Dibutyryl cAMP- or Interleukin-6-induced astrocytic differentiation enhances mannose binding lectin (MBL)-associated serine protease (MASP)-1/3 expression in C6 glioma cells.

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Correction: A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

[This corrects the article DOI: 10.1371/journal.pone.0161486.].

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

BACKGROUND: The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. METHODS: Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging. RESULTS: ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. CONCLUSIONS: Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

Stressed to death: targeting endoplasmic reticulum stress response induced apoptosis in gliomas.

Glial tumors are the main primary adult brain tumor. Even with the most advanced treatments, which include stereotactic microscope aided surgical resection, internal and external radiation therapy and local and systemic chemotherapy, median survival time for patients diagnosed with these malignancies is about 12 months. We explore here the possibility that the endoplasmic reticulum stress response (ERSR) could be a possible target to develop chemotherapeutic agents to induce toxicity in glioma cells. ERSR has the dual capacity of activating repair and/or cytotoxic mechanisms. ERSR is triggered by the accumulation of unfolded proteins in the ER. The presence of unfolded proteins in the ER regulates, via a complex biochemical cascade, the upregulation of molecular chaperones, inhibition of protein synthesis, and an increase of proteasome mediated unfolded protein degradation. ERSR in particular conditions can also contribute to cell death via activation of programmed cell death. Apoptosis activation during ERSR is usually caused by the activation of one or a combination of three biochemical cascades. Induction of these pathways ultimately leads to caspase 3 activation culminating in apoptosis. Glioma cells are in a condition of constant low grade ERSR, which possibly contributes to their resistance to treatment protocols. It is conceivable that small molecules that interact with this phenomenon ultimately could be used to modulate the system to activate apoptosis and cause gliotoxicity. We will discuss here ERSR biochemically relevant features to death mechanisms and already identified small molecules that by modulating ERSR are able to activate glioma cell death.

Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.

Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.

PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca(2+) channels in PC12 cells.

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca(2+) entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca(2+) elevation and is responsible for imparting extracellular Ca(2+) influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca(2+) pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca(2+) entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca(2+) entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca(2+) channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca(2+) mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca(2+) influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca(2+) mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca(2+) release from ER stores and extracellular Ca(2+) entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca(2+) entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca(2+) signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.

The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion.

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1(null), at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.

Astrocytes refill intracellular Ca2+ stores in the absence of cytoplasmic [Ca2+] elevation: a functional rather than a structural ability.

Capacitative Ca(2+) entry (CCE) is a phenomenon triggered by depletion of Ca(2+) content in intracellular stores (ICS). Data about this phenomenon in astrocytes are limited. We analyzed CCE in astrocytes by means of fura-2 based digital imaging. We found that in astrocytes CCE is not associated with an increase of cytosolic Ca(2+) concentration ([Ca(2+)](i)), although ICS are efficiently refilled. We used Mn(2+), thapsigargin and prolonged ATP exposure to show that CCE is not associated with cytosolic diffusion of Ca(2+) entering astrocytes. Our data suggest that the ion is being quickly sequestered in the ICS by the smooth endoplasmic reticulum Ca(2+)-ATP-ase (SERCA). Several experiments were carried out with the goal of failing the efficient uptake in the endoplasmic reticulum (ER). In fact, inhibition of SERCA activity, increased extracellular [Ca(2+)](i) or pharmacologic potentiation of CCE all caused [Ca(2+)](i) elevation during CCE, suggesting that the control of this phenomenon could have physiologic and pathological relevance. The molecular components involved in CCE have been proposed to be organized in a multi-molecular complex tethered by cytoskeleton components and arranged via a secretion coupling model. We show here that the efficient routing of Ca(2+) into the ICS in astrocytes is not affected by disruption of cytoskeleton organization or Golgi's function, but it is instead linked to the high efficiency of SERCA. We conclude that depleted ICS in astrocytes are efficiently refilled by CCE activation, although Ca(2+) influx is not accompanied by elevation of [Ca(2+)](i). This ability seems to be functional rather than structural in nature.

Specific localization of the annexin II heterotetramer in brain lipid raft fractions and its changes in spatial learning.

Annexin-II (AII) is a Ca(2+)-dependent phospholipid-binding protein that is present in both intracellular and extracellular compartments. In the present study AII immunoreactivity was found in a subpopulation of neurons in specific brain regions, including the cerebral cortex and the surface of hippocampal pyramidal neurons from adult rats. AII from synaptic membranes was detected by immunoblotting as multiple species containing the monomer (AII36) and heterotetramer (AIIt). AIIt was resistant to beta-mercaptoethanol and dithiothreitol in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but was completely reduced to monomers (36 kDa) by two-dimensional electrophoresis. AIIt resided exclusively in the detergent-resistant lipid rafts concentrated in neuronal dendrites, and its recruitment to those structures was enhanced by antibody cross-link. AII abundantly distributed on the outer leaflet of neuronal membranes and between spaces of neurons appeared to be neuronal adhesive. The formation of AIIt required synthesis of sphingolipids and cholesterol, and its stability depended on Ca2+. Increases in neuronal activities such as depolarization and learning were shown to promote formation of AIIt. Our results suggest that, via a dynamic association with dendritic lipid rafts, AII may play a role in synaptic signal transduction and remodeling. This probably involves focal adhesion and interactions with actin that are associated with brain development and memory consolidation.

Translocation of protein kinase C-betaII in astrocytes requires organized actin cytoskeleton and is not accompanied by synchronous RACK1 relocation.

Protein kinase C (PKC)-betaII is the most abundant PKC isoform in astrocytes. Upon activation, this isoform of PKC translocates from the cytosol to the plasma membrane (PM). In this study, we investigated in astrocytes the modality of PKC-betaII translocation as far as the participation of the receptor for activated C kinase-1 (RACK1) and the requirement for intact cytoskeleton in the process. In astrocytes, Western blots and immunocytochemistry coupled to confocal microscopic quantitative analysis showed that after 5 min of phorbol-12-myristate-13-acetate (PMA) exposure, native PKC-betaII, but not PKC-betaI, is relocated efficiently from the cytosol to the PM. Translocation of PKC-betaII was not associated with synchronous RACK1 relocation. Furthermore, the quantity of PM-associated PKC-betaII that co-immunoprecipitated with PM-bound RACK1 increased following PMA exposure, indicating a post activation binding of the two proteins in the PM. Because RACK1 and PKC-betaII relocation seemed not to be synchronous, we hypothesized that an intermediate interaction with the cytoskeleton was taking place. In fact, we were able to show that pharmacological disruption of actin-based cytoskeleton greatly deranged PKC-betaII translocation to the PM. The requirement for intact actin cytoskeleton was specific, because depolymerization of tubulin had no effect on the ability of the kinase to translocate to the PM. These results indicate that in astrocytes, RACK1 and PKC-betaII synchronous relocation is not essential for relocation of PKC-betaII to the PM. In addition, we show for the first time that the integrity of the actin cytoskeleton plays a specific role in PKC-betaII movements in these cells. We hypothesize that in glial cells, rapidly occurring changes of actin cytoskeleton arrangement may be involved in the fast reprogramming of PKC targeting to specific PM location to phosphorylate substrates in different cellular locations.

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system.

Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.

Transient receptor potential channel activation causes a novel form of [Ca 2+]I oscillations and is not involved in capacitative Ca 2+ entry in glial cells.

Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2+ influx triggered by intracellular Ca 2+ stores depletion, a phenomenon known as capacitative Ca 2+ entry. We studied the properties of capacitative Ca 2+ entry in astrocytes by means of single-cell Ca 2+ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2+ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr2+ and Ba2+ do not enter astrocytes during capacitative Ca 2+ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive TRPCs, when activated, initiate oscillations of cytosolic Ca 2+ concentration ([Ca 2+]i) pharmacologically and molecularly consistent with TRPC3 activation. OAG-induced [Ca 2+]i oscillations are not affected by inhibition of inositol trisphosphate (InsP3) production or blockade of the InsP3 receptor, therefore representing a novel form of [Ca 2+]i signaling. Instead, high [Ca 2+]i inhibited oscillations, by closing the OAG-sensitive channel. Also, treatment of astrocytes with antisense against TRPC3 caused a consistent decrease of the cells responding to OAG. Exogenous OAG but not endogenous DAG seems to activate TRPC3. In conclusion, in glial cells, natively expressed TRPC3s mediates a novel form of Ca 2+ signaling, distinct from capacitative Ca 2+ entry, which suggests a specific signaling function for this channel in glial cells.

Coincident elevation of cAMP and calcium influx by PACAP-27 synergistically regulates vasoactive intestinal polypeptide gene transcription through a novel PKA-independent signaling pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes calcium influx, intracellular calcium release, and elevation of cAMP in chromaffin cells. Calcium influx is required for PACAP-stimulated secretion of catecholamines and neuropeptides. The role of cAMP elevation in the action of PACAP at either sympathetic or adrenomedullary synapses, however, is unknown. Here, we show that PACAP-27-induced calcium influx through voltage-sensitive calcium channels (VSCCs), together with elevation of intracellular cAMP, was sufficient to stimulate vasoactive intestinal polypeptide (VIP) biosynthesis at least 40-fold. Combined treatment of chromaffin cells with 40 mm KCl, which elevates intracellular calcium, and 25 micrometer forskolin, which elevates intracellular cAMP, caused an increase in VIP peptide and mRNA much greater than that elicited by either agent alone, and comparable to the increase caused by 10-100 nm PACAP-27. Elevation of VIP mRNA by either KCl plus forskolin, or PACAP, (1) was independent of new protein synthesis, (2) was blocked by inhibition of calcium influx through voltage-sensitive calcium channels, (3) was calcineurin dependent, and (4) was dependent on MAP kinase activation but not activation of protein kinase A. The degree of activation of two different second-messenger pathways, calcium influx and cAMP elevation, appears to determine the magnitude of transcriptional activation of the VIP gene in chromaffin cells. Maximal stimulation of VIP biosynthesis by PACAP appears to require the coincident activation of both of these pathways.

Mitochondria regulate Ca2+ wave initiation and inositol trisphosphate signal transduction in oligodendrocyte progenitors.

Mitochondria in oligodendrocyte progenitor cells (OPs) take up and release cytosolic Ca2+ during agonist-evoked Ca2+ waves, but it is not clear whether or how they regulate Ca2+ signaling in OPs. We asked whether mitochondria play an active role during agonist-evoked Ca2+ release from intracellular stores. Ca2+ puffs, wave initiation, and wave propagation were measured in fluo-4 loaded OP processes using linescan confocal microscopy. Mitochondrial depolarization, measured by tetramethyl rhodamine ethyl ester (TMRE) fluorescence, accompanied Ca2+ puffs and waves. In addition, waves initiated only where mitochondria were localized. To determine whether energized mitochondria were necessary for wave generation, we blocked mitochondrial function with the electron transport chain inhibitor antimycin A (AA) in combination with oligomycin. AA decreased wave speed and puff probability. These effects were not due to global changes in ATP. We found that AA increased cytosolic Ca2+, markedly reduced agonist-evoked inositol trisphosphate (IP3) production, and also enhanced phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the Ca2+ dependent protein gelsolin. Thus, the reduction in puff probability and wave speed after AA treatment may be explained by competition for PIP2 between phospholipase C and gelsolin. Energized mitochondria and low cytosolic Ca2+ concentration may be required to maintain PIP2, a substrate for IP3 signal transduction.

12-hydroxyeicosatetrenoate (12-HETE) attenuates AMPA receptor-mediated neurotoxicity: evidence for a G-protein-coupled HETE receptor.

12-hydroxyeicosatetraenoic acid (12-HETE) is a neuromodulator that is synthesized during ischemia. Its neuronal effects include attenuation of calcium influx and glutamate release as well as inhibition of AMPA receptor (AMPA-R) activation. Because 12-HETE reduces ischemic injury in the heart, we examined whether it can also reduce neuronal excitotoxicity. When treated with 12-(S)HETE, cortical neuron cultures subjected to AMPA-R-mediated glutamate toxicity suffered up to 40% less damage than untreated cultures. The protective effect of 12-(S)HETE was concentration-dependent (EC50 = 88 nm) and stereostructurally selective. Maximal protection was conferred by 300 nm 12-(S)HETE; 300 nm 15-(S)HETE was similarly protective, but 300 nm 5-(S)HETE was less effective. The chiral isomer 12-(R)HETE offered no protection; neither did arachidonic acid or 12-(S)hydroperoxyeicosatetraenoic acid. Excitotoxicity was calcium-dependent, and 12-(S)HETE was demonstrated to protect by inactivating N and L (but not P) calcium channels via a pertussis toxin-sensitive mechanism. Calcium imaging demonstrated that 12-(S)HETE also attenuates glutamate-induced calcium influx into neurons via a pertussis toxin-sensitive mechanism, suggesting that it acts via a G-protein-coupled receptor. In addition, 12-(S)HETE stimulates GTPgammaS binding (indicating G-protein activation) and inhibits adenylate cyclase in forskolin-stimulated cultures over the same concentration range as it exerts its anti-excitotoxic and calcium-influx attenuating effects. These studies demonstrate that 12-(S)HETE can protect neurons from excitotoxicity by activating a G(i/o)-protein-coupled receptor, which limits calcium influx through voltage-gated channels.