Regulator Of Phosphatidylinositol 3-kinase Research Articles

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

The mechanism of Shenbing Decoction II against IgA nephropathy renal fibrosis revealed by UPLC-MS/MS, network pharmacology and experimental verification

BackgroundIgA nephropathy (IgAN) is a major and growing public health problem. Renal fibrosis plays a vital role in the progression of IgAN. This study is to investigate the mechanisms of action underlying the therapeutic effects of Shenbing Decoction II (SBDII) in IgAN renal fibrosis treatment based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), network pharmacology and experimental verification. MethodWe first used UPLC-MS/MS to explore the main compounds of SBDII, and then used network pharmacology to predict the targets and key pathways of SBDII in the treatment of IgAN renal fibrosis. Next, bovine serum albumin (BSA), lipopolysaccharide (LPS), and carbon tetrachloride (CCL4) were used to induce IgAN in rats, and then biochemical indicators, renal tissue pathology, and renal fibrosis-related indicators were examined. At the same time, part of the results predicted by network pharmacology were also verified. ResultA total of 105 compounds were identified in SBDII by UPLC-MS/MS. Network pharmacology results showed that the active compounds such as acacetin, eupatilin, and galangin may mediate the therapeutic effects of SBDII in treating IgAN by targeting tumor protein p53 (TP53) and regulating phosphatidylinositol 3-kinase (PI3K)-Akt kinase (Akt) signaling pathway. Animal experiments showed that SBDII not only significantly improved renal function and fibrosis in IgAN rats, but also significantly downregulated the expressions of p53, p-PI3K and p-Akt. ConclusionThis UPLC-MS/MS, network pharmacological and experimental study highlights that the TP53 as a target, and PI3K-Akt signaling pathway are the potential mechanism by which SBDII is involved in IgAN renal fibrosis treatment. Acacetin, eupatilin, and galangin are probable active compounds in SBDII, these results might provide valuable guidance for further studies of IgAN renal fibrosis treatment.

PTEN inhibitor and kit ligand increase in vitro activation and survival of primordial follicles in alpaca.

In mammals, activation of primordial follicles to primary follicle is a progressive and highly regulated process. There is evidence in mice that phosphatase and tensin homologue deleted on Chromosome 10 (PTEN) silencing is an important negative regulator of phosphatidylinositol 3-kinase (PI3K), which initiates activation of dormant follicles. The objective of the study was to evaluate the effect of the addition of PTEN inhibitor (bpV(HOpic)) (10 μM) and/or Kit Ligand (KL) (100 ng/mL) on the in vitro activation and survival of alpaca primordial follicles. Ovarian cortical fragments from 11 adult alpacas were cultured for 24 h in tissue culture medium (α-MEM+ ) supplemented with KL and bpV or the association of both. Subsequently, each sample was processed by classical histology and follicular counting and classification were performed. The results obtained show a reduction (p < 0.05) of primordial follicles in more than 50% in follicular tissue cultured in vitro in α-MEM+ or supplemented with bpV and/or KL versus the control (not cultured). Further, >25% increase in primary follicles in follicular tissue cultured in vitro in α-MEM+ or supplemented with KL and/or bpV versus control. However, the follicular survival rate showed a decrease of 20% in the cultured tissues, except for the α-MEM+ supplemented with KL and bpV. In conclusion, supplementation of bpV (HOpic) (10 μM) and KL (100 ng/mL) increased the activation in vitro of primordial follicles and survival after in vitro culture of alpaca ovarian tissue.

A potent bioactive fraction against colon cancer from Plectranthus vettiveroides.

This study was designed to investigate the anticancer efficacy of the organic leaf extracts of the plant, Plectranthus vettiveroides (P. vettiveroides), and to analyze the molecular mechanism of the anticancer activity. The leaf extracts were prepared by polarity-graded serial extraction of the dried leaf powder. The cytotoxic effect of the extracts was analyzed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The most active ethyl acetate extract was subjected to bioactivity-guided fractionation by column chromatography, which yielded a cytotoxic fraction designated as the P. vettiveroides fraction (PVF). The anticancer property of PVF was confirmed further by clonogenic assay. The mechanism of PVF-induced cell death was analyzed by flow cytometry and fluorescence microscopy. Additionally, the effects of PVF on apoptotic and cell survival pathways were analyzed using western immunoblot analysis. A bioactive fraction PVF, was isolated from the ethyl acetate leaf extract. PVF showed significant anticancer activity against colon cancer cells, whilst normal cells were comparatively less affected. PVF induced strong apoptotic stimuli in colorectal carcinoma cell line HCT116, involving both extrinsic and intrinsic pathways. Investigation into the molecular mechanism of anticancer activity of PVF in HCT116 cells revealed that the fraction activates the pro-apoptotic pathway via tumor suppressor protein 53 (p53) and inhibits the anti-apoptotic pathway by regulating phosphatidylinositol 3-kinase (PI3K) signaling. The findings of this study demonstrate, with mechanism-based evidence, the chemotherapeutic potential of a bioactive fraction PVF, derived from the leaves of the medicinal plant P. vettiveroides against colon cancer.

Comparative transcriptome analysis of endothelial progenitor cells of HbSS patients with and without proliferative retinopathy.

Among sickle cell anemia (SCA) complications, proliferative sickle cell retinopathy (PSCR) is one of the most important, being responsible for visual impairment in 10-20% of affected eyes. The aim of this study was to identify differentially expressed genes (DEGs) present in pathways that may be implicated in the pathophysiology of PSCR from the transcriptome profile analysis of endothelial progenitor cells. RNA-Seq was used to compare gene expression profile of circulating endothelial colony-forming cells (ECFCs) from HbSS patients with and without PSCR. Furthermore, functional enrichment analysis and protein-protein interaction (PPI) networks were performed to gain further insights into biological functions. The differential expression analysis identified 501 DEGs, when comparing the groups with and without PSCR. Furthermore, functional enrichment analysis showed associations of the DEGs in 200 biological processes. Among these, regulation of mitogen-activated protein (MAP) kinase activity, positive regulation of phosphatidylinositol 3-kinase (PI3K), and positive regulation of Signal Transducer and Activator of Transcription (STAT) receptor signaling pathway were observed. These pathways are associated with angiogenesis, cell migration, adhesion, differentiation, and proliferation, important processes involved in PSCR pathophysiology. Moreover, our results showed an over-expression of VEGFC (vascular endothelial growth factor-C) and FLT1 (Fms-Related Receptor Tyrosine Kinase 1) genes, when comparing HbSS patients with and without PSCR. These results may indicate a possible association between VEGFC and FLT1 receptor, which may activate signaling pathways such as PI3K/AKT and MAPK/ERK and contribute to the mechanisms implicated in neovascularization. Thus, our findings contain preliminary results that may guide future studies in the field, since the molecular mechanisms of PSCR are still poorly understood.

Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway

To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 μmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 μmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 μmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Compared with the control group, MYR at 50, 100 and 200 μmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.

Effects of Rosa roxburghii on insulin resistance in obese rats and its mechanisms

Objective: To investigate the effects of Rosa roxburghii on insulin resistance in obese rats and the regulation of phosphatidylinositol 3-kinase (PI3K)/ protein kinase Bβ(PKBβ/Akt2)/ glucose transporter 4(GLUT4) signaling pathway. Methods: Five-week-old male SD rats were randomly divided into normal control group (NC), model group (M), positive control group (PC), low-dose rosa roxburghii group (LD) and high-dose rosa roxburghii group (HD), with 10 rats in each group. The rats in the NC group were fed with normal diet, while those in the M, PC, LD and HD groups were fed with high-fat diet. From the 13th week, according to the dose standard of 6 ml/kg, rats in the LD group were intragastrically administered with 100 mg/kg Rosa roxburghii Tratt, the HD group were treated with 300 mg/kg Rosa roxburghii Tratt, the PC group were treated with 0.11 g/kg Chiglitazar sodium, and the NC and M groups were intragastrically administered with the same volume of normal saline. The body weight was measured every week until 20 weeks. The rats were sacrificed 24 h after the last experiment. Blood and skeletal muscle were collected. Serum total cholesterol (TC) and triglyceride (TG) contents were detected by colorimetric method, serum superoxide dismutase (SOD) activity was detected by xanthine oxidase method, serum malondialdehyde (MDA) content was detected by thiobarbituric acid method, blood glucose (FBG) value was detected by glucose oxidase method, insulin (FINS) content was detected by ELISA, and PI3K, Akt2, and GLUT4 protein and gene expressions were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with the NC group, the body weight, serum MDA, TG, TC, FBG, FINS, HOMA-IR levels in the M group were significantly increased (P＜0.01), while SOD activity, PI3K、Akt2、GLUT4 protein and mRNA expression levels were significantly increased(P＜ 0.01). Compared with group M, the body weight, serum MDA, TG, TC, FBG, FINS, and HOMA-IR were decreased significantly in LD group, HD group and PC group (P＜0.05 or P＜0.01), while SOD activity, PI3K, Akt2, GLUT4 protein and mRNA expression levels were increased significantly (P＜0.05 or P＜0.01). Conclusion: Rosa roxburghii can improve insulin resistance in obese rats by antioxidant stress and up-regulating the expressions of PI3K, Akt2, and GLUT4 proteins and genes, which may be related to the PI3K/Akt2/GLUT4 signaling pathway.

Network pharmacology analysis combined with experimental validation to explore the therapeutic mechanism of Schisandra Chinensis Mixture on diabetic nephropathy

Ethnopharmacological relevanceDiabetic nephropathy (DN) is one of the most common and serious microvascular complications of Diabetes mellitus (DM). The inflammatory response plays a critical role in DN. Schisandra Chinensis Mixture (SM) has shown promising clinical efficacy in the treatment of DN while the pharmacological mechanisms are still unclear. Aim of the studyIn this study, a network pharmacology approach and bioinformatic analysis were adopted to predict the pharmacological mechanisms of SM in DN therapy. Based on the predicted results, molecular docking and in vivo experiments were used for verification. Materials and methodsIn this study, the candidate bioactive ingredients of SM were obtained via Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and supplementing according to the literature. SM putative targets and the verified targets were acquired from TCMSP and SiwssTartgetPrediction Database. DN-related target genes were collected from GeneCards, OMIM, DisGeNET databases, and microarray data analysis. Biological function and pathway analysis were performed to further explore the pharmacological mechanisms of SM in DN therapy. The protein-protein interaction (PPI) network was established to screen the hub gene. The Receiver Operating Characteristic (ROC) analysis and the molecular docking simulations were performed to validate the potential target-drug interactions. The fingerprint spectrum of multi-components of the SM was characterized by UPLC-MS/MS. The signaling pathways associated with inflammation and hub genes were partially validated in SD rats. ResultsA total of 36 bioactive ingredients were contained, and 666 component-related targets were screened from SM, of which 50 intersected with DN targets and were considered potential therapeutic targets. GO analyses revealed that the 50 intersection targets were mainly enriched in the inflammatory response, positive regulation of angiogenesis, and positive regulation of phosphatidylinositol 3-kinase(PI3K) signaling. KEGG analyses indicated that the PI3K-Akt signaling pathway was considered as the most important pathway for SM antagonism to the occurrence and development of DN, with the highest target count enrichment. PPI network results showed that the top 15 protein targets in degree value, VEGFA, JAK2, CSF1R, NOS3, CCR2, CCR5, TLR7, FYN, BTK, LCK, PLAT, NOS2, TEK, MMP1 and MCL1, were identified as hub genes. The results of ROC analysis showed that VEGFA and NOS3 were valuable in the diagnosis of DN. The molecular docking confirmed that the core bioactive ingredients had well-binding affinity for VEGFA and NOS3. The in vivo experiments confirmed that SM significantly inhibited the over-release of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor receptor (TNF)-α in DN rats, while regulating the PI3K-AKT and VEGFA-NOS3 signaling pathways. ConclusionThis study revealed the multi-component, multi-target and multi-pathway characteristics of SM therapeutic DN. SM inhibited the inflammatory response and improved renal pathological damage in DN rats, which was related to the regulation of the PI3K-Akt and VEGFA-NOS3 signaling pathways.

Pharmacological Mechanism of Ganlu Powder in the Treatment of NASH Based on Network Pharmacology and Molecular Docking.

Nonalcoholic steatohepatitis (NASH), a progression of nonalcoholic fatty liver disease (NAFLD), is a clinical syndrome characterized by liver steatosis, inflammation, and hepatocellular damage. Ganlu powder (GLP) is a classic traditional Chinese medicine prescription that has shown favorable treatment effects on NASH. However, the underlying therapeutic mechanisms are still poorly understood. This study is aimed at exploring the potential mechanism of GLP in the treatment of NASH via network pharmacology and molecular docking. PubMed and CNKI databases were used to identify the components of GLP. Swiss and STITCH databases were employed to obtain corresponding drug targets. NASH targets were adopted from the Therapeutic Target Database (TTD), DisGeNET, DrugBank, GeneCards, and MalaCards databases. Cytoscape software was utilized to construct “drug-ingredient-target-disease” networks and the protein-protein interaction (PPI) network of GLP in NASH. AKT1 was identified as the key target. The GO functional enrichment analysis revealed that GLP might treat NASH by modulating the inflammatory response and regulating phosphatidylinositol 3-kinase signaling. The KEGG analysis showed that GLP might treat NASH by regulating the tumor necrosis factor (TNF) signal pathway by affecting the role of AKT1. According to the network pharmacology results, a virtual docking of active compounds with AKT1 was carried out, and the results indicated that the 7 components, berberine, epiberberine, jatrorrhizine, coptisine, palmatine, evodiamine, and rutecarpine, can bind stably with AKT1 and have higher binding energy than AKT1 inhibitors. The overall study findings suggest that GLP may treat NASH by regulating AKT1.

Vacuolar protein sorting-associated protein 72 homolog (VPS72) binding to lysine acetyltransferase 5 (KAT5) promotes the proliferation, invasion and migration of hepatocellular carcinoma through regulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway

ABSTRACT Hepatocellular carcinoma, a fatal malignancy that occurs in the liver, poses a major public health challenge. This paper attempted to clarify the role and mechanism of vacuolar protein sorting-associated protein 72 homolog (VPS72) in the progression of hepatocellular carcinoma. Firstly, VPS72 expression in hepatocellular carcinoma tissues and the prognostic correlation were analyzed by GEPIA2 database. Western blotting and RT-qPCR assays were used to evaluate VPS72 expression in several hepatocellular carcinoma cell lines. Then, cell proliferation was assessed by cell counting kit-8 and colony formation in HuH-7 cells with VPS72 silencing. Measurement of cell invasion and migration by transwell and wound healing assays. Next, the relationship between VPS72 and lysine acetyltransferase 5 (KAT5) was predicted by bioGRID, STRING and GEIPA2 databases, which was confirmed by Co-immunoprecipitation assay. Subsequently, KAT5 was overexpressed to explore whether VPS72 could regulate the progression of hepatocellular carcinoma by binding to KAT5. And the expression of proteins related to PI3K/AKT signaling was tested with western blotting. Results indicated that VPS72 was highly expressed in hepatocellular carcinoma tissues and cell lines and was associated with poor prognosis. VPS72 knockdown inhibited the proliferation, invasion and migration of HuH-7 cells. In addition, VPS72 could bind to KAT5. KAT5 overexpression reversed the suppressive impacts of VPS72 knockdown on the proliferation, invasion and migration in HuH-7 cells. Besides, VPS72 silencing downregulated p-PI3K and p-AKT expression, which was restored by KAT5 overexpression. Collectively, VPS72 binding to KAT5 promotes the progression of hepatocellular carcinoma through the regulation of PI3K/AKT signaling pathway.

CBX8 interacts with chromatin PTEN and is involved in regulating mitotic progression.

ObjectivesBesides its role in regulating phosphatidylinositol‐3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division.Materials and methodsImmunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle.ResultsPolycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co‐localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated‐BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells.ConclusionCBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.

Epoxyeicosatrienoic acids prevent cardiomyocytes against sepsis by A2AR-induced activation of PI3K and PPARγ

Although epoxyeicosatrienoic acids (EETs) have multiple protective effects against different diseases, whether they can improve the pathogenesis of lipopolysaccharide (LPS)-induced septic cardiac dysfunction remains unknown. We investigated the effects of EETs on the LPS-induced inflammatory response in myocardial dysfunction mice and H9c2 cardiac myocytes. Cardiac-specific CYP2J2 transgenic mice (Tr) showed improved cardiac function and reduced inflammation response after administration with LPS, while the protective effects were not observed in A2A adenosine receptor (A2AR/ADORA2A)–deficient mice (knockout/KO). In vitro, EETs prevented LPS-induced inflammation and apoptosis in the cardiomyocytes via A2AR activation. Moreover, ZM241385 (A2AR inhibitor) attenuated the cardioprotective properties of EETs. Further investigation demonstrated that A2AR signal pathway activation partly regulated phosphatidylinositol 3-kinase (PI3K) and peroxisome proliferator-activated receptor-γ (PPARγ) expression. This is the first report on EETs exerting cardioprotective effects against LPS-induced cardiomyocyte injury via A2AR activation.

Molecular Mechanism of Vitamin K2 Protection against Amyloid-β-Induced Cytotoxicity.

The pathological role of vitamin K2 in Alzheimer’s disease (AD) involves a definite link between impaired cognitive functions and decreased serum vitamin K levels. Vitamin K2 supplementation may have a protective effect on AD. However, the mechanism underlying vitamin K2 protection has not been elucidated. With the amyloid-β (Aβ) cascade hypothesis, we constructed a clone containing the C-terminal fragment of amyloid precursor protein (β-CTF/APP), transfected in astroglioma C6 cells and used this cell model (β-CTF/C6) to study the protective effect of vitamin K2 against Aβ cytotoxicity. Both cellular and biochemical assays, including cell viability and reactive oxygen species (ROS), assays assay, and Western blot and caspase activity analyses, were used to characterize and unveil the protective role and mechanism of vitamin K2 protecting against Aβ-induced cytotoxicity. Vitamin K2 treatment dose-dependently decreased the death of neural cells. The protective effect of vitamin K2 could be abolished by adding warfarin, a vitamin K2 antagonist. The addition of vitamin K2 reduced the ROS formation and inhibited the caspase-3 mediated apoptosis induced by Aβ peptides, indicating that the mechanism underlying the vitamin K2 protection is likely against Aβ-mediated apoptosis. Inhibitor assay and Western blot analyses revealed that the possible mechanism of vitamin K2 protection against Aβ-mediated apoptosis might be via regulating phosphatidylinositol 3-kinase (PI3K) associated-signaling pathway and inhibiting caspase-3-mediated apoptosis. Our study demonstrates that vitamin K2 can protect neural cells against Aβ toxicity.

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts.

This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus-oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

Phenotypic Screen with TSC-Deficient Neurons Reveals Heat-Shock Machinery as a Druggable Pathway for mTORC1 and Reduced Cilia

SUMMARYTuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficientneurons.To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors ofthe heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

Tau Reduction Prevents Key Features of Autism in Mouse Models

Autism is characterized by repetitive behaviors, impaired social interactions, and communication deficits. It is a prevalent neurodevelopmental disorder, and available treatments offer little benefit. Here, we show that genetically reducing the protein tau prevents behavioral signs of autism in two mouse models simulating distinct causes of this condition. Similar to a proportion of people with autism, both models have epilepsy, abnormally enlarged brains, and overactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/ mammalian target of rapamycin (mTOR) signaling pathway. All of these abnormalities were prevented or markedly diminished by partial or complete genetic removal of tau. We identify disinhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative PI3K regulator that tau controls, as a plausible mechanism and demonstrate that tau interacts with PTEN via tau's proline-rich domain. Our findings suggest an enabling role of tau in the pathogenesis of autism and identify tau reduction as a potential therapeutic strategy for some of the disorders that cause this condition.

Targeting EZH2 by microRNA-449a inhibits osteosarcoma cell proliferation, invasion and migration via regulation of PI3K/AKT signaling pathway and epithelial-mesenchymal transition.

Osteosarcoma (OS) is one common bone malignant tumor prevailing in young adults and children. It is increasingly recognized microRNA 449a (miR 449a) as an anti-tumor factor in various tumours. However, little is known about the biological significance of miR 449a in OS. The intent of our study was to seek the prognostic values of miR-449a in OS. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the level of miR-449a expression in 48 pairs of OS tissues and para-cancerous specimens, and the relationship between miR-449a level and clinical features of OS patient prognosis was analyzed. Moreover, we measured the miR-449a expression levels in OS cells. Transwell assay was further performed to investigate whether miR-449a influenced MG63 cell migration and invasion, which was important for malignant metastases. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated a notable decrease of miR-449a expressions in OS. The declined miR-449a expression was relevant with the poor prognosis and malignant clinicopathologic characteristics of OS patients. Thereafter, the functional assay was performed to determine the role of miR-449a in OS progression. Results of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays and transwell assays indicated that miR-449a overexpression significantly repressed OS cell proliferation, invasion, and migration. Furthermore, luciferase reporter assay showed that enhancer of zeste homolog 2 (EZH2) was a downstream target of miR-449a in OS cells. Additionally, Western blot analysis demonstrated that miR-449a exerted anti-OS functions via the regulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and epithelial-mesenchymal transition. We also indicated that miR-449a restoration could inhibit in vivo tumor growth. These results manifested that miR-449a may thus be used as a therapeutic target in OS treatments.

MicroRNA-126 enhances the biological function of endothelial progenitor cells under oxidative stress via PI3K/Akt/GSK3β and ERK1/2 signaling pathways.

Endothelial progenitor cell (EPC) transplantation is a safe and effective method to treat acute myocardial infarction (AMI). However, oxidative stress leads to the death of a large number of EPCs in the early stage of transplantation, severely weakening the therapeutic effect. Previous studies demonstrated that microRNAs regulate the biological function of EPCs. The aim of the current study was to investigate the effect of microRNA on the biological function of EPCs under oxidative stress. Quantitative reverse transcription PCR was performed to detect the expression of miR-126, miR-508-5p, miR-150, and miR-16 in EPCs from rats, among which miR-126 showed a relatively higher expression. Treatment with H2O2 decreased miR-126 expression in EPCs in a dose-dependent manner. EPCs were further transfected with miR-126 mimics or inhibitors, followed by H2O2 treatment. Overexpression of miR-126 enhanced the proliferation, migration, and tube formation of H2O2-treated EPCs. MiR-126 overexpression also inhibited reactive oxygen species and malondialdehyde levels and enhanced superoxide dismutase levels, as well as increased angiopoietin (Ang)1 expression and decreased Ang2 expression in H2O2-treated EPCs. Moreover, miR-126 participated in the regulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in EPCs, where both pathways were activated after miR-126 overexpression in H2O2-treated EPCs. Overall, we showed that miR-126 promoted the biological function of EPCs under H2O2-induced oxidative stress by activating the PI3K/Akt/GSK3β and ERK1/2 signaling pathway, which may serve as a new therapeutic approach to treat AMI.

Discovery of the PTEN Tumor Suppressor and Its Connection to the PI3K and AKT Oncogenes.

PTEN (phosphatase and tensin homolog on chromosome 10) was discovered over 20 years ago in 1997 and linked to the phosphatidylinositol 3-kinase (PI3K) and AKT oncogenes the following year. The discovery of PTEN emerged from the linked concepts of oncogenes and tumor suppressor genes that cause and prevent cancer and the fields of tumor viruses and human cancer genetics from which these two concepts arose. While much has been learned since, the initial discovery and characterization, including the discovery that PTEN is a regulator of PI3K and AKT, provide the foundation on which we continue to build our knowledge. To provide the context in which these cancer genes were discovered, background information that led to their discovery will also be discussed, which will hopefully be a useful guide for readers seeking to build on the work of others.