue from ASX-treated HFFD-fed animals showed increased tyrosine phosphorylation and decreased serine phosphorylation of insulin receptor substrates (IRS)-1 and -2. ASX increased IRS 1/2 and phosphatidylinositol 3-kinase (PI3K) association and serine phosphorylation of Akt. In addition, ASX decreased HFFD-induced serine kinases (c-jun N-terminal kinase-1 and extracellular signal-regulated kinase-1). The results suggest that ASX treatment promotes the IRS-PI3K-Akt pathway of insulin signaling by decreasing serine phosphorylation of IRS proteins, and improves glucose metabolism by modulating metabolic enzymes.

A Comprehensive Small Interfering RNA Screen Identifies Signaling Pathways Required for Gephyrin Clustering

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABA(A) receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)-Akt also activates mTOR and represses GSK3β, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF-mTOR signaling is associated with reduced GABA(A) receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K-Akt-mTOR signaling.