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Sample records for kinases regulate astrocyte

  1. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  2. Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation.

    PubMed

    Cheon, So Yeong; Cho, Kyoung Joo; Song, Juhyun; Kim, Gyung Whan

    2016-04-01

    Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke.

  3. S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase: IMPLICATIONS FOR ASTROCYTE DEVELOPMENT, ACTIVATION, AND TUMOR GROWTH.

    PubMed

    Brozzi, Flora; Arcuri, Cataldo; Giambanco, Ileana; Donato, Rosario

    2009-03-27

    S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.

  4. Extracellular regulated kinase 1/2 signaling is a critical regulator of interleukin-1β-mediated astrocyte tissue inhibitor of metalloproteinase-1 expression.

    PubMed

    Fields, Jerel; Cisneros, Irma E; Borgmann, Kathleen; Ghorpade, Anuja

    2013-01-01

    Astrocytes are essential for proper central nervous system (CNS) function and are intricately involved in neuroinflammation. Despite evidence that immune-activated astrocytes contribute to many CNS pathologies, little is known about the inflammatory pathways controlling gene expression. Our laboratory identified altered levels of tissue inhibitor of metalloproteinase (TIMP)-1 in brain lysates from human immunodeficiency virus (HIV)-1 infected patients, compared to age-matched controls, and interleukin (IL)-1β as a key regulator of astrocyte TIMP-1. Additionally, CCAAT enhancer binding protein (C/EBP)β levels are elevated in brain specimens from HIV-1 patients and the transcription factor contributes to astrocyte TIMP-1 expression. In this report we sought to identify key signaling pathways necessary for IL-1β-mediated astrocyte TIMP-1 expression and their interaction with C/EBPβ. Primary human astrocytes were cultured and treated with mitogen activated protein kinase-selective small molecule inhibitors, and IL-1β. TIMP-1 and C/EBPβ mRNA and protein expression were evaluated at 12 and 24 h post-treatment, respectively. TIMP-1 promoter-driven luciferase plasmids were used to evaluate TIMP-1 promoter activity in inhibitor-treated astrocytes. These data show that extracellular regulated kinase (ERK) 1/2-selective inhibitors block IL-1β-induced astrocyte TIMP-1 expression, but did not decrease C/EBPβ expression in parallel. The p38 kinase (p38K) inhibitors partially blocked both IL-1β-induced astrocyte TIMP-1 expression and C/EBPβ expression. The ERK1/2-selective inhibitor abrogated IL-1β-mediated increases in TIMP-1 promoter activity. Our data demonstrate that ERK1/2 activation is critical for IL-1β-mediated astrocyte TIMP-1 expression. ERK1/2-selective inhibition may elicit a compensatory response in the form of enhanced IL-1β-mediated astrocyte C/EBPβ expression, or, alternatively, ERK1/2 signaling may function to moderate IL-1β-mediated astrocyte C

  5. Trauma-induced expression of astrocytic thrombospondin-1 is regulated by P2 receptors coupled to protein kinase cascades.

    PubMed

    Tran, Minh Dinh; Furones-Alonso, Ofelia; Sanchez-Molano, Juliana; Bramlett, Helen Marie

    2012-08-22

    Thrombospondin-1 (TSP-1) is an extracellular matrix protein produced by astrocytes, which can promote synaptogenesis. The regulation of astrocytic TSP-1 involves extracellular ATP through the activation of P2Y receptors coupled to various protein kinase signaling pathways. However, not much is known about the mechanisms regulating TSP-1 expression in primary cortical astrocytes after a traumatic brain injury. Using an in-vitro model of central nervous system trauma that stimulates the release of ATP, we found that trauma-induced expression and release of TSP-1 involved purinergic signaling as both expression and release were significantly attenuated by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, a P2 receptor antagonist. Further antagonist studies with reactive blue 2 point to a role for P2Y4, as reactive blue 2 is a potent antagonist for rat P2Y4 receptors. In addition, the injury-induced expression of TSP-1 was significantly attenuated by the inhibition of extracellular signal-regulated kinase and p38/mitogen-activated protein kinase, whereas injury-induced release of TSP-1 was significantly blocked by the inhibition of extracellular signal-regulated kinase and Akt. Using an in-vivo model of a moderate parasagittal fluid-percussion brain injury, we found that TSP-1 levels were increased when compared with those in sham animals in the cortex, thalamus, and hippocampus. We conclude that TSP-1 expression after injury can be regulated by the activation of P2 receptors coupled with protein kinase signaling pathways and suggest that purinergic signaling, by regulating TSP expression, may play an important role in cell-matrix and cell-cell interactions such as those occurring during central nervous system repair.

  6. ICAM-1-induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lee, S J; Drabik, K; Van Wagoner, N J; Lee, S; Choi, C; Dong, Y; Benveniste, E N

    2000-10-15

    ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer's disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1beta and IL-1alpha protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1alpha and IL-1beta expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

  7. Extracellular signal-regulated kinase activation in spinal astrocytes and microglia contributes to cancer-induced bone pain in rats.

    PubMed

    Wang, X-W; Li, T-T; Zhao, J; Mao-Ying, Q-L; Zhang, H; Hu, S; Li, Q; Mi, W-L; Wu, G-C; Zhang, Y-Q; Wang, Y-Q

    2012-08-16

    Cancer pain, especially cancer-induced bone pain, affects the quality of life of cancer patients, and current treatments for this pain are limited. The present study demonstrates that spinal extracellular signal-regulated kinase (ERK) activation in glial cells plays a crucial role in cancer-induced bone pain. From day 4 to day 21 after the intra-tibia inoculation with Walker 256 mammary gland carcinoma cells, significant mechanical allodynia was observed as indicated by the decrease of mechanical withdrawal thresholds in the von Frey hair test. Intra-tibia inoculation with carcinoma cells induced a vast and persistent (>21 D) activation of ERK in the bilateral L2-L3 and L4-L5 spinal dorsal horn. The increased pERK1/2-immunoreactivity was observed in both Iba-1-expressing microglia and GFAP-expressing astrocytes but not in NeuN-expressing neurons. A single intrathecal injection of the selective MEK (ERK kinase) inhibitors PD98059 (10 μg) on day 12 and U0126 (1.25 and 3 μg) on day 14, attenuated the bilateral mechanical allodynia in the von Frey hair test. Altogether, our results suggest that ERK activation in spinal microglia and astrocytes is correlated with the onset of allodynia and is important for allodynia maintenance in the cancer pain model. This study indicated that inhibition of the ERK pathway may provide a new therapy for cancer-induced bone pain.

  8. Estradiol regulation of hypothalamic astrocyte adenosine 5'-monophosphate-activated protein kinase activity: role of hindbrain catecholamine signaling.

    PubMed

    Tamrakar, Pratistha; Briski, Karen P

    2015-01-01

    Recent work challenges the conventional notion that metabolic monitoring in the brain is the exclusive function of neurons. This study investigated the hypothesis that hypothalamic astrocytes express the ultra-sensitive energy gauge adenosine 5'-monophosphate-activated protein kinase (AMPK), and that the ovarian hormone estradiol (E) controls activation of this sensor by insulin-induced hypoglycemia (IIH). E- or oil (O)-implanted ovariectomized (OVX) rats were pretreated by caudal fourth ventricular administration of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) prior to sc insulin or vehicle injection. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial (VMH), arcuate (ARH), and paraventricular (PVH) nuclei and the lateral hypothalamic area (LHA), and pooled within each site for Western blot analysis of AMPK and phosphoAMPK (pAMPK) protein expression. In the VMH, baseline astrocyte AMPK and pAMPK levels were respectively increased or decreased in OVX+E versus OVX+O; these profiles did not differ between E and O rats in other hypothalamic loci. In E animals, astrocyte AMPK protein was reduced [VMH] or augmented [PVH; LHA] in response to either 6-OHDA or IIH. IIH increased astrocyte pAMPK expression in each structure in vehicle-, but not 6-OHDA-pretreated E rats. Results provide novel evidence for hypothalamic astrocyte AMPK expression and hindbrain catecholamine-dependent activation of this cell-specific sensor by hypoglycemia in the presence of estrogen. Further research is needed to determine the role of astrocyte AMPK in reactivity of these glia to metabolic imbalance and contribution to restoration of neuro-metabolic stability.

  9. Pharmacological Activation Gi/o Protein Increases Glial Cell Line-Derived Neurotrophic Factor Production through Fibroblast Growth Factor Receptor and Extracellular Signal-Regulated Kinase Pathway in Primary Cultured Rat Cortical Astrocytes.

    PubMed

    Hisaoka-Nakashima, Kazue; Matsumoto, Chie; Azuma, Honami; Taki, Sayaka; Takebayashi, Minoru; Nakata, Yoshihiro; Morioka, Norimitsu

    2017-01-01

    A significant reduction of glial cell line-derived neurotrophic factor (GDNF) has been identified in the pathophysiology of neurodegenerative and neuropsychiatric disorders. Thus, clarification of the mechanism of GDNF production, and modulating brain GDNF levels could be a novel therapeutic approach. A previous study demonstrated that antidepressant amitriptyline-induced GDNF production was significantly inhibited by pertussis toxin (PTX), a Gi/o protein inhibitor in astrocytes, the main source of GDNF in the brain. However, it is not known whether direct activation of Gi/o protein might induce GDNF expression, and what mechanisms might be involved after Gi/o protein activation. The current study investigated Gi/o protein-initiated GDNF production in rat cortical astrocytes using activators that directly activate Gi/o protein, mastoparan and compound48/80. Treatment of astrocytes with either mastoparan or compound48/80 increased GDNF mRNA expression at 3 and 6 h, and GDNF protein release at 24 h. Treatment of astrocyte with either mastoparan or compound48/80 increased brain-derived neurotrophic factor (BDNF) mRNA expression as well as GDNF. Mastoparan and compound48/80-induced GDNF mRNA expression were significantly inhibited by not only PTX, but also fibroblast growth factor receptor (FGFR) inhibitors, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. In fact, both FGFR substrate2α (FRS2α) and ERK phosphorylation were increased by treatment with either mastoparan or compound48/80, and these were significantly blocked by PTX. Thus, direct, receptor-independent Gi/o protein activation increases GDNF production through FGFR/ERK signaling pathway. The current results indicate a critical role of Gi/o signaling in the regulation of GDNF expression in astrocytes.

  10. Lateral regulation of synaptic transmission by astrocytes.

    PubMed

    Covelo, A; Araque, A

    2016-05-26

    Fifteen years ago the concept of the "tripartite synapse" was proposed to conceptualize the functional view that astrocytes are integral elements of synapses. The signaling exchange between astrocytes and neurons within the tripartite synapse results in the synaptic regulation of synaptic transmission and plasticity through an autocrine form of communication. However, recent evidence indicates that the astrocyte synaptic regulation is not restricted to the active tripartite synapse but can be manifested through astrocyte signaling at synapses relatively distant from active synapses, a process termed lateral astrocyte synaptic regulation. This phenomenon resembles the classical heterosynaptic modulation but is mechanistically different because it involves astrocytes and its properties critically depend on the morphological and functional features of astrocytes. Therefore, the functional concept of the tripartite synapse as a fundamental unit must be expanded to include the interaction between tripartite synapses. Through lateral synaptic regulation, astrocytes serve as an active processing bridge for synaptic interaction and crosstalk between synapses with no direct neuronal connectivity, supporting the idea that neural network function results from the coordinated activity of astrocytes and neurons.

  11. Curcumin exerts antinociceptive effects by inhibiting the activation of astrocytes in spinal dorsal horn and the intracellular extracellular signal-regulated kinase signaling pathway in rat model of chronic constriction injury.

    PubMed

    Ji, Feng-Tao; Liang, Jiang-Jun; Liu, Ling; Cao, Ming-Hui; Li, Feng

    2013-03-01

    Activation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain. Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor. However, whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown. This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain. Adult male Sprague-Dawley rats were randomly divided into three groups: a control (sham operated) group, and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin. Thermal and mechanical hyperalgesia thresholds were measured. The distribution and morphological changes of astrocytes were observed by immunofluorescence. Western blotting was used to detect changes in the expression of glial fibrillary acid protein (GFAP) and phosphorylated ERK. Injured rats showed obvious mechanical allodynia and thermal hyperalgesia. The number of GFAP-positive astrocytes, and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats. The soma of astrocytes also appeared hypertrophied in injured animals. Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal horn of injured compared with control rats. Curcumin reduced the injury-induced thermal and mechanical hyperalgesia, the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma, activation of GFAP and phosphorylation of ERK in the spinal dorsal horn. Curcumin can markedly alleviate nerve injury-induced neuropathic pain in rats. The analgesic effect of curcumin may be attributed to its inhibition of astrocyte hypertrophy

  12. Fisetin regulates astrocyte migration and proliferation in vitro

    PubMed Central

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-01-01

    Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro. PMID:28204814

  13. Astrocyte Regulation of Blood Flow in the Brain

    PubMed Central

    MacVicar, Brian A.; Newman, Eric A.

    2015-01-01

    Neuronal activity results in increased blood flow in the brain, a response named functional hyperemia. Astrocytes play an important role in mediating this response. Neurotransmitters released from active neurons evoke Ca2+ increases in astrocytes, leading to the release of vasoactive metabolites of arachidonic acid from astrocyte endfeet onto blood vessels. Synthesis of prostaglandin E2 (PGE2) and epoxyeicosatrienoic acids (EETs) dilate blood vessels, whereas 20-hydroxyeicosatetraenoic acid (20-HETE) constricts vessels. The release of K+ from astrocyte endfeet may also contribute to vasodilation. Oxygen modulates astrocyte regulation of blood flow. Under normoxic conditions, astrocytic Ca2+ signaling results in vasodilation, whereas under hyperoxic conditions, vasoconstriction is favored. Astrocytes also contribute to the generation of vascular tone. Tonic release of both 20-HETE and ATP from astrocytes constricts vascular smooth muscle cells, generating vessel tone. Under pathological conditions, including Alzheimer’s disease and diabetic retinopathy, disruption of normal astrocyte physiology can compromise the regulation of blood flow. PMID:25818565

  14. Astrocyte regulation of cerebral vascular tone

    PubMed Central

    Iddings, Jennifer A.

    2013-01-01

    Cerebral blood flow is controlled by two crucial processes, cerebral autoregulation (CA) and neurovascular coupling (NVC) or functional hyperemia. Whereas CA ensures constant blood flow over a wide range of systemic pressures, NVC ensures rapid spatial and temporal increases in cerebral blood flow in response to neuronal activation. The focus of this review is to discuss the cellular mechanisms by which astrocytes contribute to the regulation of vascular tone in terms of their participation in NVC and, to a lesser extent, CA. We discuss evidence for the various signaling modalities by which astrocytic activation leads to vasodilation and vasoconstriction of parenchymal arterioles. Moreover, we provide a rationale for the contribution of astrocytes to pressure-induced increases in vascular tone via the vasoconstrictor 20-HETE (a downstream metabolite of arachidonic acid). Along these lines, we highlight the importance of the transient receptor potential channel of the vanilloid family (TRPV4) as a key molecular determinant in the regulation of vascular tone in cerebral arterioles. Finally, we discuss current advances in the technical tools available to study NVC mechanisms in the brain as it relates to the participation of astrocytes. PMID:23792684

  15. Fisetin regulates astrocyte migration and proliferation in vitro.

    PubMed

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-04-01

    Fisetin (3,3',4',7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro.

  16. Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

    PubMed Central

    Aronica, Eleonora; Zurolo, Emanuele; Iyer, Anand; de Groot, Marjolein; Anink, Jasper; Carbonell, Caterina; van Vliet, Erwin A.; Baayen, Johannes C.; Boison, Detlev; Gorter, Jan A.

    2011-01-01

    Purpose Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphological feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathological hallmark of TLE. Methods Using immunocytochemistry and western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key findings In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up-regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3-4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of TLE patients. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several pro-inflammatory molecules (interleukin-1β and LPS). Significance These results suggest that dysregulation of ADK in astrocytes is a common pathological hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Whether this interaction also can play role in vivo needs to be further investigated. PMID:21635241

  17. Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells

    PubMed Central

    Cockbill, Louisa M. R.; Murk, Kai; Love, Seth; Hanley, Jonathan G.

    2015-01-01

    Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention. PMID:26466675

  18. Angiotensin III stimulates ERK1/2 mitogen-activated protein kinases and astrocyte growth in cultured rat astrocytes.

    PubMed

    Clark, Michelle A; Tran, Hsieu; Nguyen, Chinh

    2011-10-01

    Angiotensin (Ang) III is a biologically active metabolite of Ang II with similar effects and receptor binding properties as Ang II. Most Ang III studies delineate physiological effects of the peptide but, the intracellular pathways leading to the actions are unknown and are a focus of these studies. We investigated in cultured brainstem and cerebellum rat astrocytes whether Ang III stimulates ERK1/2 mitogen activated protein (MAP) kinases and astrocyte growth. Ang III significantly stimulated ERK1/2 MAP kinases in a dose- and time-dependent manner. The maximal stimulation occurred with 100 nM Ang III (2.8±0.3 and 2.3±0.1-fold over basal, in brainstem and cerebellum astrocytes, respectively). This stimulation occurred as early as 1 min, and was sustained for at least 15 min. Moreover, inhibition of the ERK1/2 MAP kinase pathway by 10 μM PD98059 attenuated Ang III-induced ERK1/2 phosphorylation. Ang III induction of ERK1/2 occurred via stimulation of the Ang AT(1) receptor since pretreatment with 10 μM Losartan, a selective AT(1) receptor blocker, prevented Ang III-induced ERK1/2 phosphorylation. The selective AT(2) Ang receptor blocker PD123319 was ineffective. Comparable to Ang II, Ang III also stimulated astrocyte growth in a concentration-dependent manner, an effect that occurred via activation of the AT(1) receptor as well. These findings suggest that Ang III has similar effects as Ang II in astrocytes since it rapidly stimulates the phosphorylation of the ERK1/2 MAP kinases and induces astrocyte proliferation through activation of the AT(1) receptor. These studies are important in establishing signaling pathways for Ang III and provide validation of the central role of Ang III.

  19. Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes.

    PubMed

    Bourguignon, Lilly Y W; Gilad, Eli; Peyrollier, Karine; Brightman, Amy; Swanson, Raymond A

    2007-05-01

    Both hyaluronan [HA, the major glycosaminoglycans in the extracellular matrix (ECM)] and CD44 (a primary HA receptor) are associated with astrocyte activation and tissue repair following central nervous system (CNS) injury. In this study we investigated the question of whether HA-CD44 interaction influences astrocyte signaling and migration. Our data indicated that HA binding to the cultured astrocytes stimulated Rac1 signaling and cytoskeleton-mediated migration. To determine the cellular and molecular basis of these events, we focused on PKN gamma, a Rac1-activated serine/threonine kinase in astrocytes. We determined that HA binding to astrocytes stimulated Rac1-dependent PKN gamma kinase activity which, in turn, up-regulated the phosphorylation of the cytoskeletal protein, cortactin, and attenuated the ability of cortactin to cross-link F-actin. Further analyses indicated that the N-terminal antiparallel coiled-coil (ACC) domains of PKN gamma interacted with Rac1, and transfection of astrocytes with PKN gamma-ACCcDNA inhibited PKN gamma activity. Over-expression of the PKN gamma-ACC domain also functions as a dominant-negative mutant to block HA/CD44-mediated PKN gamma activation of cortactin and astrocyte migration. Taken together, these findings strongly suggest that hyaluronan/CD44 interaction with Rac1-PKN gamma plays a pivotal role in cytoskeleton activation and astrocyte migration. These newly discovered HA/CD44-induced astrocyte function may provide important insight into novel therapeutic treatments for tissue repair following CNS injury.

  20. Astrocytes regulate cortical state switching in vivo

    PubMed Central

    Poskanzer, Kira E.; Yuste, Rafael

    2016-01-01

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation–dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  1. Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

    PubMed

    Lanciotti, Angela; Brignone, Maria Stefania; Visentin, Sergio; De Nuccio, Chiara; Catacuzzeno, Luigi; Mallozzi, Cinzia; Petrini, Stefania; Caramia, Martino; Veroni, Caterina; Minnone, Gaetana; Bernardo, Antonietta; Franciolini, Fabio; Pessia, Mauro; Bertini, Enrico; Petrucci, Tamara Corinna; Ambrosini, Elena

    2016-04-15

    Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

  2. Hypoxia inducible factor-2α regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors.

    PubMed

    Duan, Li-Juan; Takeda, Kotaro; Fong, Guo-Hua

    2014-01-01

    Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26(CreERT2), Tie2(Cre), and GFAP(Cre). Global Hif-2α disruption by Rosa26(CreERT2) led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2(Cre) had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP(Cre) significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP(+) mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2(+) and PDGFRα(+) astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other's development

  3. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-03-30

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.

  4. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  5. Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity

    PubMed Central

    Ormerod, Kiel G.

    2017-01-01

    Abstract Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability. PMID:28303263

  6. Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding.

    PubMed

    Kim, Jae Geun; Suyama, Shigetomo; Koch, Marco; Jin, Sungho; Argente-Arizon, Pilar; Argente, Jesús; Liu, Zhong-Wu; Zimmer, Marcelo R; Jeong, Jin Kwon; Szigeti-Buck, Klara; Gao, Yuanqing; Garcia-Caceres, Cristina; Yi, Chun-Xia; Salmaso, Natalina; Vaccarino, Flora M; Chowen, Julie; Diano, Sabrina; Dietrich, Marcelo O; Tschöp, Matthias H; Horvath, Tamas L

    2014-07-01

    We found that leptin receptors were expressed in hypothalamic astrocytes and that their conditional deletion led to altered glial morphology and synaptic inputs onto hypothalamic neurons involved in feeding control. Leptin-regulated feeding was diminished, whereas feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data reveal an active role of glial cells in hypothalamic synaptic remodeling and control of feeding by leptin.

  7. Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

    PubMed

    Diniz, Luan Pereira; Tortelli, Vanessa; Garcia, Matheus Nunes; Araújo, Ana Paula Bérgamo; Melo, Helen M; Silva, Gisele S Seixas da; Felice, Fernanda G De; Alves-Leon, Soniza Vieira; Souza, Jorge Marcondes de; Romão, Luciana Ferreira; Castro, Newton Gonçalves; Gomes, Flávia Carvalho Alcantara

    2014-12-01

    The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-β pathway. TGF-β1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. © 2014 Wiley Periodicals, Inc.

  8. Receptor-mediated regulation of neuropeptide gene expression in astrocytes.

    PubMed

    Schwartz, J P; Nishiyama, N; Wilson, D; Taniwaki, T

    1994-06-01

    One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.

  9. Astrocytes.

    ERIC Educational Resources Information Center

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  10. Astrocytes.

    ERIC Educational Resources Information Center

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  11. Phosphatidylinositol 4-phosphate 5-kinase α is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

    PubMed Central

    Kim, Bokyung; Yoon, Sarah; Kim, Yeon Joo; Liu, Tian; Woo, Joo Hong; Chwae, Yong-Joon; Joe, Eun-hye; Jou, Ilo

    2010-01-01

    In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kα is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kα mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kα knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1β and TNFα. PIP5Kα knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-κB and the degradation of IκB-α, indicating that PIP5Kα knockdown interfered with the ganglioside-activated NF-κB signaling. Together, these results suggest that PIP5Kα is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-κB activation in ganglioside-stimulated astrocytes. PMID:20720456

  12. Dexmedetomidine inhibits tumor necrosis factor-alpha and interleukin 6 in lipopolysaccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinases.

    PubMed

    Zhang, Xiaobao; Wang, Jun; Qian, Wenyi; Zhao, Jingjing; Sun, Li; Qian, Yanning; Xiao, Hang

    2014-06-01

    Astrocytes play an important role in immune regulation in the central nervous system (CNS). Dexmedetomidine (DEX) has been reported to exert anti-inflammatory effects on astrocytes stimulated by lipopolysaccharide (LPS) both in vitro and in vivo studies. However, the underlying molecular mechanisms remain poorly understood. This study was designed to evaluate the effects of DEX on tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) gene expressions in LPS-challenged astrocytes. Moreover, c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK) pathways in LPS-challenged astrocytes were also investigated. In the present study, astrocytes were stimulated with LPS in the absence and presence of various concentrations of DEX. With real-time PCR assay, we found that LPS significantly increased expressions of TNF-α and IL-6 in mRNA level; however, these effects could be attenuated by DEX. Furthermore, JNK pathway might be involved in LPS-induced astrocyte activation because JNK phosphorylation was significantly increased, and the inhibition of this pathway mediated by DEX as well as SP600125 (JNK inhibitor) decreased TNF-α and IL-6 expressions. Moreover, p38 MAPK was also activated by LPS; however, this pathway seemed to have not participated in DEX-mediated LPS-induced inflammation. These results, taken together, suggest that JNK rather than p38 MAPK signal pathway, provides the potential target for the therapeutic effects of DEX for neuronal inflammatory reactions.

  13. PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1.

    PubMed

    Lv, Youyou; Zhang, Bo; Zhai, Chunchun; Qiu, Jin; Zhang, Yue; Yao, Wenlong; Zhang, Chuanhan

    2015-12-01

    Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

  14. Interactions between Sirt1 and MAPKs regulate astrocyte activation induced by brain injury in vitro and in vivo.

    PubMed

    Li, Dan; Liu, Nan; Zhao, Hai-Hua; Zhang, Xu; Kawano, Hitoshi; Liu, Lu; Zhao, Liang; Li, Hong-Peng

    2017-03-29

    Astrocyte activation is a hallmark of traumatic brain injury resulting in neurological dysfunction or death for an overproduction of inflammatory cytokines and glial scar formation. Both the silent mating type information (Sirt1) expression and mitogen-activated protein kinase (MAPK) signal pathway activation represent a promising therapeutic target for several models of neurodegenerative diseases. We investigated the potential effects of Sirt1 upregulation and MAPK pathway pharmacological inhibition on astrocyte activation in vitro and in vivo. Moreover, we attempted to confirm the underlying interactions between Sirt1 and MAPK pathways in astrocyte activation after brain injury. The present study employs an interleukin-1β (IL-1β) stimulated primary cortical astrocyte model in vitro and a nigrostriatal pathway injury model in vivo to mimic the astrocyte activation induced by traumatic brain injury. The activation of GFAP, Sirt1, and MAPK pathways were detected by Western blot; astrocyte morphological hypertrophy was assessed using immunofluorescence staining; in order to explore the neuroprotective effect of regulation Sirt1 expression and MAPK pathway activation, the motor and neurological function tests were assessed after injury. GFAP level and morphological hypertrophy of astrocytes are elevated after injury in vitro or in vivo. Furthermore, the expressions of phosphorylated extracellular regulated protein kinases (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 activation (p-p38) are upregulated, but the Sirt1 expression is downregulated. Overexpression of Sirt1 significantly increases the p-ERK expression and reduces the p-JNK and p-p38 expressions. Inhibition of ERK, JNK, or p38 activation respectively with their inhibitors significantly elevated the Sirt1 expression and attenuated the astrocyte activation. Both the overproduction of Sirt1 and inhibition of ERK, JNK, or p38 activation can alleviate the astrocyte activation

  15. Glucocorticoid treatment of astrocytes results in temporally dynamic transcriptome regulation and astrocyte-enriched mRNA changes in vitro.

    PubMed

    Carter, Bradley S; Meng, Fan; Thompson, Robert C

    2012-12-18

    While general effects of glucocorticoids are well established, the specific cellular mechanisms by which these hormones exert tissue-dependent effects continue to be elaborated. Diseases that demonstrate altered glucocorticoid signaling have been associated with alterations in astrocytes, yet relatively little is known about the effects of glucocorticoids upon this cell type. We have analyzed mRNA expression patterns following glucocorticoid treatment of mouse primary astrocyte cultures. Microarray analysis of cortical astrocyte cultures treated with dexamethasone over an eight-point, 24 h time course identified 854 unique genes with ≥twofold change in mRNA expression at one or more time points. Clustering analysis associated subsets of these mRNA expression changes with gene ontology categories known to be impacted by glucocorticoids. Numerous mRNAs regulated by dexamethasone were also regulated by the natural ligand corticosterone; all of the mRNAs regulated ≥twofold by corticosterone were substantially attenuated by cotreatment with the glucocorticoid receptor antagonist RU486. Of the mRNAs demonstrating ≥twofold expression change in response to both glucocorticoids, 33 mRNAs were previously associated with glucocorticoid regulation, and 36 mRNAs were novel glucocorticoid targets. All genes tested by qPCR for glucocorticoid regulation in cortical astrocyte cultures were also regulated by glucocorticoids in hippocampal astrocyte cultures (18/18). Interestingly, a portion of glucocorticoid-regulated genes were astrocyte enriched; the percentage of astrocyte-enriched genes per total number of regulated genes was highest for the early time points and steadily decreased over the time course. These findings suggest that astrocytes in vitro may initially deploy cell type-specific patterns of mRNA regulatory responses to glucocorticoids and subsequently activate additional cell type-independent responses.

  16. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  17. Metabolic Interplay between Astrocytes and Neurons Regulates Endocannabinoid Action

    PubMed Central

    Viader, Andreu; Blankman, Jacqueline L.; Zhong, Peng; Liu, Xiaojie; Schlosburg, Joel E.; Joslyn, Christopher M.; Liu, Qing-Song; Tomarchio, Aaron J.; Lichtman, Aron H.; Selley, Dana E.; Sim-Selley, Laura J.; Cravatt, Benjamin F.

    2015-01-01

    Summary The endocannabinoid 2-arachidonoylglycerol (2-AG) is a retrograde lipid messenger that modulates synaptic function, neurophysiology, and behavior. 2-AG signaling is terminated by enzymatic hydrolysis—a reaction that is principally performed by monoacylglycerol lipase (MAGL). MAGL is broadly expressed throughout the nervous system, and the contributions of different brain cell types to regulating 2-AG activity have remained unclear. Here, we genetically dissect the cellular anatomy of MAGL-mediated 2-AG metabolism in the brain and show that neurons and astrocytes coordinately regulate 2-AG content and endocannabinoid-dependent forms of synaptic plasticity and behavior. We also find that astrocytic MAGL is mainly responsible for converting 2-AG to neuroinflammatory prostaglandins via a mechanism that may involve transcellular shuttling of lipid substrates. Astrocytic-neuronal interplay thus provides distributed oversight of 2-AG metabolism and function, and, through doing so, protects the nervous system from excessive CB1 receptor activation and promotes endocannabinoid crosstalk with other lipid transmitter systems. PMID:26212325

  18. Substrate-dependent regulation of ascorbate transport in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-02-26

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na{sup +}-dependent L-ascorbate transporter in the plasma membrane. The present study examined the effects of ascorbate deprivation and supplementation on the activity of the transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-({sup 14}C)ascorbate for 1 minute at 37C. They observed that the maximal uptake rate, V{sub max}, rapidly (<3 hours) increased when cultured cells were deprived of L-ascorbate. There was no change in the apparent affinity (K{sub m}) of the transport system for ascorbate. V{sub max} returned to normal following addition of L-ascorbate, but not D-isoascorbate, to the medium. The authors conclude that astrocytes adapt ascorbate transport rates to changes in substrate availability. Furthermore, the data suggest that the transport system located in the astroglial plasma membrane regulates intracellular ascorbate concentration, because changes in transport rate may compensate for regional differences and temporal fluctuations in extracellular ascorbate levels.

  19. Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes

    SciTech Connect

    Sakuma, S.; Hideyuki, S.; Akihiro, I.

    1995-07-01

    Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probable the result of an alternative splicing mechanism. The short isoform lacks a 37 amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures. 36 refs., 5 figs.

  20. Signaling molecules regulating phenotypic conversions of astrocytes and glial scar formation in damaged nerve tissues.

    PubMed

    Koyama, Yutaka

    2014-12-01

    Phenotypic conversion of astrocytes from resting to reactive (i.e., astrocytic activation) occurs in numerous brain disorders. Astrocytic activation in severely damaged brain regions often leads to glial scar formation. Because astrocytic activation and glial scar largely affect the vulnerability and tissue repair of damaged brain, numerous studies have been made to clarify mechanisms regulating the astrocytic phenotype. The phenotypic conversion is accompanied by the increased expression of intermediate filament proteins and the induction of hypertrophy in reactive astrocytes. Severe brain damage results in proliferation and migration of reactive astrocytes, which lead to glial scar formations at the injured areas. Gliogenesis from neural progenitors in the adult brain is also involved in astrocytic activation and glial scar formation. Recent studies have shown that increased expression of connexin 43, aquaporin 4, matrix metalloproteinase 9, and integrins alter the function of astrocytes. The transcription factors: STAT3, OLIG2, SMAD, NF-κB, and Sp1 have been suggested to play regulatory roles in astrocytic activation and glial scar formation. In this review, I discuss the roles of these key molecules regulating the pathophysiological functions of reactive astrocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Astrocytes negatively regulate neurogenesis through the Jagged1-mediated Notch pathway.

    PubMed

    Wilhelmsson, Ulrika; Faiz, Maryam; de Pablo, Yolanda; Sjöqvist, Marika; Andersson, Daniel; Widestrand, Asa; Potokar, Maja; Stenovec, Matjaž; Smith, Peter L P; Shinjyo, Noriko; Pekny, Tulen; Zorec, Robert; Ståhlberg, Anders; Pekna, Marcela; Sahlgren, Cecilia; Pekny, Milos

    2012-10-01

    Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin. Copyright © 2012 AlphaMed Press.

  2. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

    PubMed Central

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-01-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca2+-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca2+-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte – Syt XI – suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair. PMID:26450452

  3. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis.

    PubMed

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-04-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca(2+)-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca(2+)-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte--Syt XI--suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

  4. A pharmacological activator of AMP-activated protein kinase (AMPK) induces astrocyte stellation

    PubMed Central

    Favero, Carlita B; Mandell, James W

    2007-01-01

    AMP-activated protein kinase (AMPK) represents a key energy-sensing molecule in many cell types. Because astrocytes are key mediators of metabolic signaling in the brain, we have initiated studies on the expression and activation of AMPK in these cells. Treatment of cultured rat cortical astrocytes with a pharmacological AMPK activator, AICA-riboside (AICAR) resulted in a time- and concentration-dependent increase in phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a direct substrate. AICAR treatment also induced a transition from epithelioid to stellate morphology in a time- and concentration-dependent manner. As stellation is indicative of actin cytoskeletal reorganization, the formation of stress fibers and focal adhesions in response to AICAR was assessed. AICAR-induced stellation correlated with F-actin disassembly and focal adhesion dispersal. Furthermore, transient transfection of an activated RhoA construct prevented AICAR-induced stellation, indicating a mechanism upstream of RhoA. Use of pharmacological inhibitor compound C prevented AICAR-induced stellation demonstrating necessity of AMPK activity for the response. Our findings suggest that AMPK mediates morphological alterations of astrocytes in response to energy depletion. PMID:17706943

  5. Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

    PubMed

    Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

    2015-02-01

    Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes.

  6. NDRG2 phosphorylation provides negative feedback for SGK1-dependent regulation of a kainate receptor in astrocytes

    PubMed Central

    Matschke, Veronika; Theiss, Carsten; Hollmann, Michael; Schulze-Bahr, Eric; Lang, Florian; Seebohm, Guiscard; Strutz-Seebohm, Nathalie

    2015-01-01

    Glutamate receptors play an important role in the function of astrocytes. Among their tasks is the regulation of gliotransmission, gene expression and exocytosis of the tissue-type plasminogen activator (tPA), which has an enhancing effect on N-methyl-D-aspartate (NMDA) receptors and thus prevent over-excitation of neighboring neurons. The kainate receptor GluK2, which is expressed in neurons and astrocytes, is under tight regulation of the PI3-kinase SGK pathway as shown in neurons. SGK1 targets include N-myc downstream-regulated genes (NDRGs) 1 and 2 (NDRG1, NDRG2), proteins with elusive function. In the present study, we analyzed the effects of SGK1, NDRG1, and NDRG2 on GluK2 current amplitude and plasma membrane localization in astrocytes and heterologous expression. We demonstrate that NDRG1 and NDRG2 themselves have no effect on GluK2 current amplitudes in heterologous expressed ion channels. However, when NDRG2 is coexpressed with GluK2 and SGK1, the stimulating effect of SGK1 on GluK2 is suppressed both in heterologous expression and in astrocytes. Here, we reveal a new negative feedback mechanism, whereby GluK2 stimulation by SGK1 is regulated by parallel phosphorylation of NDRG2. This regulation of GluK2 by SGK1 and NDRG2 in astrocytes may play an important role in gliotransmission, modulation of gene expression and regulation of exocytosis of tPA. PMID:26500492

  7. AQP5 is differentially regulated in astrocytes during metabolic and traumatic injuries.

    PubMed

    Chai, Rui Chao; Jiang, Jiao Hua; Wong, Ann Yuen Kwan; Jiang, Feng; Gao, Kai; Vatcher, Greg; Hoi Yu, Albert Cheung

    2013-10-01

    Water movement plays vital roles in both physiological and pathological conditions in the brain. Astrocytes are responsible for regulating this water movement and are the major contributors to brain edema in pathological conditions. Aquaporins (AQPs) in astrocytes play critical roles in the regulation of water movement in the brain. AQP1, 3, 4, 5, 8, and 9 have been reported in the brain. Compared with AQP1, 4, and 9, AQP3, 5, and 8 are less studied. Among the lesser known AQPs, AQP5, which has multiple functions identified outside the central nervous system, is also indicated to be involved in hypoxia injury in astrocytes. In our study, AQP5 expression could be detected both in primary cultures of astrocytes and neurons, and AQP5 expression in astrocytes was confirmed in 1- to 4-week old primary cultures of astrocytes. AQP5 was localized on the cytoplasmic membrane and in the cytoplasm of astrocytes. AQP5 expression was downregulated during ischemia treatment and upregulated after scratch-wound injury, which was also confirmed in a middle cerebral artery occlusion model and a stab-wound injury model in vivo. The AQP5 increased after scratch injury was polarized to the migrating processes and cytoplasmic membrane of astrocytes in the leading edge of the scratch-wound, and AQP5 over-expression facilitated astrocyte process elongation after scratch injury. Taken together, these results indicate that AQP5 might be an important water channel in astrocytes that is differentially expressed during various brain injuries.

  8. Substrate regulation of ascorbate transport activity in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  9. Translational regulation mechanisms of aquaporin-4 supramolecular organization in astrocytes.

    PubMed

    Pisani, Francesco; Rossi, Andrea; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2011-12-01

    The two predominant isoforms of Aquaporin-4 (AQP4), AQP4-M23 and AQP4-M1, assemble in the plasma membrane to form supramolecular structures called Orthogonal Array of Particles (OAPs) whose dimension is tightly associated to the M1/M23 ratio. Here, we explore translational regulation contribution to M1/M23 expression in primary cultures of rat astrocytes, and analyze the role of M1 mRNA 5'untranslated region (5'UTR) in this mechanism. Using isoform-specific RNAi we found that in rat astrocytes primary cultures a large proportion of M23 protein derives from M1 mRNA translation. Furthermore, site-specific mutagenesis of the 5'UTR sequence of AQP4-M1 mRNA indicates that a multiple-site leaky scanning mechanism, an out-of-frame upstream ORF (uORF), and a reinitiation mechanism are able to modulate the M1/M23 ratio and consequently, OAPs formation. These mechanisms are likely to be shared by different species, including human, and they can also be assumed to play a role in those pathophysiological situations where the organization of AQP4 in supramolecular structures (OAPs) is involved. Finally, we report that, when transfected in Hela cells, the longer rat AQP4 isoform, called Mz, which is not present in human impairs OAPs formation.

  10. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  11. Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber.

    PubMed

    Min, Seul Ki; Jung, Sang Myung; Ju, Jung Hyeon; Kwon, Yeo Seon; Yoon, Gwang Heum; Shin, Hwa Sung

    2015-10-01

    Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.

  12. GPR30 regulates glutamate transporter GLT-1 expression in rat primary astrocytes.

    PubMed

    Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

    2012-08-03

    The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury.

  13. GPR30 Regulates Glutamate Transporter GLT-1 Expression in Rat Primary Astrocytes*

    PubMed Central

    Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

    2012-01-01

    The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury. PMID:22645130

  14. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    SciTech Connect

    Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  15. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

  16. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney

    2014-03-01

    Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

  17. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

    PubMed

    Hussaini, I M; Karns, L R; Vinton, G; Carpenter, J E; Redpath, G T; Sando, J J; VandenBerg, S R

    2000-07-21

    Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

  18. Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

    PubMed Central

    Nikolakopoulou, Angeliki M.; Koeppen, Jordan; Garcia, Michael; Leish, Joshua; Obenaus, Andre

    2016-01-01

    Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1loxP/yERT2-CreGFAP mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI. PMID:26928051

  19. Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury.

    PubMed

    Nikolakopoulou, Angeliki M; Koeppen, Jordan; Garcia, Michael; Leish, Joshua; Obenaus, Andre; Ethell, Iryna M

    2016-01-01

    Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1(loxP/y)ERT2-Cre(GFAP) mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI. © The Author(s) 2016.

  20. Acute and Chronic Mu Opioids Differentially Regulate Thrombospondins 1 and 2 Isoforms in Astrocytes

    PubMed Central

    2013-01-01

    Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the “reactive” state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms

  1. Acute and chronic mu opioids differentially regulate thrombospondins 1 and 2 isoforms in astrocytes.

    PubMed

    Phamduong, Ellen; Rathore, Maanjot K; Crews, Nicholas R; D'Angelo, Alexander S; Leinweber, Andrew L; Kappera, Pranay; Krenning, Thomas M; Rendell, Victoria R; Belcheva, Mariana M; Coscia, Carmine J

    2014-02-19

    Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but

  2. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  3. Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-I and insulin differs between malignant and non-neoplastic astrocytes

    SciTech Connect

    Chamaon, Kathrin; Kirches, Elmar; Kanakis, Dimitrios; Braeuninger, Stefan; Dietzmann, Knut; Mawrin, Christian . E-mail: christian.mawrin@medizin.uni-magdeburg.de

    2005-05-27

    The reasons for overexpression of the oncogene pituitary tumor transforming gene (PTTG) in tumors are still not fully understood. A possible influence of the insulin-like growth factor I (Igf-I) may be of interest, since enhanced Igf-I signalling was reported in various human tumors. We examined the influence of Igf-I and insulin on PTTG expression in human astrocytoma cells in comparison to proliferating non-neoplastic rat embryonal astrocytes. PTTG mRNA expression and protein levels were increased in malignant astrocytes treated with Igf-I or insulin, whereas in rat embryonic astrocytes PTTG expression and protein levels increased only when cells were exposed to Igf-I. Enhanced transcription did not occur after treatment with inhibitors of phosphoinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), blocking the two basic signalling pathways of Igf-I and insulin. In addition to this transcriptional regulation, both kinases directly bind to PTTG, suggesting a second regulatory route by phosphorylation. However, the interaction of endogenous PTTG with MAPK and PI3K, as well as PTTG phosphorylation were independent from Igf-I or insulin. The latter results were also found in human testis, which contains high PTTG levels as well as in nonneoplastic astrocytes. This suggest, that PI3K and MAPK signalling is involved in PTTG regulation not only in malignant astrocytomas but also in non-tumorous cells.

  4. Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca(2+) signaling.

    PubMed

    Muñoz, Manuel F; Puebla, Mariela; Figueroa, Xavier F

    2015-01-01

    Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca(2+) signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca(2+) signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca(2+) waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca(2+) entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca(2+)-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca(2+) signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca(2+) influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca(2+) signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in

  5. Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

    PubMed Central

    Muñoz, Manuel F.; Puebla, Mariela; Figueroa, Xavier F.

    2015-01-01

    Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process

  6. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

    PubMed

    Robertson, Chadia L; Srivastava, Jyoti; Siddiq, Ayesha; Gredler, Rachel; Emdad, Luni; Rajasekaran, Devaraja; Akiel, Maaged; Shen, Xue-Ning; Corwin, Frank; Sundaresan, Gobalakrishnan; Zweit, Jamal; Croniger, Colleen; Gao, Xiaoli; Ghosh, Shobha; Hylemon, Philip B; Subler, Mark A; Windle, Jolene J; Fisher, Paul B; Sarkar, Devanand

    2015-07-17

    Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis*

    PubMed Central

    Robertson, Chadia L.; Srivastava, Jyoti; Siddiq, Ayesha; Gredler, Rachel; Emdad, Luni; Rajasekaran, Devaraja; Akiel, Maaged; Shen, Xue-Ning; Corwin, Frank; Sundaresan, Gobalakrishnan; Zweit, Jamal; Croniger, Colleen; Gao, Xiaoli; Ghosh, Shobha; Hylemon, Philip B.; Subler, Mark A.; Windle, Jolene J.; Fisher, Paul B.; Sarkar, Devanand

    2015-01-01

    Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity. PMID:26070567

  8. Astrocytes Regulate GLP-1 Receptor-Mediated Effects on Energy Balance.

    PubMed

    Reiner, David J; Mietlicki-Baase, Elizabeth G; McGrath, Lauren E; Zimmer, Derek J; Bence, Kendra K; Sousa, Gregory L; Konanur, Vaibhav R; Krawczyk, Joanna; Burk, David H; Kanoski, Scott E; Hermann, Gerlinda E; Rogers, Richard C; Hayes, Matthew R

    2016-03-23

    Astrocytes are well established modulators of extracellular glutamate, but their direct influence on energy balance-relevant behaviors is largely understudied. As the anorectic effects of glucagon-like peptide-1 receptor (GLP-1R) agonists are partly mediated by central modulation of glutamatergic signaling, we tested the hypothesis that astrocytic GLP-1R signaling regulates energy balance in rats. Central or peripheral administration of a fluorophore-labeled GLP-1R agonist, exendin-4, localizes within astrocytes and neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus critical for energy balance control. This effect is mediated by GLP-1R, as the uptake of systemically administered fluorophore-tagged exendin-4 was blocked by central pretreatment with the competitive GLP-1R antagonist exendin-(9-39). Ex vivo analyses show prolonged exendin-4-induced activation (live cell calcium signaling) of NTS astrocytes and neurons; these effects are also attenuated by exendin-(9-39), indicating mediation by the GLP-1R. In vitro analyses show that the application of GLP-1R agonists increases cAMP levels in astrocytes. Immunohistochemical analyses reveal that endogenous GLP-1 axons form close synaptic apposition with NTS astrocytes. Finally, pharmacological inhibition of NTS astrocytes attenuates the anorectic and body weight-suppressive effects of intra-NTS GLP-1R activation. Collectively, data demonstrate a role for NTS astrocytic GLP-1R signaling in energy balance control. Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce food intake and are approved by the Food and Drug Administration for the treatment of obesity, but the cellular mechanisms underlying the anorectic effects of GLP-1 require further investigation. Astrocytes represent a major cellular population in the CNS that regulates neurotransmission, yet the role of astrocytes in mediating energy balance is largely unstudied. The current data provide novel evidence that astrocytes within the NTS

  9. Astrocyte regulation of sleep circuits: experimental and modeling perspectives

    PubMed Central

    Fellin, Tommaso; Ellenbogen, Jeffery M.; De Pittà, Maurizio; Ben-Jacob, Eshel; Halassa, Michael M.

    2012-01-01

    Integrated within neural circuits, astrocytes have recently been shown to modulate brain rhythms thought to mediate sleep function. Experimental evidence suggests that local impact of astrocytes on single synapses translates into global modulation of neuronal networks and behavior. We discuss these findings in the context of current conceptual models of sleep generation and function, each of which have historically focused on neural mechanisms. We highlight the implications and the challenges introduced by these results from a conceptual and computational perspective. We further provide modeling directions on how these data might extend our knowledge of astrocytic properties and sleep function. Given our evolving understanding of how local cellular activities during sleep lead to functional outcomes for the brain, further mechanistic and theoretical understanding of astrocytic contribution to these dynamics will undoubtedly be of great basic and translational benefit. PMID:22973222

  10. TNF-alpha down-regulates CXCR4 expression in primary murine astrocytes.

    PubMed

    Han, Y; Wang, J; He, T; Ransohoff, R M

    2001-01-05

    CXC chemokine receptor 4 (CXCR4) is a co-receptor for human immunodeficiency virus (HIV) infection and is believed to be involved in the pathogenesis of AIDS-associated neurologic disorders and brain tumors. The physiological roles of CXCR4 in developmental patterning of the nervous and hematopoietic system; gastrointestinal angiogenesis; and cardiac organogenesis were established by studies in gene-targeted mice. Studies on CXCR4 expression and regulation in neuroepithelial cells are fundamental for understanding its physiopathologic roles in the central nervous system (CNS). We show here that CXCR4 expression by primary mouse astrocytes is suppressed by exposure to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha caused a pronounced down-regulation of CXCR4 mRNA in a dose- and time-dependent manner. TNF-alpha-mediated decrease of CXCR4 mRNA accumulation resulted in decreased CXCR4 protein expression. As a result, the ability of stromal cell-derived factor-1alpha (SDF-1alpha) to induce activation of MAP kinases, Erk1/2 was impaired. The half life of CXCR4 mRNA in the presence and absence of TNF-alpha stimulation was comparable, suggesting that TNF-alpha down-regulated CXCR4 mRNA at the transcriptional level. These results suggest that TNF-alpha could modulate HIV and brain tumor pathogenesis and immune-mediated inflammation in the central nervous system (CNS) by regulation of CXCR4 expression.

  11. Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks

    PubMed Central

    Letellier, Mathieu; Park, Yun Kyung; Chater, Thomas E.; Chipman, Peter H.; Gautam, Sunita Ghimire; Oshima-Takago, Tomoko; Goda, Yukiko

    2016-01-01

    Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca2+ signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca2+ channels. Intracellular infusion of NMDARs or Ca2+-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites. PMID:27118849

  12. Modulating Astrocyte Transition after Stroke to Promote Brain Rescue and Functional Recovery: Emerging Targets Include Rho Kinase.

    PubMed

    Abeysinghe, Hima Charika S; Phillips, Ellie L; Chin-Cheng, Heung; Beart, Philip M; Roulston, Carli L

    2016-02-26

    Stroke is a common and serious condition, with few therapies. Whilst previous focus has been directed towards biochemical events within neurons, none have successfully prevented the progression of injury that occurs in the acute phase. New targeted treatments that promote recovery after stroke might be a better strategy and are desperately needed for the majority of stroke survivors. Cells comprising the neurovascular unit, including blood vessels and astrocytes, present an alternative target for supporting brain rescue and recovery in the late phase of stroke, since alteration in the unit also occurs in regions outside of the lesion. One of the major changes in the unit involves extensive morphological transition of astrocytes resulting in altered energy metabolism, decreased glutamate reuptake and recycling, and retraction of astrocyte end feed from both blood vessels and neurons. Whilst globally inhibiting transitional change in astrocytes after stroke is reported to result in further damage and functional loss, we discuss the available evidence to suggest that the transitional activation of astrocytes after stroke can be modulated for improved outcomes. In particular, we review the role of Rho-kinase (ROCK) in reactive gliosis and show that inhibiting ROCK after stroke results in reduced scar formation and improved functional recovery.

  13. NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement.

    PubMed

    Haruki, Hiroyo; Sano, Yasuteru; Shimizu, Fumitaka; Omoto, Masatoshi; Tasaki, Ayako; Oishi, Mariko; Koga, Michiaki; Saito, Kazuyuki; Takahashi, Toshiyuki; Nakada, Tsutomu; Kanda, Takashi

    2013-08-15

    Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for neuromyelitis optica (NMO). However, the molecular mechanism of NMO still remains unclear. The purpose of this study was to identify the possible humoral mechanisms responsible for the occurrence of astrocytic damage. Human primary astrocytes (AST) were immortalized by retroviral vectors harboring temperature-sensitive SV40 T antigen gene and AQP4 cDNA (M23), designated as hAST-AQP4. The effects of NMO sera on the content and localization of AQP4, including cytotoxicity and astrocytic morphology, were evaluated. In addition, this study examined whether the amount and localization of AQP4 protein in astrocytes were influenced by direct contact with the immortalized human brain microvascular endothelial cell line, TY09. NMO sera alone induced cytotoxicity and addition of complement had a more harmful effect on hAST-AQP4. NMO sera also decreased AQP4 mRNA and protein. NMO sera alone up-regulated TNFα and IL-6 in astrocytes and co-incubation with anti-TNFα and anti-IL-6 neutralizing antibodies blocked both the cytotoxicity and reduction of AQP4 in astrocytes. In the experiment using the in vitro BBB models, AQP4 protein mainly localized at the astrocytic membrane after co-culture with TY09, in contact with TY09. The future elucidation of factors that up-regulate AQP4 in astrocytes presumably released by blood brain barrier forming endothelial cells and that block the production of inflammatory cytokines may therefore lead to the development of a novel therapeutic strategy.

  14. A novel subtype of astrocytes expressing TRPV4 (transient receptor potential vanilloid 4) regulates neuronal excitability via release of gliotransmitters.

    PubMed

    Shibasaki, Koji; Ikenaka, Kazuhiro; Tamalu, Fuminobu; Tominaga, Makoto; Ishizaki, Yasuki

    2014-05-23

    Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca(2+) transients in astrocytes, and these Ca(2+) transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4(+) astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4(+) astrocytes. After activation, both TRPV4(+) and TRPV4(-) astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4(+) astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4(+) astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina.

    PubMed

    Gnanaguru, Gopalan; Bachay, Galina; Biswas, Saptarshi; Pinzón-Duarte, Germán; Hunter, Dale D; Brunken, William J

    2013-05-01

    Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin β2 and γ3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces β1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of β1 integrin. Finally, we show that laminins containing β2 and γ3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing β2 and γ3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

  16. Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

    PubMed Central

    Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

    2012-01-01

    Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

  17. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  18. Regulation of neuron-astrocyte metabolic coupling across the sleep-wake cycle.

    PubMed

    Petit, J-M; Magistretti, P J

    2016-05-26

    Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis.

  19. Tyrosine kinase inhibition reverses TDP-43 effects on synaptic protein expression, astrocytic function and amino acid dis-homeostasis.

    PubMed

    Heyburn, Lanier; Hebron, Michaeline L; Smith, Jacqueline; Winston, Charisse; Bechara, John; Li, Zhaoxia; Lonskaya, Irina; Burns, Mark P; Harris, Brent T; Moussa, Charbel E-H

    2016-11-01

    The trans-activating response of DNA/RNA-binding protein (TDP)-43 pathology is associated with many neurodegenerative diseases via unknown mechanisms. Here, we use a transgenic mouse model over-expressing human wild-type neuronal TDP-43 to study the effects of TDP-43 pathology on glutamate metabolism and synaptic function. We found that neuronal TDP-43 over-expression affects synaptic protein expression, including Synapsin I, and alters surrounding astrocytic function. TDP-43 over-expression is associated with an increase in glutamate and γ-amino butyric acid and reduction of glutamine and aspartate levels, indicating impairment of presynaptic terminal. TDP-43 also decreases tricarboxylic acid cycle metabolism and induces oxidative stress via lactate accumulation. Neuronal TDP-43 does not alter microglia activity or significantly changes systemic and brain inflammatory markers compared to control. We previously demonstrated that brain-penetrant tyrosine kinase inhibitors (TKIs), nilotinib and bosutinib, reduce TDP-43-induced cell death in transgenic mice. Here, we show that TKIs reverse the effects of TDP-43 on synaptic proteins, increase astrocytic function and restore glutamate and neurotransmitter balance in TDP-43 mice. Nilotinib, but not bosutinib, reverses mitochondrial impairment and oxidative metabolism. Taken together, these data suggest that TKIs can attenuate TDP-43 toxicity and improve synaptic and astrocytic function, independent of microglial or other inflammatory effects. In conclusion, our data demonstrate novel mechanisms of the effects of neuronal TDP-43 over-expression on synaptic protein expression and alteration of astrocytic function. © 2016 International Society for Neurochemistry.

  20. An atlas of human kinase regulation.

    PubMed

    Ochoa, David; Jonikas, Mindaugas; Lawrence, Robert T; El Debs, Bachir; Selkrig, Joel; Typas, Athanasios; Villén, Judit; Santos, Silvia Dm; Beltrao, Pedro

    2016-12-01

    The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

    PubMed Central

    Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney

    2014-01-01

    Blood brain barrier (BBB) breakdown is not only a consequence of, but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation. PMID:24583950

  2. δ-Opioid receptors up-regulate excitatory amino acid transporters in mouse astrocytes

    PubMed Central

    Liang, Jianfeng; Chao, Dongman; Sandhu, Harleen K; Yu, Yanbing; Zhang, Li; Balboni, Gianfranco; Kim, Dong H; Xia, Ying

    2014-01-01

    Background and Purpose Astrocytic excitatory amino acid transporters (EAATs) regulate extracellular glutamate concentrations and play a role in preventing neuroexcitotoxicity. As the δ-opioid receptor (DOP receptor) is neuroprotective against excitotoxic injury, we determined whether DOP receptor activation up-regulates EAAT expression and function. Experimental Approach We measured mRNA and protein expression of EAAT1, EAAT2 and EAAT3 in cultured mouse astrocytes exposed to a specific DOP receptor agonist (UFP-512) with or without a DOP receptor antagonist, DOP receptor siRNA or inhibitors of PKC, PKA, PI3K, p38, MAPK, MEK and ERK, and evaluated the function of EAATs by measuring glutamate uptake. Key Results Astrocytic DOP receptor mRNA and protein were suppressed by DOP receptor siRNA knockdown. DOP receptor activation increased mRNA and protein expression of EAAT1 and EAAT2, but not EAAT3, thereby enhancing glutamate uptake of astrocytes. DOP receptor-induced EAAT1 and EAAT2 expression was largely reversed by DOP receptor antagonist naltrindole or by DOP receptor siRNA knockdown, and suppressed by inhibitors of MEK, ERK and p38. DOP receptor-accelerated glutamate uptake was inhibited by EAAT blockers, DOP receptor siRNA knockdown or inhibitors of MEK, ERK or p38. In contrast, inhibitors of PKA, PKC or PI3K had no significant effect on DOP receptor-induced EAAT expression. Conclusions and Implications DOP receptor activation up-regulates astrocytic EAATs via MEK-ERK-p38 signalling, suggesting a critical role for DOP receptors in the regulation of astrocytic EAATs and protection against neuroexcitotoxicity. As decreased EAAT expression contributes to pathophysiology in many neurological diseases, including amyotrophic lateral sclerosis, our findings present a new platform for potential treatments of these diseases. PMID:25052197

  3. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 β

    PubMed Central

    2012-01-01

    Objective Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K+ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression. Methods We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64), comparing the expression in tumor patients with (n = 38) and without epilepsy (n = 26). Results Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1). Conclusions Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the

  4. Cell-type-specific expression and regulation of a c-fos-NGF fusion gene in neurons and astrocytes of transgenic mice.

    PubMed

    Onténiente, B; Horellou, P; Neveu, I; Makeh, I; Suzuki, F; Bourdet, C; Grimber, G; Colin, P; Brachet, P; Mallet, J

    1994-02-01

    A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or

  5. Spatial gradients in kinase cascade regulation.

    PubMed

    Kazmierczak, B; Lipniacki, T

    2010-11-01

    The spatiotemporal kinetics of proteins and other substrates regulate cell fate and signaling. In this study, we consider a reaction-diffusion model of interaction of membrane receptors with a two-step kinase cascade. The receptors activate the 'up-stream' kinase, which may diffuse over cell volume and activate the 'down-stream' kinase, which is also diffusing. Both kinase species and receptors are inactivated by uniformly distributed phosphatases. The positive feedback, key to the considered dynamics, arises since the up-stream kinase activates the receptors. Such a mutual interaction is characteristic for immune cell receptors. Based on the proposed model, we demonstrated that cell sensitivity (measured as a critical value of phosphatase activity at which cell maybe activated) increases with decreasing motility of receptor-interacting kinases and with increasing polarity of receptors distribution. These two effects are cooperating, the effect of receptors localisation close to one pole of the cell grows with the decreasing kinase diffusion and vanishes in the infinite diffusion limit. As the cell sensitivity increases with decreasing diffusion of receptor-interacting kinase, the overall activity of the down-stream kinase increases with its diffusion. In conclusion, the analysis of the proposed model shows that, for the fixed substrate interaction rates, spatial distribution of the surface receptors together with the motility of intracellular kinases control cell signalling and sensitivity to extracellular signals. The increase of the cell sensitivity can be achieved by (i) localisation of receptors in a small subdomain of the cell membrane, (ii) lowering the motility of receptor-interacting kinase, (iii) increasing the motility of down-stream kinases which distribute the signal over the whole cell.

  6. CD38 positively regulates postnatal development of astrocytes cell-autonomously and oligodendrocytes non-cell-autonomously.

    PubMed

    Hattori, Tsuyoshi; Kaji, Minoru; Ishii, Hiroshi; Jureepon, Roboon; Takarada-Iemata, Mika; Minh Ta, Hieu; Manh Le, Thuong; Konno, Ayumu; Hirai, Hirokazu; Shiraishi, Yoshitake; Ozaki, Noriyuki; Yamamoto, Yasuhiko; Okamoto, Hiroshi; Yokoyama, Shigeru; Higashida, Haruhiro; Kitao, Yasuko; Hori, Osamu

    2017-06-01

    Glial development is critical for the function of the central nervous system. CD38 is a multifunctional molecule with ADP-ribosyl cyclase activity. While critical roles of CD38 in the adult brain such as oxytocin release and social behavior have been reported, those in the developing brain remain largely unknown. Here we demonstrate that deletion of Cd38 leads to impaired development of astrocytes and oligodendrocytes in mice. CD38 is highly expressed in the developing brains between postnatal day 14 (P14) and day 28 (P28). In situ hybridization and FACS analysis revealed that CD38 is expressed predominantly in astrocytes in these periods. Analyses of the cortex of Cd38 knockout (Cd38(-/-) ) mice revealed delayed development of astrocytes and subsequently delayed differentiation of oligodendrocytes (OLs) at postnatal stages. In vitro experiments using primary OL cultures, mixed glial cultures, and astrocytic conditioned medium showed that astrocytic CD38 regulates the development of astrocytes in a cell-autonomous manner and the differentiation of OLs in a non-cell-autonomous manner. Further experiments revealed that connexin43 (Cx43) in astrocytes plays a promotive role for CD38-mediated OL differentiation. Finally, increased levels of NAD(+) , caused by CD38 deficiency, are likely to be responsible for the suppression of astrocytic Cx43 expression and OL differentiation. Our data indicate that CD38 is a positive regulator of astrocyte and OL development. © 2017 Wiley Periodicals, Inc.

  7. Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions.

    PubMed

    Dickens, Alex M; Tovar-Y-Romo, Luis B; Yoo, Seung-Wan; Trout, Amanda L; Bae, Mihyun; Kanmogne, Marlene; Megra, Bezawit; Williams, Dionna W; Witwer, Kennith W; Gacias, Mar; Tabatadze, Nino; Cole, Robert N; Casaccia, Patrizia; Berman, Joan W; Anthony, Daniel C; Haughey, Norman J

    2017-04-04

    Brain injury induces a peripheral acute cytokine response that directs the transmigration of leukocytes into the brain. Because this brain-to-peripheral immune communication affects patient recovery, understanding its regulation is important. Using a mouse model of inflammatory brain injury, we set out to find a soluble mediator for this phenomenon. We found that extracellular vesicles (EVs) shed from astrocytes in response to intracerebral injection of interleukin-1β (IL-1β) rapidly entered into peripheral circulation and promoted the transmigration of leukocytes through modulation of the peripheral acute cytokine response. Bioinformatic analysis of the protein and microRNA cargo of EVs identified peroxisome proliferator-activated receptor α (PPARα) as a primary molecular target of astrocyte-shed EVs. We confirmed in mice that astrocytic EVs promoted the transmigration of leukocytes into the brain by inhibiting PPARα, resulting in the increase of nuclear factor κB (NF-κB) activity that triggered the production of cytokines in liver. These findings expand our understanding of the mechanisms regulating communication between the brain and peripheral immune system and identify astrocytic EVs as a molecular regulator of the immunological response to inflammatory brain damage. Copyright © 2017, American Association for the Advancement of Science.

  8. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia

    PubMed Central

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W.; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-01-01

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. PMID:27739425

  9. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia.

    PubMed

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-10-14

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain.

  10. Astrocyte growth is regulated by neuropeptides through Tis 8 and basic fibroblast growth factor.

    PubMed Central

    Hu, R M; Levin, E R

    1994-01-01

    The important intracellular mechanisms of astrocyte growth are not well defined. Using an inhibitor of astrocyte proliferation, atrial natriuretic peptide (ANP), and the glial mitogen endothelin (ET-3), we sought a common pathway for growth regulation in these neural cells. In cultured fetal rat diencephalic astrocytes, ANP selectively and rapidly inhibited the Tis 8 immediate early gene and protein. After 4 h, ANP selectively inhibited the basic fibroblast growth factor (bFGF) gene and protein. ET-3 significantly stimulated both Tis 8 and bFGF mRNAs and protein, but also stimulated several other immediate early and growth factor/receptor genes. An antisense oligonucleotide to Tis 8 strongly prevented ET-stimulated thymidine incorporation, while the inhibitory action of ANP was enhanced. The Tis 8 antisense oligonucleotide also significantly reversed ET-stimulated bFGF transcription and enhanced the bFGF inhibition caused by ANP. In addition, an antisense oligonucleotide to bFGF significantly reversed the ET-stimulated thymidine incorporation and enhanced the ANP inhibition of DNA synthesis. The sequential modulation of Tis 8, followed by bFGF, provides a novel mechanism for both positive and negative regulation of astrocyte growth by endogenous neuropeptides. Images PMID:8163680

  11. Striatal Adenosine Signaling Regulates EAAT2 and Astrocytic AQP4 Expression and Alcohol Drinking in Mice

    PubMed Central

    Lee, Moonnoh R; Ruby, Christina L; Hinton, David J; Choi, Sun; Adams, Chelsea A; Young Kang, Na; Choi, Doo-Sup

    2013-01-01

    Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice. PMID:23032072

  12. Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

    PubMed

    Petit, Jean-Marie; Gyger, Joël; Burlet-Godinot, Sophie; Fiumelli, Hubert; Martin, Jean-Luc; Magistretti, Pierre J

    2013-10-01

    There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. 6-hour instrumental sleep deprivation (TSD). Animal sleep research laboratory. Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

  13. HCO3−-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase* ♦

    PubMed Central

    Hansen, Daniel Bloch; Garrido-Comas, Nestor; Salter, Mike; Fern, Robert

    2015-01-01

    The mechanisms of HCO3−-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H+ pump by Cl− removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3−-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS. PMID:25666621

  14. Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes.

    PubMed

    Benfenati, Valentina; Nicchia, Grazia Paola; Svelto, Maria; Rapisarda, Carmela; Frigeri, Antonio; Ferroni, Stefano

    2007-01-01

    In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

  15. Long-lasting change in brain dynamics induced by methamphetamine: enhancement of protein kinase C-dependent astrocytic response and behavioral sensitization.

    PubMed

    Narita, Minoru; Miyatake, Mayumi; Shibasaki, Masahiro; Tsuda, Makoto; Koizumi, Schuichi; Narita, Michiko; Yajima, Yoshinori; Inoue, Kazuhide; Suzuki, Tsutomu

    2005-06-01

    It is well known that long-term exposure to psychostimulants induces neuronal plasticity. Recently, accumulating evidence suggests that astrocytes may actively participate in synaptic plasticity. In this study, we found that in vitro treatment of cortical neuron/glia co-cultures with either methamphetamine (METH) or morphine (MRP) caused the activation of astrocytes via protein kinase C (PKC). Purified astrocytes were markedly activated by METH, whereas MRP had no such effect. METH, but not MRP, caused a long-lasting astrocytic activation in cortical neuron/glia co-cultures. Furthermore, MRP-induced behavioral sensitization to hyper-locomotion was reversed by 2 months of withdrawal following intermitted MRP administration, whereas behavioral sensitization to METH-induced hyper-locomotion was maintained even after 2 months of withdrawal. Consistent with this cell culture study, in vivo treatment with METH, which was associated with behavioral sensitization, caused a PKC-dependent astrocytic activation in the cingulate cortex and nucleus accumbens of mice. These findings provide direct evidence that METH induces a long-lasting astrocytic activation and behavioral sensitization through the stimulation of PKC in the rodent brain. In contrast, MRP produced a reversible activation of astrocytes via neuronal PKC and a reversibility of behavioral sensitization. This information can break through the definition of drugs of abuse and the misleading of concept that morphine produces a long-lasting neurotoxicity.

  16. Regulation of peroxisome proliferator-activated receptors (PPAR) α and -γ of rat brain astrocytes in the course of activation by toll-like receptor agonists.

    PubMed

    Chistyakov, Dmitry V; Aleshin, Stepan E; Astakhova, Alina A; Sergeeva, Marina G; Reiser, Georg

    2015-07-01

    Peroxisome proliferator-activated receptors (PPAR)-α and -γ in astrocytes play important roles in inflammatory brain pathologies. Understanding the regulation of both activity and expression levels of PPARs is an important neuroscience issue. Toll-like receptor (TLR) agonists are inflammatory stimuli that could modulate PPAR, but the mechanisms of their control in astrocytes are poorly understood. In the present study, we report that lipopolysaccharide, peptidoglycan, and flagellin, which are agonists of TLR4, TLR1/2, and TLR5, respectively, exert time- and nuclear factor kappa-light-chain-enhancer of activated B cells-dependent suppression of mRNA, protein and activity of PPARα and PPARγ. In naïve astrocytes, PPARα and PPARγ mRNA have short turnover time (half-life about 30 min for PPARα, 75 min for PPARγ) with a nearly two-fold stabilization after TLR-activation. p38 inhibition abolished TLR-induced stabilization. The levels of PPARα and PPARγ mRNA, and protein and DNA-binding activity could be modified using c-Jun N-terminal Kinase and p38 inhibitors. In addition, the expression levels of both PPARα and PPARγ isotypes were induced after inhibition of protein synthesis. This induction signifies participation of additional regulatory proteins with short life-time. They are p38-sensitive for PPARα and c-Jun N-terminal Kinase-sensitive for PPARγ. Thus, PPARα and PPARγ are regulated in astrocytes on mRNA and protein levels, mRNA stability, and DNA-binding activity during TLR-mediated responses. Astrocytes have the triad of PPARα, PPARβ/δ, and PPARγ in regulation of proinflammatory responses. Activation of Toll-like receptors (TLR) leads to PPARβ/δ overexpression, PPARα and PPARγ suppression via TLR/NF-κB pathway on mRNA, protein and activity levels. Mitogen-activated protein kinases (MAPK) p38 and JNK are involved in regulation of PPAR expression. p38 MAPK plays a special role in stabilization of PPAR mRNA. © 2015 International Society

  17. Astrocyte-derived phosphatidic acid promotes dendritic branching.

    PubMed

    Zhu, Yan-Bing; Gao, Weizhen; Zhang, Yongbo; Jia, Feng; Zhang, Hai-Long; Liu, Ying-Zi; Sun, Xue-Fang; Yin, Yuhua; Yin, Dong-Min

    2016-02-17

    Astrocytes play critical roles in neural circuit formation and function. Recent studies have revealed several secreted and contact-mediated signals from astrocytes which are essential for neurite outgrowth and synapse formation. However, the mechanisms underlying the regulation of dendritic branching by astrocytes remain elusive. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline, has been implicated in the regulation of neurite outgrowth. Here we showed that knockdown of PLD1 selectively in astrocytes reduced dendritic branching of neurons in neuron-glia mixed culture. Further studies from sandwich-like cocultures and astrocyte conditioned medium suggested that astrocyte PLD1 regulated dendritic branching through secreted signals. We later demonstrated that PA was the key mediator for astrocyte PLD1 to regulate dendritic branching. Moreover, PA itself was sufficient to promote dendritic branching of neurons. Lastly, we showed that PA could activate protein kinase A (PKA) in neurons and promote dendritic branching through PKA signaling. Taken together, our results demonstrate that astrocyte PLD1 and its lipid product PA are essential regulators of dendritic branching in neurons. These results may provide new insight into mechanisms underlying how astrocytes regulate dendrite growth of neurons.

  18. Insufficient Astrocyte-Derived Brain-Derived Neurotrophic Factor Contributes to Propofol-Induced Neuron Death Through Akt/Glycogen Synthase Kinase 3β/Mitochondrial Fission Pathway.

    PubMed

    Liu, Yanan; Yan, Yasheng; Inagaki, Yasuyoshi; Logan, Sarah; Bosnjak, Zeljko J; Bai, Xiaowen

    2017-07-01

    Growing animal evidence demonstrates that prolonged exposure to propofol during brain development induces widespread neuronal cell death, but there is little information on the role of astrocytes. Astrocytes can release neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), which can exert the protective effect on neurons in paracrine fashion. We hypothesize that during propofol anesthesia, BDNF released from developing astrocytes may not be sufficient to prevent propofol-induced neurotoxicity. Hippocampal astrocytes and neurons isolated from neonatal Sprague Dawley rats were exposed to propofol at a clinically relevant dose of 30 μM or dimethyl sulfoxide as control for 6 hours. Propofol-induced cell death was determined by propidium iodide (PI) staining in astrocyte-alone cultures, neuron-alone cultures, or cocultures containing either low or high density of astrocytes (1:9 or 1:1 ratio of astrocytes to neurons ratio [ANR], respectively). The astrocyte-conditioned medium was collected 12 hours after propofol exposure and measured by protein array assay. BDNF concentration in astrocyte-conditioned medium was quantified using enzyme-linked immunosorbent assay. Neuron-alone cultures were treated with BDNF, tyrosine receptor kinase B inhibitor cyclotraxin-B, glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021, or mitochondrial fission inhibitor Mdivi-1 before propofol exposure. Western blot was performed for quantification of the level of protein kinase B and GSK3β. Mitochondrial shape was visualized through translocase of the outer membrane 20 staining. Propofol increased cell death in neurons by 1.8-fold (% of PI-positive cells [PI%] = 18.6; 95% confidence interval [CI], 15.2-21.9, P < .05) but did not influence astrocyte viability. The neuronal death was attenuated by a high ANR (1:1 cocultures; fold change [FC] = 1.17, 95% CI, 0.96-1.38, P < .05), but not with a low ANR [1:9 cocultures; FC = 1.87, 95% CI, 1.48-2.26, P > .05

  19. Differential regulation of rice mitogen activated protein kinase kinase (MKK) by abiotic stress.

    PubMed

    Kumar, Kundan; Rao, Kudupudi Prabhakara; Sharma, Pallavi; Sinha, Alok Krishna

    2008-10-01

    Mitogen activated protein kinase cascade plays a crucial role in various biotic and abiotic stresses, hormones, cell division and developmental processes. MAP kinase kinase being integral part of this cascade performs an important function of integrating upstream signals to mitogen activated protein kinase for further appropriate cellular responses. We here report cloning of five MAP kinase kinase members from Oryza sativa indica cultivar var. Pusa Basmati 1, namely MAP kinase kinases 1, 3, 4, 6 and 10-2. All these members, except MKK10-2 possess fully canonical motif structures of MAP kinase kinase. The deduced amino acid sequence showed changes at certain position within japonica and indica variety of rice. Analysis of transcript regulation by quantitative real time PCR revealed that these five members are differentially regulated by cold, heat, salinity and drought stresses. MAP kinase kinases 4 and 6 are strongly regulated by cold and salt stresses while MAP kinase kinase 1 is regulated by salt and drought stresses. MAP kinase kinase 10-2 is regulated only by cold stress. The study provides the indication of involvement of specific MAP kinase kinase in different abiotic stress signaling and also possible cross talks that exist during the signaling processes.

  20. Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development

    PubMed Central

    Minocha, Shilpi; Valloton, Delphine; Arsenijevic, Yvan; Cardinaux, Jean-René; Guidi, Raffaella; Hornung, Jean-Pierre; Lebrand, Cécile

    2017-01-01

    The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1−/−) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors. PMID:28266561

  1. Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

    PubMed Central

    Czeredys, Magdalena; Samluk, Łukasz; Michalec, Katarzyna; Tułodziecka, Karolina; Skowronek, Krzysztof; Nałęcz, Katarzyna A.

    2013-01-01

    OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency. PMID:24349196

  2. Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes

    PubMed Central

    Zhou, Yu; Wang, Liyun; Park, Sung-Soo; Martin, Bronwen; Wang, Rui; Becker, Kevin G.; Wood, William H.; Zhang, Yongqing; Peers, Chris; Maudsley, Stuart

    2011-01-01

    The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. PMID:21738745

  3. μ and κ Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes*

    PubMed Central

    Belcheva, Mariana M.; Clark, Amy L.; Haas, Paul D.; Serna, Jannie S.; Hahn, Jason W.; Kiss, Alexi; Coscia, Carmine J.

    2005-01-01

    Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [D-ala2, mephe4, gly-ol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCε were used as a model system to study μ signaling. Evidence was gained to implicate CaM and PKCε in DAMGO stimulation of ERK. DAMGO activation of PKCε and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the μ receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCε. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids. PMID:15944153

  4. Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes.

    PubMed

    Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E

    2007-01-22

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working Global System for Mobile Communication (GSM) cell phone rated at a frequency of 1900MHz. Primary cultures were exposed to cell phone emissions for 2h. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Up-regulation occurred in both "on" and "stand-by" modes in neurons, but only in "on" mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons or astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes.

  5. Glutamate Transporter EAAT2 Expression is Up-Regulated in Reactive Astrocytes in Human Periventricular Leukomalacia

    PubMed Central

    DESILVA, TARA M.; BILLIARDS, SARAID S.; BORENSTEIN, NATALIA S.; TRACHTENBERG, FELICIA L.; VOLPE, JOSEPH J.; KINNEY, HANNAH C.; ROSENBERG, PAUL A.

    2010-01-01

    The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% ± 5.6%) compared with control white matter (21.4% ± 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation. PMID:18314905

  6. Dorsomedial hindbrain catecholamine regulation of hypothalamic astrocyte glycogen metabolic enzyme protein expression: Impact of estradiol.

    PubMed

    Tamrakar, P; Shrestha, P K; Briski, K P

    2015-04-30

    The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates

  7. Aerobic glycolysis during brain activation: adrenergic regulation and influence of norepinephrine on astrocytic metabolism.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F

    2016-07-01

    Aerobic glycolysis occurs during brain activation and is characterized by preferential up-regulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific β-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from the brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas β-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain

  8. Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

    PubMed Central

    Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E.

    2007-01-01

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Upregulation occurred in both “on” and “stand-by” modes in neurons, but only in “on” mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons and astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes. PMID:17187929

  9. c-Jun and c-Fos regulate the complement factor H promoter in murine astrocytes

    PubMed Central

    Fraczek, Laura A.; Martin, Carol B.; Martin, Brian K.

    2011-01-01

    The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimer’s disease, Parkinson’s disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241 base pair (bp) region at −416 bp to −175 bp that showed the strongest activity. Through supershift analysis it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation. PMID:21920606

  10. c-Jun and c-Fos regulate the complement factor H promoter in murine astrocytes.

    PubMed

    Fraczek, Laura A; Martin, Carol B; Martin, Brian K

    2011-10-01

    The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241 base pair (bp) region at -416 bp to -175 bp that showed the strongest activity. Through supershift analysis, it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation.

  11. mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

    PubMed Central

    Manzoni, Claudia; Mamais, Adamantios; Roosen, Dorien A.; Dihanich, Sybille; Soutar, Marc P. M.; Plun-Favreau, Helene; Bandopadhyay, Rina; Hardy, John; Tooze, Sharon A.; Cookson, Mark R.; Lewis, Patrick A.

    2016-01-01

    Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase. PMID:27731364

  12. Dexamethasone differentially regulates functional membrane properties in glioma cell lines and primary astrocytes in vitro.

    PubMed

    Hinkerohe, Daniel; Wolfkühler, Dörte; Haghikia, Aiden; Meier, Carola; Faustmann, Pedro M; Schlegel, Uwe

    2011-07-01

    Similar to astrocytes, glioma cells form a well-coupled syncytium via gap junctions. This can be influenced, for example, by activated microglia, the main inflammatory cell population within the central nervous system (CNS). Under pathological conditions such as neoplastic cell growth, microglia number and activation state are enhanced. The aim of the present study is to analyze the influence of dexamethasone (DEX) on cellular and molecular properties in glial coculture models consisting of astroglia and microglia and human and rat glioma cell lines. Primary rat glial cocultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) microglia as well as rat and human glioma cell lines (F98, C6, U87) were incubated with DEX for 24 h. DEX-treated M30 cocultures showed significant increased gap junctional intercellular communication (GJIC). DEX treatment of glioma cells resulted in depolarization of the membrane resting potential (MRP) and a significant reduction of GJIC. Furthermore, DEX reduced the amount of activated microglia in M30 cocultures. DEX had no significant effects on the tested variables in the M5 coculture. DEX differentially regulates functional membrane properties of glioma cells and astrocytes in primary glial cocultures, which might resemble steroid effects in glioma cells and adjacent glial components in vivo.

  13. PAS kinase: a nutrient sensing regulator of glucose homeostasis.

    PubMed

    DeMille, Desiree; Grose, Julianne H

    2013-11-01

    Per-Arnt-Sim (PAS) kinase (PASK, PASKIN, and PSK) is a member of the group of nutrient sensing protein kinases. These protein kinases sense the energy or nutrient status of the cell and regulate cellular metabolism appropriately. PAS kinase responds to glucose availability and regulates glucose homeostasis in yeast, mice, and man. Despite this pivotal role, the molecular mechanisms of PAS kinase regulation and function are largely unknown. This review focuses on what is known about PAS kinase, including its conservation from yeast to man, identified substrates, associated phenotypes and role in metabolic disease.

  14. Transcriptional Regulation of Human Transforming Growth Factor-α in Astrocytes.

    PubMed

    Karki, Pratap; Johnson, James; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2017-03-01

    Transforming growth factor-alpha (TGF-α) is known to play multifunctional roles in the central nervous system (CNS), including the provision of neurotropic properties that protect neurons against various neurotoxic insults. Previously, we reported that TGF-α mediates estrogen-induced enhancement of glutamate transporter GLT-1 function in astrocytes. However, the regulatory mechanism of TGF-α at the transcriptional level remains to be established. Our findings revealed that the human TGF-α promoter contains consensus sites for several transcription factors, such as NF-κB and yin yang 1 (YY1). NF-κB served as a positive regulator of TGF-α promoter activity, corroborated by observations that overexpression of NF-κB p65 increased, while mutation in the NF-κB binding sites in the TGF-α promoter reduced the promoter activity in rat primary astrocytes. Pharmacological inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC; 50 μM) or quinazoline (QNZ; 10 μM) also abolished TGF-α promoter activity, and NF-κB directly bound to its consensus site in the TGF-α promoter as evidenced by electrophoretic mobility shift assay (EMSA). Dexamethasone (DX) increased TGF-α promoter activity by activation of NF-κB. Treatment of astrocytes with 100 nM of DX for 24 h activated its glucocorticoid receptor and signaling proteins, including MAPK, PI3K/Akt, and PKA, via non-genomic pathways, to enhance TGF-α promoter activity and expression. YY1 served as a critical negative regulator of the TGF-α promoter as overexpression of YY1 decreased, while mutation of YY1 binding site in the promoter increased TGF-α promoter activity. Treatment for 3 h with 250 μM of manganese (Mn), an environmental neurotoxin, decreased astrocytic TGF-α expression by activation of YY1. Taken together, our results suggest that NF-κB is a critical positive regulator, whereas YY1 is a negative regulator of the TGF-α promoter. These findings identify potential molecular targets for

  15. Astrocyte-specific regulation of hMeCP2 expression in Drosophila

    PubMed Central

    Hess-Homeier, David L.; Fan, Chia-Yu; Gupta, Tarun; Chiang, Ann-Shyn; Certel, Sarah J.

    2014-01-01

    ABSTRACT Alterations in the expression of Methyl-CpG-binding protein 2 (MeCP2) either by mutations or gene duplication leads to a wide spectrum of neurodevelopmental disorders including Rett Syndrome and MeCP2 duplication disorder. Common features of Rett Syndrome (RTT), MeCP2 duplication disorder, and neuropsychiatric disorders indicate that even moderate changes in MeCP2 protein levels result in functional and structural cell abnormalities. In this study, we investigated two areas of MeCP2 pathophysiology using Drosophila as a model system: the effects of MeCP2 glial gain-of-function activity on circuits controlling sleep behavior, and the cell-type specific regulation of MeCP2 expression. In this study, we first examined the effects of elevated MeCP2 levels on microcircuits by expressing human MeCP2 (hMeCP2) in astrocytes and distinct subsets of amine neurons including dopamine and octopamine (OA) neurons. Depending on the cell-type, hMeCP2 expression reduced sleep levels, altered daytime/nighttime sleep patterns, and generated sleep maintenance deficits. Second, we identified a 498 base pair region of the MeCP2e2 isoform that is targeted for regulation in distinct subsets of astrocytes. Levels of the full-length hMeCP2e2 and mutant RTT R106W protein decreased in astrocytes in a temporally and spatially regulated manner. In contrast, expression of the deletion Δ166 hMeCP2 protein was not altered in the entire astrocyte population. qPCR experiments revealed a reduction in full-length hMeCP2e2 transcript levels suggesting transgenic hMeCP2 expression is regulated at the transcriptional level. Given the phenotypic complexities that are caused by alterations in MeCP2 levels, our results provide insight into distinct cellular mechanisms that control MeCP2 expression and link microcircuit abnormalities with defined behavioral deficits. PMID:25305037

  16. Tumor necrosis factor-alpha regulation of the Id gene family in astrocytes and microglia during CNS inflammatory injury.

    PubMed

    Tzeng, S F; Kahn, M; Liva, S; De Vellis, J

    1999-04-01

    The inhibitors of DNA binding (Id) gene family is highly expressed during embryogenesis and throughout adulthood in the rat central nervous system (CNS). In vitro studies suggest that the Id gene family is involved in the regulation of cell proliferation and differentiation. Recently, Id gene expression was shown to be expressed in immature and mature astrocytes during development and upregulated in reactive astrocytes after spinal cord injury. These results suggest that the Id gene family may play an important role in regulating astrocyte development and reactivity; however, the factors regulating Id expression in astrocytes remain undefined. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, is thought to play a crucial role in astrocyte/microglia activation after injury to the CNS. To determine if TNF alpha plays a role in Id gene expression, we exogenously administered TNF alpha into developing postnatal rats. We report that TNF alpha injections resulted in a rapid and transient increase in both cell number and mRNA expression for Id2 and Id3 when compared to levels observed in noninjected or control-injected animals. Id1 mRNA levels were also upregulated after TNF alpha treatment, but to a lesser degree. Significant increases in TNF alpha-induced Id2 and Id3 mRNA were observed in the ventricular/subventricular zone, cingulum and corpus callosum. TNF alpha also increased Id2 mRNA expression in the caudate putamen and hippocampus at the injection site. Id2 and Id3 mRNA+ cells were identified as GFAP+ and S100 alpha + astrocytes as well as ED1+ microglia. This is the first report to show TNF-alpha-induced modulation of the Id gene family and suggests that Id may be involved in the formation of reactive astrocytes and activated microglia in the rodent brain. These results suggest a putative role for the Id family in the molecular mechanisms regulating cellular responsiveness to TNF alpha and CNS inflammation.

  17. Down-Regulation of GABAA Receptor via Promiscuity with the Vasoactive Peptide Urotensin II Receptor. Potential Involvement in Astrocyte Plasticity

    PubMed Central

    Lecointre, Céline; Schouft, Marie-Thérèse; Leprince, Jérôme; Compère, Vincent; Morin, Fabrice; Proust, François; Gandolfo, Pierrick; Tonon, Marie-Christine; Castel, Hélène

    2012-01-01

    GABAA receptor (GABAAR) expression level is inversely correlated with the proliferation rate of astrocytes after stroke or during malignancy of astrocytoma, leading to the hypothesis that GABAAR expression/activation may work as a cell proliferation repressor. A number of vasoactive peptides exhibit the potential to modulate astrocyte proliferation, and the question whether these mechanisms may imply alteration in GABAAR-mediated functions and/or plasma membrane densities is open. The peptide urotensin II (UII) activates a G protein-coupled receptor named UT, and mediates potent vasoconstriction or vasodilation in mammalian vasculature. We have previously demonstrated that UII activates a PLC/PIPs/Ca2+ transduction pathway, via both Gq and Gi/o proteins and stimulates astrocyte proliferation in culture. It was also shown that UT/Gq/IP3 coupling is regulated by the GABAAR in rat cultured astrocytes. Here we report that UT and GABAAR are co-expressed in cerebellar glial cells from rat brain slices, in human native astrocytes and in glioma cell line, and that UII inhibited the GABAergic activity in rat cultured astrocytes. In CHO cell line co-expressing human UT and combinations of GABAAR subunits, UII markedly depressed the GABA current (β3γ2>α2β3γ2>α2β1γ2). This effect, characterized by a fast short-term inhibition followed by drastic and irreversible run-down, is not relayed by G proteins. The run-down partially involves Ca2+ and phosphorylation processes, requires dynamin, and results from GABAAR internalization. Thus, activation of the vasoactive G protein-coupled receptor UT triggers functional inhibition and endocytosis of GABAAR in CHO and human astrocytes, via its receptor C-terminus. This UII-induced disappearance of the repressor activity of GABAAR, may play a key role in the initiation of astrocyte proliferation. PMID:22563490

  18. Ndrg2, a novel gene regulated by adrenal steroids and antidepressants, is highly expressed in astrocytes.

    PubMed

    Nichols, Nancy R

    2003-12-01

    Previously, we cloned a gene from rat hippocampus that now shows homology to Ndrg2, a member of the N-myc downregulated gene (NDRG) family with putative roles in neural differentiation, synapse formation, and axon survival. Following adrenalectomy, hippocampal Ndrg2 mRNA increased in response to glucocorticoids. Ndrg2 mRNA was also upregulated by corticosterone in cerebral cortex and heart. Since Ndrg2 mRNA increased in response to glucocorticoid treatment of cultured astrocytes, we examined its cellular localization in adult brain by in situ hybridization. Ndrg2 mRNA is a prevalent message that is widely expressed throughout the brain, but is more abundant in gray matter than in white matter. Predominant mRNA expression was found in neurogenic regions of the adult brain. Furthermore, Ndrg2 mRNA in these regions was localized to GFAP-positive astrocytes or radial glia. In one of these regions, the subgranular zone of the dentate gyrus, Ndrg2 expression was decreased after adrenalectomy, and was restored to sham-operated levels by corticosterone, indicating that it is under positive regulation by glucocorticoids in vivo. Recently, another group reported that Ndr2/Ndrg2 transcripts in rat frontal cortex were decreased by chronic antidepressant treatment. Because antidepressants may alleviate symptoms of depression by reversing the effects of glucocorticoids, these data suggest that further study of Ndrg2 regulation and function in glia could contribute to understanding the pathogenesis and treatment of depression.

  19. Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes

    PubMed Central

    De Pittà, Maurizio; Goldberg, Mati; Volman, Vladislav; Berry, Hugues

    2009-01-01

    Recent years have witnessed an increasing interest in neuron–glia communication. This interest stems from the realization that glia participate in cognitive functions and information processing and are involved in many brain disorders and neurodegenerative diseases. An important process in neuron–glia communications is astrocyte encoding of synaptic information transfer—the modulation of intracellular calcium (Ca2 + ) dynamics in astrocytes in response to synaptic activity. Here, we derive and investigate a concise mathematical model for glutamate-induced astrocytic intracellular Ca2 +  dynamics that captures the essential biochemical features of the regulatory pathway of inositol 1,4,5-trisphosphate (IP3). Starting from the well-known two-variable (intracellular Ca2 +  and inactive IP3 receptors) Li–Rinzel model for calcium-induced calcium release, we incorporate the regulation of IP3 production and phosphorylation. Doing so, we extend it to a three-variable model (which we refer to as the ChI model) that could account for Ca2 +  oscillations with endogenous IP3 metabolism. This ChI model is then further extended into the G-ChI model to include regulation of IP3 production by external glutamate signals. Compared with previous similar models, our three-variable models include a more realistic description of IP3 production and degradation pathways, lumping together their essential nonlinearities within a concise formulation. Using bifurcation analysis and time simulations, we demonstrate the existence of new putative dynamical features. The cross-couplings between IP3 and Ca2 +  pathways endow the system with self-consistent oscillatory properties and favor mixed frequency–amplitude encoding modes over pure amplitude–modulation ones. These and additional results of our model are in general agreement with available experimental data and may have important implications for the role of astrocytes in the synaptic transfer of information

  20. Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity*

    PubMed Central

    Dávila, David; Fernández, Silvia; Torres-Alemán, Ignacio

    2016-01-01

    Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H2O2), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury. PMID:26631726

  1. Microtubule affinity-regulating kinase 4: structure, function, and regulation.

    PubMed

    Naz, Farha; Anjum, Farah; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2013-11-01

    MAP/Microtubule affinity-regulating kinase 4 (MARK4) belongs to the family of serine/threonine kinases that phosphorylate the microtubule-associated proteins (MAP) causing their detachment from the microtubules thereby increasing microtubule dynamics and facilitating cell division, cell cycle control, cell polarity determination, cell shape alterations, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. MARK4L is a 752-residue-long polypeptide that is divided into three distinct domains: (1) protein kinase domain (59-314), (2) ubiquitin-associated domain (322-369), and (3) kinase-associated domain (703-752) plus 54 residues (649-703) involved in the proper folding and function of the enzyme. In addition, residues 65-73 are considered to be the ATP-binding domain and Lys88 is considered as ATP-binding site. Asp181 has been proposed to be the active site of MARK4 that is activated by phosphorylation of Thr214 side chain. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation. On the other hand, the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas suggesting its involvement in cell cycle. Several biological functions are also associated with MARK4 including microtubule bundle formation, nervous system development, and positive regulation of programmed cell death. Therefore, MARK4 is considered as the most suitable target for structure-based rational drug design. Our sequence, structure- and function-based analysis should be helpful for better understanding of mechanisms of regulation of microtubule dynamics and MARK4 associated diseases.

  2. Diacylglycerol kinase θ: regulation and stability.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Petro, Elizabeth; Raben, Daniel M

    2013-01-01

    Given the well-established roles of diacylglycerol (DAG) and phosphatidic acid (PtdOH) in a variety of signaling cascades, it is not surprising that there is an increasing interest in understanding their physiological roles and mechanisms that regulate their cellular levels. One class of enzymes capable of coordinately regulating the levels of these two lipids is the diacylglycerol kinases (DGKs). These enzymes catalyze the transfer of the γ-phosphate of ATP to the hydroxyl group of DAG, which generates PtdOH while reducing DAG. As these enzymes reciprocally modulate the relative levels of these two signaling lipids, it is essential to understand the regulation and roles of these enzymes in various tissues. One system where these enzymes play important roles is the nervous system. Of the ten mammalian DGKs, eight of them are readily detected in the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-ε, and DGK-θ. Despite the increasing interest in DGKs, little is known about their regulation. We have focused some attention on understanding the enzymology and regulation of one of these DGK isoforms, DGK-θ. We recently showed that DGK-θ is regulated by an accessory protein containing polybasic regions. We now report that this accessory protein is required for the previously reported broadening of the pH profile observed in cell lysates in response to phosphatidylserine (PtdSer). Our data further reveal DGK-θ is regulated by magnesium and zinc, and sensitive to the known DGK inhibitor R599022. These data outline new parameters involved in regulating DGK-θ.

  3. Hypoosmotic volume regulation and osmolyte transport in astrocytes is blocked by an anion transport inhibitor, L-644,711.

    PubMed

    Olson, J E; Kimelberg, H K

    1995-06-05

    Cell volume, potassium content, and potassium influx were measured in rat cerebral astrocytes grown in primary culture following exposure to hypoosmotic medium containing either 3.2 mM or 50 mM potassium. Some solutions also contained 1 mM L-644,711, an anion transport inhibitor. L-644,711 inhibited volume regulation and taurine efflux induced by hypoosmotic exposure in medium containing either potassium concentration. L-644,711 also inhibited potassium uptake associated and not associated with the sodium/potassium pump. The correlation of reduced taurine efflux and volume decrease produced by L-644,711 exposure indicates the important role for this amino acid in hypoosmotic astrocyte volume regulation. However, the effects of L-644,711 on potassium transport indicate that multiple actions of this drug may be important factors in its effect on astrocyte volume regulatory mechanisms.

  4. ROCK inhibition with Y27632 promotes the proliferation and cell cycle progression of cultured astrocyte from spinal cord.

    PubMed

    Yu, Zhiyuan; Liu, Miao; Fu, Peicai; Xie, Minjie; Wang, Wei; Luo, Xiang

    2012-12-01

    Rho-associated Kinase (ROCK) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. Although its effects on astrocyte proliferation have not been well characterized, ROCK has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. In the present study, our aim was to investigate the effect of ROCK inhibition by [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (Y27632) on proliferation and DNA synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. Western blots showed that treatment of astrocytes with Y27632 increased their expression of cyclin D1, CDK4, and cyclin E, thereby causing cell cycle progression. Furthermore, Y27632-induced astrocyte proliferation was mediated through the extracellular-signal-regulated kinase signaling cascade. These results indicate the importance of ROCK in astrocyte proliferation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Sigma 1 receptor regulates ERK activation and promotes survival of optic nerve head astrocytes

    PubMed Central

    Zhao, Jing; Mysona, Barbara A.; Wang, Jing; Gonsalvez, Graydon B.; Smith, Sylvia B.; Bollinger, Kathryn E.

    2017-01-01

    The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death. PMID:28898265

  6. Glutamine synthetase stability and subcellular distribution in astrocytes are regulated by γ-aminobutyric type B receptors.

    PubMed

    Huyghe, Deborah; Nakamura, Yasuko; Terunuma, Miho; Faideau, Mathilde; Haydon, Philip; Pangalos, Menelas N; Moss, Stephen J

    2014-10-17

    Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

  7. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  8. Astrocytes optimize synaptic fidelity

    NASA Astrophysics Data System (ADS)

    Nadkarni, Suhita; Jung, Peter; Levine, Herbert

    2007-03-01

    Most neuronal synapses in the central nervous system are enwrapped by an astrocytic process. This relation allows the astrocyte to listen to and feed back to the synapse and to regulate synaptic transmission. We combine a tested mathematical model for the Ca^2+ response of the synaptic astrocyte and presynaptic feedback with a detailed model for vesicle release of neurotransmitter at active zones. The predicted Ca^2+ dependence of the presynaptic synaptic vesicle release compares favorably for several types of synapses, including the Calyx of Held. We hypothesize that the feedback regulation of the astrocyte onto the presynaptic terminal optimizes the fidelity of the synapse in terms of information transmission.

  9. Interlukin-18 Is a Pivot Regulatory Factor on Matrix Metalloproteinase-13 Expression and Brain Astrocytic Migration.

    PubMed

    Chen, Jia-Hong; Tsai, Chon-Haw; Lin, Hsiao-Yun; Huang, Chien-Fang; Leung, Yuk-Man; Lai, Sheng-Wei; Tsai, Cheng-Fang; Chang, Pei-Chun; Lu, Dah-Yuu; Lin, Chingju

    2016-11-01

    The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

  10. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  11. Regulation of proinflammatory cytokines gene expression by nociceptin/orphanin FQ in the spinal cord and the cultured astrocytes.

    PubMed

    Fu, X; Zhu, Z-H; Wang, Y-Q; Wu, G-C

    2007-01-05

    Peripheral inflammation induces central sensitization characterized by the development of allodynia and hyperalgesia to thermal stimuli. Recent evidence suggests that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the development of behavioral hypersensitivity after nerve injury or peripheral inflammation. The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (ORL1 receptor), has been demonstrated to play an important role in modulation of nociceptive signals. In the present study, we investigated: (1) astrocyte activation and proinflammatory cytokine expression at the lumbar spinal cord following intraplantar administration of complete Freund's adjuvant (CFA) in rats; (2) the mechanism of N/OFQ on nociception modulation, the relationship between N/OFQ and cytokines in the rat CNS in vivo and in vitro. The results showed: (1) CFA-induced peripheral inflammation evoked robust astrocyte activation and proinflammatory cytokines spinally; (2) down-regulation of cytokine mRNA transcripts by intrathecal administration of N/OFQ, the effects produced by N/OFQ were abolished by combination with ORL1 receptor-specific antagonist [Nphe(1)]N/OFQ(1-13)NH2; (3) ORL1 receptor was expressed on astrocytes of rat spinal cord; (4) cytokine gene expression was inhibited in astrocyte cultures exposed to N/OFQ, the inhibiting effects of N/OFQ were significantly blocked by [Nphe(1)]N/OFQ(1-13)NH2. The present data demonstrated that astrocyte activation and enhanced cytokine expression at the CNS had a role in eliciting behavioral hypersensitivity; the anti-nociception function of N/OFQ might be dependent on cytokines derived from astrocytes, the effects were attributable to the ORL1 receptor pathway.

  12. Astrocytes regulate developmental changes in the chloride ion gradient of embryonic rat ventral spinal cord neurons in culture

    PubMed Central

    Li, Yong-Xin; Schaffner, Anne E; Walton, Marc K; Barker, Jeffery L

    1998-01-01

    Embryonic rat ventral spinal cord neurons were dissociated at day 15 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of type I astrocytes; or (iii) PDL in astrocyte-conditioned medium (ACM) to examine the influence of astroglia on the regulation of GABAA receptor/Cl− channel properties. Potentiometric oxonol dye recordings of intact cells indicated that embryonic neurons were uniformly depolarized by muscimol. The depolarizing effects disappeared in cells dissociated during the early postnatal period and recovered in culture for 24 h. Similar recordings using the calcium-imaging dye fura-2 AM revealed that GABA or muscimol triggered a sustained rise in cytosolic Ca2+ (Cac2+) in embryonic neurons that was dependent on extracellular Ca2+, blocked by bicuculline and nifedipine and sensitive to changes in extracellular chloride. The incidence and amplitude of the Ca2+ response decreased with time in vitro and was accelerated in neurons cultured on astrocytes compared with those on PDL. Perforated patch-clamp recordings revealed that GABA depolarized neurons in a Cl−-dependent and bicuculline-sensitive manner. Both the resting membrane potential and the GABA equilibrium potential became more hyperpolarized with time in vitro. Astrocytes and ACM accelerated the transformation of GABAergic potential responses from depolarizing to hyperpolarizing. The change occurred over the first 4 days in co-culture or in ACM but took more than 2 weeks in neurons cultured on PDL alone. The intrinsic, elementary properties of GABAA receptor/Cl− channels including open time and unitary conductance changed independently of the presence of astrocytes or ACM. Mean open time of the dominant kinetic component decreased and conductance increased with time in vitro. In sum, astrocytes accelerate the developmental change in the Cl− ion gradient extrinsic to GABAA receptor/Cl− channels, which is critical for triggering Ca2+ entry, without influencing parallel changes in

  13. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    PubMed Central

    Vlasova-St. Louis, Irina; Bohjanen, Paul R.

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  14. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs.

    PubMed

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-25

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.

  15. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

    PubMed

    Ard, Ryan; Mulatz, Kirk; Pomoransky, Julia L; Parks, Robin J; Trinkle-Mulcahy, Laura; Bell, John C; Gee, Stephen H

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

  16. HIV-1-Infected and/or Immune Activated Macrophages Regulate Astrocyte SDF-1 Production Through IL-1β

    PubMed Central

    PENG, HUI; ERDMANN, NATHAN; WHITNEY, NICHOLAS; DOU, HUANGYU; GORANTLA, SANTHI; GENDELMAN, HOWARD E.; GHORPADE, ANUJA; ZHENG, JIALIN

    2007-01-01

    Stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 play important roles in the pathogenesis of human immunodeficiency virus type one (HIV-1)-associated dementia (HAD) by serving as a HIV-1 co-receptor and affecting cell migration, virus-mediated neurotoxicity, and neurodegeneration. However, the underlying mechanisms regulating SDF-1 production during disease are not completely understood. In this report we investigated the role of HIV-1 infected and immune competent macrophage, the principal target cell and mediator of neuronal injury and death in HAD, in regulating SDF-1 production by astrocytes. Our data demonstrated that astrocytes are the primary cell type expressing SDF-1 in the brain. Immune-activated or HTV-1-infected human monocyte-derived-macrophage (MDM) conditioned media (MCM) induced a substantial increase in SDF-1 production by human astrocytes. This SDF-1 production was directly dependent on MDM IL-1β following both viral and immune activation. The MCM-induced production of SDF-1 was prevented by IL-1β receptor antagonist (IL-1Ra) and IL-1β siRNA treatment of human MDM. These laboratory observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, reactive astrocytes showed a significant increase in SDF-1 expression, as observed by immunocytochemical staining. Similarly, SDF-1 mRNA levels were increased in the encephalitic region as measured by real time RT-PCR, and correlated with IL-1β mRNA expression. These observations provide direct evidence that IL-1β, produced from HIV-1-infected and/or immune competent macrophage, induces production of SDF-1 by astrocytes, and as such contribute to ongoing SDF-1 mediated CNS regulation during HAD. PMID:16944452

  17. Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

    SciTech Connect

    Spilsbury, Alison; Vauzour, David; Spencer, Jeremy P.E.; Rattray, Marcus

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or TNF

  18. ROLE OF C/EBP-β, p38 MAPK and MKK6 IN IL-1β MEDIATED C3 GENE REGULATION IN ASTROCYTES

    PubMed Central

    Maranto, Jeffrey; Rappaport, Jay; Datta, Prasun K.

    2011-01-01

    Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK and C/EBP-β isoforms (LAP1, LAP2 or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, i.e., MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas, overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAPT235A showed reduction in IL-1β mediated C3 promoter activation. These results suggest that the p38α MAPK and MKK6 play prominent roles in IL-1β and C/EBP-β mediated C3 gene expression in astrocytes. PMID:21308746

  19. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  20. A tale of two stories: astrocyte regulation of synaptic depression and facilitation.

    PubMed

    De Pittà, Maurizio; Volman, Vladislav; Berry, Hugues; Ben-Jacob, Eshel

    2011-12-01

    Short-term presynaptic plasticity designates variations of the amplitude of synaptic information transfer whereby the amount of neurotransmitter released upon presynaptic stimulation changes over seconds as a function of the neuronal firing activity. While a consensus has emerged that the resulting decrease (depression) and/or increase (facilitation) of the synapse strength are crucial to neuronal computations, their modes of expression in vivo remain unclear. Recent experimental studies have reported that glial cells, particularly astrocytes in the hippocampus, are able to modulate short-term plasticity but the mechanism of such a modulation is poorly understood. Here, we investigate the characteristics of short-term plasticity modulation by astrocytes using a biophysically realistic computational model. Mean-field analysis of the model, supported by intensive numerical simulations, unravels that astrocytes may mediate counterintuitive effects. Depending on the expressed presynaptic signaling pathways, astrocytes may globally inhibit or potentiate the synapse: the amount of released neurotransmitter in the presence of the astrocyte is transiently smaller or larger than in its absence. But this global effect usually coexists with the opposite local effect on paired pulses: with release-decreasing astrocytes most paired pulses become facilitated, namely the amount of neurotransmitter released upon spike i+1 is larger than that at spike i, while paired-pulse depression becomes prominent under release-increasing astrocytes. Moreover, we show that the frequency of astrocytic intracellular Ca(2+) oscillations controls the effects of the astrocyte on short-term synaptic plasticity. Our model explains several experimental observations yet unsolved, and uncovers astrocytic gliotransmission as a possible transient switch between short-term paired-pulse depression and facilitation. This possibility has deep implications on the processing of neuronal spikes and resulting

  1. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    PubMed

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  2. LIM kinases: function, regulation and association with human disease.

    PubMed

    Scott, Rebecca W; Olson, Michael F

    2007-06-01

    The LIM kinase family consists of just two members: LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2). With uniquely organised signalling domains, LIM kinases are regulated by several upstream signalling pathways, principally acting downstream of Rho GTPases to influence the architecture of the actin cytoskeleton by regulating the activity of the cofilin family proteins cofilin1, cofilin2 and destrin. Although the LIM kinases are very homologous, particularly when comparing kinase domains, there is emerging evidence that each may be subject to different regulatory pathways and may contribute to both distinct and overlapping cellular and developmental functions. Normal central nervous system development is reliant upon the presence of LIMK1, and its deletion has been implicated in the development of the human genetic disorder Williams syndrome. Normal testis development, on the other hand, is disrupted by the deletion of LIMK2. In addition, the possible involvement of each kinase in cardiovascular disorders as well as cancer has recently emerged. The LIM kinases have been proposed to play an important role in tumour-cell invasion and metastasis; fine-tuning the balance between phosphorylated and non-phosphorylated cofilin may be a significant determinant of tumour-cell metastatic potential. In this review, we outline the structure, regulation and function of LIM kinases and their functions at cellular and organismal levels, as well as their possible contributions to human disease.

  3. Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy

    PubMed Central

    Hubbard, Jacqueline A.; Szu, Jenny I.; Yonan, Jennifer M.; Binder, Devin K.

    2016-01-01

    Astrocytes regulate extracellular glutamate and water homeostasis through the astrocyte-specific membrane proteins glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4), respectively. The role of astrocytes and the regulation of GLT1 and AQP4 in epilepsy are not fully understood. In this study, we investigated the expression of GLT1 and AQP4 in the intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used real-time polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analysis at 1, 4, 7, and 30 days after kainic acid-induced status epilepticus (SE) to determine hippocampal glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes), GLT1, and AQP4 expression changes during the development of epilepsy (epileptogenesis). Following IHKA, all mice had SE and progressive increases in GFAP immunoreactivity and GFAP protein expression out to 30 days post-SE. A significant initial increase in dorsal hippocampal GLT1 immunoreactivity and protein levels were observed 1 day post SE and followed by a marked downregulation at 4 and 7 days post SE with a return to near control levels by 30 days post SE. AQP4 dorsal hippocampal protein expression was significantly downregulated at 1 day post SE and was followed by a gradual return to baseline levels with a significant increase in ipsilateral protein levels by 30 days post SE. Transient increases in GFAP and AQP4 mRNA were also observed. Our findings suggest that specific molecular changes in astrocyte glutamate transporters and water channels occur during epileptogenesis in this model, and suggest the novel therapeutic strategy of restoring glutamate and water homeostasis. PMID:27155358

  4. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation

    PubMed Central

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M.; Echeverria, Valentina; Barreto, George E.

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions. PMID:27445795

  5. Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

    PubMed Central

    Yin, Zhaoobao; Aschner, Judy L.; Santos, Ana Paula dos; Aschner, Michael

    2008-01-01

    Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson’s disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague–Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethylrhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (ΔΨm). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 hrs) also significantly (p<0.01) dissipates the ΔΨm in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways. PMID:18313649

  6. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  7. Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy.

    PubMed

    Obara-Michlewska, Marta; Pannicke, Thomas; Karl, Anett; Bringmann, Andreas; Reichenbach, Andreas; Szeliga, Monika; Hilgier, Wojciech; Wrzosek, Antoni; Szewczyk, Adam; Albrecht, Jan

    2011-12-01

    Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

  8. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  9. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms.

    PubMed

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-04-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  10. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  11. The Nrf2-Inducible Antioxidant Defense in Astrocytes can be Both Up- and Down-Regulated by Activated Microglia: Involvement of p38 MAPK

    PubMed Central

    CORREA, FERNANDO; LJUNGGREN, ELIN; MALLARD, CARINA; NILSSON, MICHAEL; WEBER, STEPHEN G.; SANDBERG, MATS

    2012-01-01

    The effects of microglia-conditioned medium (MCM) on the inducible Nrf2 system in astrocyte-rich cultures were investigated by determination of glutathione (GSH) levels, γglutamylcysteine ligase (γGCL) activity, the protein levels of Nrf2, Keap1, the modulatory subunit of γGCL (γGCL-M) and activated MAP kinases (ERK1/2, JNK and p38). Microglia were either cultured for 24 h in serum-free culture medium to achieve microglia-conditioned medium from non-activated cells (MCM0), used as control condition, or activated with different concentrations (0.1–1,000 ng mL–1)of lipopolysaccharide (LPS) to produce MCM0.1–1,000. Acute exposure (24 h) to MCM100 increased GSH, γGCL activity, the protein levels of γGCL-M, Nrf2, and activated JNK and ERK1/2 in astrocyte-rich cultures. In contrast, treatment with MCM10 for 24 h decreased components of the Nrf2 system in parallel with activation of p38 MAPK. Stimulation of the Nrf2 system by tBHQ was partly intact after 24 h but blocked after 72 h treatment with MCM10 and MCM100. This down-regulation after 72 h correlated with activation of p38 MAPK and lack of ERK1/2 and JNK activation. The negative effects were partly reversed by an inhibitor of p38 which restored tBHQ mediated protection against oxidative stress. In conclusion, the study showed a negative effect of MCM10 on the inducible anti-oxidant defense in astrocyte-rich cultures at both 24 and 72 h that correlated with activation of p38 and was partly reversed by a p38 inhibitor. A transient protective effect of MCM100 on astrocyte-rich cultures against H2O2 toxicity was observed at 24 h which coincided with activation of JNK and ERK1/2. PMID:21351160

  12. Structure and dynamic regulation of Src-family kinases.

    PubMed

    Engen, J R; Wales, T E; Hochrein, J M; Meyn, M A; Banu Ozkan, S; Bahar, I; Smithgall, T E

    2008-10-01

    Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the down-regulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family.

  13. Time-of-day regulates subcellular trafficking, tripartite synaptic localization and polyadenylation of the astrocytic Fabp7 mRNA

    PubMed Central

    Gerstner, Jason R.; Vanderheyden, William M.; LaVaute, Timothy; Westmark, Cara J.; Rouhana, Labib; Pack, Allan I.; Wickens, Marv; Landry, Charles F.

    2012-01-01

    The astrocyte brain fatty acid binding protein (Fabp7) has previously been shown to have a coordinated diurnal regulation of mRNA and protein throughout mouse brain, and an age-dependent decline in protein expression within synaptoneurosomal fractions. Mechanisms that control time-of-day changes in expression and trafficking Fabp7 to the perisynaptic process are not known. In this study, we confirmed an enrichment of Fabp7 mRNA and protein in the astrocytic perisynaptic compartment, and observed a diurnal change in the intracellular distribution of Fabp7 mRNA in molecular layers of hippocampus. Northern blotting revealed a coordinated time-of-day dependent oscillation for the Fabp7 mRNA poly(A) tail throughout murine brain. Cytoplasmic polyadenylation element-(CPE-) binding protein (CPEB1) regulates subcellular trafficking and translation of synaptic plasticity-related mRNAs. Here we show that Fabp7 mRNA co-immunoprecipitated with CPEB1 from primary mouse astrocyte extracts, and its 3′UTR contains phylogenetically conserved CPEs capable of regulating translation of reporter mRNAs during Xenopus oocyte maturation. Given that Fabp7 expression is confined to astrocytes and neural progenitors in adult mouse brain, the synchronized cycling pattern of Fabp7 mRNA is therefore novel of known CPE-regulated transcripts. These results implicate circadian, sleep and/or metabolic control of CPEB-mediated subcellular trafficking and localized translation of Fabp7 mRNA in the tripartite synapse of mammalian brain. PMID:22279223

  14. P2Y2 receptor up-regulation induced by guanosine or UTP in rat brain cultured astrocytes.

    PubMed

    Ballerini, P; Di Iorio, P; Caciagli, F; Rathbone, M P; Jiang, S; Nargi, E; Buccella, S; Giuliani, P; D'Alimonte, I; Fischione, G; Masciulli, A; Romano, S; Ciccarelli, R

    2006-01-01

    Among P2 metabotropic ATP receptors, P2Y2 subtype seems to be peculiar as its upregulation triggers important biological events in different cells types. In non-stimulated cells including astrocytes, P2Y2 receptors are usually expressed at levels lower than P2Y1 sites, however the promoter region of the P2Y2 receptors has not yet been studied and little is known about the mechanisms underlying the regulation of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and naturally occurring agonist for P2Y2 sites, but also guanosine induced an up-regulation of astrocyte P2Y2 receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside since in our previous studies it was reported to be released by cultured astrocytes and to exert different neuroprotective effects. UTP and guanosine-evoked P2Y2 receptor up-regulation in rat brain cultured astrocytes was linked to an increased P2Y2-mediated intracellular calcium response, thus suggesting an increased P2Y2 activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-mediated P2Y2 up-regulation, thus indicating that de novo transcription was required. The effect of UTP and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface receptor mRNAs. The results show that ERK1-2/MAPK pathway play a key role in the P2Y2 receptor up-regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in guanosine-induced transcriptional activation of P2Y2 mRNA and that increased intracellular calcium levels and PKC activation may also mediate P2Y2 receptor up-regulation triggered by UTP. The extracellular release of ATP under physiological and pathological conditions has been widely studied. On the contrary, little is known about the release of

  15. Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways.

    PubMed

    Zhang, Yingmei; Xue, Dongbo; Wang, Xiaochun; Lu, Ming; Gao, Bo; Qiao, Xin

    2014-01-01

    The aim of this study was to identify agents that regulate autophagy. A total of 544 differentially expressed genes were screened from the intersection set of GSE2435 and GSE31040, which was obtained from the Gene Expression Omnibus database and 19 differentially expressed kinases were selected according to a 'protein kinase database'. Gene ontology‑biological process (GO-BP) enrichment analysis revealed that the 19 kinases were mainly associated with phosphorylation. The protein-protein interaction network exhibited 30 differentially expressed genes that interacted with BRAF, and GO-BP enrichment analysis showed the function of these genes were mainly involved in cell death and apoptosis. The kinase-kinase inhibitor regulatory network identified16 kinase inhibitors that specifically inhibited BRAF. Previous studies indicated that sorafenib is capable of regulating autophagy and regorafenib has also been reported; however, there have been no studies regarding the regulation of autophagy by afatinib, selumetinib, PD318088, axitinib, TAK-733, GDC-0980, GSK2126458, PLX-4720, AS703026, trametinib, GDC-0941 and PF-04217903. Thus, these kinase inhibitors are potential targets for further study on the regulation of autophagy in the future.

  16. [Quercetin regulates cell cycle-related gene expression in a model of glucose-oxygen deprivation in astrocytes].

    PubMed

    Yao, Fang; Zhang, Lanlan; Yuan, Zhaohu; Zeng, Yong; Wu, Bingyi

    2013-09-01

    To study the effect of quercetin on gene expression in astrocytes after glucose-oxygen deprivation and the underlying mechanism. The primary cultured astrocytes were randomly divided into glucose-oxygen deprivation group (only treated with glucose-oxygen deprivation for 4 hours) and glucose-oxygen deprivation combined with quercetin-treated group (glucose-oxygen deprivation for 4 hours combined with quercetin treatment for 24 hours). Their mRNA expressions were analyzed by the large-scale oligo microarray. The differential genes obtained were further confirmed by real-time quantitative PCR (qRT-PCR). Compared with the glucose-oxygen deprivation group, the glucose-oxygen deprivation combined with quercetin-treated group presented the changes in the expressions of 31 genes that were related to cell cycle, of which 5 genes were up-regulated and 26 were down-regulated. Six of those differential genes were confirmed by qRT-PCR and the result of their differential expressions was consistent with that by large-scale oligo microarray. Quercetin can regulate some of cell cycle-related genes in astrocytes after glucose-oxygen deprivation.

  17. Regulation of the syncytin-1 promoter in human astrocytes by multiple sclerosis-related cytokines

    SciTech Connect

    Mameli, Giuseppe . E-mail: viross@uniss.it; Astone, Vito; Khalili, Kamel; Serra, Caterina; Sawaya, Bassel E.; Dolei, Antonina

    2007-05-25

    Syncytin-1 has a physiological role during early pregnancy, as mediator of trophoblast fusion into the syncytiotrophoblast layer, hence allowing embryo implantation. In addition, its expression in nerve tissue has been proposed to contribute to the pathogenesis of multiple sclerosis (MS). Syncytin-1 is the env glycoprotein of the ERVWE1 component of the W family of human endogenous retroviruses (HERV), located on chromosome 7q21-22, in a candidate region for genetic susceptibility to MS. The mechanisms of ERVWE1 regulation in nerve tissue remain to be identified. Since there are correlations between some cytokines and MS outcome, we examined the regulation of the syncytin-1 promoter by MS-related cytokines in human U-87MG astrocytic cells. Using transient transfection assays, we observed that the MS-detrimental cytokines TNF{alpha}, interferon-{gamma}, interleukin-6, and interleukin-1 activate the ERVWE1 promoter, while the MS-protective interferon-{beta} is inhibitory. The effects of cytokines are reduced by the deletion of the cellular enhancer domain of the promoter that contains binding sites for several transcription factors. In particular, we found that TNF{alpha} had the ability to activate the ERVWE1 promoter through an NF-{kappa}B-responsive element located within the enhancer domain of the promoter. Electrophoretic mobility shift and ChIP assays showed that TNF{alpha} enhances the binding of the p65 subunit of NF-{kappa}B, to its cognate site within the promoter. The effect of TNF{alpha} is abolished by siRNA directed against p65. Taken together, these results illustrate a role for p65 in regulating the ERVWE1 promoter and in TNF{alpha}-mediated induction of syncytin-1 in multiple sclerosis.

  18. Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

    SciTech Connect

    Gu, Jun; Ni, Yingjie; Xu, Lin; Xu, Hongliang; Cai, Zhengdong

    2016-01-22

    Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in the spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.

  19. Regulation of heart muscle pyruvate dehydrogenase kinase

    PubMed Central

    Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

    1974-01-01

    1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate

  20. Kinase cascades regulating entry into apoptosis.

    PubMed Central

    Anderson, P

    1997-01-01

    All cells are constantly exposed to conflicting environment cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. In addition to survival factor withdrawal, cell death can be triggered by environmental stresses such as heat, UV light, and hyperosmolarity or by dedicated death receptors (e.g., FAS/APO-1 and tumor necrosis factor [TNF] receptors) that are counterparts of growth factor or survival receptors at the cell surface. One of the ways that cells integrate conflicting exogenous stimuli is by phosphorylation (or dephosphorylation) of cellular constituents by interacting cascades of serine/threonine and tyrosine protein kinases (and phosphatases). Survival factors (e.g., growth factors and mitogens) activate receptor tyrosine kinases and selected mitogen-activated, cyclin-dependent, lipid-activated, nucleic acid-dependent, and cyclic AMP-dependent kinases to promote cell survival and proliferation, whereas environmental stress (or death factors such as FAS/APO-1 ligand and TNF-alpha) activates different members of these kinase families to inhibit cell growth and, under some circumstances, promote apoptotic cell death. Because individual kinase cascades can interact with one another, they are able to integrate conflicting exogenous stimuli and provide a link between cell surface receptors and the biochemical pathways leading to cell proliferation or cell death. PMID:9106363

  1. The insulin-like growth factor I receptor regulates glucose transport by astrocytes.

    PubMed

    Hernandez-Garzón, Edwin; Fernandez, Ana M; Perez-Alvarez, Alberto; Genis, Laura; Bascuñana, Pablo; Fernandez de la Rosa, Ruben; Delgado, Mercedes; Angel Pozo, Miguel; Moreno, Estefania; McCormick, Peter J; Santi, Andrea; Trueba-Saiz, Angel; Garcia-Caceres, Cristina; Tschöp, Matthias H; Araque, Alfonso; Martin, Eduardo D; Torres Aleman, Ignacio

    2016-11-01

    Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971. © 2016 Wiley Periodicals, Inc.

  2. Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes

    PubMed Central

    Ajit, Deepa; Simonyi, Agnes; Li, Runting; Chen, Zihong; Hannink, Mark; Fritsche, Kevin L.; Mossine, Valeri V.; Smith, Robert E.; Dobbs, Thomas K.; Luo, Rensheng; Folk, William R.; Gu, Zezong; Lubahn, Dennis B.; Weisman, Gary A.; Sun, Grace Y.

    2016-01-01

    The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemiscals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promotor activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells. PMID:27166148

  3. Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor.

    PubMed

    Van Wagoner, N J; Oh, J W; Repovic, P; Benveniste, E N

    1999-07-01

    In the CNS, astrocytes are a major inducible source of interleukin-6 (IL-6). Although IL-6 has beneficial effects in the CNS because of its neurotrophic properties, its overexpression is generally detrimental, adding to the pathophysiology associated with CNS disorders. Many factors have been shown to induce IL-6 expression by astrocytes, particularly the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). However, the role of IL-6 in its own regulation in astrocytes has not been determined. In this study, we examined the influence of IL-6 alone or in combination with TNF-alpha or IL-1beta on IL-6 expression. IL-6 alone had no effect on IL-6 expression; however, the addition of the soluble IL-6 receptor (sIL-6R) induced IL-6 transcripts. Addition of TNF-alpha or IL-1beta plus IL-6/sIL-6R led to synergistic increases in IL-6 expression. This synergy also occurred in the absence of exogenously added IL-6, attributable to TNF-alpha- or IL-1beta-induced endogenous IL-6 protein production. IL-6 upregulation seen in the presence of TNF-alpha or IL-1beta plus IL-6/sIL-6R was transcriptional, based on nuclear run-on analysis. Experiments were extended to other IL-6 family members to determine their role in IL-6 regulation in astrocytes. Oncostatin M (OSM) induced IL-6 alone and synergized with TNF-alpha for enhanced expression. These results demonstrate that IL-6/sIL-6R and OSM play an important role in the regulation of IL-6 expression within the CNS, particularly in conjunction with the proinflammatory cytokines TNF-alpha and IL-1beta.

  4. Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

    PubMed

    Ajit, Deepa; Simonyi, Agnes; Li, Runting; Chen, Zihong; Hannink, Mark; Fritsche, Kevin L; Mossine, Valeri V; Smith, Robert E; Dobbs, Thomas K; Luo, Rensheng; Folk, William R; Gu, Zezong; Lubahn, Dennis B; Weisman, Gary A; Sun, Grace Y

    2016-07-01

    The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells. Copyright © 2016 Elsevier Ltd

  5. Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

    PubMed Central

    Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

    2013-01-01

    The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in

  6. Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop*

    PubMed Central

    Quintero, Omar A.; Unrath, William C.; Stevens, Stanley M.; Manor, Uri; Kachar, Bechara; Yengo, Christopher M.

    2013-01-01

    Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells. PMID:24214986

  7. p53-independent mechanisms regulate the P2-MDM2 promoter in adult astrocytic tumours.

    PubMed

    Dimitriadi, M; Poulogiannis, G; Liu, L; Bäcklund, L M; Pearson, D M; Ichimura, K; Collins, V P

    2008-10-07

    The MDM2 gene is amplified and/or overexpressed in about 10% of glioblastomas and constitutes one of a number of ways the p53 pathway is disrupted in these tumours. MDM2 encodes a nuclear phosphoprotein that regulates several cell proteins by binding and/or ubiquitinating them, with p53 being a well-established partner. MDM2 has two promoters, P1 and P2 that give rise to transcripts with distinct 5' untranslated regions. Transcription from P2 is believed to be controlled by p53 and a single-nucleotide polymorphism (SNP309, T>G) in P2 is reported to be associated with increased risk for, and early development of, malignancies. The use of P1 and P2 has not been investigated in gliomas. We used RT-PCR to study P1- and P2-MDM2 transcript expression in astrocytic tumours, xenografts and cell lines with known MDM2, TP53 and p14(ARF) gene status. Both promoters were used in all genetic backgrounds including the use of the P2 promoter in TP53 null cells, indicating a p53-independent induction of transcription. Transcripts from the P1 promoter formed a greater proportion of the total MDM2 transcripts in tumours with MDM2 amplification, despite these tumours having two wild-type TP53 alleles. Examination of SNP309 in glioblastoma patients showed a borderline association with survival but no apparent correlation with age at diagnosis nor with TP53 and p14(ARF) status of their tumours. Our findings also indicate that elevated MDM2 mRNA levels in tumours with MDM2 amplification are preferentially driven by the P1 promoter and that the P2 promoter is not only regulated by p53 but also by other transcription factor(s).

  8. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  9. DJ-1 deficiency impairs glutamate uptake into astrocytes via the regulation of flotillin-1 and caveolin-1 expression

    PubMed Central

    Kim, Jin-Mo; Cha, Seon-Heui; Choi, Yu Ree; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2016-01-01

    Parkinson’s disease (PD) is a common chronic and progressive neurodegenerative disorder. Although the cause of PD is still poorly understood, mutations in many genes including SNCA, parkin, PINK1, LRRK2, and DJ-1 have been identified in the familial forms of PD. It was recently proposed that alterations in lipid rafts may cause the neurodegeneration shown in PD. Here, we observe that DJ-1 deficiency decreased the expression of flotillin-1 (flot-1) and caveolin-1 (cav-1), the main protein components of lipid rafts, in primary astrocytes and MEF cells. As a mechanism, DJ-1 regulated flot-1 stability by direct interaction, however, decreased cav-1 expression may not be a direct effect of DJ-1, but rather as a result of decreased flot-1 expression. Dysregulation of flot-1 and cav-1 by DJ-1 deficiency caused an alteration in the cellular cholesterol level, membrane fluidity, and alteration in lipid rafts-dependent endocytosis. Moreover, DJ-1 deficiency impaired glutamate uptake into astrocytes, a major function of astrocytes in the maintenance of CNS homeostasis, by altering EAAT2 expression. This study will be helpful to understand the role of DJ-1 in the pathogenesis of PD, and the modulation of lipid rafts through the regulation of flot-1 or cav-1 may be a novel therapeutic target for PD. PMID:27346864

  10. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  11. Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

    PubMed Central

    Paulsen, Friedrich; Hammer, Christian M.

    2014-01-01

    The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation. PMID:24718314

  12. Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures

    PubMed Central

    Jayakumar, Arumugam R.; Curtis, Kevin M.; Panickar, Kiran S.; Shamaladevi, Nagarajarao; Norenberg, Michael D.

    2016-01-01

    Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure. PMID:27918421

  13. Acute treatment with 17beta-estradiol attenuates astrocyte-astrocyte and astrocyte-neuron communication.

    PubMed

    Rao, Shilpa P; Sikdar, Sujit Kumar

    2007-12-01

    Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control.

  14. Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation12

    PubMed Central

    Berezovsky, Artem D.; Poisson, Laila M.; Cherba, David; Webb, Craig P.; Transou, Andrea D.; Lemke, Nancy W.; Hong, Xin; Hasselbach, Laura A.; Irtenkauf, Susan M.; Mikkelsen, Tom; deCarvalho, Ana C.

    2014-01-01

    The high-mobility group–box transcription factor sex-determining region Y–box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications. PMID:24726753

  15. Sox2 promotes malignancy in glioblastoma by regulating plasticity and astrocytic differentiation.

    PubMed

    Berezovsky, Artem D; Poisson, Laila M; Cherba, David; Webb, Craig P; Transou, Andrea D; Lemke, Nancy W; Hong, Xin; Hasselbach, Laura A; Irtenkauf, Susan M; Mikkelsen, Tom; deCarvalho, Ana C

    2014-03-01

    The high-mobility group-box transcription factor sex-determining region Y-box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  17. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  18. SNAT3-mediated glutamine transport in perisynaptic astrocytes in situ is regulated by intracellular sodium.

    PubMed

    Todd, Alison C; Marx, Mari-Carmen; Hulme, Sarah R; Bröer, Stefan; Billups, Brian

    2017-06-01

    The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pHi and Nai+. Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (KM of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na(+) ]i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na(+) ]i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na(+) ]i . These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic Nai+ signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling. © 2017 Wiley Periodicals, Inc.

  19. Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes

    PubMed Central

    Tham, Daniel Kai Long; Joshi, Bharat; Moukhles, Hakima

    2016-01-01

    The water-permeable channel aquaporin-4 (AQP4) is highly expressed in perivascular astrocytes of the mammalian brain and represents the major conduit for water across the blood-brain barrier. Within these cells, AQP4 is found in great quantities at perivascular endfoot sites but is detected in lesser amounts at the membrane domains within the brain parenchyma. We had previously established that this polarization was regulated by the interaction between dystroglycan (DG), an extracellular matrix receptor that is co-expressed with AQP4, and the laminin that is contained within the perivascular basal lamina. In the present study, we have attempted to describe the mechanisms that underlie this regulation, using primary astrocyte cultures. Via biotinylation, we found that the cell-surface expression of AQP4 is DG-dependent and is potentiated by laminin. We also determined that this laminin-dependent increase occurs not through an upregulation of total AQP4 levels, but rather from a redirection of AQP4 from an intracellular, EEA-1-associated pool to the cell surface. We then demonstrated an association between DG and dynamin and showed that dynamin functioned in conjunction with clathrin to regulate surface AQP4 amounts. Furthermore, we observed that DG preferentially binds to the inactive forms of dynamin, suggesting that this interaction was inhibitory for AQP4 endocytosis. Finally, we showed that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4. Our data therefore indicate that the dual interation of DG with laminin and dynamin is involved in the regulation of AQP4 internalization, leading to its asymmetric enrichment at perivascular astrocyte endfeet. PMID:27788222

  20. Protein kinaseregulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  1. Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

    PubMed Central

    Akimoto, K; Nakaya, M; Yamanaka, T; Tanaka, J; Matsuda, S; Weng, Q P; Avruch, J; Ohno, S

    1998-01-01

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression. PMID:9761742

  2. Regulation and Function of the Ipl1/Aurora Kinase

    DTIC Science & Technology

    2004-05-01

    S., et al., The Budding Yeast Iplip/Aurora Protein Kinase Regulates Mitotic Spindle Disassembly. I. Cell Biol., 2003. 160: 329-339. 9. Desai, A., et...Two Sacchromyces cerevisiae kinesin-related gene-products required for mitotic spindle assembly. J. Cell Biol., 1992. 118:109-120 9 Appendix Training ...destabilizing kinesin, are delayed in spindle breakdown similar to ipll mutants. It is possible that Ipllp regulates spindle breakdown by directly regulating

  3. Tenascin-C regulates proliferation and migration of cultured astrocytes in a scratch wound assay.

    PubMed

    Nishio, T; Kawaguchi, S; Yamamoto, M; Iseda, T; Kawasaki, T; Hase, T

    2005-01-01

    Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Astrocytes are known to play major roles in wound healing in the CNS. To elucidate the roles of TNC in wound closure by astrocytes, we have examined the morphological changes of cultured astrocytes in a scratch wound assay and measured the content of soluble TNC released into the medium. We have also localized the expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP), vimentin and integrin beta1. After wounding, glial cells rapidly released the largest TNC isoform and proliferated in the border zones. Subsequently, they became polarized with unidirectional processes and finally migrated toward the denuded area. The proliferating border zone cells and pre-migratory cells intensely expressed TNC mRNA, TNC-, vimentin-, GFAP- and integrin beta1-like immunoreactivity, while the migratory cells showed generally reduced expression except the front. Exogenous TNC enhanced cell proliferation and migration, while functional blocking with anti-TNC or anti-integrin beta1 antibody reduced both of them. These results suggest that mechanical injury induces boundary astrocytes to produce and release TNC that promotes cell proliferation and migration via integrin beta1 in an autocrine/paracrine fashion.

  4. Astrocyte development: A Guide for the Perplexed.

    PubMed

    Molofsky, Anna Victoria; Deneen, Benjamin

    2015-08-01

    Astrocytes are the predominant cell type in the brain and perform key functions vital to CNS physiology, including blood brain barrier formation and maintenance, synaptogenesis, neurotransmission, and metabolic regulation. To fully understand the contributions of astrocytes to brain function, it will be important to bridge the existing gap between development and physiology. In this review, we provide an overview of Astrocyte development, including recent insights into molecular mechanisms of astrocyte specification, regional patterning and proliferation. This developmental perspective is complemented with recent findings that describe the functional maturation of astrocytes and their prospective diversity. Future progress in understanding Astrocyte development will depend on the development of astrocyte- stage specific markers and tools for manipulating astrocytes without affecting neuron production. Ultimately, a mechanistic approach to Astrocyte development will be crucial to developing new treatments for the many neurodevelopmental, neurodegenerative, neuroimmune, and neoplastic diseases involving astrocyte dysfunction.

  5. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

    PubMed

    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  6. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice

    PubMed Central

    Berta, Temugin; Liu, Yen-Chin; Xu, Zhen-Zhong; Ji, Ru-Rong

    2013-01-01

    Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA−/−) mice and astrocyte cultures. tPA−/− mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10 mg/kg/day, s.c.), is abrogated in tPA−/− mice. Chronic morphine induces tPA expression in GFAP-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of IBA-1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance. PMID:23707980

  7. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice.

    PubMed

    Berta, T; Liu, Y-C; Xu, Z-Z; Ji, R-R

    2013-09-05

    Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.

  8. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    PubMed

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Glucocorticoid receptor β regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of β-catenin/TCF transcriptional activity.

    PubMed

    Yin, Ying; Zhang, Xiufen; Li, Zaiwang; Deng, Lingxiao; Jiao, Guoqing; Zhang, Bin; Xie, Ping; Mu, Huijun; Qiao, Weizhen; Zou, Jian

    2013-11-01

    Astrocytes react to central nervous system (CNS) injury and participate in gliotic responses, imparting negative, as well as positive effects on axonal regeneration. Despite the considerable biochemical and morphological changes astrocytes undergo following insult, and the known influence of steroids on glial activation, details surrounding glucocorticoid receptor expression and activity are lacking. Such mechanistic information is essential for advancing and enhancing therapies in the treatment of CNS injuries. Using an in vitro wound-healing assay, we found glucocorticoid receptor β (GRβ), not GRα, is upregulated and acts as a regulator of gliosis after injury. In addition, our results suggest that GRβ interacts with β-catenin and is a necessary component for proliferation and migration in both injured astrocytes and glioma cells. Further analysis indicated GRβ/β-catenin interaction as a key modulator of astrocyte reactivity through sustained Wnt/β-catenin/TCF signaling in its dominant-negative effect on GRα mediated trans-repression by a GSK-3β-independent manner. These findings expand our knowledge of the mechanism of GRβ action in promoting astrocyte proliferation and migration following injury and in glioma. This information furthers our understanding the function of glucocorticoid receptor in CNS injury and disease, as well as in the basic biochemical responses astrocytes undergo in response to injury and glioma pathogenesis.

  10. Purines regulate adult brain subventricular zone cell functions: contribution of reactive astrocytes.

    PubMed

    Boccazzi, Marta; Rolando, Chiara; Abbracchio, Maria P; Buffo, Annalisa; Ceruti, Stefania

    2014-03-01

    Brain injuries modulate activation of neural stem cells (NSCs) in the adult brain. In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, contribute to reactive gliosis, and possibly directly affect subventricular zone (SVZ) cell functioning. Among eNTs and derived metabolites, the P2Y1 receptor agonist ADP strongly promotes astrogliosis and might also influence SVZ progenitor activity. Here, we tested the ability of the stable P2Y1 agonist adenosine 5'-O-(2-thiodiphosphate) (ADPβS) to control adult NSC functions both in vitro and in vivo, with a focus on the possible effects exerted by reactive astrocytes. In the absence of growth factors, ADPβS promoted proliferation and differentiation of SVZ progenitors. Moreover, ADPβS-activated astrocytes markedly changed the pattern of released cytokines and chemokines, and strongly modulated neurosphere-forming capacity of SVZ progenitors. Notably, a significant enhancement in proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were shifted to normal medium. In vivo, ADPβS administration in the lateral ventricle of adult mice by osmotic minipumps caused diffused reactive astrogliosis, and a strong response of SVZ progenitors. Indeed, proliferation of glial fibrillary acidic protein-positive NSCs increased and led to a significant expansion of SVZ transit-amplifying progenitors and neuroblasts. Lineage tracing experiments performed in the GLAST::CreERT2;Rosa-YFP transgenic mice further demonstrated that ADPβS promoted proliferation of glutamate/aspartate transporter-positive progenitors and sustained their progression toward the generation of rapidly dividing progenitors. Altogether, our results show that the purinergic system crucially affects SVZ progenitor activities both directly and through the involvement of reactive astrocytes.

  11. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

    PubMed

    Baer, Matthew L; Henderson, Scott C; Colello, Raymond J

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.

  12. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

    PubMed Central

    Baer, Matthew L.; Henderson, Scott C.; Colello, Raymond J.

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  13. Structure, Regulation, Signaling, and Targeting of Abl Kinases in Cancer

    PubMed Central

    2012-01-01

    Abl kinases are prototypic cytoplasmic tyrosine kinases and are involved in a variety of chromosomal aberrations in different cancers. This causes the expression of Abl fusion proteins, such as Bcr-Abl, that are constitutively activated and drivers of tumorigenesis. Over the past decades, biochemical and functional studies on the molecular mechanisms of Abl regulation have gone hand in hand with progression of our structural understanding of autoinhibited and active Abl conformations. In parallel, Abl oncoproteins have become prime molecular targets for cancer therapy, using adenosine triphosphate (ATP)–competitive kinase inhibitors, such as imatinib. Abl-targeting drugs serve as a paradigm for our understanding of kinase inhibitor action, specificity, and resistance development. In this review article, I will review the molecular mechanisms that are responsible for the regulation of Abl kinase activity and how oncogenic Abl fusions signal. Furthermore, past and ongoing efforts to target Abl oncoproteins using ATP-competitive and allosteric inhibitors, as well as future possibilities using combination therapy, will be discussed. PMID:23226581

  14. Kinase Regulation by Hydrophobic Spine Assembly in Cancer

    PubMed Central

    Ahuja, Lalima G.; Meharena, Hiruy S.; Kannan, Natarajan; Kornev, Alexandr P.

    2014-01-01

    A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation. PMID:25348715

  15. Kinase regulation by hydrophobic spine assembly in cancer.

    PubMed

    Hu, Jiancheng; Ahuja, Lalima G; Meharena, Hiruy S; Kannan, Natarajan; Kornev, Alexandr P; Taylor, Susan S; Shaw, Andrey S

    2015-01-01

    A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation.

  16. Expression and function of membrane regulators of complement on rat astrocytes in culture.

    PubMed Central

    Rogers, C A; Gasque, P; Piddlesden, S J; Okada, N; Holers, V M; Morgan, B P

    1996-01-01

    Human astrocytes express CD59, decay accelerating factor and membrane cofactor protein to restrict the damaging effect of complement (C) activation on their cell surface. 5I2 antigen (5I2 Ag) is the functional analogue of the latter two proteins in rats. We here demonstrate the surface expression on rat astrocytes of CD59 and 5I2 Ag and use sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting to confirm their identity and to quantify expression. Rat CD59 (MW 20,000) was expressed at 720 x 10(3) molecules per cell and 5I2 Ag (MW 58,000 and 64,000) at 625 x 10(3) molecules per cell. Reverse transcription-polymerase chain reaction using specific oligonucleotide primers demonstrated expression of mRNA for each protein. Twenty-four-hour stimulation with inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukins-1 beta, -2 and -6) or phorbol myristate acetate had no significant effect on the level of expression of either protein as determined by Western blotting. Lysis caused by classical pathway activation of C in human or rat serum was enhanced by blocking the function of CD59 and 5I2 Ag on rat astrocytes with monoclonal antibodies. Images Figure 2 Figure 3 Figure 4 PMID:8707343

  17. Relationship between L-glutamate-regulated intracellular Na+ dynamics and ATP hydrolysis in astrocytes.

    PubMed

    Magistretti, P J; Chatton, J-Y

    2005-01-01

    Glutamate uptake into astrocytes and the resulting increase in intracellular Na+ (Na+(i)) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na+/K+-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg2+ (Mg2+(i)), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg2+(i) (i.e. decreased ATP), which was reversible after glutamate washout. Na+(i) and ATP changes were then directly compared by simultaneous Na+(i) and Mg2+ imaging. Glutamate increased both parameters with different rates and blocking the Na+/K+-ATPase during the glutamate-evoked Na+(i) response, resulted in a drop of Mg2+(i) levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na+ homeostasis and ATP levels in astrocytes.

  18. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    PubMed

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

  19. ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex

    PubMed Central

    Lalo, U.; Palygin, O.; Verkhratsky, A.; Grant, S. G. N.; Pankratov, Y.

    2016-01-01

    Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca2+-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca2+-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity. PMID:27640997

  20. Sevoflurane Inhibits Glutamate-Aspartate Transporter and Glial Fibrillary Acidic Protein Expression in Hippocampal Astrocytes of Neonatal Rats Through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) Pathway.

    PubMed

    Wang, Wei; Lu, Rui; Feng, Da-Yun; Zhang, Hui

    2016-07-01

    The mechanisms underlying general anesthesia-induced neurotoxicity are unclear. Astrocytes have been recognized as important contributors to neuronal development. Until now, the response of the astrocytes to neonatal general anesthetic exposure has been unreported. Postnatal day 7 rats received 2.5% sevoflurane for 6 hours. Expressions of glial fibrillary acidic protein (GFAP) and glutamate-aspartate transporter (GLAST) and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway were detected on days 1, 3, 7, and 14 after sevoflurane inhalation. In addition, cultured astrocytes were exposed to 2.5% sevoflurane for 2 hours and GFAP, GLAST expressions, and JAK/STAT phosphorylation were evaluated. Furthermore, we pharmacologically disrupted JAK/STAT signaling in vivo by treatment with the JAK/STAT inhibitor AG490 and in vitro by treatment with JAK inhibitor I to detect the consequent expression of GFAP and GLAST. Sevoflurane induced a robust decrease of GFAP and GLAST expression in hippocampal tissue compared with sham control groups at 1 to 14 days after sevoflurane exposure. Immunohistochemistry showed colocalization of GFAP, GLAST, and pSTAT3 in the hippocampal CA1 region. Western blot analysis also revealed a significant decrease of pJAK1, pJAK2, and pSTAT3 in the sevoflurane group. In vitro study showed that GFAP, GLAST, pJAK1, pJAK2, and pSTAT3 expressions in cultured astrocytes were remarkably decreased at 24 to 48 hours after sevoflurane treatment. Either AG490 or JAK inhibitor I significantly decreased expressions of GFAP and GLAST in hippocampus or cultured astrocytes. Astrocytic GLAST was inhibited by sevoflurane in the hippocampus of neonatal rats. Inactivation of the JAK/STAT pathway possibly contributes to this effect of sevoflurane. Astrocytic dysfunction induced by sevoflurane may contribute to its neurotoxicity in the developing brain.

  1. Spinal astrocytic c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the amitriptyline analgesic efficacy in painful peripheral neuropathies.

    PubMed

    Sanna, Maria Domenica; Ghelardini, Carla; Galeotti, Nicoletta

    2017-03-05

    Several drugs and agents are currently used for the treatment of neuropathic pain. Among them amitriptyline, a tricyclic antidepressant drug, represent a first line treatment. Despite its well-documented clinical efficacy, amitriptyline is ineffective in some animal models of neuropathic pain. The aim of this study was to investigate into amitriptyline poor efficacy in neuropathic pain and to determine the role of c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the analgesic effects of this drug. Experiments were performed in mice with painful peripheral neuropathies due to the antiretroviral agent 2,3-dideoxycytidine (ddC), and with the partial sciatic nerve injury produced in the spared nerve injury model (SNI). In mice subjected to SNI and antiretroviral treatment, amitriptyline did not attenuate mechanical allodynia and thermal hyperalgesia. Conversely, intrathecal injection of the JNK inhibitor SP600125 prevented SNI and ddC-induced nociceptive behavior and, its inactive dose co-administrated with amitriptyline induced an antinociceptive effect. Western blotting analysis showed an upregulation of p-JNK in the lumbar spinal cord of SNI and ddC-exposed mice, that was further enhanced after amitriptyline administration. Additionally, amitriptyline further promoted astrocyte activation in neuropathic mice, as illustrated by the increased expression of glial fibrillary acidic protein (GFAP), that was attenuated by intrathecal injection of the JNK inhibitor. These data indicate astrocyte JNK activation as counteracting pathway to amitriptyline analgesic response. Targeting the JNK pathway in spinal astroglia may present an efficient way to improve the analgesic efficacy of amitriptyline in the neuropathic pain treatment.

  2. Kinases, tails and more: regulation of PTEN function by phosphorylation.

    PubMed

    Fragoso, Rita; Barata, João T

    2015-05-01

    Phosphorylation regulates the conformation, stability, homo- and heterotypic protein interactions, localization, and activity of the tumor suppressor PTEN. From a simple picture, at the beginning of this millennium, recognizing that CK2 phosphorylated PTEN at the C-terminus and thereby impacted on PTEN stability and activity, research has led to a significantly more complex scenario today, where for instance GSK3, Plk3, ATM, ROCK or Src-family kinases are also gaining the spotlight in this evolving play. Here, we review the current knowledge on the kinases that phosphorylate PTEN, and on the impact that specific phosphorylation events have on PTEN function. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. A molecular ruler regulates cytoskeletal remodelling by the Rho kinases

    PubMed Central

    Truebestein, Linda; Elsner, Daniel J.; Fuchs, Elisabeth; Leonard, Thomas A.

    2015-01-01

    The Rho-associated coiled-coil kinases (ROCK) are essential regulators of the actin cytoskeleton; however, the structure of a full-length ROCK is unknown and the mechanisms by which its kinase activity is controlled are not well understood. Here we determine the low-resolution structure of human ROCK2 using electron microscopy, revealing it to be a constitutive dimer, 120 nm in length, with a long coiled-coil tether linking the kinase and membrane-binding domains. We find, in contrast to previous reports, that ROCK2 activity does not appear to be directly regulated by binding to membranes, RhoA, or by phosphorylation. Instead, we show that changing the length of the tether modulates ROCK2 function in cells, suggesting that it acts as a molecular ruler. We present a model in which ROCK activity is restricted to a discrete region of the actin cytoskeleton, governed by the length of its coiled-coil. This represents a new type of spatial control, and hence a new paradigm for kinase regulation. PMID:26620183

  4. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    PubMed

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  5. Heterogeneity of reactive astrocytes

    PubMed Central

    Anderson, Mark A.; Ao, Yan; Sofroniew, Michael V.

    2014-01-01

    Astrocytes respond to injury and disease in the central nervous system (CNS) with a process referred to as reactive astrogliosis. Recent progress demonstrates that reactive astrogliosis is not a simple all-or-none phenomenon, but is a finely gradated continuum of changes that range from reversible alterations in gene expression and cell hypertrophy, to scar formation with permanent tissue rearrangement. There is now compelling evidence that reactive astrocytes exhibit a substantial potential for heterogeneity at multiple levels, including gene expression, cell morphology, topography (distance from lesions), CNS regions, local (among neighboring cells), cell signaling and cell function. Structural and functional changes are regulated in reactive astrocytes by many different potential signaling events that occur in a context dependent manner. It is noteworthy that different stimuli of astrocyte reactivity can lead to similar degrees of GFAP upregulation while causing substantially different changes in transcriptome profiles and cell function. Thus, it is not possible to equate simple and uniform measures such as cell hypertrophy and upregulation of GFAP expression with a single, uniform concept of astrocyte reactivity. Instead, it is necessary to recognize the considerable potential for heterogeneity and determine the functional implications of astrocyte reactivity in a context specific manner as regulated by specific signaling events. PMID:24361547

  6. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    SciTech Connect

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  7. Bidirectional Regulation of Neutrophil Migration by MAP Kinases

    PubMed Central

    Liu, Xiaowen; Ma, Bo; Malik, Asrar B.; Tang, Haiyang; Yang, Tao; Sun, Bo; Wang, Gang; Minshall, Richard D.; Li, Yan; Zhao, Yong; Ye, Richard D.; Xu, Jingsong

    2012-01-01

    To kill invading bacteria, neutrophils must interpret spatial cues, migrate, and reach target sites. Although initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we report that two mitogen-activated protein kinases played opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase (Erk) potentiated G protein-coupled receptor kinase GRK2 activity and inhibited neutrophil migration, whereas p38 MAPK acted as a non-canonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 MAPK controls neutrophil “stop” and “go” behaviors, ensuring neutrophils precisely reach their final destination as the first line of host-defense. PMID:22447027

  8. Mnk Kinases in Cytokine Signaling and Regulation of Cytokine Responses

    PubMed Central

    Joshi, Sonali; Platanias, Leonidas C.

    2013-01-01

    The kinases Mnk1 and Mnk2 are activated downstream of the p38 MAPK and MEK/ERK signaling pathways. Extensive work over the years has shown that these kinases control phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and regulate engagement of other effector elements, including hnRNPA1 and PSF. Mnk kinases are ubiquitously expressed and play critical roles in signaling for various cytokine receptors, while there is emerging evidence that they have important functions as mediators of pro-inflammatory cytokine production. In this review the mechanisms of activation of MNK pathways by cytokine receptors are addressed and their roles in diverse cytokine-dependent biological processes are reviewed. The clinical-translational implications of such work and the relevance of future development of specific MNK inhibitors for the treatment of malignancies and auto-immune disorders are discussed. PMID:23710261

  9. Sex hormone-dependent attenuation of EAE in a transgenic mouse with astrocytic expression of the RNA regulator HuR.

    PubMed

    Wheeler, Crystal; Nabors, L Burt; Barnum, Scott; Yang, Xiuhua; Hu, Xianzhen; Schoeb, Trenton R; Chen, Dongquan; Ardelt, Agnieszka A; King, Peter H

    2012-05-15

    In experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative diseases, astrocytes play an important role in promoting or attenuating the inflammatory response through induction of different cytokines and growth factors. HuR plays a major role in regulating many of these factors by modulating RNA stability and translational efficiency. Here, we engineered transgenic mice to express HuR in astrocytes using the human glial fibrillary acidic protein promoter and found that female transgenic mice had significantly less clinical disability and histopathological changes in the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our findings support a role for the astrocyte and posttranscriptional regulation in hormonally-mediated attenuation of EAE.

  10. Sex Hormone-Dependent Attenuation of EAE in a Transgenic Mouse with Astrocytic Expression of the RNA Regulator HuR

    PubMed Central

    Wheeler, Crystal; Nabors, L. Burt; Barnum, Scott; Yang, Xiuhua; Hu, Xianzhen; Schoeb, Trenton; Chen, Dongquan; Ardelt, Anise A.; King, Peter H.

    2012-01-01

    In experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative diseases, astrocytes play an important role in promoting or attenuating the inflammatory response through induction of different cytokines and growth factors. HuR plays a major role in regulating many of these factors by modulating RNA stability and translational efficiency. Here, we engineered transgenic mice to express HuR in astrocytes using the human glial fibrillary acidic protein promoter and found that female transgenic mice had significantly less clinical disability and histopathological changes in the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our findings support a role for the astrocyte and posttranscriptional regulation in hormonally-mediated attenuation of EAE. PMID:22445740

  11. Cytosolic phospholipase A2 regulates alcohol-mediated astrocyte inflammatory responses in HIV-associated neurocognitive disorders

    PubMed Central

    Pandey, R; Ghorpade, A

    2015-01-01

    Alcohol (EtOH) abuse and HIV-1 infection remain leading public health problems not only in the United States but also across the world. Alcohol abusers have a significantly greater risk of HIV-1 infection than non-drinkers globally. In the United States, prevalence of EtOH abuse is over two-fold higher in HIV-1-positive individuals than that of the general population. Although alcohol abusers show neurodegeneration, exacerbated neuroinflammation and oxidative damage, the mechanism(s) by which EtOH regulates astrocyte inflammatory responses in HIV-associated neurocognitive disorders is unknown. Thus, we explored signaling pathway(s) involved in EtOH-mediated activation of human astrocytes with HIV-1 and subsequent alterations in their inflammatory functions. Alcohol exposure altered the morphology of astrocytes, proinflammatory responses and induced cytotoxicity in a dose-dependent manner. Time-dependent changes were also evaluated. EtOH and HIV-1 cotreatment decreased cell viability and proliferation, while increasing apoptosis and mitochondrial depolarization. EtOH and HIV-1 together increased the levels of proinflammatory molecules, interleukin-1β, tumor necrosis factor-α, CXCL8, tissue inhibitor of metalloproteinases-1 and more importantly, arachidonic acid, a known downstream target of cytosolic phospholipase A2 (cPLA2). Consistent with this observation, phospho-cPLA2 levels were augmented in HIV-1 and EtOH cotreatment as compared with HIV-1 or EtOH alone. Cyclooxygenase 2 was upregulated as measured by real-time PCR and western blot, whereas cotreatment of HIV-1 and EtOH decreased cytochrome P450-2E1 levels as compared with EtOH alone. Furthermore, we confirmed that blocking cPLA2 with arachidonyl tri floro methyl ketone, a cPLA2-specific inhibitor, effectively prevented cPLA2 phosphorylation and downstream outcomes. Thus, the present findings suggest that cPLA2 has a critical role in alcohol and HIV-induced astrocyte inflammation. In the future, cPLA2

  12. Kinase regulation by sulfur and selenium containing compounds.

    PubMed

    Sanmartín, Carmen; Plano, Daniel; Font, María; Palop, Juan Antonio

    2011-05-01

    Kinases are enzymes that are involved in a wide-range of cellular targets such as cell proliferation, metabolism, survival and apoptosis. Aberrations in the activity of the kinases have been linked to many human diseases such as diabetes, inflammation and cancer. The discovery of more than 518 kinases encoded by the human genome has spurred the development of rapid screening techniques for potential drugs against these enzymes and these have been identified as interesting targets for medicinal chemistry programs, especially in cancer therapy. On the other hand, sulfur and selenium have been increasingly recognized as essential elements in biology and medicine. Converging data from epidemiological and clinical studies have highlighted these elements as effective chemopreventive agents, particularly against various types of cancer (prostate, lung, breast, leukemia, colon, skin, lymphome, thyroid, pancreas, liver). These elements act through a wide range of potential mechanisms where one identified signal pathway event is kinase modulation, which is common for the two elements and emerges as a valid target. The kinases modulated by sulfur and selenium derivatives include MAP, ERK, JNK, Akt, Cdc2, Cyclin B1 and Cdc25c amongst others. Although both of the elements in question are in the same group in the periodic table and have similar biochemistries, there are relevant differences related to redox potentials, stabilities, oxidation states and anticancer activity. Literature data suggest that the replacement of sulfur by selenium in established cancer chemopreventive agents results in more effective chemopreventive analogs. In view of the multi-target kinase mechanisms in preventing cellular transformation, as well as the differences and similarities between them, in this review we focus on the development of new structures that contain selenium and/or sulfur and discuss our understanding of the regulation of antitumoral effects with emphasis on kinase modulation

  13. Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex.

    PubMed Central

    Pawelczyk, T; Olson, M S

    1992-01-01

    The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo. PMID:1463442

  14. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  15. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

    PubMed

    Honsa, Pavel; Valny, Martin; Kriska, Jan; Matuskova, Hana; Harantova, Lenka; Kirdajova, Denisa; Valihrach, Lukas; Androvic, Peter; Kubista, Mikael; Anderova, Miroslava

    2016-09-01

    NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531.

  16. DJ-1 associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes.

    PubMed

    Kim, Kwang Soo; Kim, Jin Soo; Park, Ji-Young; Suh, Young Ho; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2013-12-01

    Parkinson's disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

  17. Cell fate regulation governed by a repurposed bacterial histidine kinase

    SciTech Connect

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  18. Cell fate regulation governed by a repurposed bacterial histidine kinase

    DOE PAGES

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; ...

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

  19. Spatially coordinated kinase signaling regulates local axon degeneration.

    PubMed

    Chen, Mark; Maloney, Janice A; Kallop, Dara Y; Atwal, Jasvinder K; Tam, Stephen J; Baer, Kristin; Kissel, Holger; Kaminker, Joshua S; Lewcock, Joseph W; Weimer, Robby M; Watts, Ryan J

    2012-09-26

    In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3β, we show that the β isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3β in axon degeneration.

  20. Regulation of Aurora-A kinase on the mitotic spindle.

    PubMed

    Kufer, Thomas A; Nigg, Erich A; Silljé, Herman H W

    2003-12-01

    The error-free segregation of duplicated chromosomes during cell division is essential for the maintenance of an intact genome. This process is brought about by a highly dynamic bipolar array of microtubules, the mitotic spindle. The formation and function of the mitotic spindle during M-phase of the cell cycle is regulated by protein phosphorylation, involving multiple protein kinases and phosphatases. Prominent among the enzymes implicated in spindle assembly is the serine/threonine-specific protein kinase Aurora-A. In several common human tumors, Aurora-A is overexpressed, and deregulation of this kinase was shown to result in mitotic defects and aneuploidy. Moreover, recent genetic evidence directly links the human Aurora-A gene to cancer susceptibility. Several of the physiological substrates of Aurora-A presumably await identification, but recent studies are beginning to shed light on the regulation of this critical mitotic kinase. Here, we review these findings with particular emphasis on the role of TPX2, a prominent spindle component implicated in a Ran-GTP-mediated spindle assembly pathway.

  1. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  2. The molecular regulation of Janus kinase (JAK) activation

    PubMed Central

    2014-01-01

    The Janus Kinase (JAK) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2, was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haematopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organisation of the component FERM-SH2, pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain containing proteins, SOCS (Suppressors of Cytokine Signalling) proteins and Lnk. These recent advances highlight the diversity of regulatory mechanisms utilised by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  3. Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

    PubMed Central

    Bermingham, Daniel P.

    2016-01-01

    Modulation of neurotransmission by the monoamines dopamine (DA), norepinephrine (NE), and serotonin (5-HT) is critical for normal nervous system function. Precise temporal and spatial control of this signaling in mediated in large part by the actions of monoamine transporters (DAT, NET, and SERT, respectively). These transporters act to recapture their respective neurotransmitters after release, and disruption of clearance and reuptake has significant effects on physiology and behavior and has been linked to a number of neuropsychiatric disorders. To ensure adequate and dynamic control of these transporters, multiple modes of control have evolved to regulate their activity and trafficking. Central to many of these modes of control are the actions of protein kinases, whose actions can be direct or indirectly mediated by kinase-modulated protein interactions. Here, we summarize the current state of our understanding of how protein kinases regulate monoamine transporters through changes in activity, trafficking, phosphorylation state, and interacting partners. We highlight genetic, biochemical, and pharmacological evidence for kinase-linked control of DAT, NET, and SERT and, where applicable, provide evidence for endogenous activators of these pathways. We hope our discussion can lead to a more nuanced and integrated understanding of how neurotransmitter transporters are controlled and may contribute to disorders that feature perturbed monoamine signaling, with an ultimate goal of developing better therapeutic strategies. PMID:27591044

  4. Src tyrosine kinase regulates adhesion and chemotaxis in Waldenstrom Macroglobulinemia

    PubMed Central

    Ngo, Hai T.; Azab, Abdel Kareem; Farag, Mena; Jia, Xiaoying; Melhem, Molly M.; Runnels, Judith; Roccaro, Aldo M.; Azab, Feda; Sacco, Antonio; Leleu, Xavier; Anderson, Kenneth C.; Ghobrial, Irene M.

    2009-01-01

    Purpose Waldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, WM is still incurable. Src tyrosine kinase was shown to play a central role in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival, in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration and survival in WM. Experimental Design We have tested the expression of Src tyrosine kinase in WM and normal cells, and tested the effect of its specific inhibitor AZD-530 on adhesion, migration, cell cycle and survival of WM cell line and patient samples. Moreover, we tested its effect on sytockeletal and cell cycle signaling in WM. Results We demonstrated that Src is over expressed in WM cells compared to control B cells. And that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration and cytosekletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G1 cell cycle arrest; however, it had minimal effect on survival of WM cells, and no significant effect on survival of normal cells. Conclusions Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with WM. PMID:19755386

  5. Blocking transient receptor potential vanilloid 2 channel in astrocytes enhances astrocyte-mediated neuroprotection after oxygen-glucose deprivation and reoxygenation.

    PubMed

    Zhang, Han; Xiao, Jun; Hu, Zheng; Xie, Minjie; Wang, Wei; He, Dan

    2016-10-01

    Astrocytes play important roles in homeostatic regulation in the central nervous system and are reported to influence the outcome of ischemic injury. Regulating Ca(2+) signaling of astrocytes is a promising strategy for stroke therapy. Herein, we report for the first time that transient receptor potential vanilloid 2 (TRPV2), a Ca(2+) -permeable channel that is important in osmotic balance regulation, expresses in rat cortical astrocytes by immunofluorescence. Moreover, oxygen-glucose deprivation and reoxygenation (OGD/R) treatment enhanced the expression. The TRPV2 is functional because Ca(2+) imaging showed that activating the TRPV2 channel in cultured astrocytes increased intracellular Ca(2+) level and the increment of intracellular Ca(2+) level expanded when astrocytes were treated with OGD/R. Staining with 5-ethynyl-2'-deoxyuridine (EdU) revealed that while blocking the TRPV2, it promoted the proliferation of astrocytes. Additionally, blocking the TRPV2 in astrocytes increased the synthesis of nerve growth factor (NGF) mRNA and the secretion of NGF by real-time PCR and enzyme-linked immunosorbent assay respectively. We further found that the increased secretion of NGF could be reversed by c-JunN-terminalkinase (JNK) inhibitor and blocking the TRPV2 caused the phosphorylation of JNK. These indicated that blocking the TRPV2 induced NGF secretion via the mitogen-activated protein kinase (MAPK)-JNK signaling pathway. As the promoted proliferation of astrocytes and secretion of NGF were reported to have neuroprotective effects in the early stage of stroke, we concluded that targeting the TRPV2 channel in astrocytes might be a potential new therapeutic strategy in ischemic stroke.

  6. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  7. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    PubMed Central

    Johnson, Adam J.; Wu, Ming J.

    2016-01-01

    The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport. PMID:28009816

  8. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    PubMed Central

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  9. Protein kinase A regulates the osteogenic activity of Osterix.

    PubMed

    He, Siyuan; Choi, You Hee; Choi, Joong-Kook; Yeo, Chang-Yeol; Chun, ChangJu; Lee, Kwang Youl

    2014-10-01

    Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

  10. Morin Suppresses Astrocyte Activation and Regulates Cytokine Release in Bone Cancer Pain Rat Models.

    PubMed

    Jiang, Wei; Wang, Ying; Sun, Wei; Zhang, Mengyuan

    2017-09-01

    As inflammatory and immune responses are involved in pathophysiology of debilitating neuropathic pain, reagents that can modulate these two responses may have therapeutic potential. Morin, derived from the moraceae family of plants, benefits inflammation-related diseases, but its antinociceptive effects on cancer pain remain elusive. In the present study, we investigated antinociceptive effects of morin on bone cancer pain using a rat model, where rats were subject to implantation of Walker 256 mammary gland carcinoma cells into the tibia. Morin (5-20 mg/kg) dose-dependently attenuated behavioral hypersensitivities, including mechanical allodynia and free movement pain, which was accompanied by downregulation of astrocyte marker glial fibrillary acidic protein in the spinal cord in cancer-bearing rats. Treatment with morin also induced reduction of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and upregulation of an antiinflammatory cytokine IL-10. Furthermore, intrathecal injection of AM630 (an antagonist of cannabinoid receptor 2, CB2 ), but not naloxone (an antagonist of opioid receptors), significantly blocked morin attenuation of behavioral hypersensitivities. Taken together, these results suggest that morin suppresses astrocyte activation and neuro-inflammation induced by bone cancer pain and its antinociceptive effects on bone cancer pain may be associated with activation of CB2 receptors in the spinal cord. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Dietary β-carotene regulates interleukin-1β-induced expression of apolipoprotein E in astrocytes isolated from stroke-prone spontaneously hypertensive rats.

    PubMed

    Yamagata, Kazuo; Nakayama, Chika; Suzuki, Koichi

    2013-01-01

    Stroke-prone spontaneously hypertensive rats (SHRSP) have an abnormality in cholesterol synthesis, but the pathological relevance of this to stroke and related neuronal disorders is not yet clear. The induction of astrocyte-derived cholesterol transportation to neurons by apolipoprotein E (apoE) promotes neuronal repair after brain injuries such as stroke. Such repair is reduced by interleukin-1 beta (IL-1β) and stroke conditions. Furthermore, fibroblast growth factor 1 (FGF1) regulates the production of apoE-cholesterol-rich high density lipoproteins (HDL) and induces gliosis of astrocytes. On the other hand, high levels of plasma carotenoids reduce the risk of ischemic stroke. Thus, we investigated the expression of apoE in primary astrocytes that had been treated with IL-1β or β-carotene. In addition, we compared the expression levels of Apoe genes in astrocytes from SHRSP/Izm and normal control rats, Wistar-Kyoto rats (WKY/Izm) following hypoxia/reoxygenation (H/R). The expression levels of genes and proteins were investigated by RT-PCR, Western blotting (WB), and immunofluorescence analysis. IL-1β decreased the expression levels of the Apoe gene. Conversely, β-carotene significantly enhanced the expression levels of genes related to cholesterol regulation, including Abca1, Abcg1, Hmgcr as well as Apoe. During H/R, the gene expression levels of Apoe were decreased in the SHRSP/Izm rats in comparison with the WKY/Izm rats. These results suggest that IL-1β decreases Apoe expression levels, whereas β-carotene strongly elevates Apoe levels and inhibits FGF1-mediated gliosis of astrocytes. Furthermore, under hypoxic stress, astrocytes isolated from SHRSP/Izm rats displayed altered regulation of Apoe compared with those from WKY/Izm rats. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Spatial regulation of Raf kinase signaling by RKTG

    PubMed Central

    Feng, Lin; Xie, Xiaoduo; Ding, Qiurong; Luo, Xiaolin; He, Jing; Fan, Fengjuan; Liu, Weizhong; Wang, Zhenzhen; Chen, Yan

    2007-01-01

    Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway. PMID:17724343

  13. A Model for p38MAPK-Induced Astrocyte Senescence.

    PubMed

    Mombach, José C M; Vendrusculo, Bruno; Bugs, Cristhian A

    2015-01-01

    Experimental evidence indicates that aging leads to accumulation of senescent cells in tissues and they develop a secretory phenotype (also known as SASP, for senescence-associated secretory phenotype) that can contribute to chronic inflammation and diseases. Recent results have showed that markers of senescence in astrocytes from aged brains are increased in brains with Alzheimer's disease. These studies strongly involved the stress kinase p38MAPK in the regulation of the secretory phenotype of astrocytes, yet the molecular mechanisms underlying the onset of senescence and SASP activation remain unclear. In this work, we propose a discrete logical model for astrocyte senescence determined by the level of DNA damage (reparable or irreparable DNA strand breaks) where the kinase p38MAPK plays a central role in the regulation of senescence and SASP. The model produces four alternative stable states: proliferation, transient cycle arrest, apoptosis and senescence (and SASP) computed from its inputs representing DNA damages. Perturbations of the model were performed through gene gain or loss of functions and compared with results concerning cultures of normal and mutant astrocytes showing agreement in most cases. Moreover, the model allows some predictions that remain to be tested experimentally.

  14. Age-Dependent Neurochemical Remodeling of Hypothalamic Astrocytes.

    PubMed

    Santos, Camila Leite; Roppa, Paola Haack Amaral; Truccolo, Pedro; Fontella, Fernanda Urruth; Souza, Diogo Onofre; Bobermin, Larissa Daniele; Quincozes-Santos, André

    2017-10-04

    The hypothalamus is a crucial integrative center in the central nervous system, responsible for the regulation of homeostatic activities, including systemic energy balance. Increasing evidence has highlighted a critical role of astrocytes in orchestrating hypothalamic functions; they participate in the modulation of synaptic transmission, metabolic and trophic support to neurons, immune defense, and nutrient sensing. In this context, disturbance of systemic energy homeostasis, which is a common feature of obesity and the aging process, involves inflammatory responses. This may be related to dysfunction of hypothalamic astrocytes. In this regard, the aim of this study was to evaluate the neurochemical properties of hypothalamic astrocyte cultures from newborn, adult, and aged Wistar rats. Age-dependent changes in the regulation of glutamatergic homeostasis, glutathione biosynthesis, amino acid profile, glucose metabolism, trophic support, and inflammatory response were observed. Additionally, signaling pathways including nuclear factor erythroid-derived 2-like 2/heme oxygenase-1 p38 mitogen-activated protein kinase, nuclear factor kappa B, phosphatidylinositide 3-kinase/Akt, and leptin receptor expression may represent putative mechanisms associated with the cellular alterations. In summary, our findings indicate that as age increases, hypothalamic astrocytes remodel and exhibit changes in their neurochemical properties. This process may play a role in the onset and/or progression of metabolic disorders.

  15. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  16. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  17. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase.

    PubMed

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W Todd; Tonks, Nicholas K

    2015-06-26

    Despite significant evidence to the contrary, the view that phosphatases are "nonspecific" still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as "erasers" that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of "nonspecific phosphatases." We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    PubMed

    Haolong, Cong; Du, Ning; Hongchao, Tian; Yang, Yang; Wei, Zhang; Hua, Zhang; Wenliang, Zhang; Lei, Song; Po, Tien

    2013-01-01

    Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  19. ABL Tyrosine Kinases: Evolution of Function, Regulation, and Specificity*

    PubMed Central

    Colicelli, John

    2010-01-01

    ABL-family proteins comprise one of the best conserved branches of the tyrosine kinases. Each ABL protein contains an SH3-SH2-TK (Src homology 3–Src homology 2–tyrosine kinase) domain cassette, which confers autoregulated kinase activity and is common among nonreceptor tyrosine kinases. This cassette is coupled to an actin-binding and -bundling domain, which makes ABL proteins capable of connecting phosphoregulation with actin-filament reorganization. Two vertebrate paralogs, ABL1 and ABL2, have evolved to perform specialized functions. ABL1 includes nuclear localization signals and a DNA binding domain through which it mediates DNA damage-repair functions, whereas ABL2 has additional binding capacity for actin and for microtubules to enhance its cytoskeletal remodeling functions. Several types of posttranslational modifications control ABL catalytic activity, subcellular localization, and stability, with consequences for both cytoplasmic and nuclear ABL functions. Binding partners provide additional regulation of ABL catalytic activity, substrate specificity, and downstream signaling. Information on ABL regulatory mechanisms is being mined to provide new therapeutic strategies against hematopoietic malignancies caused by BCR-ABL1 and related leukemogenic proteins. PMID:20841568

  20. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  1. Focal adhesion kinase negatively regulates neuronal insulin resistance.

    PubMed

    Gupta, Amit; Bisht, Bharti; Dey, Chinmoy Sankar

    2012-06-01

    Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.

  2. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  3. Lim kinase regulates the development of olfactory and neuromuscular synapses.

    PubMed

    Ang, Lay-Hong; Chen, Weitao; Yao, Ying; Ozawa, Rie; Tao, Enxiang; Yonekura, Junichiro; Uemura, Tadashi; Keshishian, Haig; Hing, Huey

    2006-05-01

    Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system. To unravel the cellular and molecular functions of Limk, we have either knocked out or activated the Limk gene in Drosophila. At the neuromuscular junction, loss of Limk leads to enlarged terminals, while increasing the activity of Limk leads to stunted terminals with fewer synaptic boutons. In the antennal lobe, loss of Limk abolishes the ability of p21-activated kinase (Pak) to alter glomerular development. In contrast, increase in Limk function leads to ectopic glomeruli, a phenotype suppressible by the coexpression of a hyperactive Cofilin gene. These results establish Limk as a critical regulator of Cofilin function and synapse development, and a downstream effector of Pak in vivo.

  4. Rho kinase regulates neurite outgrowth of hippocampal neurons via calcium dependent cytoskeleton regulation

    PubMed Central

    Ji, Zhisheng; Cai, Zhenbin; Zhang, Jifeng; Liu, Nannuan; Chen, Jing; Tan, Minghui; Lin, Hongsheng; Guo, Guoqing

    2017-01-01

    Objective: To investigate whether calcium is involved in downstream signal transduction in neurite outgrowth regulated by Rho kinase. Methods: In vitro primary hippocampal neurons were cultured and treated with Rho kinase agonist (LPA) or antagonist (Y-27632). Then, the cytoskeleton and neurite outgrowth were observed. After addition of calcium antagonist BAPTA/AM to reduce intracellular calcium, the cytoskeleton distribution and neurite outgrowth were observed. Results: The activation or inhibition of Rho kinase could significantly alter the number and length of neurites of hippocampal neurons. Rho kinase regulated the cytoskeleton to regulate the neurite outgrowth, and LPA could significantly increase intracellular calcium. After BAPTA/AM treatment, the length and branch number of neurites of neurons reduced markedly. BAPTA/AM was able to reduce intracellular calcium and decrease neuronal cytoskeleton. Treatment with both BAPTA/AM and LPA could stop the retraction of neurites, but the length and branch number of neurites remained unchanged after treatment with Y-27632 and LPA. Conclusion: Calcium may affect the cytoskeleton arrangement to regulate neurite outgrowth, and calcium is involved in the downstream signal transduction of Rho kinase regulated neurite outgrowth of hippocampal neurons. PMID:28337305

  5. Protein Kinase C-α–Mediated Regulation of Low-Density Lipoprotein Receptor–Related Protein and Urokinase Increases Astrocytoma Invasion

    PubMed Central

    Amos, Samson; Mut, Melike; diPierro, Charles G.; Carpenter, Joan E.; Xiao, Aizhen; Kohutek, Zachary A.; Redpath, Gerard T.; Zhao, Yunge; Wang, Jiahu; Shaffrey, Mark E.; Hussaini, Isa M.

    2008-01-01

    Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor–related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-α (PKC-α) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-α small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate–induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-α/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-α and PI3K could provide a new treatment paradigm for glioblastomas. PMID:17974965

  6. Brassinosteroid regulated kinases (BRKs) that mediate brassinosteroid signal transduction and uses thereof

    DOEpatents

    Wang, Zhi-Yong; Tang, Wenqiang

    2013-09-24

    The present invention identifies a novel family of kinases regulated by brassinosteroids, referred to as BRKs (brassinosteroid regulated kinases) or BSKs (brassinosteroid signaling kinases). The present invention provides methods for modulating the response of a plant cell to a brassinosteroid using BRKs.

  7. P2X7 receptors stimulate AKT phosphorylation in astrocytes

    PubMed Central

    Jacques-Silva, Maria C; Rodnight, Richard; Lenz, Guido; Liao, Zhongji; Kong, Qiongman; Tran, Minh; Kang, Yuan; Gonzalez, Fernando A; Weisman, Gary A; Neary, Joseph T

    2004-01-01

    Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′-triphosphate (UTP) and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism. PMID:15023862

  8. Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.

    PubMed

    Shirakawa, Hisashi; Sakimoto, Shinya; Nakao, Kenji; Sugishita, Aiko; Konno, Masakazu; Iida, Shota; Kusano, Ayaka; Hashimoto, Emina; Nakagawa, Takayuki; Kaneko, Shuji

    2010-09-29

    Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own

  9. Casein Kinase 1 Functions as both Penultimate and Ultimate Kinase in Regulating Cdc25A Destruction

    PubMed Central

    Honaker, Yuchi; Piwnica-Worms, Helen

    2010-01-01

    The Cdc25A protein phosphatase drives cell cycle transitions by activating cyclin-dependent protein kinases. Failure to regulate Cdc25A leads to deregulated cell cycle progression, bypass of cell cycle checkpoints and genome instability. Ubiquitin-mediated proteolysis plays an important role in balancing Cdc25A levels. Cdc25A contains a DS82G motif whose phosphorylation is targeted by β-TrCP E3 ligase during interphase. Targeting of β-TrCP to Cdc25A requires phosphorylation of serines 79 (S79) and 82 (S82). Here, we report that casein kinase 1 alpha (CK1α) phosphorylates Cdc25A on both S79 and S82 in a hierarchical manner requiring prior phosphorylation of serine 76 by Chk1 or GSK-3β. This facilitates β-TrCP binding and ubiquitin-mediated proteolysis of Cdc25A throughout interphase and following exposure to genotoxic stress. The priming of Cdc25A by at least three kinases (Chk1, GSK-3β, CK1α), some of which also require priming, ensures diverse extra- and intra-cellular signals interface with Cdc25A to precisely control cell division. PMID:20348946

  10. Circular RNA HIPK2 regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124-2HG.

    PubMed

    Huang, Rongrong; Zhang, Yuan; Han, Bing; Bai, Ying; Zhou, Rongbin; Gan, Guangming; Chao, Jie; Hu, Gang; Yao, Honghong

    2017-10-03

    Circular RNAs are a subclass of noncoding RNAs in mammalian cells; however, whether these RNAs are involved in the regulation of astrocyte activation is largely unknown. Here, we have shown that the circular RNA HIPK2 (circHIPK2) functions as an endogenous microRNA-124 (MIR124-2HG) sponge to sequester MIR124-2HG and inhibit its activity, resulting in increased sigma non-opioid intracellular receptor 1 (SIGMAR1/OPRS1) expression. Knockdown of circHIPK2 expression significantly inhibited astrocyte activation via the regulation of autophagy and endoplasmic reticulum (ER) stress through the targeting of MIR124-2HG and SIGMAR1. These findings were confirmed in vivo in mouse models, as microinjection of a circHIPK2 siRNA lentivirus into mouse hippocampi inhibited astrocyte activation induced by methamphetamine or lipopolysaccharide (LPS). These findings provide novel insights regarding the specific contribution of circHIPK2 to astrocyte activation in the context of drug abuse as well as for the treatment of a broad range of neuroinflammatory disorders.

  11. Genetic dys-regulation of astrocytic glutamate transporter EAAT2 and its implications in neurological disorders and manganese toxicity

    PubMed Central

    Karki, Pratap; Smith, Keisha; Johnson, James; Aschner, Michael; Lee, Eunsook

    2014-01-01

    Astrocytic glutamate transporters, the excitatory amino acid transporter (EAAT) 2 and EAAT1 [glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) in rodents, respectively], are the main transporters for maintaining optimal glutamate levels in the synaptic clefts by taking up more than 90% of glutamate from extracellular space thus preventing excitotoxic neuronal death. Reduced expression and function of these transporters, especially EAAT2, has been reported in numerous neurological disorders, including amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, schizophrenia and epilepsy. The mechanism of down-regulation of EAAT2 in these diseases has yet to be fully established. Genetic as well as transcriptional dys-regulation of these transporters by various modes, such as single nucleotide polymorphisms (SNPs) and epigenetics, resulting in impairment of their functions, might play an important role in the etiology of neurological diseases. Consequently, there has been an extensive effort to identify molecular targets for enhancement of EAAT2 expression as a potential therapeutic approach. Several pharmacological agents increase expression of EAAT2 via NF-κB and CREB at the transcriptional level. However, the negative regulatory mechanisms of EAAT2 have yet to be identified. Recent studies, including those from our laboratory, suggest that the transcriptional factor yin yang 1 (YY1) plays a critical role in the repressive effects of various neurotoxins, such as manganese (Mn), on EAAT2 expression. In this review, we will focus on transcriptional epigenetics, and translational regulation of EAAT2. PMID:25064045

  12. Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression

    PubMed Central

    Strutz-Seebohm, Nathalie; Seebohm, Guiscard; Shumilina, Ekaterina; Mack, Andreas F; Wagner, Hans-Joachim; Lampert, Angelika; Grahammer, Florian; Henke, Guido; Just, Lothar; Skutella, Thomas; Hollmann, Michael; Lang, Florian

    2005-01-01

    Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid-dependent genes include the serum- and glucocorticoid-inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3-kinase) pathway by growth factors such as insulin-like growth factor-1 (IGF1) or tumour growth factor β (TGF-β). Previously, a fourfold higher expression of SGK1 has been observed in fast-learning rats as compared with slow-learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 μg day−1 for 8–20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate-induced currents following treatment with dexamethasone (1 μm) and TGF-β (1 μm). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate-induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids. PMID:15774535

  13. The noble gas argon modifies extracellular signal-regulated kinase 1/2 signaling in neurons and glial cells.

    PubMed

    Fahlenkamp, Astrid V; Rossaint, Rolf; Haase, Hajo; Al Kassam, Hussam; Ryang, Yu-Mi; Beyer, Cordian; Coburn, Mark

    2012-01-15

    Recently, the noble gas argon has been identified as a potent neuroprotective agent, but little is known about its cellular effects. In this in vitro study, we investigated argon's influence on the extracellular signal-regulated kinase (ERK) 1/2, a ubiquitous enzyme with numerous functions in cell proliferation and survival. Primary neuronal and astroglial cell cultures and the microglial cell line BV-2 were exposed to 50 vol.% argon. Further possible effects were studied following stimulation of microglia with 50 ng/ml LPS. ERK 1/2 activation was assessed by phosphorylation state-specific western blotting, cytokine levels by real-time PCR and western blotting. Total phosphotyrosine phosphatase activity was examined with p-nitrophenylphosphate. After 30 min exposure, argon significantly activated ERK 1/2 signaling in microglia. Enhanced phosphorylation of ERK 1/2 was also found in astrocytes and neurons following argon exposure, but it lacked statistical significance. In microglia, argon did not substantially interfere with LPS-induced ERK1/2 activation and inflammatory cytokine induction. Addition of the MEK-Inhibitor U0126 abolished the induced ERK 1/2 phosphorylation. Cellular phosphatase activity and the inactivation of phosphorylated ERK 1/2 were not altered by argon. In conclusion, argon enhanced ERK 1/2 activity in microglia via the upstream kinase MEK, probably through a direct mode of activation. ERK 1/2 signaling in astrocytes and neurons in vitro was also influenced, although not with statistical significance. Whether ERK 1/2 activation by argon affects cellular functions like differentiation and survival in the brain in vivo will have to be determined in future experiments. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Ack kinase regulates CTP synthase filaments during Drosophila oogenesis.

    PubMed

    Strochlic, Todd I; Stavrides, Kevin P; Thomas, Sam V; Nicolas, Emmanuelle; O'Reilly, Alana M; Peterson, Jeffrey R

    2014-11-01

    The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.

  15. Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation.

    PubMed Central

    Le Good, J Ann; Brindley, David N

    2004-01-01

    The regulation of protein kinase C (PKC)zeta in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCzeta. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCzeta Delta PSS), but mutants designed to mimic phosphorylated PKCzeta had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCzeta. The T560A mutant also protected PKCzeta against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCzeta Delta PSS, T410E, PKCzeta Delta PSS T410/560E, PKCzeta and T560A. Expressed PKCzeta activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCzeta activity (PKCzeta Delta PSS, PKCzeta Delta PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation. PMID:14580237

  16. TRPM7 is regulated by halides through its kinase domain

    PubMed Central

    Yu, Haijie; Zhang, Zheng; Lis, Annette; Penner, Reinhold; Fleig, Andrea

    2013-01-01

    Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg2+), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg2+ ([Mg2+]i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg2+ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg2+]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells. PMID:23471296

  17. Regulation of astrocyte pathology by fluoxetine prevents the deterioration of Alzheimer phenotypes in an APP/PS1 mouse model.

    PubMed

    Qiao, Jinping; Wang, Junhui; Wang, Hongxing; Zhang, Yanbo; Zhu, Shenghua; Adilijiang, Abulimiti; Guo, Huining; Zhang, Ruiguo; Guo, Wei; Luo, Gang; Qiu, Yiqing; Xu, Haiyun; Kong, Jiming; Huang, Qingjun; Li, Xin-Min

    2016-02-01

    Studies have implicated astrocytic dysfunction in Alzheimer's disease (AD). However, the role of astrocytes in the pathophysiology and treatment of the disease is poorly characterized. Here, we identified astrocytes as independent key factors involved in several Alzheimer-like phenotypes in an APP/PS1 mouse model, including amyloid pathology, altered neuronal and synaptic properties, and impaired cognition. In vitro astrocytes from APP/PS1 mice induced synaptotoxicity as well as reduced dendritic complexity and axonal branching of hippocampal neurons. These astrocytes produced high levels of soluble β-amyloid (Aβ) which could be significantly inhibited by fluoxetine (FLX) via activating serotonin 5-HT2 receptors. FLX could also protect hippocampal neurons against astrocyte-induced neuronal damage in vitro. In the same APP/PS1 mice, FLX inhibited activation of astrocytes, lowered Aβ products, ameliorated neurotoxicity, and improved behavioral performance. These findings may provide a basis for the clinical application of FLX in patients, and may also lay the groundwork for exploration of other novel astrocyte-based therapies of AD.

  18. REGULATION OF MEMORY – FROM THE ADRENAL MEDULLA TO LIVER TO ASTROCYTES TO NEURONS1

    PubMed Central

    Gold, Paul E.

    2014-01-01

    Epinephrine, released into blood from the adrenal medulla in response to arousing experiences, is a potent enhancer of learning and memory processing. This review examines mechanisms by which epinephrine exerts its effects on these cognitive functions. Because epinephrine is largely blocked from moving from blood to brain, it is likely that the hormone's effects on memory are mediated by peripheral actions. A classic effect of epinephrine is to act at the liver to break down glycogen stores, resulting in increased blood glucose levels. The increase in blood glucose provides additional energy substrates to the brain to buttress the processes needed for an experience to be learned and remembered. In part, it appears that the increased glucose may act in the brain in a manner akin to that evident in the liver, engaging glycogenolysis in astrocytes to provide an energy substrate, in this case lactate, to augment neuronal functions. Together, the findings reveal a mechanism underlying modulation of memory that integrates the physiological functions of multiple organ systems to support brain processes. PMID:24406469

  19. Astrocyte Elevated Gene-1 (AEG-1): a multifunctional regulator of normal and abnormal physiology

    PubMed Central

    Yoo, Byoung Kwon; Emdad, Luni; Lee, Seok-Geun; Su, Zao-zhong; Santhekadur, Prasanna; Chen, Dong; Gredler, Rachel; Fisher, Paul B.; Sarkar, Devanand

    2011-01-01

    Since its initial identification and cloning in 2002, Astrocyte Elevated Gene-1 (AEG-1), also known as metadherin (MTDH), 3D3 and LYsine-RIch CEACAM1 co-isolated (LYRIC), has emerged as an important oncogene that is overexpressed in all cancers analyzed so far. Examination of a large cohort of patient samples representing diverse cancer indications has revealed progressive increase in AEG-1 expression with stages and grades of the disease and an inverse relationship between AEG-1 expression level and patient prognosis. AEG-1 functions as a bona fide oncogene by promoting transformation. In addition, it plays a significant role in invasion, metastasis, angiogenesis and chemoresistance, all important hallmarks of an aggressive cancer. AEG-1 is also implicated in diverse physiological and pathological processes, such as development, inflammation, neurodegeneration, migraine and Huntington disease. AEG-1 is a highly basic protein with a transmembrane domain and multiple nuclear localization signals and it is present in the cell membrane, cytoplasm, nucleus, nucleolus and endoplasmic reticulum. In each location, AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes the combination of which contributes to its pleiotrophic properties. The present review provides a snapshot of the current literature along with future perspectives on this unique molecule. PMID:21256156

  20. Up-regulation of reactivity and survival genes in astrocytes after exposure to short duration overpressure.

    PubMed

    Vandevord, Pamela J; Leung, Lai Yee; Hardy, Warren; Mason, Matthew; Yang, King H; King, Albert I

    2008-04-04

    Gurdjian et al. proposed decades ago that pressure gradients played a major factor in neuronal injury due to impact. In the late 1950s, their experiments on concussion demonstrated that the principal factor in the production of concussion in animals was the sudden increase of intracranial pressure accompanying head injury. They reported the increase in pressure severity correlated with an increase in 'altered cells' resulting in animal death. More recently, Hardy et al. (2006) demonstrated the presence of transient pressure pulses with impact conditions. These studies indicate that short duration overpressure should be further examined as a mechanism of traumatic brain injury (TBI). In the present study, we designed and fabricated a barochamber that simulated overpressure noted in various head injury studies. We tested the effect of overpressure on astrocytes. Expressions of apoptotic, reactivity and survival genes were examined at 24, 48 and 72 h post-overpressure exposure. At 24 h, we found elevated levels of reactivity and survival gene expression. By 48 h, a decreased expression of apoptotic genes was demonstrated. This study reinforces the hypothesis that transient pressure acts to instigate the cellular response displayed following TBI.

  1. WNK kinases regulate thiazide-sensitive Na-Cl cotransport.

    PubMed

    Yang, Chao-Ling; Angell, Jordan; Mitchell, Rose; Ellison, David H

    2003-04-01

    Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.

  2. Astrocytes in Migration.

    PubMed

    Zhan, Jiang Shan; Gao, Kai; Chai, Rui Chao; Jia, Xi Hua; Luo, Dao Peng; Ge, Guo; Jiang, Yu Wu; Fung, Yin-Wan Wendy; Li, Lina; Yu, Albert Cheung Hoi

    2017-01-01

    Cell migration is a fundamental phenomenon that underlies tissue morphogenesis, wound healing, immune response, and cancer metastasis. Great progresses have been made in research methodologies, with cell migration identified as a highly orchestrated process. Brain is considered the most complex organ in the human body, containing many types of neural cells with astrocytes playing crucial roles in monitoring normal functions of the central nervous system. Astrocytes are mostly quiescent under normal physiological conditions in the adult brain but become migratory after injury. Under most known pathological conditions in the brain, spinal cord and retina, astrocytes are activated and become hypertrophic, hyperplastic, and up-regulating GFAP based on the grades of severity. These three observations are the hallmark in glia scar formation-astrogliosis. The reactivation process is initiated with structural changes involving cell process migration and ended with cell migration. Detailed mechanisms in astrocyte migration have not been studied extensively and remain largely unknown. Here, we therefore attempt to review the mechanisms in migration of astrocytes.

  3. Regulation of macropinocytosis by p21-activated kinase-1.

    PubMed

    Dharmawardhane, S; Schürmann, A; Sells, M A; Chernoff, J; Schmid, S L; Bokoch, G M

    2000-10-01

    The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

  4. Creatine kinase in cell cycle regulation and cancer.

    PubMed

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK.

  5. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    PubMed Central

    Lou, Sunny S.; Diz-Muñoz, Alba; Weiner, Orion D.; Fletcher, Daniel A.

    2015-01-01

    Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. PMID:25918227

  6. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism.

    PubMed

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan; Maillet, Jean-Christian; Fottinger, Alexandra; Foley, Tanya; Byham, Michèle-Renée; Iqbal, Tasfia Ahmed; Yoneda, Atsuko; Couchman, John R; Parks, Robin J; Gee, Stephen H

    2012-10-01

    Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

  7. Tricornered Kinase Regulates Synapse Development by Regulating the Levels of Wiskott-Aldrich Syndrome Protein.

    PubMed

    Natarajan, Rajalaxmi; Barber, Kara; Buckley, Amanda; Cho, Phillip; Egbejimi, Anuoluwapo; Wairkar, Yogesh P

    2015-01-01

    Precise regulation of synapses during development is essential to ensure accurate neural connectivity and function of nervous system. Many signaling pathways, including the mTOR (mechanical Target of Rapamycin) pathway operate in neurons to maintain genetically determined number of synapses during development. mTOR, a kinase, is shared between two functionally distinct multi-protein complexes- mTORC1 and mTORC2, that act downstream of Tuberous Sclerosis Complex (TSC). We and others have suggested an important role for TSC in synapse development at the Drosophila neuromuscular junction (NMJ) synapses. In addition, our data suggested that the regulation of the NMJ synapse numbers in Drosophila largely depends on signaling via mTORC2. In the present study, we further this observation by identifying Tricornered (Trc) kinase, a serine/threonine kinase as a likely mediator of TSC signaling. trc genetically interacts with Tsc2 to regulate the number of synapses. In addition, Tsc2 and trc mutants exhibit a dramatic reduction in synaptic levels of WASP, an important regulator of actin polymerization. We show that Trc regulates the WASP levels largely, by regulating the transcription of WASP. Finally, we show that overexpression of WASP (Wiskott-Aldrich Syndrome Protein) in trc mutants can suppress the increase in the number of synapses observed in trc mutants, suggesting that WASP regulates synapses downstream of Trc. Thus, our data provide a novel insight into how Trc may regulate the genetic program that controls the number of synapses during development.

  8. Growth factors and steroid mediated regulation of cytoskeletal protein expression in serum-deprived primary astrocyte cultures.

    PubMed

    Bramanti, Vincenzo; Bronzi, Daniela; Tomassoni, Daniele; Costa, Antonino; Raciti, Giuseppina; Avitabile, Marcello; Amenta, Francesco; Avola, Roberto

    2008-12-01

    In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them together. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressful conditions.

  9. A functional coupling between extrasynaptic NMDA receptors and A-type K+ channels under astrocyte control regulates hypothalamic neurosecretory neuronal activity

    PubMed Central

    Naskar, Krishna; Stern, Javier E

    2014-01-01

    Neuronal activity is controlled by a fine-tuned balance between intrinsic properties and extrinsic synaptic inputs. Moreover, neighbouring astrocytes are now recognized to influence a wide spectrum of neuronal functions. Yet, how these three key factors act in concert to modulate and fine-tune neuronal output is not well understood. Here, we show that in rat hypothalamic magnocellular neurosecretory cells (MNCs), glutamate NMDA receptors (NMDARs) are negatively coupled to the transient, voltage-gated A-type K+ current (IA). We found that activation of NMDARs by extracellular glutamate levels influenced by astrocyte glutamate transporters resulted in a significant inhibition of IA. The NMDAR–IA functional coupling resulted from activation of extrasynaptic NMDARs, was calcium- and protein kinase C-dependent, and involved enhanced steady-state, voltage-dependent inactivation of IA. The NMDAR–IA coupling diminished the latency to the first evoked spike in response to membrane depolarization and increased the total number of evoked action potentials, thus strengthening the neuronal input/output function. Finally, we found a blunted NMDA-mediated inhibition of IA in dehydrated rats. Together, our findings support a novel signalling mechanism that involves a functional coupling between extrasynaptic NMDARs and A-type K+ channels, which is influenced by local astrocytes. We show this signalling complex to play an important role in modulating hypothalamic neuronal excitability, which may contribute to adaptive responses during a sustained osmotic challenge such as dehydration. PMID:24835172

  10. Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

    PubMed Central

    Bevensee, Mark O.; Apkon, Michael; Boron, Walter F.

    1997-01-01

    In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na+-driven influx of HCO3− causes the increase in intracellular pH (pHi) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3−. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl− by incubating them in a Cl−-free solution for an average of ∼11 min. We verified the depletion with the Cl−-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl−-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3−. Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl−. Therefore, the transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/ HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the presence/absence of CO2/HCO3− and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO2/HCO3− solutions contained 20% CO2 and 68 mM HCO3−, pH 7.3, to maximize the HCO3− flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3− elicited an equivalent HCO3− efflux of 281 μM s−1. The HCO3− influx elicited by returning external Na+ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/ HCO3−. Thus, the Na

  11. Tyrosine Kinase Inhibitors Regulate OPG through Inhibition of PDGFRβ

    PubMed Central

    Tay, Mei Lin; Lin, Jian-Ming; Bava, Usha; Callon, Karen; Cornish, Jillian; Naot, Dorit; Grey, Andrew

    2016-01-01

    Nilotinib and imatinib are tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). In vitro, imatinib and nilotinib inhibit osteoclastogenesis, and in patients they reduce levels of bone resorption. One of the mechanisms that might underlie these effects is an increase in the production of osteoprotegerin (OPG). In the current work we report that platelet-derived growth factor receptor beta (PDGFRβ) signaling regulates OPG production in vitro. In addition, we have shown that TKIs have effects on RANKL signaling through inhibition of the PDGFRβ and other target receptors. These findings have implications for our understanding of the mechanisms by which TKIs affect osteoclastogenesis, and the role of PDGFRβ signaling in regulating osteoclastogenesis. Further studies are indicated to confirm the clinical effects of PDGFRβ-inhibitors and to elaborate the intracellular pathways that underpin these effects. PMID:27737004

  12. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  13. Protein Kinase D Regulates Cell Death Pathways in Experimental Pancreatitis

    PubMed Central

    Yuan, Jingzhen; Liu, Yannan; Tan, Tanya; Guha, Sushovan; Gukovsky, Ilya; Gukovskaya, Anna; Pandol, Stephen J.

    2012-01-01

    Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-κB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK) in pancreatic acinar cells. Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins, caspases, receptor-interacting protein kinase 1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis. PMID:22470346

  14. Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

    PubMed Central

    Matsushima, Shouji; Kuroda, Junya; Zhai, Peiyong; Liu, Tong; Ikeda, Shohei; Nagarajan, Narayani; Yokota, Takashi; Kinugawa, Shintaro; Hsu, Chiao-Po; Li, Hong; Tsutsui, Hiroyuki

    2016-01-01

    NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2– production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling. PMID:27525436

  15. Molecular mechanisms of astrocyte-induced synaptogenesis.

    PubMed

    Baldwin, Katherine T; Eroglu, Cagla

    2017-08-01

    Astrocytes are morphologically complex cells that perform a wide variety of critical functions in the brain. As a structurally and functionally integrated component of the synapse, astrocytes secrete proteins, lipids, and small molecules that bind neuronal receptors to promote synaptogenesis and regulate synaptic connectivity. Additionally, astrocytes are key players in circuit formation, instructing the formation of synapses between distinct classes of neurons. This review highlights recent publications on the topic of astrocyte-mediated synaptogenesis, with a focus on the molecular mechanisms through which astrocytes orchestrate the formation of synaptic circuits. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    PubMed

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat.

  17. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  18. p38 MAP kinase regulates circadian rhythms in Drosophila.

    PubMed

    Vrailas-Mortimer, Alysia D; Ryan, Sarah M; Avey, Matthew J; Mortimer, Nathan T; Dowse, Harold; Sanyal, Subhabrata

    2014-12-01

    The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways.

  19. Pyruvate Kinase M2: A Potential Target for Regulating Inflammation

    PubMed Central

    Alves-Filho, Jose C.; Pålsson-McDermott, Eva M.

    2016-01-01

    Pyruvate kinase (PK) is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signaling pathways, affecting both the enzymatic activity of PKM2 as a PK and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for this protein as a therapeutic target in inflammatory disorders. PMID:27148264

  20. Acetate metabolism does not reflect astrocytic activity, contributes directly to GABA synthesis, and is increased by silent information regulator 1 activation.

    PubMed

    Rowlands, Benjamin D; Klugmann, Matthias; Rae, Caroline D

    2017-03-01

    [(13) C]Acetate is known to label metabolites preferentially in astrocytes rather than neurons and it has consequently been used as a marker for astrocytic activity. Recent discoveries suggest that control of acetate metabolism and its contributions to the synthesis of metabolites in brain is not as simple as first thought. Here, using a Guinea pig brain cortical tissue slice model metabolizing [1-(13) C]D-glucose and [1,2-(13) C]acetate, we investigated control of acetate metabolism and the degree to which it reflects astrocytic activity. Using a range of [1,2-(13) C]acetate concentrations, we found that acetate is a poor substrate for metabolism and will inhibit metabolism of itself and of glucose at concentrations in excess of 2 mmol/L. By activating astrocytes using potassium depolarization, we found that use of [1,2-(13) C]acetate to synthesize glutamine decreases significantly under these conditions showing that acetate metabolism does not necessarily reflect astrocytic activity. By blocking synthesis of glutamine using methionine sulfoximine, we found that significant amount of [1,2-(13) C]acetate are still incorporated into GABA and its metabolic precursors in neurons, with around 30% of the GABA synthesized from [1,2-(13) C]acetate likely to be made directly in neurons rather than from glutamine supplied by astrocytes. Finally, to test whether activity of the acetate metabolizing enzyme acetyl-CoA synthetase is under acetylation control in the brain, we incubated slices with the AceCS1 deacetylase silent information regulator 1 (SIRT1) activator SRT 1720 and showed consequential increased incorporation of [1,2-(13) C]acetate into metabolites. Taken together, these data show that acetate metabolism is not directly nor exclusively related to astrocytic metabolic activity, that use of acetate is related to enzyme acetylation and that acetate is directly metabolized to a significant degree in GABAergic neurons. Changes in acetate metabolism should be

  1. Arginine vasopressin regulated ASCT1 expression in astrocytes from stroke-prone spontaneously hypertensive rats and congenic SHRpch1_18 rats.

    PubMed

    Yamagata, K; Yamamoto, M; Kawakami, K; Ohara, H; Nabika, T

    2014-05-16

    In stroke-prone spontaneously hypertensive rats (SHRSP/Izm), ischemia induces swelling of astrocytes, a process that subsequently leads to neuronal death. Following ischemic insult, arginine vasopressin (AVP) can induce edema and l-serine released by astrocytes supports the survival of neuronal cells. The purpose of this study was to examine whether AVP contributed to the regulation of l-serine production following ischemic stroke. Here, we used cultured astrocytes from SHRSP/Izm rats and Wistar Kyoto rats (WKY/Izm) to examine whether AVP changed the production of l-serine and/or altered gene expression levels of the neural amino acid transporter (Slc1a4), 3-phosphoglycerate dehydrogenase (Phgdh) and serine racemase (SRR). Furthermore, using astrocytes from the congenic rat SHRpch1_18 strain having quantitative trait loci (QTL) of stroke, we examined expression of those genes under conditions of hypoxia and reoxygenation (H/R). The expression levels of ASCT1 protein, the genes described above and l-serine levels were determined by Western blotting (WB), RT-PCR, real-time quantitative RT-PCR and HPLC. AVP increased the production of l-serine and the expression of Slc1a4 in WKY/Izm and SHRSP/Izm astrocytes. The production of l-serine and the expression of Slc1a4 were lower in SHRSP/Izm than in WKY/Izm cells. This difference was not seen with Phgdh. In the SHRpch1_18 strain, the expression of Slc1a4 and Phgdh significantly decreased after H/R. AVP-mediated enhanced expression of ASCT1 was blocked by the addition of bumetanide. These results suggest that the AVP-mediated attenuated expression of ASCT1 in astrocytes is associated with reduced l-serine production in SHRSP/Izm astrocytes. We hypothesize that reduction of gene expression by AVP might be related to the induction of stroke in the SHRpch1_18 rat strain. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain*

    PubMed Central

    Tassin, Tara C.; Benavides, David R.; Plattner, Florian; Nishi, Akinori; Bibb, James A.

    2015-01-01

    Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission. PMID:25971971

  3. Structural plasticity and catalysis regulation of a thermosensor histidine kinase

    PubMed Central

    Albanesi, Daniela; Martín, Mariana; Trajtenberg, Felipe; Mansilla, María C.; Haouz, Ahmed; Alzari, Pedro M.; de Mendoza, Diego; Buschiazzo, Alejandro

    2009-01-01

    Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases. PMID:19805278

  4. Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

    PubMed

    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

    Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

  5. Parkin Regulates the Activity of Pyruvate Kinase M2*

    PubMed Central

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  6. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  7. Localization and regulation of mouse pantothenate kinase 2.

    PubMed

    Leonardi, Roberta; Zhang, Yong-Mei; Lykidis, Athanasios; Rock, Charles O; Jackowski, Suzanne

    2007-10-02

    Coenzyme A (CoA) biosynthesis is initiated by pantothenate kinase (PanK) and CoA levels are controlled through differential expression and feedback regulation of PanK isoforms. PanK2 is a mitochondrial protein in humans, but comparative genomics revealed that acquisition of a mitochondrial targeting signal was limited to primates. Human and mouse PanK2 possessed similar biochemical properties, with inhibition by acetyl-CoA and activation by palmitoylcarnitine. Mouse PanK2 localized in the cytosol, and the expression of PanK2 was higher in human brain compared to mouse brain. Differences in expression and subcellular localization should be considered in developing a mouse model for human PanK2 deficiency.

  8. MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

    PubMed Central

    Fanger, G R; Johnson, N L; Johnson, G L

    1997-01-01

    MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli. PMID:9305638

  9. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    PubMed

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

  10. Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms

    PubMed Central

    Chen, Feng; Yu, Yanfang; Haigh, Steven; Johnson, John; Lucas, Rudolf; Stepp, David W.; Fulton, David J. R.

    2014-01-01

    NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes

  11. Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

    PubMed Central

    Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

    2016-01-01

    The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

  12. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice

    PubMed Central

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3−/− mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3−/− mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  13. Rho kinase acts as a downstream molecule to participate in protein kinaseregulation of vascular reactivity after hemorrhagic shock in rats.

    PubMed

    Li, Tao; Zhu, Yu; Zang, Jia-tao; Peng, Xiao-yong; Lan, Dan; Yang, Guang-ming; Xu, Jing; Liu, Liang-ming

    2014-09-01

    Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

  14. Effect of FK506 and cyclosporine A on the expression of BDNF, tyrosine kinase B and p75 neurotrophin receptors in astrocytes exposed to simulated ischemia in vitro.

    PubMed

    Gabryel, Bozena; Bernacki, Jacek

    2009-07-01

    We investigated whether the immunosuppressive drugs, FK506 and cyclosporine A, increase BDNF protein and/or mRNA expression in ischemic astrocytes and if an increase could be related to changes in the nuclear expression of p-CREB, p-Erk1/2 and p-Akt. The influence of these immunosuppressants on protein and mRNA levels of TrkB and p75(NTR) receptors was also examined. On day 21, cultures of rat astrocytes were subjected to ischemic conditions simulated in vitro (combined oxygen glucose deprivation, OGD) for 8h and exposed to FK506 (10-1000nM) and cyclosporine A (0.25-10microM). FK506 and cyclosporine A (at 1000nM and 0.25microM, respectively) stimulated the expression and release of BDNF in cultured rat cerebral cortical astrocytes exposed to OGD. The immunosuppressants at these doses simultaneously increased p-CREB and p-Erk1/2 expression in the nuclear fraction of astrocytes. The results RT-PCR and Western blot analysis provided further evidence of a modulating influence of the drugs on the expression of trkB and p75(NTR) genes and their protein products in ischemic astrocytes.

  15. ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes.

    PubMed

    Liu, Na; Stoica, George; Yan, Mingshan; Scofield, Virginia L; Qiang, Wenan; Lynn, William S; Wong, Paul K Y

    2005-12-01

    ATM kinase, the product of the ataxia telangiectasia mutated (Atm) gene, is activated by genomic damage. ATM plays a crucial role in cell growth and development. Here we report that primary astrocytes isolated from ATM-deficient mice grow slowly, become senescent, and die in culture. However, before reaching senescence, these primary Atm(-/-) astrocytes, like Atm(-/-) lymphocytes, show increased spontaneous DNA synthesis. These astrocytes also show markers of oxidative stress and endoplasmic reticulum (ER) stress, including increased levels of heat shock proteins (HSP70 and GRP78), malondialdehyde adducts, Cu/Zn superoxide dismutase, procaspase 12 cleavage, and redox-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In addition, HSP70 and ERK1/2 phosphorylation are upregulated in the cerebella of ATM-deficient mice. This increase in ERK1/2 phosphorylation is seen primarily in cerebellar astrocytes, or Bergmann glia, near degenerating Purkinje cells. ERK1/2 activation and astrogliosis are also found in other parts of the brain, for example, the cortex. We conclude that ATM deficiency induces intrinsic growth defects, oxidative stress, ER stress, and ERKs activation in astrocytes.

  16. Regulation and functions of sphingosine kinases in the brain

    PubMed Central

    Bryan, Lauren; Kordula, Tomasz; Spiegel, Sarah; Milstien, Sheldon

    2009-01-01

    It has long been known that sphingolipids, especially sphingomyelin, a principal component of myelin, are highly enriched in the central nervous system and are structural components of all eukaryotic cell membranes. In the last few years, substantial evidence has accumulated from studies of many types of cells demonstrating that in addition to their structural roles, their breakdown products form a new class of signaling molecules with potent and myriad regulatory effects on essentially every cell in the body. While the sphingolipid metabolites sphingosine and its precursor ceramide have been associated with cell growth arrest and apoptosis, sphingosine-1-phosphate (S1P) enhances proliferation, differentiation, and cell survival as well as regulates many physiological and pathological processes. The relative levels of these three interconvertible sphingolipid metabolites, and thus cell fate, are strongly influenced by the activity of sphingosine kinases, of which there are two isoforms, designated SphK1 and SphK2, the enzymes that phosphorylate sphingosine to produce S1P. Not much is yet known of the importance of S1P in the central nervous system. Therefore, this review is focused on current knowledge of regulation of SphK1 and SphK2 on both transcriptional and post-translational levels and the functions of these isozymes and their product S1P and its receptors in the central nervous system. PMID:18485923

  17. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells

    SciTech Connect

    Orlando, Kelly A.; Stone, Nicole L.; Pittman, Randall N. . E-mail: pittman@pharm.med.upenn.edu

    2006-01-01

    During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

  18. Coordinated regulation of endocannabinoid-mediated retrograde synaptic suppression in the cerebellum by neuronal and astrocytic monoacylglycerol lipase

    PubMed Central

    Liu, Xiaojie; Chen, Yao; Vickstrom, Casey R.; Li, Yan; Viader, Andreu; Cravatt, Benjamin F.; Liu, Qing-song

    2016-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic depression including depolarization-induced suppression of excitation (DSE) and inhibition (DSI). 2-AG is degraded primarily by monoacylglycerol lipase (MAGL), which is expressed in neurons and astrocytes. Using knockout mice in which MAGL is deleted globally or selectively in neurons or astrocytes, we investigated the relative contribution of neuronal and astrocytic MAGL to the termination of DSE and DSI in Purkinje cells (PCs) in cerebellar slices. We report that neuronal MAGL plays a predominant role in terminating DSE at climbing fiber (CF) to PC synapses, while both neuronal and astrocytic MAGL significantly contributes to the termination of DSE at parallel fiber (PF) to PC synapses and DSI at putative Stellate cell to PC synapses. Thus, DSE and DSI at different synapses is not uniformly affected by global and cell type-specific knockout of MAGL. Additionally, MAGL global knockout, but not cell type-specific knockout, caused tonic activation and partial desensitization of the CB1 receptor at PF-PC synapses. This tonic CB1 activation is mediated by 2-AG since it was blocked by the diacylglycerol lipase inhibitor DO34. Together, these results suggest that both neuronal and astrocytic MAGL contribute to 2-AG clearance and prevent CB1 receptor over-stimulation in the cerebellum. PMID:27775008

  19. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

    PubMed

    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  20. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

    PubMed Central

    Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

  1. Regulation of multidrug resistance protein 1 by tumor necrosis factor alpha in cultured glial cells: involvement of nuclear factor-kappaB and c-Jun N-terminal kinase signaling pathways.

    PubMed

    Ronaldson, Patrick T; Ashraf, Tamima; Bendayan, Reina

    2010-04-01

    Pharmacotherapy of brain HIV-1 infection may be limited by ABC transporters [i.e., P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1)] that export antiretroviral drugs from HIV-1 brain cellular targets (i.e., astrocytes, microglia). Using an in vitro astrocyte model of an HIV-1 associated inflammatory response, our laboratory has shown that cytokines [i.e., tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6], which are secreted in response to HIV-1 envelope glycoprotein gp120 exposure, can decrease P-gp functional expression; however, it is unknown whether these same cytokines can alter expression and/or activity of other ABC transporters (i.e., Mrp1). In primary cultures of rat astrocytes, Mrp1 expression was increased by TNF-alpha (2.7-fold) but was not altered by IL-1 beta or IL-6. Cellular retention of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an Mrp substrate, was reduced in TNF-alpha-treated astrocytes, suggesting increased Mrp-mediated transport. Pharmacologic inhibition of nuclear factor-kappaB (NF-kappaB) signaling with SN50 prevented both TNF-alpha release and Mrp1 expression changes in astrocytes triggered with gp120; however, SN50 did not attenuate Mrp1 expression in cells triggered with TNF-alpha. In contrast, Mrp1 functional expression was not altered in the presence of gp120 or TNF-alpha when astrocyte cultures were pretreated with 1,9-pyrazoloanthrone (SP600125), an established c-Jun N-terminal kinase (JNK) inhibitor. SP600125 did not affect TNF-alpha release from cultured astrocytes triggered with gp120. Mrp1 mRNA expression was increased after treatment with gp120 (1.6-fold) or TNF-alpha (1.7-fold), suggesting altered Mrp1 gene transcription. These data suggest that gp120 and TNF-alpha can up-regulate Mrp1 expression in cultured astrocytes. Furthermore, our results imply that both NF-kappaB and JNK signaling are involved in Mrp1 regulation during an HIV-1 associated inflammatory response.

  2. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

    PubMed Central

    Montes de Oca Balderas, Pavel; Aguilera, Penélope

    2015-01-01

    Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase. PMID:25954808

  3. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.

    PubMed

    Montes de Oca Balderas, Pavel; Aguilera, Penélope

    2015-01-01

    Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.

  4. Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3

    SciTech Connect

    Zeliadt, Nicholette A.; Mauro, Laura J.; Wattenberg, Elizabeth V.

    2008-11-01

    Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

  5. mTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms.

    PubMed

    Ge, Yejing; Wu, Ai-Luen; Warnes, Christine; Liu, Jianming; Zhang, Chongben; Kawasome, Hideki; Terada, Naohiro; Boppart, Marni D; Schoenherr, Christopher J; Chen, Jie

    2009-12-01

    Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.

  6. IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

    PubMed

    Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

    2013-01-01

    Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

  7. Regulation of Src and Csk Nonreceptor Tyrosine Kinases in the Filasterean Ministeria vibrans

    PubMed Central

    2015-01-01

    The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2–kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals. PMID:24520931

  8. Regulation of Src and Csk nonreceptor tyrosine kinases in the filasterean Ministeria vibrans.

    PubMed

    Schultheiss, Kira P; Craddock, Barbara P; Suga, Hiroshi; Miller, W Todd

    2014-03-04

    The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2-kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals.

  9. PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

    PubMed

    Boccalandro, Hernán E; De Simone, Silvia N; Bergmann-Honsberger, Ariane; Schepens, Isabelle; Fankhauser, Christian; Casal, Jorge J

    2008-01-01

    Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

  10. Regulation of protein kinase C by the cytoskeletal protein calponin.

    PubMed

    Leinweber, B; Parissenti, A M; Gallant, C; Gangopadhyay, S S; Kirwan-Rhude, A; Leavis, P C; Morgan, K G

    2000-12-22

    Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

  11. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  12. Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity*

    PubMed Central

    Chichili, Gurunadh R.; Cail, Robert C.; Rodgers, William

    2012-01-01

    The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C16)-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C16-anchored DiO with didodecyl (C12)-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation. PMID:22613726

  13. The Role of Specific Mitogen-Activated Protein Kinase Signaling Cascades in the Regulation of Steroidogenesis

    PubMed Central

    Manna, Pulak R.; Stocco, Douglas M.

    2011-01-01

    Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models. PMID:21637381

  14. Hypothalamic AMP-Activated Protein Kinase Regulates Glucose Production

    PubMed Central

    Yang, Clair S.; Lam, Carol K.L.; Chari, Madhu; Cheung, Grace W.C.; Kokorovic, Andrea; Gao, Sun; Leclerc, Isabelle; Rutter, Guy A.; Lam, Tony K.T.

    2010-01-01

    OBJECTIVE The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production. RESEARCH DESIGN AND METHODS To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D157A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1312 [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo. RESULTS Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production. CONCLUSIONS These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo. PMID:20682691

  15. IκB Kinase 2 Regulates TPL-2 Activation of Extracellular Signal-Regulated Kinases 1 and 2 by Direct Phosphorylation of TPL-2 Serine 400

    PubMed Central

    Roget, Karine; Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Gantke, Thorsten; Yang, Huei-Ting; Janzen, Julia; Morrice, Nick; Abbott, Derek

    2012-01-01

    Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription. PMID:22988300

  16. Multifaceted roles for astrocytes in spreading depolarization

    PubMed Central

    Seidel, Jessica L.; Escartin, Carole; Ayata, Cenk; Bonvento, Gilles; Shuttleworth, C. William

    2015-01-01

    Spreading depolarizations (SD) are coordinated waves of synchronous depolarization, involving large numbers of neurons and astrocytes as they spread slowly through brain tissue. The recent identification of SDs as likely contributors to pathophysiology in human subjects has led to a significant increase in interest in SD mechanisms, and possible approaches to limit the numbers of SDs or their deleterious consequences in injured brain. Astrocytes regulate many events associated with SD. SD initiation and propagation is dependent on extracellular accumulation of K+ and glutamate, both of which involve astrocytic clearance. SDs are extremely metabolically demanding events, and signaling through astrocyte networks is likely central to the dramatic increase in regional blood flow that accompanies SD in otherwise healthy tissues. Astrocytes may provide metabolic support to neurons following SD, and may provide a source of adenosine that inhibits neuronal activity following SD. It is also possible that astrocytes contribute to the pathophysiology of SD, as a consequence of excessive glutamate release, facilitation of NMDA receptor activation, brain edema due to astrocyte swelling, or disrupted coupling to appropriate vascular responses after SD. Direct or indirect evidence has accumulated implicating astrocytes in many of these responses, but much remains unknown about their specific contributions, especially in the context of injury. Conversion of astrocytes to a reactive phenotype is a prominent feature of injured brain, and recent work suggests that the different functional properties of reactive astrocytes could be targeted to limit SDs in pathophysiological conditions. PMID:26301517

  17. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP PNP.

    PubMed

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M; Ho, Thau F; Maley, Kathleen; Randall, Tanya; Head, Martha S; Koretke, Kristin K; Schnackenberg, Christine G

    2007-12-01

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 A crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The alphaC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a beta-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel beta-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.

  18. A developmentally regulated MAP kinase activated by hydration in tobacco pollen.

    PubMed Central

    Wilson, C; Voronin, V; Touraev, A; Vicente, O; Heberle-Bors, E

    1997-01-01

    A novel mitogen-activated protein (MAP) kinase signaling pathway has been identified in tobacco. This pathway is developmentally regulated during pollen maturation and is activated by hydration during pollen germination. Analysis of different stages of pollen development showed that transcriptional and translational induction of MAP kinase synthesis occurs at the mid-bicellular stage of pollen maturation. However, the MAP kinase is stored in an inactive form in the mature, dry pollen grain. Kinase activation is very rapid after hydration of the dry pollen, peaking at approximately 5 min and decreasing thereafter. Immunoprecipitation of the kinase activity by an anti-phosphotyrosine antibody is consistent with the activation of a MAP kinase. The kinetics of activation suggest that the MAP kinase plays a role in the activation of the pollen grain after hydration rather than in pollen tube growth. PMID:9401129

  19. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function.

    PubMed

    Falsig, Jeppe; Pörzgen, Peter; Lund, Søren; Schrattenholz, André; Leist, Marcel

    2006-02-01

    Upon injury, astrocytes assume an activated state associated with the release of inflammatory mediators. To model this, we stimulated murine primary astrocytes with a complete inflammatory cytokine mix consisting of TNF-alpha, IL-1beta and IFN-gamma. We analysed the transcriptional response of 480 genes at 4 and 16 h after stimulation on a chip designed to give a representative overview over the inflammation-relevant part of the transcriptome of macrophage-like cells. The list of the 182 genes found to be significantly regulated in astrocytes revealed an intriguing co-ordinate regulation of genes linked to the biological processes of antiviral/antimicrobial defence, antigen presentation and facilitation of leucocyte invasion. The latter group was characterized by very high up-regulations of chemokine genes. We also identified regulations of a thymidylate kinase and an interferon-regulated protein with a tetratricopeptide motive, both up to now only known from macrophages. The transcriptional regulations were confirmed on the protein level by a proteomic analysis. These findings taken together suggest that activated astrocytes in brain behave similarly in many respects to inflamed macrophages in the periphery.

  20. Protein kinase regulation of a cloned epithelial Na+ channel

    PubMed Central

    1996-01-01

    We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC- expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be

  1. From in silico astrocyte cell models to neuron-astrocyte network models: A review.

    PubMed

    Oschmann, Franziska; Berry, Hugues; Obermayer, Klaus; Lenk, Kerstin

    2017-02-08

    The idea that astrocytes may be active partners in synaptic information processing has recently emerged from abundant experimental reports. Because of their spatial proximity to neurons and their bidirectional communication with them, astrocytes are now considered as an important third element of the synapse. Astrocytes integrate and process synaptic information and by doing so generate cytosolic calcium signals that are believed to reflect neuronal transmitter release. Moreover, they regulate neuronal information transmission by releasing gliotransmitters into the synaptic cleft affecting both pre- and postsynaptic receptors. Concurrent with the first experimental reports of the astrocytic impact on neural network dynamics, computational models describing astrocytic functions have been developed. In this review, we give an overview over the published computational models of astrocytic functions, from single-cell dynamics to the tripartite synapse level and network models of astrocytes and neurons.

  2. The Y’s that bind: negative regulators of Src family kinase activity in platelets

    PubMed Central

    NEWMAN, D. K.

    2015-01-01

    Summary Members of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity. PMID:19630799

  3. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  4. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  5. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development

    PubMed Central

    Dickerman, Benjamin K.; White, Christine L.; Kessler, Patricia M.; Sadler, Anthony J.; Williams, Bryan R.G.; Sen, Ganes C.

    2015-01-01

    The murine double-stranded RNA-binding protein RAX and the human homolog PACT were originally characterized as activators of protein kinase R (PKR). Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax−/− mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eIF2α (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax−/− mice. Generating rax−/− mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax−/− defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax−/− mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax−/− defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21WAF1/CIP1. These results demonstrate that PKR kinase activity is required for onset of the rax−/− phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development. PMID:26414443

  6. Astrocyte calcium signaling: the third wave.

    PubMed

    Bazargani, Narges; Attwell, David

    2016-02-01

    The discovery that transient elevations of calcium concentration occur in astrocytes, and release 'gliotransmitters' which act on neurons and vascular smooth muscle, led to the idea that astrocytes are powerful regulators of neuronal spiking, synaptic plasticity and brain blood flow. These findings were challenged by a second wave of reports that astrocyte calcium transients did not mediate functions attributed to gliotransmitters and were too slow to generate blood flow increases. Remarkably, the tide has now turned again: the most important calcium transients occur in fine astrocyte processes not resolved in earlier studies, and new mechanisms have been discovered by which astrocyte [Ca(2+)]i is raised and exerts its effects. Here we review how this third wave of discoveries has changed our understanding of astrocyte calcium signaling and its consequences for neuronal function.

  7. BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases.

    PubMed

    Chiarini, Anna; Dal Pra, Ilaria; Gottardo, Rossella; Bortolotti, Federica; Whitfield, James F; Armato, Ubaldo

    2005-03-01

    Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.

  8. Glycogen serves as an energy source that maintains astrocyte cell proliferation in the neonatal telencephalon.

    PubMed

    Gotoh, Hitoshi; Nomura, Tadashi; Ono, Katsuhiko

    2017-06-01

    Large amounts of energy are required when cells undergo cell proliferation and differentiation for mammalian neuronal development. Early neonatal mice face transient starvation and use stored energy for survival or to support development. Glycogen is a branched polysaccharide that is formed by glucose, and serves as an astrocytic energy store for rapid energy requirements. Although it is present in radial glial cells and astrocytes, the role of glycogen during development remains unclear. In the present study, we demonstrated that glycogen accumulated in glutamate aspartate transporter (GLAST)+ astrocytes in the subventricular zone and rostral migratory stream. Glycogen levels markedly decreased after birth due to the increase of glycogen phosphorylase, an essential enzyme for glycogen metabolism. In primary cultures and in vivo, the inhibition of glycogen phosphorylase decreased the proliferation of astrocytic cells. The number of cells in the G1 phase increased in combination with the up-regulation of cyclin-dependent kinase inhibitors or down-regulation of the phosphorylation of retinoblastoma protein (pRB), a determinant for cell cycle progression. These results suggest that glycogen accumulates in astrocytes located in specific areas during the prenatal stage and is used as an energy source to maintain normal development in the early postnatal stage.

  9. Differential induction of heme oxygenase and other stress proteins in cultured hippocampal astrocytes and neurons by inorganic lead.

    PubMed

    Cabell, Leigh; Ferguson, Charles; Luginbill, Deana; Kern, Marcey; Weingart, Adam; Audesirk, Gerald

    2004-07-01

    We examined the effects of exposure to inorganic lead (Pb2+) on the induction of stress proteins in cultured hippocampal neurons and astrocytes, with particular emphasis on the induction of heme oxygenase-1 (HO-1). In radiolabeled neuronal cultures, Pb2+ exposure had no significant effect on the synthesis of any protein at any concentration (up to 250 microM) or duration of exposure (up to 4 days). In radiolabeled astrocyte cultures, however, Pb2+ exposure (100 nM to 100 microM; 1-4 days) increased synthesis of proteins with approximate molecular weights of 23, 32, 45, 57, 72, and 90 kDa. Immunoblot experiments showed that Pb2+ exposure (100 nM to 10 microM, 1-14 days) induces HO-1 synthesis in astrocytes, but not in neurons; this is probably the 32-kDa protein. The other heme oxygenase isoform, HO-2, is present in both neurons and astrocytes, but is not inducible by Pb2+ at concentrations up to 100 microM. HO-1 can be induced by a variety of stimuli. We found that HO-1 induction in astrocytes is increased by combined exposure to Pb2+ and many other stresses, including heat, nitric oxide, H2O2, and superoxide. One of the stimuli that may induce HO-1 is oxidative stress. Lead exposure causes oxidative stress in many cell types, including astrocytes. Induction of HO-1 by Pb2+ is reduced by the hydroxyl radical scavengers dimethylthiourea (DMTU) and mannitol, but not by inhibitors of calmodulin, calmodulin-dependent protein kinases, protein kinase C, or extracellular signal-regulated kinases (ERK). Therefore, we conclude that oxidative stress is an important mechanism by which Pb2+ induces HO-1 synthesis in astrocytes.

  10. Astrocytic Actions on Extrasynaptic Neuronal Currents

    PubMed Central

    Pál, Balázs

    2015-01-01

    In the last few decades, knowledge about astrocytic functions has significantly increased. It was demonstrated that astrocytes are not passive elements of the central nervous system (CNS), but active partners of neurons. There is a growing body of knowledge about the calcium excitability of astrocytes, the actions of different gliotransmitters and their release mechanisms, as well as the participation of astrocytes in the regulation of synaptic functions and their contribution to synaptic plasticity. However, astrocytic functions are even more complex than being a partner of the “tripartite synapse,” as they can influence extrasynaptic neuronal currents either by releasing substances or regulating ambient neurotransmitter levels. Several types of currents or changes of membrane potential with different kinetics and via different mechanisms can be elicited by astrocytic activity. Astrocyte-dependent phasic or tonic, inward or outward currents were described in several brain areas. Such currents, together with the synaptic actions of astrocytes, can contribute to neuromodulatory mechanisms, neurosensory and -secretory processes, cortical oscillatory activity, memory, and learning or overall neuronal excitability. This mini-review is an attempt to give a brief summary of astrocyte-dependent extrasynaptic neuronal currents and their possible functional significance. PMID:26696832

  11. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells.

    PubMed

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P

    2014-03-28

    Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer's disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  12. HIV-1 Tat Promotes Lysosomal Exocytosis in Astrocytes and Contributes to Astrocyte-mediated Tat Neurotoxicity.

    PubMed

    Fan, Yan; He, Johnny J

    2016-10-21

    Tat interaction with astrocytes has been shown to be important for Tat neurotoxicity and HIV/neuroAIDS. We have recently shown that Tat expression leads to increased glial fibrillary acidic protein (GFAP) expression and aggregation and activation of unfolded protein response/endoplasmic reticulum (ER) stress in astrocytes and causes neurotoxicity. However, the exact molecular mechanism of astrocyte-mediated Tat neurotoxicity is not defined. In this study, we showed that neurotoxic factors other than Tat protein itself were present in the supernatant of Tat-expressing astrocytes. Two-dimensional gel electrophoresis and mass spectrometry revealed significantly elevated lysosomal hydrolytic enzymes and plasma membrane-associated proteins in the supernatant of Tat-expressing astrocytes. We confirmed that Tat expression and infection of pseudotyped HIV.GFP led to increased lysosomal exocytosis from mouse astrocytes and human astrocytes. We found that Tat-induced lysosomal exocytosis was tightly coupled to astrocyte-mediated Tat neurotoxicity. In addition, we demonstrated that Tat-induced lysosomal exocytosis was astrocyte-specific and required GFAP expression and was mediated by ER stress. Taken together, these results show for the first time that Tat promotes lysosomal exocytosis in astrocytes and causes neurotoxicity through GFAP activation and ER stress induction in astrocytes and suggest a common cascade through which aberrant astrocytosis/GFAP up-regulation potentiates neurotoxicity and contributes to neurodegenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes.

    PubMed

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  14. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    SciTech Connect

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  15. AOP-1 interacts with cardiac-specific protein kinase TNNI3K and down-regulates its kinase activity.

    PubMed

    Feng, Yan; Liu, Dong-Qing; Wang, Zhen; Liu, Zhao; Cao, Hui-Qing; Wang, Lai-Yuan; Shi, Na; Meng, Xian-Min

    2007-11-01

    In the present study, a yeast two-hybrid screening system was used to identify the interaction partners of cardiac troponin I-interacting kinase (TNNI3K) that might serve as regulators or targets, and thus in turn to gain some insights on the roles of TNNI3K. After screening the adult heart cDNA library with a bait construct encoding the ANK motif of TNNI3K, antioxidant protein 1 (AOP-1) was isolated. The interaction between TNNI3K and AOP-1 was confirmed by the in vitro binding assay and coexpression experiments in vivo. The colocalization of TNNI3K and AOP-1 was clarified by confocal immunofluorescence. Moreover, coexpression of AOP-1 inhibited TNNI3K kinase activity in the in vitro kinase assay.

  16. Bioenergetic Mechanisms in Astrocytes May Contribute to Amyloid Plaque Deposition and Toxicity*

    PubMed Central

    Fu, Wen; Shi, Diya; Westaway, David; Jhamandas, Jack H.

    2015-01-01

    Alzheimer disease (AD) is characterized neuropathologically by synaptic disruption, neuronal loss, and deposition of amyloid β (Aβ) protein in brain structures that are critical for memory and cognition. There is increasing appreciation, however, that astrocytes, which are the major non-neuronal glial cells, may play an important role in AD pathogenesis. Unlike neurons, astrocytes are resistant to Aβ cytotoxicity, which may, in part, be related to their greater reliance on glycolytic metabolism. Here we show that, in cultures of human fetal astrocytes, pharmacological inhibition or molecular down-regulation of a main enzymatic regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3), results in increased accumulation of Aβ within and around astrocytes and greater vulnerability of these cells to Aβ toxicity. We further investigated age-dependent changes in PFKFB3 and astrocytes in AD transgenic mice (TgCRND8) that overexpress human Aβ. Using a combination of Western blotting and immunohistochemistry, we identified an increase in glial fibrillary acidic protein expression in astrocytes that paralleled the escalation of the Aβ plaque burden in TgCRND8 mice in an age-dependent manner. Furthermore, PFKFB3 expression also demonstrated an increase in these mice, although at a later age (9 months) than GFAP and Aβ. Immunohistochemical staining showed significant reactive astrogliosis surrounding Aβ plaques with increased PFKFB3 activity in 12-month-old TgCRND8 mice, an age when AD pathology and behavioral deficits are fully manifested. These studies shed light on the unique bioenergetic mechanisms within astrocytes that may contribute to the development of AD pathology. PMID:25814669

  17. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

    PubMed

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2016-12-16

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes.

    PubMed Central

    Conant, K; Ma, M; Nath, A; Major, E O

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection has been associated with an increase in the binding of the transcription factor NF-kappa B to its consensus sequence in the viral promoter. Using cultures of primary human fetal astrocytes, we show that exogenous HIV-1 Tat protein, which has been demonstrated to be released from infected cells, is associated with an increase in the binding of this transcription factor to an HIV-1 long terminal repeat kappa B sequence. This effect occurs rapidly and is independent of new protein synthesis. We also demonstrate that extracellular Tat protein is associated with an increase in protein kinase C activity. If Tat functions similarly in other cell types, such findings could relate to some of this protein's previously described physiological effects. These effects include Tat's ability to upregulate the synthesis of specific cytokines and to act as a growth factor. PMID:8627654

  19. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  20. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  1. A MAP Kinase Kinase Interacts with SymRK and Regulates Nodule Organogenesis in Lotus japonicus[C][W

    PubMed Central

    Chen, Tao; Zhu, Hui; Ke, Danxia; Cai, Kai; Wang, Chao; Gou, Honglan; Hong, Zonglie; Zhang, Zhongming

    2012-01-01

    The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis. PMID:22353370

  2. Recent Progress on Liver Kinase B1 (LKB1): Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    PubMed Central

    Gan, Ren-You; Li, Hua-Bin

    2014-01-01

    Liver kinase B1 (LKB1), known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK)-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK), salt-inducible kinase (SIK), sucrose non-fermenting protein-related kinase (SNRK) and brain selective kinase (BRSK) signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers. PMID:25244018

  3. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  4. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  5. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    PubMed Central

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease. PMID:26973524

  6. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

  7. Sequential conformational transitions and α-helical supercoiling regulate a sensor histidine kinase.

    PubMed

    Berntsson, Oskar; Diensthuber, Ralph P; Panman, Matthijs R; Björling, Alexander; Gustavsson, Emil; Hoernke, Maria; Hughes, Ashley J; Henry, Léocadie; Niebling, Stephan; Takala, Heikki; Ihalainen, Janne A; Newby, Gemma; Kerruth, Silke; Heberle, Joachim; Liebi, Marianne; Menzel, Andreas; Henning, Robert; Kosheleva, Irina; Möglich, Andreas; Westenhoff, Sebastian

    2017-08-18

    Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases.Sensor histidine kinases (SHK) consist of sensor, linker and kinase modules and different models for SHK signal transduction have been proposed. Here the authors present nano- to millisecond time-resolved X-ray scattering measurements, which reveal a structural mechanism for kinase domain activation in SHK.

  8. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  9. Creating Order from Chaos: Cellular Regulation by Kinase Anchoring

    PubMed Central

    Scott, John D.; Dessauer, Carmen W.; Tasken, Kjetil

    2012-01-01

    Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems. PMID:23043438

  10. Astrocytic P2Y(1) receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia.

    PubMed

    Kuboyama, Kazuya; Harada, Hideki; Tozaki-Saitoh, Hidetoshi; Tsuda, Makoto; Ushijima, Kazuo; Inoue, Kazuhide

    2011-09-01

    After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.

  11. Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase.

    PubMed Central

    Chiariello, M; Gomez, E; Gutkind, J S

    2000-01-01

    Mitogen-activated protein (MAP) kinases, p42(MAPK) and p44(MAPK), are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G(1)/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G(1) phase, once the known mechanisms by which MAP kinase controls G(1) progression, accumulation of G(1) cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control. PMID:10903150

  12. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed Central

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-01-01

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed. Images Fig. 4. PMID:2829841

  13. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-12-15

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.

  14. Protein Kinase N2 Regulates AMP-Kinase Signaling and Insulin Responsiveness of Glucose Metabolism in Skeletal Muscle.

    PubMed

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-07-18

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. As skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. While Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, while stimulating fatty acid oxidation and incorporation into triglycerides, and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC1α and SREBP1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017, American Journal of Physiology-Endocrinology and Metabolism.

  15. Activated spinal astrocytes are involved in the maintenance of chronic widespread mechanical hyperalgesia after cast immobilization

    PubMed Central

    2014-01-01

    Background In the present study, we examined spinal glial cell activation as a central nervous system mechanism of widespread mechanical hyperalgesia in rats that experienced chronic post-cast pain (CPCP) 2 weeks after cast immobilization. Activated spinal microglia and astrocytes were investigated immunohistologically in lumbar and coccygeal spinal cord segments 1 day, 5 weeks, and 13 weeks following cast removal. Results In the lumbar cord, astrocytes were activated after microglia. Astrocytes also were activated after microglia in the coccygeal cord, but with a delay that was longer than that observed in the lumbar cord. This activation pattern paralleled the observation that mechanical hyperalgesia occurred in the hindleg or the hindpaw before the tail. The activating transcription factor 3 (ATF3) immune response in dorsal root ganglia (DRG) on the last day of cast immobilization suggested that nerve damage might not occur in CPCP rats. The neural activation assessed by the phosphorylated extracellular signal-regulated kinase (pERK) immune response in DRG arose 1 day after cast removal. In addition, L-α-aminoadipate (L-α-AA), an inhibitor of astrocyte activation administered intrathecally 5 weeks after cast removal, inhibited mechanical hyperalgesia in several body parts including the lower leg skin and muscles bilaterally, hindpaws, and tail. Conclusions These findings suggest that activation of lumbar cord astrocytes is an important factor in widespread mechanical hyperalgesia in CPCP. PMID:24456903

  16. Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms

    PubMed Central

    Perjés, Ábel; Skoumal, Réka; Tenhunen, Olli; Kónyi, Attila; Simon, Mihály; Horváth, Iván G.; Kerkelä, Risto; Ruskoaho, Heikki; Szokodi, István

    2014-01-01

    Background Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin. Methods and Results In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity. Conclusions Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure. PMID:24695532

  17. Ste20-related proline/alanine-rich kinase: A novel regulator of intestinal inflammation

    PubMed Central

    Yan, Yutao; Merlin, Didier

    2008-01-01

    Recently, inflammatory bowel disease (IBD) has been the subject of considerable research, with increasing attention being paid to the loss of intestinal epithelial cell barrier function as a mechanism of pathogenesis. Ste20-related proline/alanine-rich kinase (SPAK) is involved in regulating barrier function. SPAK is known to interact with inflammation-related kinases (such as p38, JNK, NKCC1, PKCtheta;, WNK and MLCK), and with transcription factor AP-1, resulting in diverse biological phenomena, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and regulation of chloride transport. This review examines the involvement of Ste20-like kinases and downstream mitogen-activated protein kinases (MAPKs) pathways in the pathogenesis and control of intestinal inflammation. The primary focus will be on the molecular features of intestinal inflammation, with an emphasis on the interaction between SPAK and other molecules, and the effect of these interactions on homeostatic maintenance, cell volume regulation and increased cell permeability in intestinal inflammation. PMID:18985800

  18. PERK pathway is involved in oxygen-glucose-serum deprivation-induced NF-kB activation via ROS generation in spinal cord astrocytes.

    PubMed

    Liu, Jinbo; Du, Lijian

    2015-11-13

    Mitochondrial dysfunction is a direct target of hypoxic/ischemic stress in astrocytes, which results in the increased production of reactive oxygen species (ROS). Previous reports showed that ROS can activate NF-kB in spinal cord astrocytes, which occurs as a secondary injury during the pathological process of spinal cord injury (SCI). Protein kinase RNA (PKR)-like ER kinase (PERK) plays an important role in mitochondrial dysfunction. To elucidate the specific role of PERK in hypoxic/ischemic-induced NF-kB activation in spinal astrocytes, we utilized an in vitro oxygen-glucose deprivation (OGD) model, which showed an enhanced formation of ROS and NF-kB activation. Knockdown of PERK resulted in reduced activation of PERK and ROS generation in astrocytes under OGD conditions. Notably, the knockdown of PERK also induced NF-kB activation in astrocytes. These data suggest that PERK is required for the hypoxic/ischemic-induced-dependent regulation of ROS and that it is involved in NF-kB activation in the astrocytes.

  19. Down-Regulation of the Met Receptor Tyrosine Kinase by Presenilin-dependent Regulated Intramembrane Proteolysis

    PubMed Central

    Foveau, Bénédicte; Ancot, Frédéric; Leroy, Catherine; Petrelli, Annalisa; Reiss, Karina; Vingtdeux, Valérie; Giordano, Silvia; Fafeur, Véronique

    2009-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) acts through the membrane-anchored Met receptor tyrosine kinase to induce invasive growth. Deregulation of this signaling is associated with tumorigenesis and involves, in most cases, overexpression of the receptor. We demonstrate that Met is processed in epithelial cells by presenilin-dependent regulated intramembrane proteolysis (PS-RIP) independently of ligand stimulation. The proteolytic process involves sequential cleavage by metalloproteases and the γ-secretase complex, leading to generation of labile fragments. In normal epithelial cells, although expression of cleavable Met by PS-RIP is down-regulated, uncleavable Met displayed membrane accumulation and induced ligand-independent motility and morphogenesis. Inversely, in transformed cells, the Met inhibitory antibody DN30 is able to promote Met PS-RIP, resulting in down-regulation of the receptor and inhibition of the Met-dependent invasive growth. This demonstrates the original involvement of a proteolytic process in degradation of the Met receptor implicated in negative regulation of invasive growth. PMID:19297528

  20. Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase

    PubMed Central

    Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, Peter

    2011-01-01

    Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process. PMID:21807898

  1. Lack of Csk-mediated negative regulation in a unicellular SRC kinase.

    PubMed

    Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, Iñaki; Miller, W Todd

    2012-10-16

    Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.

  2. Up-regulation of CB2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis.

    PubMed

    Fernández-Trapero, María; Espejo-Porras, Francisco; Rodríguez-Cueto, Carmen; Coates, Joan R; Pérez-Díaz, Carmen; de Lago, Eva; Fernández-Ruiz, Javier

    2017-01-09

    Targeting the CB2 receptor afforded neuroprotection in SOD1(G93A) mutant mice, a model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors were facilitated by their up-regulation in the spinal cord in SOD1(G93A) mutant mice. Herein, we have investigated whether a similar CB2 receptor up-regulation, as well as parallel changes in other endocannabinoid elements, are evident in the spinal cord of dogs with degenerative myelopathy (DM), caused from mutations in the superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem spinal cords from unaffected and DM-affected dogs. Tissues were used first to confirm the loss of motor neurons using Nissl staining, which was accompanied by glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated possible differences in the expression of endocannabinoid genes measured by qPCR between DM-affected and control dogs. We found no changes in the CB1 receptor (also found with CB1 receptor immunostaining) as well as in NAPE-PLD, DAGL, FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly elevated in DM-affected dogs determined by qPCR and Western-blotting, results reconfirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling co-localized with GFAP but not Iba-1, indicating up-regulation of CB2 receptors on astrocytes in DM-affected dogs. In summary, our results demonstrated a marked up-regulation of CB2 receptors occurring in the spinal cord in canine DM, which was concentrated in activated astrocytes. Such receptors may be used as a potential target to enhance the neuroprotective effects exerted by these glial cells.

  3. Extracellular signal-regulated protein kinase activation in spinal cord contributes to pain hypersensitivity in a mouse model of type 2 diabetes.

    PubMed

    Xu, Xiang; Chen, Hui; Ling, Bing-Yu; Xu, Lan; Cao, Hong; Zhang, Yu-Qiu

    2014-02-01

    Painful peripheral neuropathy is a common complication of diabetes mellitus. The symptom of pain can become a major factor that decreases the quality of life of patients with diabetes, while effective treatment is lacking. In the present study, we aimed to investigate the changes of pain threshold in the early stage of diabetes in db/db mice, an animal model of type 2 diabetes mellitus, and the underlying molecular mechanisms. We found that (1) db/db mice (with a leptin receptor-null mutation and characterized by obesity and hyperglycemia) showed hypersensitivity to mechanical and thermal stimuli at the early stage of diabetes; (2) phosphorylated extracellular signal-regulated kinase (pERK), but not total ERK in the spinal cord and dorsal root ganglia in db/db mice significantly increased compared with wild-type mice. The increased pERK immunoreactivity occurred in both NeuN-expressing neurons and GFAP-expressing astrocytes, but not in Iba-1-expressing microglia; (3) both single and consecutive (for 5 days) intrathecal injections of U0126 (2 nmol per day), a selective MEK (an ERK kinase) inhibitor beginning at 8 weeks of age, attenuated the bilateral mechanical allodynia in the von-Frey test and heat hyperalgesia in Hargreave's test; and (4) db/db mice also displayed increased nocifensive behavior during the formalin test, and this was blocked by intrathecal injection of U0126. Also, the expression of pERK1 and pERK2 was upregulated following the formalin injection. Our results suggested that the activation of ERK in spinal neurons and astrocytes is correlated with pain hypersensitivity of the type 2 diabetes animal model. Inhibiting the ERK pathway may provide a new therapy for pain control in type 2 diabetes.

  4. Integrated stress response of vertebrates is regulated by four eIF2α kinases

    PubMed Central

    Taniuchi, Shusuke; Miyake, Masato; Tsugawa, Kazue; Oyadomari, Miho; Oyadomari, Seiichi

    2016-01-01

    The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates. PMID:27633668

  5. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  6. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed Central

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-01-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo. PMID:9001227

  7. Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms.

    PubMed

    Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol; Manne, Bhanu Kanth; Badolia, Rachit; Borgognone, Alessandra; Tsygankov, Alexander Y; McKenzie, Steven E; Kunapuli, Satya P

    2013-10-04

    Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

  8. Post-Transcriptional Regulation of MKK4-A Stress Signaling Kinase Implicated in the Regulation of Metastatic Growth

    DTIC Science & Technology

    2006-04-01

    REPORTABLE OUTCOMES: Reportable outcomes that have resulted from this research to include: DISSERTATION: ictoria L. Robinson : Post-transcriptional...manuscript by Robinson et al. is provided in the following Appendix. TRANSLATIONAL REGULATION OF MAP KINASE KINASE 4 (MKK4) EXPRESSION Victoria L... Robinson ‡, Ore Shalhav‡, Kristen Otto‡, Tomoko Kawai§, Myriam Gorospe§, and Carrie W. Rinker-Schaeffer‡¶ From the ‡Department of Surgery, Section of

  9. Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.

    PubMed

    Stork, Tobias; Sheehan, Amy; Tasdemir-Yilmaz, Ozge E; Freeman, Marc R

    2014-07-16

    Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.

  10. Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase.

    PubMed

    Elsholz, Alexander K W; Wacker, Sarah A; Losick, Richard

    2014-08-01

    We report that the Bacillus subtilis exopolysaccharide (EPS) is a signaling molecule that controls its own production. EPS synthesis depends on a tyrosine kinase that consists of a membrane component (EpsA) and a kinase component (EpsB). EPS interacts with the extracellular domain of EpsA, which is a receptor, to control kinase activity. In the absence of EPS, the kinase is inactivated by autophosphorylation. The presence of EPS inhibits autophosphorylation and instead promotes the phosphorylation of a glycosyltransferase in the biosynthetic pathway, thereby stimulating the production of EPS. Thus, EPS production is subject to a positive feedback loop that ties its synthesis to its own concentration. Tyrosine kinase-mediated self-regulation could be a widespread feature of the control of exopolysaccharide production in bacteria.

  11. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases.

    PubMed

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X Edward; West, Graham M; Kovach, Amanda; Tan, M H Eileen; Suino-Powell, Kelly M; He, Yuanzheng; Xu, Yong; Chalmers, Michael J; Brunzelle, Joseph S; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R; Melcher, Karsten; Xu, H Eric

    2012-01-06

    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.

  12. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    PubMed Central

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    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. PMID:22116026

  13. Rho-associated coiled-coil containing kinases (ROCK): structure, regulation, and functions.

    PubMed

    Julian, Linda; Olson, Michael F

    2014-01-01

    Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase. (1)(-) (5) They belong to the AGC family of serine/threonine kinases (6) and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK.

  14. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    SciTech Connect

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2014-10-02

    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.

  15. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    PubMed

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  16. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  17. Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    PubMed Central

    Banning, Antje; Ockenga, Wymke; Finger, Fabian; Siebrasse, Philipp; Tikkanen, Ritva

    2012-01-01

    Flotillin-1 and flotillin-2 are important regulators of signal transduction pathways such as growth factor signaling. Flotillin expression is increased under pathological conditions such as neurodegenerative disorders and cancer. Despite their importance for signal transduction, very little is known about the transcriptional regulation of flotillins. Here, we analyzed the expression of flotillins at transcriptional level and identified flotillins as downstream targets of the mitogen activated kinases ERK1/2. The promoter activity of flotillins was increased upon growth factor stimulation in a MAPK dependent manner. Overexpression of serum response factor or early growth response gene 1 resulted in increased flotillin mRNA and protein expression. Furthermore, both promoter activity and expression of endogenous flotillins were increased upon treatment with retinoic acid or by overexpression of the retinoid X receptor and its binding partners RARα and PPARγ. Our data indicate that the expression of flotillins, which can be detected in all cultured cells, is fine-tuned in response to various external stimuli. This regulation may be critical for the outcome of signaling cascades in which flotillins are known to be involved. PMID:23029064

  18. Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

    PubMed

    Monsivais, Diana; Dyson, Matthew T; Yin, Ping; Navarro, Antonia; Coon, John S; Pavone, Mary Ellen; Bulun, Serdar E

    2016-05-01

    To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. Case-control. University research setting. Premenopausal women. Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) β. Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERβ-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERβ based on small interfering RNA knockdown and chromatin immunoprecipitation of ERβ followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. ERβ leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  19. IL-17 induces reactive astrocytes and up-regulation of vascular endothelial growth factor (VEGF) through JAK/STAT signaling

    PubMed Central

    You, Tao; Bi, Yihui; li, Jun; Zhang, Mingkai; Chen, Xuezhou; Zhang, Keke; Li, Jun

    2017-01-01

    Spinal cord injury is a grave neurological disability resulting in neuron degeneration and permanent paralysis. The inflammation triggered by the injury would promote the spinal cord lesion in turn. Activated astrocytes during inflammatory response could promote glial scar formation and contribute to the progression of the spinal cord injury. Interleukin 17 (IL-17) was upregulated in inflammatory responses to contusion or compression of the spinal cord. in this study, IL-17 could induce reactive astrocytes which was indicated by a well-known hallmark glial fibrillary acidic protein (GFAP) in vitro and in vivo. Moreover, we demonstrated that the upregulation of VEGF was induced by IL-17 human astrocytoma cells. In our further investigation, IL-17 induced the expression of VEGF in spinal cord injury by activating JAK/STAT signaling pathway both in vitro and in vivo. In addition, we also found that IL-17 significantly changed tissue preservation and residual urine volumes and blood-spinal cord-barrier integrity in vivo. This newly found IL-17-JAK/STAT-VEGF axis improves our understanding of the molecular mechanism of spinal cord injury during inflammatory response and provides another potential target of spinal cord injury. PMID:28281545

  20. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  1. Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

    PubMed Central

    Damarla, Mahendra; Hasan, Emile; Boueiz, Adel; Le, Anne; Pae, Hyun Hae; Montouchet, Calypso; Kolb, Todd; Simms, Tiffany; Myers, Allen; Kayyali, Usamah S.; Gaestel, Matthias; Peng, Xinqi; Reddy, Sekhar P.; Damico, Rachel; Hassoun, Paul M.

    2009-01-01

    Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling. PMID:19240800

  2. Protein Kinase Pathways That Regulate Neuronal Survival and Death

    DTIC Science & Technology

    2004-08-01

    kinase 3 inactivation. Char- sants, phenytoin and carbamazepine. J. Pharmacol. Exp. Ther 286, acterization of dominant-negative mutant of PKB. J. Biol...Asted, A. A. Ansari. 1995. Fetal neural transplantation for Parkinson and A. Bjorklund. 1986. Behavioral effects of human fetal do- disease. In...Bilateral fetal mesencephalic 63. Yayon, A., M. Klagsbrun, J. D. Esko, P. Leder, and D. M. grafting in two patients with Parkinsonism induced by 1-methyl

  3. Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase.

    PubMed

    Núñez, Andrés; Franco, Alejandro; Soto, Teresa; Vicente, Jero; Gacto, Mariano; Cansado, José

    2010-12-31

    In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.

  4. Fission Yeast Receptor of Activated C Kinase (RACK1) Ortholog Cpc2 Regulates Mitotic Commitment through Wee1 Kinase*

    PubMed Central

    Núñez, Andrés; Franco, Alejandro; Soto, Teresa; Vicente, Jero; Gacto, Mariano; Cansado, José

    2010-01-01

    In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G1/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G2/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G1/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes. PMID:20974849

  5. Regulation of therapeutic resistance in cancers by receptor tyrosine kinases

    PubMed Central

    Chen, Mei-Kuang; Hung, Mien-Chie

    2016-01-01

    In response to DNA damage lesions due to cellular stress, DNA damage response (DDR) pathways are activated to promote cell survival and genetic stability or unrepaired lesion-induced cell death. Current cancer treatments predominantly utilize DNA damaging agents, such as irradiation and chemotherapy drugs, to inhibit cancer cell proliferation and induce cell death through the activation of DDR. However, a portion of cancer patients is reported to develop therapeutic resistance to these DDR-inducing agents. One significant resistance mechanism in cancer cells is oncogenic kinase overexpression, which promotes cell survival by enhancing DNA damage repair pathways and evading cell cycle arrest. Among the oncogenic kinases, overexpression of receptor tyrosine kinases (RTKs) is reported in many of solid tumors, and numerous clinical trials targeting RTKs are currently in progress. As the emerging trend in cancer treatment combines DNA damaging agents and RTK inhibitors, it is important to understand the substrates of RTKs relative to the DDR pathways. In addition, alteration of RTK expression and their phosphorylated substrates can serve as biomarkers to stratify patients for combination therapies. In this review, we summarize the deleterious effects of RTKs on the DDR pathways and the emerging biomarkers for personalized therapy. PMID:27186434

  6. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis.

    PubMed

    Jones, Nathaniel G; Wang, Qiuling; Sibley, L David

    2017-02-01

    Toxoplasma gondii is an apicomplexan parasite that secretes a large number of protein kinases and pseudokinases from its rhoptry organelles. Although some rhoptry kinases (ROPKs) act as virulence factors, many remain uncharacterized. In this study, predicted ROPKs were assessed for bradyzoite expression then prioritized for a reverse genetic analysis in the type II strain Pru that is amenable to targeted disruption. Using CRISPR/Cas9, we engineered C-terminally epitope tagged ROP21 and ROP27 and demonstrated their localization to the parasitophorous vacuole and cyst matrix. ROP21 and ROP27 were not secreted from microneme, rhoptry, or dense granule organelles, but rather were located in small vesicles consistent with a constitutive pathway. Using CRISPR/Cas9, the genes for ROP21, ROP27, ROP28, and ROP30 were deleted individually and in combination, and the mutant parasites were assessed for growth and their ability to form tissue cysts in mice. All knockouts lines were normal for in vitro growth and bradyzoite differentiation, but a combined ∆rop21/∆rop17 knockout led to a 50% reduction in cyst burden in vivo. Our findings question the existing annotation of ROPKs based solely on bioinformatic techniques and yet highlight the importance of secreted kinases in determining the severity of chronic toxoplasmosis.

  7. Ligand-Induced Asymmetry in Histidine Sensor Kinase Complex Regulates Quorum Sensing

    SciTech Connect

    Neiditch,M.; Federle, M.; Pompeani, A.; Kelly, R.; Swem, D.; Jeffrey, P.; Bassler, B.; Hughson, F.

    2006-01-01

    Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

  8. Per-Arnt-Sim Kinase (PASK): An Emerging Regulator of Mammalian Glucose and Lipid Metabolism.

    PubMed

    Zhang, Dan-dan; Zhang, Ji-gang; Wang, Yu-zhu; Liu, Ying; Liu, Gao-lin; Li, Xiao-yu

    2015-09-07

    Per-Arnt-Sim Kinase (PASK) is an evolutionarily-conserved nutrient-responsive protein kinase that regulates lipid and glucose metabolism, mitochondrial respiration, phosphorylation, and gene expression. Recent data suggests that mammalian PAS kinase is involved in glucose metabolism and acts on pancreatic islet α/β cells and glycogen synthase (GS), affecting insulin secretion and blood glucose levels. In addition, PASK knockout mice (PASK-/-) are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet, implying that PASK may be a new target for metabolic syndrome (MetS) treatment as well as the cellular nutrients and energy sensors-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and the targets of rapamycin (m-TOR). In this review, we will briefly summarize the regulation of PASK on mammalian glucose and lipid metabolism and its possible mechanism, and further explore the potential targets for MetS therapy.

  9. Per-Arnt-Sim Kinase (PASK): An Emerging Regulator of Mammalian Glucose and Lipid Metabolism

    PubMed Central

    Zhang, Dan-dan; Zhang, Ji-gang; Wang, Yu-zhu; Liu, Ying; Liu, Gao-lin; Li, Xiao-yu

    2015-01-01

    Per-Arnt-Sim Kinase (PASK) is an evolutionarily-conserved nutrient-responsive protein kinase that regulates lipid and glucose metabolism, mitochondrial respiration, phosphorylation, and gene expression. Recent data suggests that mammalian PAS kinase is involved in glucose metabolism and acts on pancreatic islet α/β cells and glycogen synthase (GS), affecting insulin secretion and blood glucose levels. In addition, PASK knockout mice (PASK-/-) are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet, implying that PASK may be a new target for metabolic syndrome (MetS) treatment as well as the cellular nutrients and energy sensors—adenosine monophosphate (AMP)-activated protein kinase (AMPK) and the targets of rapamycin (m-TOR). In this review, we will briefly summarize the regulation of PASK on mammalian glucose and lipid metabolism and its possible mechanism, and further explore the potential targets for MetS therapy. PMID:26371032

  10. WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters*

    PubMed Central

    Rinehart, Jesse; Vázquez, Norma; Kahle, Kristopher T.; Hodson, Caleb A.; Ring, Aaron M.; Gulcicek, Erol E.; Louvi, Angeliki; Bobadilla, Norma A.; Gamba, Gerardo; Lifton, Richard P.

    2011-01-01

    NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl−]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl−-dependent 86Rb+ uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl− accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO2 enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling. PMID:21733846

  11. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

    PubMed Central

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  12. Malaria Protein Kinase CK2 (PfCK2) Shows Novel Mechanisms of Regulation

    PubMed Central

    Graciotti, Michele; Alam, Mahmood; Solyakov, Lev; Schmid, Ralf; Burley, Glenn; Bottrill, Andrew R.; Doerig, Christian; Cullis, Paul; Tobin, Andrew B.

    2014-01-01

    Casein kinase 2 (protein kinase CK2) is a conserved eukaryotic serine/theronine kinase with multiple substrates and roles in the regulation of cellular processes such as cellular stress, cell proliferation and apoptosis. Here we report a detailed analysis of the Plasmodium falciparum CK2, PfCK2, demonstrating that this kinase, like the mammalian orthologue, is a dual specificity kinase able to phosphorylate at both serine and tyrosine. However, unlike the human orthologue that is auto-phosphorylated on tyrosine within the activation loop, PfCK2 shows no activation loop auto-phosphorylation but rather is auto-phosphorylated at threonine 63 within subdomain I. Phosphorylation at this site in PfCK2 is shown here to regulate the intrinsic kinase activity of PfCK2. Furthermore, we generate an homology model of PfCK2 in complex with the known selective protein kinase CK2 inhibitor, quinalizarin, and in so doing identify key co-ordinating residues in the ATP binding pocket that could aid in designing selective inhibitors to PfCK2. PMID:24658579

  13. Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices

    PubMed Central

    Sasaki, Takuya; Ishikawa, Tomoe; Abe, Reimi; Nakayama, Ryota; Asada, Akiko; Matsuki, Norio; Ikegaya, Yuji

    2014-01-01

    Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level. PMID:24710057

  14. A mechanism for regulation of chloroplast LHC II kinase by plastoquinol and thioredoxin.

    PubMed

    Puthiyaveetil, Sujith

    2011-06-23

    State transitions are acclimatory responses to changes in light quality in photosynthesis. They involve the redistribution of absorbed excitation energy between photosystems I and II. In plants and green algae, this redistribution is produced by reversible phosphorylation of the chloroplast light harvesting complex II (LHC II). The LHC II kinase is activated by reduced plastoquinone (PQ) in photosystem II-specific low light. In high light, when PQ is also reduced, LHC II kinase becomes inactivated by thioredoxin. Based on newly identified amino acid sequence features of LHC II kinase and other considerations, a mechanism is suggested for its redox regulation.

  15. [Involvement of cyclin-dependent kinase CDK1/CDC28 in regulation of cell cycle].

    PubMed

    Koltovaya, N A

    2013-07-01

    Cyclin-dependent kinases (CDKs) are a family of enzymes essential for the progression of the cells through the cell cycle in eukaryotes. Moreover, genetic stability-maintaining processes, such as checkpoint control and DNA repair, require the phosphorylation of a wide variety of target substrates by CDK. In budding yeast Saccharomyces cerevisiae, the key role in the cell cycle progression is played by CDK1/CDC28 kinase. This enzyme is the most thoroughly investigated. In this review the involvement of CDC28 kinase in regulation of the cell cycle is discussed in the light of newly obtained data.

  16. Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

    PubMed Central

    Weijman, Johannes F.; Kumar, Abhishek; Jamieson, Sam A.; King, Chontelle M.; Caradoc-Davies, Tom T.; Ledgerwood, Elizabeth C.; Murphy, James M.

    2017-01-01

    Apoptosis signal-regulating kinases (ASK1–3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling. PMID:28242696

  17. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    PubMed Central

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  18. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases.

    PubMed

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.

  19. Human astrocytes in the diseased brain.

    PubMed

    Dossi, Elena; Vasile, Flora; Rouach, Nathalie

    2017-02-13

    Astrocytes are key active elements of the brain that contribute to information processing. They not only provide neurons with metabolic and structural support, but also regulate neurogenesis and brain wiring. Furthermore, astrocytes modulate synaptic activity and plasticity in part by controlling the extracellular space volume, as well as ion and neurotransmitter homeostasis. These findings, together with the discovery that human astrocytes display contrasting characteristics with their rodent counterparts, point to a role for astrocytes in higher cognitive functions. Dysfunction of astrocytes can thereby induce major alterations in neuronal functions, contributing to the pathogenesis of several brain disorders. In this review we summarize the current knowledge on the structural and functional alterations occurring in astrocytes from the human brain in pathological conditions such as epilepsy, primary tumours, Alzheimer's disease, major depressive disorder and Down syndrome. Compelling evidence thus shows that dysregulations of astrocyte functions and interplay with neurons contribute to the development and progression of various neurological diseases. Targeting astrocytes is thus a promising alternative approach that could contribute to the development of novel and effective therapies to treat brain disorders.

  20. Astrocytes conspire with neurons during progression of neurological disease

    PubMed Central

    McGann, James C.; Lioy, Daniel T.; Mandel, Gail

    2012-01-01

    As astrocytes are becoming recognized as important mediators of normal brain function, studies into their roles in neurological disease have gained significance. Across mouse models for neurodevelopmental and neurodegenerative diseases, astrocytes are considered key regulators of disease progression. In Rett syndrome and Parkinson’s disease, astrocytes can even initiate certain disease phenotypes. Numerous potential mechanisms have been offered to explain these results, but research into the functions of astrocytes in disease is just beginning. Crucially, in vivo verification of in vitro data is still necessary, as well as a deeper understanding of the complex and relatively unexplored interactions between astrocytes, oligodendrocytes, microglia, and neurons. PMID:22475461

  1. Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone.

    PubMed

    Zhen, X; Wei, L; Wu, Q; Zhang, Y; Chen, Q

    2001-02-16

    Parathyroid hormone (PTH) and its related peptide regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. However, the intracellular pathway for transducing PTH/PTH-related peptide signals in chondrocytes remains unclear. Here, we show that this pathway is mediated by mitogen-activated protein kinase (MAPK) p38. Incubation of hypertrophic chondrocytes with PTH (1-34) induces an inhibition of p38 kinase activity in a time- and dose-dependent manner. Inhibition of protein kinase C prevents PTH-induced p38 MAPK inhibition, whereas inhibition of protein kinase A has no effect. Thus, protein kinase C, but not protein kinase A, is required for the inhibition of p38 MAPK by PTH. Treatment of hypertrophic chondrocytes by PTH or by p38 MAPK inhibitor SB203580 up-regulates Bcl-2, suggesting that Bcl-2 lies downstream of p38 MAPK in the PTH signaling pathway. Inhibition of p38 MAPK in hypertrophic chondrocytes by either PTH, SB303580, or both together leads to a decrease of hypertrophic marker type X collagen mRNA and an increase of the expression of prehypertrophic marker cartilage matrix protein. Therefore, inhibition of p38 converts a hypertrophic cell phenotype to a prehypertrophic one, thereby preventing precocious chondrocyte hypertrophy. Taken together, these data suggest a major role for p38 MAPK in transmitting PTH signals to regulate chondrocyte differentiation.

  2. Astrocyte Elevated Gene-1 Is a Novel Modulator of HIV-1-associated Neuroinflammation via Regulation of Nuclear Factor-κB Signaling and Excitatory Amino Acid Transporter-2 Repression*

    PubMed Central

    Vartak-Sharma, Neha; Gelman, Benjamin B.; Joshi, Chaitanya; Borgamann, Kathleen; Ghorpade, Anuja

    2014-01-01

    Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV)-1 and tumor necrosis factor (TNF)-α-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic aggressive tumors. However, little is known about its role in astrocyte responses during HIV-1 central nervous system (CNS) infection and whether it contributes toward the development of HIV-associated neurocognitive disorders (HAND). Therefore, in this study, we investigated changes in AEG-1 CNS expression in HIV-1-infected brain tissues and elucidated a potential mechanism of AEG-1-mediated regulation of HAND. Immunoblotting and immunohistochemical analyses of HIV-1 seropositive and HIV-1 encephalitic human brain tissues revealed significantly elevated levels of AEG-1 protein. Immunohistochemical analyses of HIV-1 Tat transgenic mouse brain tissues also showed a marked increase in AEG-1 staining. Similar to in vivo observations, cultured astrocytes expressing HIV-1 Tat also revealed AEG-1 and cytokine up-regulation. Astrocytes treated with HAND-relevant stimuli, TNF-α, interleukin (IL)-1β, and HIV-1, also significantly induced AEG-1 expression and nuclear translocation via activation of the nuclear factor (NF)-κB pathway. Co-immunoprecipitation studies demonstrated IL-1β- or TNF-α-induced AEG-1 interaction with NF-κB p65 subunit. AEG-1 knockdown decreased NF-κB activation, nuclear translocation, and transcriptional output in TNF-α-treated astrocytes. Moreover, IL-1β treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2, increased expression of excitatory amino acid transporter 2 repressor ying yang 1, and reduced glutamate clearance, a major transducer of excitotoxic neuronal damage. Findings from this study identify a novel transcriptional co-factor function of AEG-1 and further implicate AEG-1 in HAND-associated neuroinflammation. PMID:24855648

  3. Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix.

    PubMed

    Choi, You Hee; Choi, Jun-Ha; Oh, Jae-Wook; Lee, Kwang-Youl

    2013-03-08

    Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

  4. Haloperidol and Clozapine Differentially Affect the Expression of Arrestins, Receptor Kinases, and Extracellular Signal-Regulated Kinase Activation

    PubMed Central

    Ahmed, Mohamed Rafiuddin; Gurevich, Vsevolod V.; Dalby, Kevin N.; Benovic, Jeffrey L.; Gurevich, Eugenia V.

    2009-01-01

    Dopamine and other G protein-coupled receptors (GPCRs) represent the major target of antipsychotic drugs. GPCRs undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. Arrestins and GRKs are major regulators of GPCR signaling. We elucidated changes in expression of two arrestins and four GRKs following chronic (21 days) treatment with haloperidol (1 mg/kg i.p.) or clozapine (20 mg/kg i.p.) 2 or 24 h after the last injection in 11 brain regions. Haloperidol decreased GRK3 in ventrolateral caudate-putamen and transiently down-regulated GRK5 in globus pallidus and caudal caudate-putamen. Clozapine also caused a short-term suppression of the GRK5 expression in the caudal caudate-putamen and globus pallidus, but, unlike haloperidol, elevated GRK5 in the caudal caudate-putamen after 24 h. Unlike haloperidol, clozapine decreased arrestin2 and GRK3 in hippocampus and GRK3 in globus pallidus but increased arrestin2 in the core of nucleus accumbens and ventrolateral caudate-putamen and GRK2 in prefrontal cortex. Clozapine, but not haloperidol, induced long-term activation of extracellular signal-regulated kinase (ERK) 2 in ventrolateral caudate-putamen and transient in prefrontal cortex. The data demonstrate that haloperidol and clozapine differentially affect the expression of arrestins and GRKs and ERK activity, which may play a role in determining their clinical profile. PMID:18178904

  5. Regulation of cyclic AMP formation in cultures of human foetal astrocytes by beta 2-adrenergic and adenosine receptors.

    PubMed

    Woods, M D; Freshney, R I; Ball, S G; Vaughan, P F

    1989-09-01

    Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.

  6. A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

    PubMed

    Chatton, J Y; Marquet, P; Magistretti, P J

    2000-11-01

    The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

  7. Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.

    PubMed

    Fiory, Francesca; Oriente, Francesco; Miele, Claudia; Romano, Chiara; Trencia, Alessandra; Alberobello, Anna Teresa; Esposito, Iolanda; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2004-03-19

    We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.

  8. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  9. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    PubMed Central

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  10. IKKβ regulates essential functions of the vascular endothelium through kinase-dependent and -independent pathways

    PubMed Central

    Ashida, Noboru; SenBanerjee, Sucharita; Kodama, Shohta; Foo, Shi Yin; Coggins, Matthew; Spencer, Joel A.; Zamiri, Parisa; Shen, Dongxiao; Li, Ling; Sciuto, Tracey; Dvorak, Ann; Gerszten, Robert E.; Lin, Charles P.; Karin, Michael; Rosenzweig, Anthony

    2011-01-01

    Vascular endothelium provides a selective barrier between the blood and tissues, participates in wound healing and angiogenesis, and regulates tissue recruitment of inflammatory cells. Nuclear factor (NF)-κB transcription factors are pivotal regulators of survival and inflammation, and have been suggested as potential therapeutic targets in cancer and inflammatory diseases. Here we show that mice lacking IKKβ, the primary kinase mediating NF-κB activation, are smaller than littermates and born at less than the expected Mendelian frequency in association with hypotrophic and hypovascular placentae. IKKβ-deleted endothelium manifests increased vascular permeability and reduced migration. Surprisingly, we find that these defects result from loss of kinase-independent effects of IKKβ on activation of the serine-threonine kinase, Akt. Together, these data demonstrate essential roles for IKKβ in regulating endothelial permeability and migration, as well as an unanticipated connection between IKKβ and Akt signalling. PMID:21587235

  11. Signaling, Regulation, and Specificity of the Type II p21-activated Kinases*

    PubMed Central

    Ha, Byung Hak; Morse, Elizabeth M.; Turk, Benjamin E.; Boggon, Titus J.

    2015-01-01

    The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases. PMID:25855792

  12. Regulation of Greatwall kinase by protein stabilization and nuclear localization

    PubMed Central

    Yamamoto, Tomomi M; Wang, Ling; Fisher, Laura A; Eckerdt, Frank D; Peng, Aimin

    2014-01-01

    Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry. PMID:25483093

  13. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

  14. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  15. Integrin-mediated Ras–Extracellular Regulated Kinase (ERK) Signaling Regulates Interferon γ Production in Human Natural Killer Cells

    PubMed Central

    Mainiero, Fabrizio; Gismondi, Angela; Soriani, Alessandra; Cippitelli, Marco; Palmieri, Gabriella; Jacobelli, Jordan; Piccoli, Mario; Frati, Luigi; Santoni, Angela

    1998-01-01

    Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 integrin–mediated Ras activation. In addition, we demonstrate that ligation of β1 integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors. PMID:9763606

  16. Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

    PubMed Central

    Ely, B; Pittard, J

    1979-01-01

    Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme. PMID:222728

  17. Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of hematopoietic cell lines and primary megakaryocytic progenitors.

    PubMed

    Lannutti, Brian J; Drachman, Jonathan G

    2004-05-15

    In this study we demonstrate that thrombopoietin (TPO)-stimulated Src family kinases (SFKs) inhibit cellular proliferation and megakaryocyte differentiation. Using the Src kinase inhibitors pyrolopyrimidine 1 and 2 (PP1, PP2), we show that TPO-dependent proliferation of BaF3/Mpl cells was enhanced at concentrations that are specific for SFKs. Similarly, proliferation is increased after introducing a dominant-negative form of Lyn into BaF3/Mpl cells. Murine marrow cells from Lyn-deficient mice or wild-type mice cultured in the presence of the Src inhibitor, PP1, yielded a greater number of mature megakaryocytes and increased nuclear ploidy. Truncation and targeted mutation of the Mpl cytoplasmic domain indicate that Y112 is critical for Lyn activation. Examining the molecular mechanism for this antiproliferative effect, we determined that SFK inhibitors did not affect tyrosine phosphorylation of Janus kinase 2 (JAK2), Shc, signal transducer and activator of transcription (STAT)5, or STAT3. In contrast, pretreatment of cells with PP2 increased Erk1/2 (mitogen-activated protein kinase [MAPK]) phosphorylation and in vitro kinase activity, particularly after prolonged TPO stimulation. Taken together, our results show that Mpl stimulation results in the activation of Lyn kinase, which appears to limit the proliferative response through a signaling cascade that regulates MAPK activity. These data suggest that SFKs modify the rate of TPO-induced proliferation and are likely to affect cell cycle regulation during megakaryocytopoiesis.

  18. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    PubMed

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  19. β-Adrenergic stimulation activates protein kinase Cε and induces extracellular signal-regulated kinase phosphorylation and cardiomyocyte hypertrophy.

    PubMed

    Li, Lin; Cai, Hongyan; Liu, Hua; Guo, Tao

    2015-06-01

    The cardiac adrenergic signaling pathway is important in the induction of cardiac hypertrophy. The cardiac adrenergic pathway involves two main branches, phospholipase C (PLC)/protein kinase C (PKC) and the adenylate cyclase (cAMPase)/protein kinase A (PKA) signaling pathways. It is hypothesized that PLC/PKC and cAMPase/PKA are activated by the α‑adrenergic receptor (αAR) and the β‑adrenergic receptor (βAR), respectively. Previous studies have demonstrated that exchange protein directly activated by cAMP (Epac), a guanine exchange factor, activates phospholipase Cε. It is possible that there are βAR‑activated PKC pathways mediated by Epac and PLC. In the present study, the role of Epac and PLC in βAR activated PKC pathways in cardiomyocytes was investigated. It was found that PKCε activation and translocation were induced by the βAR agonist, isoproterenol (Iso). Epac agonist 8‑CPT‑2'OMe‑cAMP also enhanced PKCε activation. βAR stimulation activated PKCε in the cardiomyocytes and was regulated by Epac. Iso‑induced change in PKCε was not affected in the cardiomyocytes, which were infected with adenovirus coding rabbit muscle cAMP‑dependent protein kinase inhibitor. However, Iso‑induced PKCε activation was inhibited by the PLC inhibitor, U73122. The results suggested that Iso‑induced PKCε activation was independent of PKA, but was regulated by PLC. To further investigate the downstream signal target of PKCε activation, the expression of phosphorylated extracellular signal‑regulated kinase (pERK)1/2 and the levels of ERK phosphorylation was analyzed. The results revealed that Iso‑induced PKCε activation led to an increase in the expression of pERK1/2. ERK phosphorylation was inhibited by the PKCε inhibitor peptide. Taken together, these data demonstrated that the βAR is able to activate PKCε dependent on Epac and PLC, but independent of PKA.

  20. Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases, Which Control Stomata Development in Arabidopsis thaliana*

    PubMed Central

    Khan, Mamoona; Rozhon, Wilfried; Bigeard, Jean; Pflieger, Delphine; Husar, Sigrid; Pitzschke, Andrea; Teige, Markus; Jonak, Claudia; Hirt, Heribert; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module. PMID:23341468

  1. Lipopolysaccharide Activation of the TPL-2/MEK/Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Cascade Is Regulated by IκB Kinase-Induced Proteolysis of NF-κB1 p105†

    PubMed Central

    Beinke, S.; Robinson, M. J.; Hugunin, M.; Ley, S. C.

    2004-01-01

    The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate