Patched1 regulates hedgehog signaling at the primary cilium

Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. We investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localized to cilia and inhibited Smoothened (Smo) by preventing its accumulation within cilia. When Shh bound to Ptc1, Ptc1 left the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function.     

Deconstructing the hedgehog pathway in development and disease

The Hedgehog (Hh) family of secreted signaling proteins is a master regulator of cell fate determination in metazoans, contributing to both pattern formation during embryonic development and postembryonic tissue homeostasis. In a universally used mode of action, graded distribution of Hh protein induces differential cell fate in a dose-dependent manner in cells that receive Hh. Though much of this pathway has been elucidated from genetically based studies in model organisms, such as Drosophila and mice, the importance of Hh-mediated signaling in humans is clearly evident from malformations and a broad range of cancers that arise when the pathway is corrupted.     

alphaE-catenin controls cerebral cortical size by regulating the hedgehog signaling pathway

During development, cells monitor and adjust their rates of accumulation to produce organs of predetermined size. We show here that central nervous system-specific deletion of the essential adherens junction gene, alphaE-catenin, causes abnormal activation of the hedgehog pathway, resulting in shortening of the cell cycle, decreased apoptosis, and cortical hyperplasia. We propose that alphaE-catenin connects cell-density-dependent adherens junctions with the developmental hedgehog pathway and that this connection may provide a negative feedback loop controlling the size of developing cerebral cortex.     

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.     

Diverse psychotomimetics act through a common signaling pathway

Three distinct classes of drugs: dopaminergic agonists (such as D-amphetamine), serotonergic agonists (such as LSD), and glutamatergic antagonists (such as PCP) all induce psychotomimetic states in experimental animals that closely resemble schizophrenia symptoms in humans. Here we implicate a common signaling pathway in mediating these effects. In this pathway, dopamine- and an adenosine 3',5'-monophosphate (cAMP)-regulated phospho-protein of 32 kilodaltons (DARPP-32) is phosphorylated or dephosphorylated at three sites, in a pattern predicted to cause a synergistic inhibition of protein phosphatase-1 and concomitant regulation of its downstream effector proteins glycogen synthesis kinase-3 (GSK-3), cAMP response element-binding protein (CREB), and c-Fos. In mice with a genetic deletion of DARPP-32 or with point mutations in phosphorylation sites of DARPP-32, the effects of D-amphetamine, LSD, and PCP on two behavioral parameters-sensorimotor gating and repetitive movements-were strongly attenuated.     

Wnt-LRP6信号通路激活REST蛋白的表达并发挥神经保护作用

为检测以LRP6受体为激活开关的Wnt-β-catenin信号通路与PrP106-126毒性多肽引起的神经元损伤之间的相互关系,并研究该信号通路与神经保护性蛋白REST的相关性,从而进一步阐明Wnt-LRP6-REST对毒性多肽引起的神经元损伤的影响。利用Wnt信号通路的激活剂Licl、抑制剂DKK1或PrP106-126毒性多肽刺激原代神经元,通过免疫印迹检测神经保护性蛋白REST及Wnt信号通路下游相关蛋白的变化情况,通过激光共聚焦观察REST与Wnt信号通路正相关蛋白β-catenin的共定位情况。分别构建LRP6的过表达质粒pCMV-C-HALRP和干扰质粒pGPH1/GFP/Neo-LRP6-Rat shRNA-1和shRNA-2,将已经构建好的质粒转染进入原代神经元。通过免疫印迹检测PrP106-126毒性多肽对Wnt信号通路标志性蛋白(β-catenin及GSK3β)的影响,检测LRP6的过表达或干扰对REST或Wnt信号通路下游蛋白的影响。通过免疫荧光观察LRP6对由毒性多肽诱导的神经纤维损伤的影响,用流式细胞仪检测相应的细胞活力。由LRP6受体激活的Wnt-β-catenin信号通路在一定程度上正向调控神经保护性蛋白REST的表达,LRP6在信号调节的过程中起到了关键作用,并且LRP6对由PrP106-126毒性多肽引起的神经元损伤有缓解作用,LRP6的过表达增加了神经元细胞的活力,起到了神经保护作用。     

拟南芥泛素连接酶RING1A参与ABA信号途径的调控

拟南芥基因AtRING1A(Arabidopsis thaliana really interesting new gene 1A)编码的蛋白RING1A是多梳抑制复合体1(PRC1)中的核心组分.RING1A协同两个锌原子,执行泛素连接酶的功能,能募集靶蛋白并将其泛素化,从而使其被降解.为了研究AtRING1A在拟南芥中的功能和表达水平,ring1a突变体以及35S启动子的过表达植株被用来进行一系列分析.结果表明RING1A蛋白定位在细胞核中,并且在花中表达水平最高.AtRING1A过表达植株增强了对植物激素脱落酸(ABA)的耐受性,且在ABA胁迫下,AtRING1A可以促进ABA响应基因的表达.上述分析初步显示,AtRING1A响应植物激素ABA的信号,参与ABA诱导的细胞应答通路,并且可以增加拟南芥的ABA耐性.通过对AtRING1A基因的功能研究,为后续研究RING家族基因的功能及作用机制提供了依据.     

Patterning of the zebrafish retina by a wave of sonic hedgehog activity

The Drosophila retina is patterned by a morphogenetic wave driven by the Hedgehog signaling protein. Hedgehog, secreted by the first neurons, induces neuronal differentiation and hedgehog expression in nearby uncommitted cells, thereby propagating the wave. Evidence is presented here that the zebrafish Hedgehog homolog, Sonic Hedgehog, is also expressed in the first retinal neurons, and that Sonic Hedgehog drives a wave of neurogenesis across the retina, strikingly similar to the wave in Drosophila. The conservation of this patterning mechanism is unexpected, given the highly divergent structures of vertebrate and invertebrate eyes, and supports a common evolutionary origin of the animal visual system.     

β-伴大豆球蛋白通过p38/JNK MAPK信号通路引起IPEC-J2细胞损伤

采用细胞体外培养技术,研究不同浓度7S(β-伴大豆球蛋白)对IPEC-J2(猪小肠上皮细胞)的影响。实验分为6组,A(对照组)、B(1 mg·mL^-1 7S)、C(5 mg·mL^-1 7S)、D(10 mg·mL^-1 7S)、E(5 mg·mL^-1 7S+1 μmol·L^-1 SP600125)、F(5 mg·mL^-1 7S+1 μmol·L^-1 SB202190),24 h后,用CCK-8法检测细胞存活率,收集细胞用ELISA法检测NO、5-HT、IL-6和IL-10含量,用Western blot法检测p-JNK、p-p38、Bcl-2蛋白表达量,用PCR法检测Bad、Bax、Bcl-2、Bcl-x_L和Caspase-3 mRNA相对表达量。检测结果表明:C组和D组的IPEC-J2细胞活性极显著降低(P<0.01),E组和F组的细胞活性显著高于C组(P<0.05),说明7S促进炎性细胞因子NO、5-HT和IL-6的分泌,降低IL-10的分泌,添加抑制剂使细胞因子NO、5-HT和IL-6分泌减少,IL-10的分泌增多;同时7S促进p-JNK、p-p38蛋白表达,降低Bcl-2蛋白表达,添加抑制剂可抑制p-JNK、p-p38蛋白表达,使Bcl-2蛋白表达增高。Bcl-2/Bax mRNA比值随7S浓度增加而降低,Bax/Bcl-x_l比值随7S浓度增加而升高,Caspase-3 mRNA相对表达量随7S浓度增加而升高。添加抑制剂后Bcl-2/Bax比值增高,Bax/Bcl-x_l比值降低,Caspase-3 mRNA降低。结果表明,7S通过p38/JNK MAPK信号通路引起仔猪肠上皮细胞损伤。     

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.     

Autophagy and the nutritional signaling pathway

During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1) and VPS34 (which encodes a class III phosphatidylinositol (PtdIns) 3-kinase) complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs). Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin) and AMP-activated protein kinase (AMPK). AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.     

Illumination of the melanopsin signaling pathway

In mammals, a small population of intrinsically photosensitive retinal ganglion cells (ipRGCs) plays a key role in the regulation of nonvisual photic responses, such as behavioral responses to light, pineal melatonin synthesis, pupillary light reflex, and sleep latency. These ipRGCs also express melanopsin (Opn4), a putative opsin-family photopigment that has been shown to play a role in mediating these nonvisual photic responses. Melanopsin is required for the function of this inner retinal pathway, but its precise role in generating photic responses has not yet been determined. We found that expression of melanopsin in Xenopus oocytes results in light-dependent activation of membrane currents through the Galpha(q)/Galpha(11) G protein pathway, with an action spectrum closely matching that of melanopsin-expressing ipRGCs and of behavioral responses to light in mice lacking rods and cones. When coexpressed with arrestins, melanopsin could use all-trans-retinaldehyde as a chromophore, which suggests that it may function as a bireactive opsin. We also found that melanopsin could activate the cation channel TRPC3, a mammalian homolog of the Drosophila phototransduction channels TRP and TRPL. Melanopsin therefore signals more like an invertebrate opsin than like a classical vertebrate rod-and-cone opsin.     

斑马鱼中Wnt信号通过调控侧线基板形成影响侧线发育机制

鱼类侧线系统源自胚胎期的侧线基板,侧线基板的特化受到多种信号的综合作用。利用转基因斑马鱼hsp:wnt8a-egfp和hsp:dkk1-egfp,增强或抑制Wnt信号,通过YO-PRO-1染色和毛细胞计数,研究斑马鱼中Wnt信号对侧线系统发育的影响。热激诱导Dkk1过表达后,斑马鱼侧线系统的神经丘不能形成。碱性磷酸酶染色和整体原位杂交结果表明:Wnt信号是神经丘毛细胞前体细胞以及侧线基板标记基因Eya1和Six2b表达所必需的。综上结果:Wnt信号可能通过调控侧线基板形成影响斑马鱼的侧线发育。     

Regulators of cerebellar granule cell development act through specific signaling pathways

The proper development of the central nervous system depends upon a finely tuned balance between cell proliferation and programmed cell death (PCD). Although PCD was initially believed to depend solely on the inability of certain neurons to obtain access to a limited supply of trophic factors, it has become apparent that the local production of death signals is also critical. In this Viewpoint, we discuss several pathways implicated in the survival of cerebellar granule cells- both pathways that protect from apoptosis and pathways that promote apoptosis-and describe how these disparate pathways converge on the final common mediators of PCD. Information on other important pathways implicated in granule cell survival may be found in the Connections Maps.     

Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.     

不同类型日粮对泌乳奶牛乳腺组织胰岛素通路及JAK2-STAT信号通路影响

研究不同类型日粮对奶牛血清激素、乳腺激素受体及其下游相关基因影响.试验选用10头健康泌乳中期荷斯坦奶牛,随机分成2组,分别饲喂不同类型粗饲料日粮,根据粗饲料来源不同,分为单一秸秆型(CS组)与苜蓿、青贮型(MF组).试验预饲期2周,正饲期4周.于正饲期最后1d清晨采集乳静脉血液以及乳腺组织样本.正饲期最后3d记录产奶量,并按早晚4:6比例采集奶样,使用乳成分分析仪测定乳蛋白.采用ELISA(酶联免疫吸附试验)方法和放射性免疫法(RIA)测定血清胰岛素、瘦素、脂联素、胰岛素样生长因子-1、生长激素、催乳素和雌二醇等激素含量,采用qRT-PCR方法测定乳腺相关激素受体及胰岛素下游通路相关基因mRNA表达丰度.结果表明,MF组奶牛血清中胰岛素和脂联素含量显著高于CS组(P＜o.05),其他激素含量并没有显著差异.MF组奶牛乳腺组织中INSR、PRLR、CSF2RB受体,IRS1、IRS4、S6K、JAK2基因mRNA表达量较CS组显著上调(P＜0.05).结果表明,饲喂苜蓿青贮粗饲料型日粮能提高血清中胰岛素和脂联素浓度,并能激活奶牛乳腺胰岛素信号通路与JAK2-STAT通路中部分重要基因表达,从而提高奶牛对饲料利用、促进脂代谢与蛋白质合成,对提高奶牛乳蛋白产量有重要意义.     

铁皮石斛多糖影响Notch信号通路相关分子改善矽肺纤维化的研究

为观察铁皮石斛多糖(Dendrobium officinale polysaccharide, DOP)通过Notch信号通路抗矽肺纤维化的作用,将48只雄性昆明小鼠按随机数字表法分为对照组和造模组,造模组小鼠滴注50.0 mg·mL^-1 SiO₂混悬液200μL来制作矽肺纤维化小鼠动物模型。将造模成功后的小鼠随机分为模型组、吡非尼酮(pirfenidone, PFD)组以及DOP低、中、高剂量(125、250、500 mg·kg^-1)组,每组8只。PFD组和DOP低、中、高剂量组分别灌胃给药,对照组和模型组灌胃等量的生理盐水,连续给药28 d后检测相关指标。HE和Masson染色检测小鼠肺组织的病理形态学变化;免疫荧光定量PCR (qRT-PCR)检测肺组织中Notch1、Ⅲ型胶原纤维(Col-Ⅲ)、Hes1 mRNA表达;免疫组织化学法检测肺组织中Notch1、Col-Ⅲ、Hes1蛋白表达。Westren blot检测肺组织中Notch1、Col-Ⅲ、Hes1蛋白相对表达量的变化。结果表明：与对照组相比,模型组小鼠矽...