Mechanism of liponecrosis,a distinct mode of programmed cell death

An exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA) elicits “liponecrosis," a mode of programmed cell death (PCD) which differs from the currently known PCD subroutines. Here, we report the following mechanism for liponecrotic PCD. Exogenously added POA is incorporated into POA-containing phospholipids that then amass in the endoplasmic reticulum membrane, mitochondrial membranes and the plasma membrane. The buildup of the POA-containing phospholipids in the plasma membrane reduces the level of phosphatidylethanolamine in its extracellular leaflet, thereby increasing plasma membrane permeability for small molecules and committing yeast to liponecrotic PCD. The excessive accumulation of POA-containing phospholipids in mitochondrial membranes impairs mitochondrial functionality and causes the excessive production of reactive oxygen species in mitochondria. The resulting rise in cellular reactive oxygen species above a critical level contributes to the commitment of yeast to liponecrotic PCD by: (1) oxidatively damaging numerous cellular organelles, thereby triggering their massive macroautophagic degradation; and (2) oxidatively damaging various cellular proteins, thus impairing cellular proteostasis. Several cellular processes in yeast exposed to POA can protect cells from liponecrosis. They include: (1) POA oxidation in peroxisomes, which reduces the flow of POA into phospholipid synthesis pathways; (2) POA incorporation into neutral lipids, which prevents the excessive accumulation of POA-containing phospholipids in cellular membranes; (3) mitophagy, a selective macroautophagic degradation of dysfunctional mitochondria, which sustains a population of functional mitochondria needed for POA incorporation into neutral lipids; and (4) a degradation of damaged, dysfunctional and aggregated cytosolic proteins, which enables the maintenance of cellular proteostasis.     

Glutamate receptor signaling interplay modulates stress-sensitive mitogen-activated protein kinases and neuronal cell death

Glutamate receptors modulate multiple signaling pathways, several of which involve mitogen-activated protein (MAP) kinases, with subsequent physiological or pathological consequences. Here we report that stimulation of the N-methyl-D-aspartate (NMDA) receptor, using platelet-activating factor (PAF) as a messenger, activates MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase, in primary cultures of hippocampal neurons. Activation of the metabotropic glutamate receptor (mGluR) blocks this NMDA-signaling through PAF and MAP kinases, and the resultant cell death. Recombinant PAF-acetylhydrolase degrades PAF generated by NMDA-receptor activation; the hetrazepine BN50730 (an intracellular PAF receptor antagonist) also inhibits both NMDA-stimulated MAP kinases and neuronal cell death. The finding that the NMDA receptor-PAF-MAP kinase signaling pathway is attenuated by mGluR activation highlights the exquisite interplay between glutamate receptors in the decision making process between neuronal survival and death.     

In vivo functions of mitogen-activated protein kinases: conclusions from knock-in and knock-out mice

Multicellular organisms achieve intercellular communication by means of signalling molecules whose effect on the target cell is mediated by signal transduction pathways. Such pathways relay, amplify and integrate signals to elicit appropriate biological responses. Protein kinases form crucial intermediate components of numerous signalling pathways. One group of protein kinases, the mitogen-activated protein kinases (MAP kinases) are kinases involved in signalling pathways that respond primarily to mitogens and stress stimuli. In vitro studies revealed that the MAP kinases are implicated in several cellular processes, including cell division, differentiation, cell survival/apoptosis, gene expression, motility and metabolism. As such, dysfunction of specific MAP kinases is associated with diseases such as cancer and immunological disorders. However, the genuine in vivo functions of many MAP kinases remain elusive. Genetically modified mouse models deficient in a specific MAP kinase or expressing a constitutive active or a dominant negative variant of a particular MAP kinase offer valuable tools for elucidating the biological role of these protein kinases. In this review, we focus on the current status of MAP kinase knock-in and knock-out mouse models and their phenotypes. Moreover, examples of the application of MAP kinase transgenic mice for validating therapeutic properties of specific MAP kinase inhibitors, and for investigating the role of MAP kinase in pathogen-host interactions will be discussed.     

Protective Effects of mGluR5 Positive Modulators Against Traumatic Neuronal Injury Through PKC-Dependent Activation of MEK/ERK Pathway

Several previous studies utilizing selective pharmacological antagonists have demonstrated that type 5 metabotropic glutamate receptors (mGluR5) are potential therapeutic targets for the treatment of numerous disorders of the central nervous system, but the role of mGluR5 activation in traumatic brain injury (TBI) is not fully understood. Here in an in vitro TBI model, the mGluR5 agonist (RS)-2-chloro-5- hydroxyphenylglycine (CHPG) and the positive allosteric modulators 3-cyano-N-(1,3- diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) were used to investigate the neuroprotective potency of mGluR5 activation. Data showed that CHPG and CDPPB suppressed the increase of LDH release and caspase-3 activation induced by traumatic neuronal injury in a dose-dependent manner, and the salutary effects were also present when these compounds were added 1 h after injury. Western blot was used to examine the activation of three members of mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase (p38). CHPG and CDPPB enhanced the activation of ERK after traumatic neuronal injury, and PD98059 and U0126, two selective MEK/ERK inhibitors, partly revised the protective effects. Furthermore, we also investigated the role of protein kinase C (PKC) in CHPG and CDPPB-induced neuroprotection. With the pretreatment of chelerythrine chloride, a PKC inhibitor, the surpressing effects of CHPG and CDPPB on traumatic injury-evoked LDH release and caspase-3 activation were blocked. All of these findings extended the protective role of mGluR5 activation in an in vitro model of TBI and suggested that these protective effects might be mediated by the PKC-dependent activation of MEK/ERK pathway. These results may have important implications for the development of mGluR5 modulators to treat TBI.     

PDZ结构域蛋白CAL缓解鱼藤酮引起的MN9D细胞损伤

PDZ(PSD95-DLG1-ZO1) 域蛋白囊性纤维化跨膜电导调节相关配体(CAL)与Ⅰ组代谢型谷氨酸受体(mGluRⅠ)相互作用并且调节其下游信号.近年发现,mGluRⅠ与帕金森病密切相关.然而,CAL蛋白是否在帕金森病中发挥作用,目前尚未见报道.本文选用线粒体复合物Ⅰ 抑制剂鱼藤酮处理小鼠多巴胺能神经元细胞系MN9D,建立帕金森病细胞模型,探讨CAL在鱼藤酮刺激多巴胺能神经元过程中的作用及可能机制.结果显示,鱼藤酮引起CAL蛋白表达减少,过表达CAL蛋白可以部分缓解鱼藤酮引起的MN9D细胞活力下降、细胞凋亡及c-Jun N端激酶(JNK)磷酸化.加入JNK抑制剂SP600125,鱼藤酮引起的细胞活力下降同样有所恢复.提示CAL蛋白可能通过调节JNK信号通路保护多巴胺能神经元.     

Nitric oxide: promoter or suppressor of programmed cell death?

Nitric oxide (NO) is a short-lived gaseous free radical that predominantly functions as a messenger and effector molecule. It affects a variety of physiological processes, including programmed cell death (PCD) through cyclic guanosine monophosphate (cGMP)-dependent and-independent pathways. In this field, dominant discoveries are the diverse apoptosis networks in mammalian cells, which involve signals primarily via death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) that recruit caspases as effector molecules. In plants, PCD shares some similarities with animal cells, but NO is involved in PCD induction via interacting with pathways of phytohormones. NO has both promoting and suppressing effects on cell death, depending on a variety of factors, such as cell type, cellular redox status, and the flux and dose of local NO. In this article, we focus on how NO regulates the apoptotic signal cascade through protein S-nitrosylation and review the recent progress on mechanisms of PCD in both mammalian and plant cells.     

Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells

Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling.     

MAPK signaling in inflammation-associated cancer development

Mitogen-activated protein (MAP) kinases comprise a family of protein-serine/threonine kinases, which are highly conserved in protein structures from unicellular eukaryotic organisms to multicellular organisms, including mammals. These kinases, including ERKs, JNKs and p38s, are regulated by a phosphorelay cascade, with a prototype of three protein kinases that sequentially phosphorylate one another. MAPKs transduce extracellular signals into a variety of cellular processes, such as cell proliferation, survival, death, and differentiation. Consistent with their essential cellular functions, MAPKs have been shown to play critical roles in embryonic development, adult tissue homeostasis and various pathologies. In this review, we discuss recent findings that reveal the profound impact of these pathways on chronic inflammation and, particularly, inflammation-associated cancer development.     

Critical Temporal Modulation of Neuronal Programmed Cell Injury

1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD).2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 M) or SIN-1 (300 M) directly induced PCD.3. Neurons positive for PCD increased from 22 ± 3% (untreated) to 72 ± 3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72 ± 3% (NO only) to 25 ± 3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure.4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.     

Upregulation of the expression of prodeath serine/threonine protein kinase for programmed cell death by steroid hormone 20-hydroxyecdysone

Serine/threonine protein kinases phosphorylate protein substrates to initiate further cellular events. Different serine/threonine protein kinases have varied functions despite their highly conserved homology. We propose prodeath-S/TK, a prodeath serine/threonine protein kinase from the lepidopteran insect Helicoverpa armigera, promotes programmed cell death (PCD) during metamorphosis. Prodeath-S/TK is expressed in various tissues with a high expression level during molting and metamorphosis by 20-hydroxyecdysone (20E) induction. Prodeath-S/TK is localized in the larval midgut during metamorphosis. Prodeath-S/TK knockdown by injecting dsRNA into larval hemocoel suppresses the 20E-induced metamorphosis and PCD, as well as downregulates a set of genes involved in the PCD and 20E signaling pathway. 20E upregulates prodeath-S/TK expression through its nuclear receptor EcR-B1 and USP1. Prodeath-S/TK overexpression in the epidermal cell line leads to PCD with DNA fragmentation and the activation of caspases 3 and 7. Prodeath-S/TK plays role in the cytoplasm. The N-terminal and C-terminal sequences of prodeath-S/TK determine its subcellular location. These data indicate that prodeath-S/TK participates in PCD by regulating gene expression in the 20E signaling pathway.     

Targeting WNT, protein kinase B, and mitochondrial membrane integrity to foster cellular survival in the nervous system

Targeting essential cellular pathways that determine neuronal and vascular survival can foster a successful therapeutic platform for the treatment of a wide variety of degenerative disorders in the central nervous system. In particular, oxidative cellular injury can precipitate several nervous system disorders that may either be acute in nature, such as during cerebral ischemia, or more progressive and chronic, such as during Alzheimer disease. Apoptotic injury in the brain proceeds through two distinct pathways that ultimately result in the early externalization of membrane phosphatidylserine (PS) residues and the late induction of genomic DNA fragmentation. Degradation of DNA may acutely impact cellular survival, while the exposure of membrane PS residues can lead to microglial phagocytosis of viable cells, cellular inflammation, and thrombosis in the vascular system. Through either independent or common pathways, the Wingless/Wnt pathway and the serine-threonine kinase Akt serve central roles in the maintenance of cellular integrity and the prevention of the phagocytic disposal of cells "tagged" by PS exposure. By selectively governing the activity of specific downstream substrates that include GSK-3beta, Bad, and beta-catenin, Wnt and Akt serve to foster neuronal and vascular survival and block the induction of programmed cell death. Novel to Akt is its capacity to protect cells from phagocytosis through the direct modulation of membrane PS exposure. Intimately linked to the activation of Wnt signaling and Akt is the maintenance of mitochondrial membrane potential and the regulation of Bcl-xL, mitochondrial energy metabolism, and cytochrome c release that can lead to specific cysteine protease activation.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.     

Protein kinase signaling cascades in CNS trauma

Advances in our understanding of the signaling pathways and cellular functions regulated by protein kinase cascades have paved the way to study their role in the response of brain and spinal cord to traumatic injury. Mechanical forces imparted by trauma stimulate mitogen-activated protein kinases and protein kinase B/Akt as well as cause changes in the state of phosphorylation of glycogen synthase kinase-3beta. Extracellular ATP released by mechanical strain stimulates P2 purinergic receptors that are coupled to these protein kinase signaling pathways. These kinases regulate gene expression, cell survival, proliferation, differentiation, growth arrest, and apoptosis, thereby affecting cell fate, repair and plasticity after trauma. Elucidation of the molecular responses of protein kinase cascades to mechanical strain and the genes regulated by these signaling pathways may lead to therapeutic opportunities to minimize losses in motor skills and cognitive function caused by trauma to the central nervous system.     

细胞凋亡中的Caspase家族

保守的Asp特异性半胱氨酸蛋白酶(Caspase)家族是哺乳动物细胞中程序性死亡(PCD)的介导者和执行者. 原凋亡信号首先活化不同的Caspase启始因子,再由启始因子激活级联下游的Caspase效应分子,最终由效应分子特异地水解细胞中的一系列底物而导致细胞解体.Caspase家族是整个PCD过程的关键元件,它们通过与众多蛋白质(激活因子或抑制因子)的相互作用来调控细胞的生死存亡.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.