An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1β signaling pathway

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1β and TNFα signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1β-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1β-dependent manner. Overexpression of FBXW5 inhibited IL-1β-induced activation of JNK/p38 MAPKs and NF-κB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1β stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1β signaling pathway.     

Induction of cardiac fibroblast lysyl oxidase by TGF-β1 requires PI3K/Akt, Smad3, and MAPK signaling

Lysyl oxidase (LOX) is a key extracellular enzyme responsible for the post-translational modification of collagens I and III to form mature fibrillar collagen. Increased expression of LOX is associated with fibrosis and cardiac dysfunction, yet little is known about the regulation of LOX in the heart. In this study, the cell signaling pathways responsible for the regulation of LOX expression by transforming growth factor (TGF)-β1 were assessed. Adult cardiac fibroblasts were isolated from male Sprague-Dawley rat hearts by enzymatic digestion. Fibroblasts were grown in DMEM with 10% FBS until approximately 80% confluent, growth arrested for 24h, and then treated with TGF-β1 (0-10 ng/ml), in the absence or presence of inhibitors of (1) PI3K (wortmannin), (2) Smad3 (SIS3), (3) p38-MAPK (PD169316), (4) JNK (SP600125) and (5) ERK1/2 (PD98059). TGF-β1 treatment significantly upregulated LOX mRNA and protein expression in cardiac fibroblasts, as well as activity in the cell-conditioned media. Concomitant increases in collagen types I and III, and bone morphogenic protein (BMP-1) expression were found in response to TGF-β1. The increase of LOX protein in response to TGF-β1 was prevented by inhibitors of PI3K, Smad3, p38-MAPK, JNK and ERK1/2. Blockade of PI3K also decreased TGF-β1 induced phosphorylation of Smad3, suggesting that the PI3K/Akt and Smad pathways may be integrated in TGF-β1 signaling. Further studies are warranted to address the regulation of LOX in the normal and diseased heart, and how this critical extracellular enzyme may be targeted for clinical benefit.     

TL1A induces the expression of TGF-β-inducible gene h3 (βig-h3) through PKC, PI3K, and ERK in THP-1 cells

βig-h3, an extracellular matrix protein involved in various biological processes including cellular growth, differentiation, adhesion, migration, and angiogenesis, has been shown to be elevated in various inflammatory processes. Death receptor 3 (DR3), a member of the TNF-receptor superfamily that is expressed on T cells and macrophages, is involved in the regulation of inflammatory processes through interaction with its cognate ligand, TNF-like ligand 1A (TL1A). In order to find out whether the TL1A-induced inflammatory activation of macrophages is associated with the up-regulation of βig-h3 expression, the human acute monocytic leukemia cell line (THP-1) was stimulated with either recombinant human TL1A- or DR3-specific monoclonal antibodies. Stimulation of DR3 up-regulated the intracellular levels as well as the secretion of βig-h3. Utilization of various inhibitors and Western blot analysis revealed that activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), phosphoinositide kinase-3 (PI3K), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is required for TL1A-induced βig-h3 expression. PKC appears to be the upstream regulator of PI3K since the presence of PKC inhibitor blocked the phosphorylation of AKT without affecting ERK phosphorylation. On the other hand, suppression of either PI3K or ERK activity resulted in the suppression of IκB phosphorylation. These findings indicate that TL1A can regulate the inflammatory processes through modulation of the βig-h3 expression through two separate pathways, one through PKC and PI3K and the other through ERK, which culminates at NF-κB activation.     

Gab2, via PI-3K, regulates ARF1 in FcεRI-mediated granule translocation and mast cell degranulation

Mast cells are major players in allergic responses. IgE-dependent activation through FcεR leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI3K or SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) binding sites. Examination of these mutants showed that both binding sites were required for the degranulation and anaphylaxis response but not for cytokine production or contact hypersensitivity. Furthermore, the PI3K, but not the SHP2, binding site was important for granule translocation during degranulation. We also identified a small GTPase, ADP-ribosylation factor (ARF)1, as the downstream target of PI3K that regulates granule translocation. FcεRI stimulation induced ARF1 activation, and this response was dependent on Fyn and the PI3K binding site of Gab2. ARF1 activity was required for FcεRI-mediated granule translocation. These data indicated that Fyn/Gab2/PI3K/ARF1-mediated signaling is specifically involved in granule translocation and the anaphylaxis response.     

Polycomb Repressive Complex 2 and H3K27me3 Cooperate with H3K9 Methylation To Maintain Heterochromatin Protein 1α at Chromatin

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.     

Calmodulin and IQGAP1 activation of PI3Kα and Akt in KRAS,HRAS and NRAS-driven cancers

Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP₃. CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS- and NRAS-driven cancers.     

四倍体刺槐无性系K2和K3的组织培养

1997年,北京林业大学从韩国引进了具有速生和饲料用途的刺槐(Robinia pseudoacacia L.)四倍体优良无性系,目前已在全国各省区试推广.与普通刺槐相比,四倍体刺槐具有叶大、速生等优点,且较普通刺槐有更强的适应性,耐干旱、贫瘠、烟尘及盐碱能力强,成林快,是水土保持、防风固沙及退耕还林的良好树种,可作为西北地区造林的先锋树种.     

The H3K27 demethylase UTX-1 regulates C. elegans lifespan in a germline-independent, insulin-dependent manner

Aging is accompanied by alterations in epigenetic marks that control chromatin states, including histone acetylation and methylation. Enzymes that reversibly affect histone marks associated with active chromatin have recently been found to regulate aging in Caenorhabditis elegans. However, relatively little is known about the importance for aging of histone marks associated with repressed chromatin. Here, we use a targeted RNAi screen in C. elegans to identify four histone demethylases that significantly regulate worm lifespan, UTX‐1, RBR‐2, LSD‐1, and T26A5.5. Interestingly, UTX‐1 belongs to a conserved family of histone demethylases specific for lysine 27 of histone H3 (H3K27me3), a mark associated with repressed chromatin. Both utx‐1 knockdown and heterozygous mutation of utx‐1 extend lifespan and increase the global levels of the H3K27me3 mark in worms. The H3K27me3 mark significantly drops in somatic cells during the normal aging process. UTX‐1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin‐FoxO signaling pathway. These findings identify the H3K27me3 histone demethylase UTX‐1 as a novel regulator of worm lifespan in somatic cells.     

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes

Background

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.

Methods

Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.

Results

The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.

General significance

This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.     

Synthesis of β-1, 3-Glucan and β-1, 3 and β-1, 4-Mixed Glucan from UDP-α-d-Glucose

A particulate enzyme preparation from Phaseolus aureus (mung bean) seedlings catalyzed the synthesis of a water insoluble β-1,3-glucan from UDP-α-d-glucose (UDPG) at high concentrations (0.4~20 mm) and an alkaline insoluble β-1,3 and β-1,4-mixed glucan from UDPG at a low concentration (8.5 µm).

Furthermore, the two kinds of β-glucan synthetases which were investigated with two reaction systems at high and low concentrations of UDPG had different properties in optimal pH, stability of enzyme activity, and metallic ion requirement.     

84K杨PagC3H3基因表达模式分析

木质素作为木材的主要组成成分,通常是由3种单体聚合而成,在其生物合成过程中,共有10个酶家族参与负责将苯丙胺酸转化为单体木质素,其中C3H是在对-香豆酰辅酶A（p-coumaroyl CoA）到咖啡酰辅酶A（caffeoyl CoA）的羟基化过程和G/S单体形成中的关键控制酶类,探究PagC3H3基因表达模式,对于进一步了解该基因功能具有重要意义。该研究通过定量PCR对PagC3H3基因的组织特异性表达进行分析;克隆得到了长度为2 035 bp的PagC3H3的启动子序列,预测含有多个顺式作用元件;同时,将获得的PagC3H3的启动子序列构建植物表达载体pBI121-PagC3H3pro：：GUS,进行拟南芥瞬时转化,结果显示PagC3H3基因在84K杨的根、中部茎节和基部茎节中的表达量较高;瞬时转化拟南芥,GUS染色表明：在下胚轴和根中GUS活性较强,由此推测PagC3H3基因在木质素合成过程中发挥作用。     

缺失plcg1基因引起成纤维细胞PI—3K信号上调

磷脂酶C－γ1（phospholipase C－γ1,PLC－γ1）与磷脂酰肌醇3－激酶（phosphatidylinositol-3 kinase,PI-3K）是生长因子调控细胞生长与增殖的两个重要信号中介。为探讨PLC－γ1在表皮生长因子（EGF）介导的细胞分裂信号中的代偿机制,用磷脂酶C（phospholipase C,PLC）特异性抑制剂U73122及PI－3K牧场划必抑制剂wortmannin处理剔除PLC－δ1基因plcg1(PLC-γ1^-/-）及野生型（PLC－γ1^ / )小鼠胚胎成纤维细胞,发现未经处理情况下两种细胞的克隆形成能力、细胞活力及EGF引起的DNA合成能力相似,且均可被U73122与wortmannin抑制,但与PLC－γ1^ / 细胞相比,PLC－γ1^－／－更依赖于PI－3K,而对PLC的依赖性却减小。Western印迹也表明EGF刺激后PI－3K的p85α亚单位酷氨酸磷酸化程度比野生型显著增高,PI－3K信号通路的激活出现上调,且PLC－γ1^-/-中无基近亲PLC－γ2的代偿表达。因此PLC－γ1^-/-中PLC－γ1的功能可能被PI－3K通路代偿,而PLC－γ2或其他PLC同工酶并不代偿其功能。结果表明EGF介导的信号能路的冗余性及PLC－γ1信号通路的可代偿性。     

Nucleosome compaction facilitates HP1γ binding to methylated H3K9

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1''s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg²⁺ or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.     

Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.