Cytokine and intracellular signaling regulation of tissue factor expression in astrocytes

There is evidence that inflammatory cytokines such as IL-1beta, TNFalpha, and IL-6 are involved in the pathogenesis of cerebrovascular disorders including stroke. One action of cytokines that contributes to diseases in peripheral tissues is upregulation of the procoagulant receptor tissue factor (TF). In the CNS, astrocytes are the primary cells that express TF; although little is known about how TF is regulated in these cells. Experiments were performed to evaluate the effect of cytokine treatment on TF activity in primary cultures of murine cortical astrocytes and in the human astrocytoma cell line (CCF). IL-1beta treatment induced a 2.5-fold increase in TF activity in the primary astrocytes and a 3-fold induction in the astrocytoma cells. TNFalpha treatment induced a 2.5-fold increase in TF activity in both the primary astrocytes and astrocytoma cells. IL-6 upregulated TF activity 2-fold in primary astrocytes, however, it had no effect on TF activity in the astrocytoma cells. The signaling pathways regulating TF expression in these cells were examined by using staurosporine, a broad spectrum inhibitor of serine-threonine protein kinases, and by examining the effects of intermediates in the sphingomyelin signaling pathway. Staurosporine inhibited IL-1beta-induced TF activity in the primary astrocytes but did not effect IL-1beta- or TNFalpha-induced TF activity in the astrocytoma cells. TF activity in the astrocytoma cells was upregulated 1.5-fold over constitutive levels by a ceramide analogue or the enzyme sphingomyelinase, however the ceramide analogue had no effect on TF activity in the primary astrocytes. These results suggest inflammatory cytokines can upregulate TF activity in astrocytes and the astrocytoma CCF cell line although the two cell types appear to utilize different signaling pathways to mediate TF expression. Further studies will be important to more completely define the signaling regulation of TF in astrocytes since alterations in brain TF levels may play a key role in CNS pathophysiology.     

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.     

Novel dual-reporter preclinical screen for antiastrocytoma agents identifies cytostatic and cytotoxic compounds

Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma, there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective antitumor agents. The authors have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential antitumor treatments that specifically target astrocytoma.     

Modulation of lysine transport in cultured rat astrocytes and astrocytoma cells

In the previous paper, it was shown that the transport of lysine into astrocytes and astrocytoma cells obeys the classical enzyme kinetics. Although unmodulated lysine transport into both normal rat astrocytes and rat astrocytoma cells is somewhat slower than needed for observed growth in the culture, it is capable of a large degree of enhancement. Insulin increases the Vmax for lysine influx in astrocytes tenfold and in astrocytoma cells fivefold. Glutathione produces a Vmax enhancement of 80% for astrocytes and 70% for astrocytoma cells. gamma-Glutamyl hydrazide is a weak inhibitor of lysine transport. Diethyl maleate appears to break down the regulation of lysine transport and allows a large increase in lysine influx in both cell types studied. Basic amino acid analogues canaline and S-aminoethylcysteine are not potent inhibitors of lysine transport. Lysine efflux kinetics are slower for C6 cells than for astrocytes; this difference is abolished by diethyl maleate and by dithiothreitol.     

Direct cloning of astrocytes from primary culture without previous immortalization

Summary In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.     

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.     

The mechanism of action of MPTP-induced neuroinflammation and its modulation by melatonin in rat astrocytoma cells,C6

Abstract

The 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of Parkinson's disease (PD), but its mechanism of action on astrocytes is not understood. The effect of melatonin on MPTP-induced neuroinflammation in astrocytes is also not known. The present study demonstrated that MPTP treatment of rat astrocytoma cells, C6 for 24 h significantly increased nitrative and oxidative stress and intracellular calcium (Ca²⁺⁺) level. MPTP also activated phosphorylated p38 mitogen activated protein kinase (P-p38 MAPK) and up-regulated expressions of inflammatory proteins. Moreover, MPTP modulated mRNA expressions of pro-inflammatory cytokine genes via activating nuclear factor kappa-B (NF-kB) translocation. Treatment of melatonin with MPTP reversed all these MPTP-induced changes. Study with deprenyl demonstrates that MPTP is inducing neuroinflammation in astrocytoma cells. The present findings elucidated the molecular mechanism of MPTP-induced neuroinflammation and its modulation by melatonin in astrocytoma cells (C6).     

Endothelins Stimulate c-fos and Nerve Growth Factor Expression in Astrocytes and Astrocytoma

Abstract: Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c- fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c- fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c- fos and nerve growth factor, and (b) phorbol esterevoked down-regulation of protein kinase C completely abolishes the c- fos induction by endothelin, but not that by the β-adrenergic agonist isoproterenol, a known activator of the cyclic AMP-dependent pathway. Our results support the hypothesis that c- fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.     

Quadratic variance models for adaptively preprocessing SELDI-TOF mass spectrometry data

Background  

Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) is a proteomics tool for biomarker discovery and other high throughput applications. Previous studies have identified various areas for improvement in preprocessing algorithms used for protein peak detection. Bottom-up approaches to preprocessing that emphasize modeling SELDI data acquisition are promising avenues of research to find the needed improvements in reproducibility.     

Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility

Originally thought of as a stomach-derived endocrine peptide acting via its receptors in the central nervous system to stimulate food intake and growth hormone expression, ghrelin and its receptor (growth hormone secretagogue receptor (GHS-R)) are widely expressed in a number of organ systems, including cancer cells. However, the direct functional role of ghrelin and its receptor in tumors of central nervous system origin remains to be defined. Here, we demonstrate that the human astrocytoma cell lines U-118, U-87, CCF-STTG1, and SW1088 express 6-, 11-, 15-, and 29-fold higher levels of GHS-R compared with primary normal human astrocytes. The ligation of GHS-R by ghrelin on these cells resulted in an increase in intracellular calcium mobilization, protein kinase C activation, actin polymerization, matrix metalloproteinase-2 activity, and astrocytoma motility. In addition, ghrelin led to actin polymerization and membrane ruffling on cells, with the specific co-localization of the small GTPase Rac1 with GHS-R on the leading edge of the astrocytoma cells and imparting the tumor cells with a motile phenotype. Disruption of the endogenous ghrelin/GHS-R pathway by RNA interference resulted in diminished motility, matrix metalloproteinase activity, and Rac expression, whereas tumor cells stably overexpressing GHS-R exhibited increased cell motility. The relevance of ghrelin and GHS-R expression was verified in clinically relevant tissues from 20 patients with oligodendrogliomas and grade II-IV astrocytomas. Analysis of a central nervous system tumor tissue microarray revealed that strong GHS-R and ghrelin expression was significantly more common in high grade tumors compared with low grade ones. Together, these findings suggest a novel role for the ghrelin/GHS-R axis in astrocytoma cell migration and invasiveness of cancers of central nervous system origin.     

Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.     

Involvement of selenoprotein P in protection of human astrocytes from oxidative damage

Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP. We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein, which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx.     

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.     

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II–IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.     

Mass Spectrometrical Analysis of Galectin Proteins in Primary Rat Cerebellar Astrocytes

Galectins are a family of animal lectins with specificity for β-galactosides and are involved in a host of cellular activities, ranging from development to cancer. The molecules are expressed by neural and non-neural cells intracellularly as well as extracellularly. Using two-dimensional gel electrophoresis coupled to tandem mass spectrometry, the present work aimed to identify and characterize galectins in primary rat cerebellar astrocytes. The protein-chemical method identified nine spots representing two members of the galectin family, namely galectin-1 and galectin-3. These findings suggest that high abundant expression of galectin in astrocytes is limited to the two abundant galectin family members. As these family members are linked to human astrocytic tumors, their reliable detection in astrocytes by proteomic techniques would enable us to further understand their role in neural development, injury, and regeneration in general and astrocytoma in particular.     

Relative Expression of mRNAS Coding for Glutaminase Isoforms in CNS Tissues and CNS Tumors

Glutaminase (GA) in mammalian tissues occurs in three isoforms: LGA (liver-type), KGA (kidney-type) and GAC (a KGA variant). Our previous study showed that human malignant gliomas (WHO grades III and IV) lack expression of LGA mRNA but are enriched in GAC mRNA relative to KGA mRNA. Here we analyzed the expression of mRNAs coding for the three isoforms in the biopsy material derived from other central nervous system tumors of WHO grades I–III. Non-neoplastic resective epileptic surgery samples served as control, as did cultured rat astrocytes and neurons. The GAC mRNA/KGA mRNA expression ratio was as a rule higher in the neoplastic than in control tissues, irrespective of the cell type dominating in the tumor or tumor malignancy. LGA mRNA expression was relatively very low in cultured astrocytes, and very low to absent in astrocytoma pilocyticum, ependymoma and subependymal giant cell astrocytoma (SEGA), tumors of astrocytic origin. LGA mRNA expression was almost as high as that of KGA and GAC mRNA in cultured neurons and epileptic surgery samples which were enriched in neurons. LGA mRNA was also relatively high in ganglioglioma which contains a discernable proportion of neuronal cells, and in oligodendroglioma. The results show that low expression of LGA mRNA is a feature common to normal astrocytes and astroglia-derived tumor cells or ependymomas and can be considered as a cell-type, rather than a malignancy marker.     

Activation of human macrophages by amyloid-beta is attenuated by astrocytes

In Alzheimer's disease, neuritic amyloid-beta plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-beta can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-beta(1-42) stimulate these macrophages to the production of superoxide anions and TNF-alpha. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-beta(1-42). In cocultures with astrocytes and amyloid-beta(1-42)-stimulated macrophages, decreased levels of both superoxide anion and TNF-alpha were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-beta(1-42) to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-beta(1-42) to astrocytoma cells and pretreatment of astrocytes with amyloid-beta(1-16) prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-beta(1-42)-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-alpha secretion, the activation state of NF-kappaB was investigated in macrophages and astrocytoma cells after amyloid-beta(1-42) treatment. Interestingly, although activation of NF-kappaB could not be detected in amyloid-beta-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-beta stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-beta(1-42) by interfering with amyloid-beta(1-42) binding to macrophages.