Inhibition of autophagy in mitotic animal cells

In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1–/– embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation .     

Augmentation of lipolysis in adipocytes from fed rats, but not from starved rats, by inhibition of rolipram-sensitive phosphodiesterase 4

The sensitivity of adipocytes to lipolytic agents is increased after starvation. In this study, we found that LY294002, an inhibitor of phosphatidylinositol-3 kinase (PI3K), in the concentration of more than 50 microM potentiates lipolysis induced by adenosine deaminase in adipocytes from fed rats (f-adipocytes), but not from starved rats (s-adipocytes). It also enhanced the sensitivity to lipolytic action of isoproterenol in f-adipocytes much more than s-adipocytes. The target of LY294002 may be an anti-lipolytic regulator expressed in response to food intake. Since another PI3K inhibitor, wortmannin, or a phosphodiesterase 3 (PDE3) inhibitor, cilostamide, failed to cause any specific effect to f-adipocytes, the PI3K-PDE3B pathway cannot be a target of LY294002. We found that LY294002 inhibits efficiently the cytoplasmic PDE activity of adipocytes. Rolipram, a specific inhibitor of PDE4, also inhibited the cytoplasmic PDE and caused a preferential increase of lipolysis in f-adipocytes. LY294002 blunted the actions of rolipram on lipolysis and the PDE activity. LY294002 accelerated protein kinase A activation. These data suggest that the rolipram-sensitive PDE4 is an anti-lipolytic enzyme expressed according to food intake. LY294002 may potentiate lipolysis through inhibition of the PDE4.     

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.     

中华按蚊含LY结构域的胰蛋白酶基因的分子特征及表达模式分析

【目的】在全基因组鉴定中华按蚊Anopheles sinensis 含LY结构域的胰蛋白酶(LY-trypsin)基因,探究其分子特征、表达模式以及系统发育关系。【方法】以NCBI数据库中冈比亚按蚊An. gambiae、埃及伊蚊Aedes aegypti、黑腹果蝇Drosophila melanogaster和致倦库蚊Culex quinquefasciatus胰蛋白酶家族基因的氨基酸序列为询问序列,通过本地Blast搜索鉴定中华按蚊基因组中胰蛋白酶基因,将中华按蚊LY-trypsin基因依据其结构域特征及系统发生关系进行命名LY-trypsin。运用生物信息学方法预测中华按蚊LY-trypsin基因的结构、在scaffold的定位、结构域、系统发育关系,及其在中华按蚊不同发育时期、成蚊不同组织和吸血前后雌成蚊中的表达模式。【结果】在中华按蚊全基因组上共鉴定得到27个中华按蚊LY-trypsin基因;在黑腹果蝇、冈比亚按蚊、致倦库蚊和埃及伊蚊基因组中未鉴定到LY-trypsin基因。中华按蚊27个LY-trypsin基因分别编码329~1 125个氨基酸,分子量在36.8~125.5 kD之间,等电点在4.73~8.94间。其中, 20个LY-trypsin具有信号肽,信号肽长度在10~62个氨基酸之间。这27个中华按蚊LY-trypsin基因的外显子数为1~5个,内含子长62~20 093 bp。这27个中华按蚊LY-trypsin基因被定位在11个scaffold上,其编码蛋白均有YWTD保守基序(LY基序);其中16个LY-trypsin基因所编码的氨基酸序列具有含丝氨酸、组氨酸和天冬氨酸催化三联体的活性位点。系统发育结果表明,27个中华按蚊LY-trypsin基因聚类为4个分支。在不同发育阶段,半数以上的中华按蚊LY-trypsin基因显示出相似的表达模式,在幼虫阶段均高水平表达;在中华按蚊不同成蚊组织中,LY-trypsin基因均有不同程度的表达;吸血前后雌蚊中仅有个别基因表达,并未发现表达特异性。【结论】本研究在中华按蚊基因组中首次鉴定出LY trypsin基因,并揭示了这些LY-trypsin基因的分子特征和表达模式,为LY-trypsin基因的进一步研究提供了信息框架。     

Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway

In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called ‘vascular niche’. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.     

Differential effects of γ-secretase and BACE1 inhibition on brain Aβ levels in vitro and in vivo

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of β-amyloid peptide (Aβ) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the Aβ producing enzymes γ-secretase and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the Aβ-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo , comparing it to the well characterized γ-secretase inhibitor LY450139. We sampled interstitial fluid Aβ from awake APPswe/PS1dE9 AD mice by in vivo Aβ microdialysis. In addition, we measured levels of endogenous brain Aβ extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar Aβ-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of Aβ in both in vivo models, we were unable to show any Aβ-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC₅₀ value several fold. In contrast, significant reduction of 40–50% of interstitial fluid Aβ and wildtype cortical Aβ was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased Aβ40 levels, compared with γ-secretase inhibition by LY450139.     

Autocrine activation of MAPK signaling mediates intrinsic tolerance to androgen deprivation in LY6D prostate cancer cells

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