Activation Of Phosphoinositol 3-kinase Research Articles

PIK3R3 is a candidate regulator of platelet count in people of Bangladeshi ancestry

BackgroundBlood platelets are mediators of atherothrombotic disease and are regulated by complex sets of genes. Association studies in European ancestry populations have already detected informative platelet regulatory loci. Studies in other ancestries can potentially reveal new associations because of different allele frequencies, linkage structures, and variant effects. ObjectivesTo reveal new regulatory genes for platelet count (PLT). MethodsGenome-wide association studies (GWAS) were performed in 20,218 Bangladeshi and 9198 Pakistani individuals from the Genes & Health study. Loci significantly associated with PLT underwent fine-mapping to identify candidate genes. ResultsOf 1588 significantly associated variants (P < 5 × 10−8) at 20 loci in the Bangladeshi analysis, most replicated findings in prior transancestry GWAS and in the Pakistani analysis. However, the Bangladeshi locus defined by rs946528 (chr1:46019890) did not associate with PLT in the Pakistani analysis but was in the same linkage disequilibrium block (r2 ≥ 0.5) as PLT-associated variants in prior East Asian GWAS. The single independent association signal was refined to a 95% credible set of 343 variants spanning 8 coding genes. Functional annotation, mapping to megakaryocyte regulatory regions, and colocalization with blood expression quantitative trait loci identified the likely mediator of the PLT phenotype to be PIK3R3 encoding a regulator of phosphoinositol 3-kinase (PI3K). ConclusionAbnormal PI3K activity in the vessel wall is already implicated in the pathogenesis of atherothrombosis. Our identification of a new association between PIK3R3 and PLT provides further mechanistic insights into the contribution of the PI3K pathway to platelet biology.

T cell extrinsic mechanics explaining the T cell hyperactivation seen in Activated PI3K δ Syndrome

Abstract Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many surface receptors expressed by T cells and has been implicated in the control of CD4+ T cell helper differentiation, activation, and proliferation. APDS patients have increased PI3K activity leading to several immune manifestations with the some of the most common features being lymphoproliferation, respiratory tract infections, Th2-related pathologies, and impaired antibody responses. We previously analysed a CRISPR/Cas9 mouse model (Pik3cdE1020K). This showed intrinsic changes impacting on T cell function (increased Th2 cytokines and poor Tfh function) but also extrinsic factors driving a decrease in frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation. Here we investigated these extrinsic components further. We found that PI3K B cells are driving Tfh differentiation. We also explored the role of PI3K in maintaining Treg function and identity and determined if Treg dysregulation was driving T cell hyperactivation. These studies reveal an important role of PI3K in modulating T cell differentiation, activation, and function through both T cell intrinsic and extrinsic mechanisms. An improved understanding of T cell hyperactivation can provide insight into the lymphoproliferation that affects most APDS patients. Overall, these results reveal the potential to dissect the mechanisms underlying disease, providing grounds to define new drug targets and ultimately improve the clinical management of these patients.

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment

BackgroundOncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood.MethodsWe use an in...

T cell dysfunction in Activated PI3K δ Syndrome explained by cell intrinsic and extrinsic components

Abstract Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many T cell receptors and has been implicated in the control of CD4+ T cell helper differentiation, activation and proliferation. APDS patients have increased PI3K activity that leads to several immune manifestations including lymphoproliferation, Th2-related pathologies and impaired Ab responses. Analysis of a CRISPR/Cas9 mouse model (Pik3cdE1020K) showed a decreased frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation, as well as changes in cytokine expression and T cell help. Our studies investigated CD4+ T cell intrinsic and extrinsic alterations contributing to the dysregulation of the immune response. We identified cell intrinsic features resulting in increased IL-4 and IL-5, explaining the Th2 mediated disease in these patients. Additionally, cell intrinsic changes in Tfh generation and function were shown in the in vivo mouse model, contributing to an impaired ability to sustain GCs and alter isotype switching. Furthermore, additional studies have shown that the overactive T cells result from both intrinsic and extrinsic factors, with contributions from both dysregulated T cells and B cells exerting effects on T cell activation. Together, these studies reveals an important role of PI3K in modulating T cell differentiation and function through both T cell-intrinsic and extrinsic mechanisms. This provides new insights into the APDS pathophysiology with potential to improve the clinical management of these patients.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release.

Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5β1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5β1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.

Abstract 706: In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade

Abstract Known genomic correlates of response to immunotherapy do not perfectly predict clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. We hypothesize that somatic, cancer-associated mutations account for heterogeneity in spontaneous response to tumors and response to immunotherapy. We have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapy. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations. Tumor-bearing animals were treated with anti-PD-1. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A&amp;gt;G, increased in representation in anti-PD-1 treated tumors but not in immunodeficient animals, suggesting that activity of the mutant allele conferred selective growth advantage in the setting of tumor immunity. This mutation encodes a constitutively active mutant catalytic domain, PIK3CA H1047R. MC38 tumors homogeneously expressing H1047R and implanted into wild-type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene regressed. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry. Single-cell RNA-seq of tumor-infiltrating immune cells revealed a population of myeloid cells expressing known immune inhibitory proteins that differentially enriched in PIK3CA H1047R-expressing tumors. Our data suggest that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment. As such, activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy, and these studies provide a rationale for therapeutic combination trials of PI3K inhibition with checkpoint blockade and other myeloid-targeting immunotherapies. Citation Format: Natalie B. Collins, Brian C. Miller, Kevin Bi, Rose Al Abosy, Kathleen Yates, Yashaswi Shrestha, John Doench, Jesse Boehm, W Nicholas Haining. In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 706.

β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3-N-Glycan Complex.

Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N-glycans was found to be colocalized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc-branching formation on β4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N-glycans promoted β4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N-glycan-mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N-glycan-mediated tumorigenesis. These findings provide a novel role for N-glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3-N-glycan complex.Implications:N-Glycosylation of β4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3-N-glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR.

PI3K\u03b4 contributes to ER stress-associated asthma through ER-redox disturbances: the involvement of the RIDD\u2013RIG-I\u2013NF-\u03baB axis

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD–RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.

Abstract A16: Defining molecular mechanisms of resistance to tumor immunity

Abstract Recent success of immune checkpoint blockade solidifies the importance of the immune system in the defense against cancer. The clinical impact of the immune response is, however, very heterogeneous, with some patients achieving dramatic responses while others fail to respond. Known genomic correlates of response to immunotherapy are not perfectly predictive of clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. I hypothesize somatic acquired mutations of individual tumors may account for heterogeneity in the spontaneous response to tumors and response to immunotherapy. I have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to discover new mechanisms of immune resistance, define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapeutic strategies. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations from the Pan Cancer analysis within The Cancer Genome Atlas (TCGA). These cell lines form tumors when implanted subcutaneously in immunocompetent animals. Tumor-bearing animals were then subjected to immunotherapy with either therapeutic vaccination or checkpoint blockade with anti-PD-1. Barcode relative representation was measured by next generation sequencing at the time of tumor implantation and at the time of tumor harvest post-immunotherapy. Barcoded mutant ORFs that confer immune resistance increased in representation under immune pressure in comparison to untreated or immunodeficient animals. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A&amp;gt;G, consistently increased in representation in both B16 and MC38 immunotherapy-treated tumors. This mutation encodes a constitutively active mutant catalytic domain of PI3K, PIK3CA H1047R. MC38 tumors homogenously expressing PIK3CA H1047R and implanted into wild type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene or non-scoring mutation in PI3K regressed after treatment with anti-PD-1. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry of tumor infiltrating lymphocytes. I conclude that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion. As such, activation of PI3K may be useful as a predictor of resistance to immunotherapy. Importantly, these findings also provide a rationale for therapeutic combination trials of immune checkpoint blockade and PI3K inhibition. Citation Format: Natalie B. Collins, Robert Manguso, Hans Pope, W. Nicholas Haining. Defining molecular mechanisms of resistance to tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A16.

Cytokine-induced macropinocytosis in macrophages is regulated by 14-3-3ζ through its interaction with serine-phosphorylated coronin 1.

The induction of macropinocytosis in macrophages during an inflammatory response is important for clearance of pathogenic microbes as well as the generation of appropriate immune responses. Recent data suggest that cytokine stimulation of macrophages induces macropinocytosis through phosphorylation of the protein coronin 1, thereby redistributing coronin 1 from the cell cortex to the cytoplasm followed by the activation of phosphoinositol-3 (PI-3) kinase. However, how coronin 1 phosphorylation regulates these processes remains unclear. We here define an essential role for 14-3-3ζ in cytokine-induced and coronin-1-dependent macropinocytosis in macrophages. We found that, upon stimulation, phosphorylated coronin 1 transiently associated with 14-3-3ζ and receptor of activated C kinase 1 (RACK1). Importantly, downregulation of 14-3-3ζ, but not RACK1, prevented relocation of coronin 1, as well as the induction of PI-3 kinase activity and thereby macropinocytosis upon cytokine stimulation. Together these data define an essential role for 14-3-3ζ in the regulation of macropinocytosis in macrophages upon cytokine stimulation through modulation of the localization of coronin 1.

Upregulation of PI3K/AKT/mTOR, FABP5 and PPARβ/δ in Human Psoriasis and Imiquimod-induced Murine Psoriasiform Dermatitis Model.

Psoriasis is a common, and currently incurable chronic immune-mediated skin disease (1), with incompletely understood etiology partially due to the unavailability of animal models that can emulate major features of the disease. The phospho-inositol-3 kinase (PI3K)/protein kinase B(Akt) and mammalian target of rapamycin (mTOR) signaling which regulate metabolism, cell proliferation, survival, apoptosis, and frequently deregulated in diverse cancers (2, 3), has recently emerged as a clinically relevant target for inflammatory diseases including psoriasis (4, 5). This pathway is tightly regulated through feedback loops in part via 2 mTOR complexes, (C1 and 2), with linkage through Akt. PI3K activation triggers the phosphorylation of a 3-hydroxyl group, which then activates Akt kinase and promotes keratinocyte hyperproliferation and inhibit differentiation, as observed in psoriasis (4). Initial clinical data suggest that secondgeneration inhibitors (targeting both mTOR and PI3-K kinases) provide therapeutic benefit for psoriasis (6). Additionally, psoriasis-associated epidermal-type fatty acid-binding protein (FABP-5) involved in cytosolic fatty acid-transport, as well as the peroxisome proliferatoractivated receptor β/δ (PPAR β/δ), a ligand-activated transcription factor and member of the nuclear hormone receptors superfamily, are upregulated in psoriasis (7–9). The goal of this study was to assess whether mTOR, its upstream (PI3K and Akt) and downstream S6K1 (specifically S6Ser235/236 and S6Ser240/244) targets, as well as FABP5 and PPARβ/δ, are overexpressed in inflamed toll-like receptor-7/8 ligand imiquimod (IMQ)induced Balb/c mouse skin lesions and, whether their expressions simulate those observed in inflamed skin lesions from untreated psoriatic patients compared with matched controls.

Presynaptic Leptin Action Suppresses Excitatory Synaptic Transmission onto Ventral Tegmental Area Dopamine Neurons

Leptin is an adipocyte-derived cytokine that can act in the brain to suppress feeding and maintain energy homeostasis. Additionally, leptin activates its receptors in the ventral tegmental area (VTA), a critical site for neuroadaptations to rewarding stimuli, to modulate reward-seeking behaviors. Although leptin can decrease intrinsic excitability of dopamine neurons in the VTA, it is unknown whether leptin can modulate excitatory synaptic transmission in this brain region. Because plasticity of glutamatergic synapses onto VTA neurons can encode predictive information about reward, we hypothesized that leptin can decrease excitatory synaptic transmission onto dopamine neurons. Using whole-cell patch clamp electrophysiology in mouse midbrain slices, we tested the effects of leptin on evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) or N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs) onto VTA dopamine neurons. Leptin depressed both AMPAR and NMDAR EPSCs in VTA dopamine neurons and reduced frequency but not amplitude of mini EPSCs. Bath application of the MEK1/2 inhibitor U0126 did not alter leptin-induced suppression of AMPAR EPSCs. However, external, but not internal, application of the phosphoinositol 3-kinase (PI3K) or Janus kinase 2 (Jak2) tyrosine kinase inhibitors abolished leptin-induced synaptic depression. This study demonstrates that leptin causes a presynaptic inhibition of the probability of glutamate release onto VTA dopamine neurons. This synaptic inhibition requires Jak2 and PI3K activation. Leptin-induced weakening of synaptic strength onto dopamine cells may underlie its inhibitory effects on appetitive behavior for rewarding stimuli.

Insulin in the ventral tegmental area reduces hedonic feeding and suppresses dopamine concentration via increased reuptake

Mesolimbic dopamine (DA) signaling has been implicated in the incentive, reinforcing and motivational aspects of food intake. Insulin receptors are expressed on dopaminergic neurons of the ventral tegmental area (VTA), and insulin may act in the VTA to suppress feeding. However, the neural mechanisms underlying insulin effects in the VTA are poorly understood. Here, we measured the effects of insulin on evoked DA concentration in the VTA using fast-scan cyclic voltammetry. Insulin concentration-dependently reduced evoked somatodendritic DA in the VTA, requiring activation of phosphoinositol 3-kinase and mTOR signaling. Insulin depression of somatodendritic DA was abolished in the presence of a selective DA transporter (DAT) inhibitor, GBR 12909, as well as in VTA slices of DAT knockout mice, suggesting that insulin upregulated the number or function of DAT to reduce DA concentration. Finally, insulin administered to the VTA depressed sated feeding of sweetened high-fat food. Taken together, these results indicate that insulin depresses DA concentration in the VTA via increased reuptake of DA through DAT. Insulin-mediated decrease of DA in the VTA may suppress salience of food once satiety is reached.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

P38MAPK, ERK and PI3K Signaling Pathways Are Involved in C5a-Primed Neutrophils for ANCA-Mediated Activation

BackgroundThe complement system is one of the important contributing factors in the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the signaling pathways of C5a-mediated priming of human neutrophils for ANCA-induced neutrophil activation.Methodology/Principal FindingsThe effects of the p38 mitogen-activated protein kinase (p38MAPK) inhibitor (SB202190), extracellular signal-regulated kinase (ERK) inhibitor (PD98059), c-Jun N-terminal kinase (JNK) inhibitor (6o) and phosphoinositol 3-kinase (PI3K) inhibitor (LY294002) were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of membrane-bound PR3 (mPR3) on neutrophils. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 254.8±67.1, which decreased to 203.6±60.3, 204.4±36.7, 202.4±49.9 and 188±47.9 upon pre-incubation with SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.05), respectively. For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with above-mentioned inhibitors. The lactoferrin concentration increased in C5a-primed neutrophils induced by MPO or PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with above-mentioned three inhibitors. mPR3 expression increased from 923.3±182.4 in untreated cells to 1278.3±299.3 after C5a treatment and decreased to 1069.9±188.9, 1100±238.2, 1092.3±231.8 and 1053.9±200.3 by SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.01), respectively.Conclusions/SignificanceActivation of p38MAPK, ERK and PI3K are important steps in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA.

A novel role for insulin resistance in the connection between obesity and postmenopausal breast cancer

The past two decades have seen a drastic increase in obesity rates in Western societies and emerging countries. As such, it has become increasingly important to understand the molecular mechanisms by which obesity affects the risk of developing associated co-morbidities. The present study aimed at identifying the effect of insulin on breast cancer and breast epithelial cells, reflective of obesity-associated hyperinsulinaemia, as a molecular explanation for the increased risk of oestrogen receptor-negative postmenopausal breast cancer in obese women. Both of the examined breast cancer cell lines (MDA‑MB-231 and SK-BR-3) showed intact insulin signalling (insulin receptor phosphorylation and activation of phosphoinositol-3 kinase and mitogen-activated protein kinase cell signalling pathways), with MDA-MB-231 cells showing aberrantly amplified insulin signalling. Insulin did not induce a physiologically significant change in proliferation or apoptosis in either cell line. MDA-MB-231 cells showed decreased cell proliferation and increased S-phase population, while SK-BR-3 cells showed increased cyclinD and cyclinE gene expression and increased necrosis after insulin treatment. Hyperinsulinaemia may not be a universal mechanism by which obesity affects breast cancer progression. Normal breast epithelial cells (MCF-10A) showed intact insulin signalling, increased cell proliferation and reduced apoptosis after insulin treatment, suggesting cell growth and survival-promoting effects of insulin on these cells. Thus, hyperinsulinaemia may affect breast cancer aetiology rather than progression and this finding may provide a novel molecular mechanism for the role of insulin in the promotion of increased postmenopausal breast cancer risk in obese women.

Phosphoinositol 3-kinase, a novel target molecule for the inhibitory effects of juglone on TPA-induced cell transformation

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 µM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.

Review:Novel roles of nuclear angiotensin receptors and signaling mechanisms

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.

Impaired Keratinocyte Proliferative and Clonogenic Potential in Transgenic Mice Overexpressing 14-3-3σ in the Epidermis

The 14-3-3 protein family controls diverse biochemical processes through interaction with phosphorylated consensus sequences in protein targets. Its epithelial specific member, 14-3-3σ, also known as stratifin, is highly expressed in differentiated keratinocytes, and in vitro evidence indicates that 14-3-3σ downregulation leads to keratinocyte immortalization. To define the role of 14-3-3σ in skin homeostasis in vivo, we generated transgenic mice overexpressing 14-3-3σ in proliferating keratinocytes of the epidermis and hair follicle. Transgenic animals show decreased epidermal thickness and hair follicle density associated with reduced number of proliferating keratinocytes and decreased levels of keratins 14, 5, and 15. Primary keratinocytes isolated from transgenic mice manifest reduced proliferation and migration. Moreover, clonogenicity assessment and label-retaining analysis reveal a reduction in keratinocyte progenitor cell number in transgenic mice. Response to IGF-1 is strongly impaired in cultured transgenic keratinocytes compared with wild-type cells. Consistently, activation of phosphoinositol 3-kinase (PI3K), AKT, and Rac1, all IGF-1 downstream mediators, is reduced. Our results demonstrate that 14-3-3σ controls the in vivo epidermal proliferation-differentiation switch by reducing proliferative potential and forcing keratinocytes to exit the cell cycle, and that this effect associates with inhibition of the IGF-1 pathway.

PI3Kγ deletion reduces variability in the in vivo osteolytic response induced by orthopaedic wear particles

Orthopedic wear particles activate a number of intracellular signaling pathways associated with inflammation in macrophages and we have previously shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is one of the signal transduction pathways that mediates the in vitro activation of macrophages by orthopedic wear particles. Since PI3Kγ is primarily responsible for PI3K activity during inflammation, we hypothesized that PI3Kγ mediates particle-induced osteolysis in vivo. Our results do not strongly support the hypothesis that PI3Kγ regulates the overall amount of particle-induced osteolysis in the murine calvarial model. However, our results strongly support the conclusion that variability in the amount of particle-induced osteolysis between individual mice is reduced in the PI3Kγ(-/-) mice. These results suggest that PI3Kγ contributes to osteolysis to different degrees in individual mice and that the mice, and patients, that are most susceptible to osteolysis may be so, in part, due to an increased contribution from PI3Kγ.