PI3K-dependent Phosphorylation Of Akt Research Articles

11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

Leveraging molecular structure and bioactivity with chemical language models for de novo drug design

Generative chemical language models (CLMs) can be used for de novo molecular structure generation by learning from a textual representation of molecules. Here, we show that hybrid CLMs can additionally leverage the bioactivity information available for the training compounds. To computationally design ligands of phosphoinositide 3-kinase gamma (PI3Kγ), a collection of virtual molecules was created with a generative CLM. This virtual compound library was refined using a CLM-based classifier for bioactivity prediction. This second hybrid CLM was pretrained with patented molecular structures and fine-tuned with known PI3Kγ ligands. Several of the computer-generated molecular designs were commercially available, enabling fast prescreening and preliminary experimental validation. A new PI3Kγ ligand with sub-micromolar activity was identified, highlighting the method’s scaffold-hopping potential. Chemical synthesis and biochemical testing of two of the top-ranked de novo designed molecules and their derivatives corroborated the model’s ability to generate PI3Kγ ligands with medium to low nanomolar activity for hit-to-lead expansion. The most potent compounds led to pronounced inhibition of PI3K-dependent Akt phosphorylation in a medulloblastoma cell model, demonstrating efficacy of PI3Kγ ligands in PI3K/Akt pathway repression in human tumor cells. The results positively advocate hybrid CLMs for virtual compound screening and activity-focused molecular design.

IFITM3 Acts As a PIP3 Scaffold in TCR-PI3K Signaling

Background: Phosphatidylinositol 3-kinase (PI3K) signaling is an indispensable element of T-cell receptor (TCR), co-stimulatory receptor, and cytokine receptor activation in T cells and in the oncogenic signaling underlying T-cell malignancies. We recently identified interferon-inducible transmembrane protein 3 (IFITM3) as an unexpected but essential component of antigen receptor signaling and malignant transformation in B cells. In contrast to its previously characterized role as an interferon-stimulated gene which restricts viral entry through endosomal membranes, IFITM3 also directly binds to the lipid PIP3 - the product of PI3K - at the B cell surface and amplifies PI3K-mediated signal transduction. Whether IFITM3 serves an analogous function in other lymphocytes is currently unknown. Here, we demonstrate that IFITM3 is also required for robust PI3K-driven signaling and proliferation in primary and transformed human T cells. Results: We found that resting primary human T cells express low levels of IFITM3 but robustly upregulate IFITM3 protein following CD3/CD28 stimulation. To test whether IFITM3 participates in TCR signaling, we analyzed IFITM3 and PIP3 subcellular localization in Jurkat cells. Proximity ligation assays revealed that IFITM3 co-localizes with CD3δ at the cell surface, indicating recruitment of IFITM3 to TCR signaling complexes. Moreover, in the absence of IFITM3, a fluorescent reporter of PIP3 localization failed to redistribute from a diffuse cytosolic pattern to discrete foci at the cell surface upon TCR stimulation. We further explored whether these aberrant PIP3 dynamics were associated with broader defects in PIP3-dependent signaling axes important in T cells. In accord with prior findings in B cells, both Jurkat and primary T cells lacking IFITM3 expression exhibited diminished TCR signaling, including Ca2+ flux and PI3K-dependent AKT phosphorylation, and impaired cell proliferation relative to controls. These results demonstrate that T cells require IFITM3 for optimal antigen receptor- and PI3K-driven signaling. Mechanistically, IFITM3 is phosphorylated by Src family kinases at Y20, promoting retention at the plasma membrane, and specifically binds to PIP3, a highly negatively charged phospholipid, through basic residues in its conserved intracellular loop (CIL). We assessed the functional relevance of these residues in T cells by comparing the effects of wild-type and mutant IFITM3 variants. Overexpression of wild-type IFITM3 or a mutant with constitutive plasma membrane localization (Y20E) increased TCR-induced AKT phosphorylation, but a CIL mutant incapable of binding to PIP3 (K83A/K104A) lacked this activity and instead compromised cell viability and growth. Primary T cells expressing CIL-mutant IFITM3 were more apoptotic and demonstrated impaired cell cycle progression and proliferation, highlighting PIP3 binding as an essential feature of IFITM3 in T cell biology. Beyond normal T cells, PI3K signaling also enables the transformation of malignant T cells, with PI3K inhibitors emerging as promising agents in the treatment of peripheral T cell lymphomas (PTCLs). To determine whether IFITM3 contributes to the oncogenic signaling in PTCLs, we developed disease models incorporating both the expression of PTCL-derived oncogenes in T cells and the capability to assess candidate genes as functional players in pathogenesis. In a mouse bone marrow chimera system, conditional expression (via CD4-Cre) of a lentivirus-delivered oncogene (e.g., ITK-SYK) gave rise to circulating transgenic donor T cells and oncogene-dependent morbidity and mortality. In a humanized mouse system, a T cell-specific promoter controlled transgene expression upon transduction of CD34+ cord blood progenitors. In a separate approach, transduction of mature human T cells differentiated in vitro from CD34+ cells permitted a constitutive promoter. These results establish tractable systems to assess IFITM3 as a requisite factor for oncogenic signaling in PTCLs. Conclusion: We conclude that IFITM3 is essential for antigen receptor and oncogenic signaling not only in B cells but also in T cells. These findings identify a novel component of TCR signaling and suggest a shared mechanism for lymphocyte dependency on IFITM3 through scaffolding the lipid PIP3, a mechanism which can be leveraged to amplify oncogenic signaling in T cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity.

Infection triggers expansion and effector differentiation of T cells specific for microbial antigens in association with metabolic reprograming. We found that the glycolytic enzyme lactate dehydrogenase A (LDHA) is induced in CD8+ T effector cells through phosphoinositide 3-kinase (PI3K) signaling. In turn, ablation of LDHA inhibits PI3K-dependent phosphorylation of Akt and its transcription factor target Foxo1, causing defective antimicrobial immunity. LDHA deficiency cripples cellular redox control and diminishes adenosine triphosphate (ATP) production in effector T cells, resulting in attenuated PI3K signaling. Thus, nutrient metabolism and growth factor signaling are highly integrated processes, with glycolytic ATP serving as a rheostat to gauge PI3K-Akt-Foxo1 signaling in the control of T cell immunity. Such a bioenergetic mechanism for the regulation of signaling may explain the Warburg effect.

Anti-diabetic effect and mechanism of Kursi Wufarikun Ziyabit in L6 rat skeletal muscle cells

Kursi Wufarikun Ziyabit (KWZ) is a traditional prescription that used in folk tea drinking for its health care effect in treatment of type 2 diabetes mellitus (T2DM) in central Asia. However, the underlying mechanism of KWZ in T2DM has not been investigated extensively. This study designed to observe the effect of KWZ on glucose consumption and assess the molecular mechanism on associated proteins in insulin signaling and ER stress pathway in L6 rat skeletal muscle cells. The results showed that, KWZ exhibited proteins of PTP-1B and α-glycosidase inhibitory activity in vitro. No cytotoxicity of KWZ was found on L6 cell line. The best effect of glucose consumption of cells was shown at 6.25 μg/mL after KWZ treatment for 12 h. Expression of PTP-1B protein was inhibited by KWZ in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by KWZ. Moreover, the insulin-mediated induction of IRS-1 and GSK-3 were also activated by KWZ. Western blot results indicated that KWZ significantly improved the levels of ER stress proteins, which reduced the expression of GRP78, enhanced the expression of the PERK, eIF2α and XBP1s. The activation of PERK/eIF2α was likely consequence of GRP78 inhibition, and this might be beneficial for improving the stability of ER and alleviating insulin resistance. These results suggest that KWZ might be serving as the potential drug for the prevention and treatment of T2DM.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

Anti-diabetic effect of three new norditerpenoid alkaloids in vitro and potential mechanism via PI3K/Akt signaling pathway.

Diabetes is a metabolic disease with the characteristic of high blood glucose (hyperglycemia). In our previous study, we found that nigelladines A-C (compounds A-C), three norditerpenoid alkaloids from the seeds of Nigella glandulifera Freyn (Ranunculaceae) exhibited protein of tyrosine phosphatase 1B (PTP1B) inhibitory activity in vitro. In the present study, we further investigated their anti-diabetes activities in L6 moytubes and illuminated the mechanisms of action of compounds A-C. Several parameters of glucose metabolism such as glucose consumption, glycogen content and hexokinase activity were increased by compounds A-C. The results suggested that compounds A-C improved glucose metabolism through promoting synthesis of glycogen. Expression of PTP1B protein was inhibited by compounds A-C in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by compounds A-C and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the insulin-mediated induction of insulin receptor substrate-1 (IRS-1) and glycogen synthase kinase-3β (GSK-3β) were also suppressed by wortmannin. Western blot results indicated that compounds A-C-induced IRS-1/Akt activation was likely a consequence of PTP1B inhibition. Compounds A-C promoted glycogen synthesis through Akt-mediated GSK3 phosphorylation. Therefore, activation of PI3K/Akt insulin signaling pathway and suppression of PTP1B is the molecular mechanism that contributes to the anti-diabetic effect of compounds A-C in cellular models. The three alkaloids potentially serve as lead compounds for the development of antidiabetic drugs.

PI3K inhibitor LY294002, as opposed to wortmannin, enhances AKT phosphorylation in gemcitabine-resistant pancreatic cancer cells.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinaseB (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.

Nephrin, a transmembrane protein, is involved in pancreatic beta-cell survival signaling

Nephrin, a cell surface signaling receptor, regulates podocyte function in health and disease. We study the role of nephrin in β-cell survival signaling. We report that in mouse islet β-cells and the mouse pancreatic beta-cell line (βTC-6 cells) nephrin is associated and partly co-localized with PI3-kinase. Incubation of cells with functional anti-nephrin antibodies induced nephrin clustering at the plasma membrane, nephrin phosphorylation and recruitment of PI3-kinase to nephrin thus resulting in increased PI3K-dependent Akt phosphorylation and augmented phosphorylation/inhibition of pro-apoptotic Bad and FoxO. Nephrin silencing abolished Akt activation and increased susceptibility of cells to apoptosis. High glucose impaired nephrin signaling, increased nephrin internalization and up-regulated PKCα expression. Interestingly, a marked decrease in nephrin expression and phosphorylated Akt was observed in pancreatic islets of db/db lepr−/− diabetic mice. Our findings revealed that nephrin is involved in β-cell survival and suggest that glucose-induced changes in nephrin signaling may contribute to gradual pancreatic β-cell loss in type 2 diabetes.

Abstract LB-37: P-REX1 is a novel PTEN-interacting protein that activates PI3K signaling in breast cancer.

Abstract The mechanism(s) underlying regulation of the tumor suppressor phosphatase PTEN, which negatively regulates phosphatidylinositol 3-kinase (PI3K) signaling, remains unclear. The PI3K/AKT pathway regulates cancer cell growth, survival, and metabolism. Since PTEN is expressed and PI3K is hyperactivated in the majority of breast cancers, we hypothesized that PTEN is inhibited by protein interactions, thus derepressing PI3K signaling. A proteomic screen to identify novel PTEN-binding proteins in breast cancer cells revealed interaction with P-REX1, a known Rac guanine exchange factor (GEF). A separate proteomic experiment to identify PTEN/PI3K-regulated proteins showed that P-REX1 levels are decreased upon loss of PTEN and increased upon inhibition of PI3K. PTEN/P-REX1 interaction and the effects of pharmacological PI3K pathway inhibitors on P-REX1 levels were validated by immunoprecipitation/immunoblot analyses. In reverse-phase protein array analysis of lysates from 597 primary human breast tumors, levels of P-REX1 protein were positively correlated with PTEN protein, and inversely correlated with phospho-AKT-T308 (both p<0.005). Comparing diverse types of carcinomas (n=2,009 from International Genomics Consortium's expO) and cancer cell lines (n=807 from Cancer Cell Line Encyclopedia), PREX1 mRNA levels were highest in ER+ and HER2+ breast cancer. In another series of 1,293 epithelial tumors (from The Cancer Genome Atlas), PREX1 is amplified or mutated in 6.2% of cases, and in 5% of breast cancers. Since multiple genomic lesions that can activate the PI3K pathway are known to co-exist in cancer cells, we tested whether PREX1 lesions co-exist with other PI3K pathway-activating lesions. Among genes encoding proteins implicated in growth factor receptor/PI3K/PTEN signaling and phosphatidylinositol metabolism, we found a significant enrichment for PREX1 mutation/amplification in 54/79 (68%) genes across 1,523 carcinomas. Overexpression and RNAi knockdown experiments revealed that P-REX1 increases steady-state and insulin-like growth factor-1 (IGF-1)-induced PI3K-dependent AKT phosphorylation in ER+ breast cancer cells. Studies are underway to identify the mechanistic role of P-REX1 in PI3K/PTEN signaling, and to determine whether P-REX1 directly inhibits PTEN. Our findings suggest that 1) P-REX1 is an activator of the PI3K/AKT pathway, 2) expression and mutation patterns implicate PREX1 in PI3K activation, and 3) neutralizing P-REX1 effects on PTEN/PI3K may be a novel therapeutic approach to selectively abrogate PI3K signaling in ER+ and HER2+ breast cancers while sparing normal tissues. Citation Format: Lloye M. Dillon, Jennifer R. Bean, Justin M. Balko, W. Hayes McDonald, David B. Friedman, Ana M. Gonzalez-Angulo, Gordon B. Mills, Carlos L. Arteaga, Todd W. Miller. P-REX1 is a novel PTEN-interacting protein that activates PI3K signaling in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-37. doi:10.1158/1538-7445.AM2013-LB-37

Abstract P5-07-03: Gallotannins and Gallic acid From Mango Fruit (Mangifera Indica L) Suppress Breast Cancer Tumor Growth by Targeting Phosphatidylinositol3-kinase (PI3K)-Akt-NF-kB Pathway and Associated microRNAs

Abstract The cytotoxic and anti-inflammatory properties of polyphenolics have been demonstrated in numerous types of cancer and the objective of this research was to understand the post-transcriptional targets involved in anti-inflammatory and pro-apoptotic pathways in the breast cancer cell line BT474, treated with a polyphenolic extract (2.5–40µg/ml) rich in gallotannins and gallic acid from mango fruit in vitro and in athymic nude mice bearing BT474 cells as xenografts. Results show that cell proliferation as well as tumor size and weight in vivo was decreased by polyphenolics 60% and 70% in vitro and vivo, respectively. NF-kB protein and mRNA expression as well as activation were decreased in a dose-dependent manner (0.6 fold). The extract also inhibited PI3K-dependent phosphorylation of AKT and expression and this was accompanied by down-regulation of miRNA 126 of BT474 (2.25 fold) and in vivo. NFκB-dependent genes involved in apoptosis-inhibition, metastasis and angiogenesis, such as survivin, VCAM-1, and VEGF were also decreased, in vitro and in vivo. A microRNA-array was also used to investigate whether microRNAs with target regions within affected genes were affected by the polyphenolic extract and results show that in addition to miR-126, miR-let-7a and miR-let-7b among others were significantly increased by the extract. In summary the anti-carcinogenic and anti-inflammatory activity of mango polyphenolics in breast cancer cells were at least in part due to targeting miR-126 -Phosphatidylinositol 3-kinase (PI3K)-Akt-NF-kB which plays an important role in the proliferative/ inflammatory phenotype exhibited in this breast cancer cell line. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-03.

Cytotoxicity of pomegranate polyphenolics in breast cancer cells in vitro and vivo: potential role of miRNA-27a and miRNA-155 in cell survival and inflammation

Several studies have demonstrated that polyphenolics from pomegranate (Punica granatum L.) are potent inhibitors of cancer cell proliferation and induce apoptosis, cell cycle arrest, and also decrease inflammation in vitro and vivo. There is growing evidence that botanicals exert their cytotoxic and anti-inflammatory activities, at least in part, by decreasing specificity protein (Sp) transcription factors. These are overexpressed in breast tumors and regulate genes important for cancer cell survival and inflammation such as the p65 unit of NF-κB. Moreover, previous studies have shown that Pg extracts decrease inflammation in lung cancer cell lines by inhibiting phosphatidylinositol-3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibiting the activation of NF-kB in vivo. The objective of this study was to investigate the roles of miR-27a-ZBTB10-Sp and miR-155-SHIP-1-PI3K on the anti-inflammatory and cytotoxic activity of pomegranate extract. Pg extract (2.5-50μg/ml) inhibited growth of BT-474 and MDA-MB-231 cells but not the non-cancer MCF-10F and MCF-12F cells. Pg extract significantly decreased Sp1, Sp3, and Sp4 as well as miR-27a in BT474 and MDA-MB-231 cells and increased expression of the transcriptional repressor ZBTB10. A significant decrease in Sp proteins and Sp-regulated genes was also observed. Pg extract also induced SHIP-1 expression and this was accompanied by downregulation of miRNA-155 and inhibition of PI3K-dependent phosphorylation of AKT. Similar results were observed in tumors from nude mice bearing BT474 cells as xenografts and treated with Pg extract. The effects of antagomirs and knockdown of SHIP-1 by RNA interference confirmed that the anti-inflammatory and cytotoxic effects of Pg extract were partly due to the disruption of both miR-27a-ZBTB10 and miR-155-SHIP-1. In summary, the anticancer activities of Pg extract in breast cancer cells were due in part to targeting microRNAs155 and 27a. Both pathways play an important role in the proliferative/inflammatory phenotype exhibited by these cell lines.

Protein Kinase G and Focal Adhesion Kinase Converge on Src/Akt/β-Catenin Signaling Module in Osteoblast Mechanotransduction

Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.

Polyphenolic Extract from Mangifera indica is Cytotoxic in ER + BT474 Breast Cancer Cells and Targets the SHIP‐1‐Phosphatidylinositol3‐kinase (PI3K)‐Akt through microRNA‐155

The objective of this research was to understand the post‐transcriptional targets involved in anti‐inflammatory and pro‐apoptotic pathways in estrogen receptor positive (ER+) breast cancer cells BT474 treated with a polyphenolic extract (2.5–40μg/ml) rich in gallotannins and galic acid from Mangifera Indica L in vitro and in nude mice with BT474 cells as xenografts. Results show that cell proliferation as well as tumor size and weight in vivo was decreased by polyphenolics. NF‐kB protein and mRNA expression as well as activation were decreased in a dose‐dependent manner accompanied by increased expression of IKKα/β kinases, and impaired IκB degradation.The extract also inhibited PI3K dependent phosphorylation of AKT and increased in SHIP‐1 expression and this was accompanied by down‐regulation of miRNA 155 of BT474 (0.6 fold) and in vivo. Results were confirmed by using a mimic for miR‐155 which in part reversed the effects of the extracts. NFκB‐dependent genes involved in anti‐apoptosis, metastasis and angiogenesis, such as survivin, VCAM‐1, and VEGF were also decreased.In summary the anti‐carcinogenic and anti‐inflammatory activity of mango polyphenolics in breast cancer cells were at least in part due to targeting miR‐155‐SHIP‐1‐Phosphatidylinositol 3‐kinase (PI3K)‐Akt‐NF‐kB which plays an important role in the proliferative/inflammatory phenotype exhibited in this breast cancer cell line.Grant Funding Source : NIH

DA6034 promotes gastric epithelial cell migration and wound‐healing through the mTOR pathway

7-Carboxymethyloxy-3',4',5-trimethoxy flavone (DA6034), a synthetic derivative of eupatilin, has a protective effect on gastric mucosa against various ulcerogens, and is currently in the phase III clinical trial in the treatment of peptic ulcer disease. Cell migration and/or growth plays a role in the repair process of gastric ulcer, so this study investigated the effect of DA6034 on the movement and proliferation of gastric epithelial cells and its associated signaling pathway. The migration of AGS or SNU484 human gastric epithelial cells was shown by scratch-induced wound healing and transwell assays, and the proliferation of the cells was assessed by FACS and proliferation assays. Treatment of DA6034 promoted the migration of gastric epithelial cells in a concentration-dependent manner. DA6034 treatment facilitated the phosphorylation of mTOR that led to an increase in the activity of S6K1, indicating its ability to activate mTOR and S6K1. Rapamycin aborted the wound-healing effect of DA6034, which supported the role of mTOR activation in the wound-healing process. In addition, DA6034 treatment increased PI3K-dependent Akt phosphorylation, which was necessary for the enhancement of cell migration. DA6034, however, did not stimulate the proliferation of gastric epithelial cells, being consistent with no activation of ERK1/2 by the agent. DA6034 has the ability to heal scratch wounds, which may result from an increase in gastric epithelial cell migration as mediated by PI3K-Akt-dependent activation of mTOR and S6K1. Our finding may be of help in understanding the molecular basis of the anti-ulcer effect of DA6034.

Phosphorylation of Akt and ERK1/2 Is Required for VEGF-A/VEGFR2-Induced Proliferation and Migration of Lymphatic Endothelium

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.

Multiple protein kinase pathways mediate amplified IL-6 release by human lung fibroblasts co-exposed to nickel and TLR-2 agonist, MALP-2

Microbial stimuli and atmospheric particulate matter (PM) interact to amplify the release of inflammatory and immune-modulating cytokines. The basis of this interaction, however, is not known. Cultured human lung fibroblasts (HLF) were used to determine whether various protein kinase pathways were involved in the release of IL-6 following combined exposure to the PM-derived metal, Ni, and M. fermentans-derived macrophage-activating lipopeptide 2 (MALP-2), a toll-like receptor 2 agonist. Synergistic release of IL-6 by MALP-2 and NiSO 4 was obvious after 8 h of co-stimulation and correlated with a late phase accumulation of IL-6 mRNA. Ni and MALP-2, alone or together, all led to rapid and transient phosphorylations of ERK 1/2 and JNK/SAPK of similar magnitude. p38 phosphorylation, however, was observed only after prolonged treatment of cells with both stimuli together. A constitutive level of PI3K-dependent Akt phosphorylation remained unchanged by Ni and/or MALP-2 exposure. IL-6 induced by Ni/MALP-2 co-exposure was partially dependent on activity of HIF-1α and COX-2 as shown by targeted knockdown using siRNA. IL-6 release in response to Ni/MALP-2 was partially sensitive to pharmacological inhibition of ERK 1/2, p38, and PI3K signaling. The protein kinase inhibitors had minimal or no effects on Ni/MALP-2-induced accumulation of HIF-1α protein, however, COX-2 expression and, more markedly PGE 2 production, were suppressed by LY294002, SB203580, and U0126. Thus, Ni/MALP-2 interactions involve multiple protein kinase pathways (ERK 1/2, p38, and PI3K) that modulate events downstream from the early accumulation of HIF-1α to promote IL-6 gene expression directly or secondarily, through COX-2-derived autocrine products like PGE 2.

Pasteurella multocida toxin is a potent activator of anti-apoptotic signalling pathways

Toxigenic Pasteurella multocida strains produce a 146 kDa protein toxin (PMT) that due to its high mitogenic activity is thought to possess carcinogenic properties. PMT affects several signal transduction pathways related to cancer by constitutively stimulating heterotrimeric G proteins. Downstream of Galpha(q), Galpha(13) and Galpha(i), the toxin activates the small GTPase RhoA, MAP kinases and signal transducer and activator of transcription (STAT) proteins. PMT also stimulates Gbetagamma signalling and activates phosphoinositide 3-kinase (PI3K)-related pathways, which play a crucial role in proliferation and apoptosis. We show that treatment of HEK293 cells with PMT inhibits staurosporine-mediated apoptosis through PI3K-dependent phosphorylation of Akt and constitutive expression of Pim-1 kinase. Simultaneous activation of these survival kinases allows the activation of pro-survival pathways, such as GSK3beta, Mcl-1, Bcl-xL and Bcl-2, as well as the downregulation of apoptotic signals by Bax or Puma. Only the combined inhibition of Akt and Pim reverses the PMT-induced protection from staurosporine-induced apoptosis. In addition, we show that apoptosis induced by tumour chemotherapeutic agents is blocked by PMT in human cancer cell lines. Our data indicate that PMT is a highly potent anti-apoptotic agent, which supports the view of a carcinogenic potential of the toxin.

Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia

Insulin is neuroprotective following transient global brain ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear.Objective: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a.Methods: We utilized rats subjected to 10 minutes of global brain ischemia, with or without insulin administered at the onset of reperfusion.Results: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusion nor insulin induced any change in the phosphorylation or subcellular localization of FoxO3a, Bim or Bad. A single bolus of high-dose insulin reduced CA1 neuronal cell death and thus represents a potential therapeutic intervention for global brain ischemia.Discussion: These results reveal that proximal elements of a known cell-survival pathway are triggered by high-dose insulin during early reperfusion. Insulin induces robust PI3K-dependent phosphorylation of Akt by 30 minute reperfusion and results in improvement of hippocampal structure and function. However, the Akt substrates FoxO3a, Bim and Bad do not undergo corresponding changes in phosphorylation or subcellular localization in this model of global brain ischemia. The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified.

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization.

The recognition of bacterial lipoproteins by toll-like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2-dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (myeloid differentiation primary response gene 88) and Mal/TIRAP (MyD88-adapter-like/TIR-domain-containing adaptor protein). TLR2-dependent, but MyD88-independent responses have not been described yet. We report here on a novel-signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3-kinase (PI3K), p85alpha, in an inducible fashion. The Mal-p85alpha interaction drives PI3K-dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP(3)) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP(3) formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP(3) generation and macrophage polarization.