Gene Expression In Astrocytes Research Articles

Chronic endothelin exposure inhibits connexin43 expression in cultured cortical astroglia

Severe brain lesions are accompanied by sustained increases in endothelin (ET) levels, which in turn profoundly affect brain microcirculation and neural cell function. A known response of astrocytes to acute increases in ET levels is the rapid and transient closure of gap junctions and the subsequent decrease of gap junction-mediated intercellular communication (GJIC). Because evidence exists that the loss of GJIC alters astrocytic gene expression, we analyzed the effects of chronic ET exposure on astrocytic gap junction coupling. We found that within 24 hr, cultured cortical astrocytes respond to low nanomolar concentrations (2-10 nM) of either ET-1 or ET-3 with a robust inhibition of connexin (Cx)43 expression, the major junctional protein in astrocytes, and a subsequent decline of GIJC. We further observed that in the continuous presence of ETs, Cx43 expression remained inhibited for at least 7 days. In addition, a similar decrease of Cx43 expression occurred in cultured spinal cord astrocytes maintained with ET-1 for 3 days. Applying ETs in combination with the highly selective ETA and ETB receptor antagonists, BQ123 and BQ788, respectively, revealed that the inhibitory influences on astrocytic Cx43 expression depend on activation of ETB receptors. We suggest that the observed ET-dependent inhibition of Cx43 expression and the resulting decline of GJIC might represent a major pathway by which ETs regulate astrocytic gene expression in the injured brain.

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-α (TNF-α). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na +/K +-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.

Platelet-activating factor enhances the expression of nerve growth factor in normal human astrocytes under hypoxia

Nerve growth factor (NGF) is required for the survival of neurons. We have addressed the effect of platelet-activating factor (PAF), one of the mediators of ischemic injury of the brain, on NGF expression in astrocytes. Normal human astrocytes in culture were stimulated with PAF, and levels of NGF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). PAF increased the expressions of NGF mRNA and protein in astrocytes in time- and concentration-dependent manners. After 48-h stimulation, 10 nmol/L PAF increased the levels of NGF protein in astrocyte-conditioned medium by 1.4-fold. The PAF-induced stimulation of NGF expression was further enhanced (2.1-fold of the control) in the cells under hypoxic culture condition. BN52021 (Ginkgolide B), an antagonist for PAF binding sites, suppressed the effect of PAF. We conclude that PAF enhances NGF gene expression in human astrocytes, and the PAF-induced increase in the expression of NGF under hypoxia may benefit the protection of the nervous tissue by promoting neuronal survival.

Interplay between HIV-1 Vpr and Sp1 Modulates p21WAF1 Gene Expression in Human Astrocytes

The Vpr (viral protein R) of human immunodeficiency virus, type 1, which is expressed during the late stage of the viral infection, has received special attention because of its ability to control transcription of the human immunodeficiency virus, type 1, long terminal repeat and to influence cell cycle progression. Here we demonstrate that Vpr has the ability to regulate transcription of the cyclin-dependent kinase inhibitor, p21(WAF1) (p21), one of the key regulators of the cell cycle, in human astrocytic cells. The results from transcription assays demonstrated that Vpr augments promoter activity of p21 through the GC-rich region located between nucleotides -84 and -74 with respect to the +1 transcription start site. Activation of p21 by Vpr required cooperativity of Sp1, which binds to the DNA sequence spanning -84 to -74. Results from bandshift assay revealed an increased level of Sp1 DNA binding activity in the presence of Vpr. Furthermore, Vpr was able to associate with Sp1 via the zinc finger domain located in the C-terminal region of Sp1. Functional studies revealed that the cooperativity between Vpr and Sp1 requires the zinc finger domain at the C terminus and the glutamine-rich domain at the N terminus of Sp1. Expression of p53 further enhanced the level of Vpr-Sp1-mediated transcription activation of p21 through the sequence spanning -84 to -74 and increased the DNA binding activity of Sp1 in the presence of Vpr. Results from glutathione S-transferase pull-down assay showed the association of Vpr with p53 in extracts containing Sp1. Altogether, the outcome of our functional and binding studies suggested that the physical interaction of Vpr with Sp1 and p53 could modulate transcriptional activity of p21.

Dual role of cAMP in iNOS expression in glial cells and macrophages is mediated by differential regulation of p38-MAPK/ATF-2 activation and iNOS stability

We reported previously that cAMP analogues or cAMP synthesis activator (forskolin; FSK) inhibit lipopolysaccharide (LPS)-induced inducible nitric-oxide systase (iNOS) gene expression in astrocytes, while they enhance that in macrophages. Here, we report that the FSK-mediated inhibition of iNOS expression in C6 glial cells is due to its reduced transcriptional activity, while the FSK-mediated enhancement of iNOS expression in RAW264.7 macrophages is a result of increased stability of iNOS protein without transcriptional enhancement. The LPS/interferon-γ (IFN)-induced iNOS transcription was inhibited by FSK via inhibition of p38-MAPK/ATF-2 activity in glial cells while it was not affected in macrophages. In both cell types, proteasome activities were required for the spontaneous degradation of iNOS protein, and the inhibition of proteasome activity by MG132 after maximum increase of iNOS protein levels further enhanced iNOS protein induction by LPS/IFN, suggesting the involvement of proteasome in iNOS degradation. More importantly, the iNOS protein levels were equalized by the MG132 posttreatment in macrophages treated with LPS/IFN alone and along with FSK, and ubiquitinated iNOS protein levels were reduced by FSK posttreatment, suggesting that the FSK-mediated inhibition of ubiquitination of iNOS protein and the following increased stability of iNOS protein are one of the mechanisms of cAMP-pathway-mediated enhancement of iNOS gene expression in macrophages. To our knowledge, this is the first evidence that cAMP regulates iNOS expression at the posttranslational level in macrophages.

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes ( n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.

DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure.

There is clinical and experimental evidence that elevated intraocular pressure (IOP), a mechanical stress, is involved in the pathogenesis of glaucomatous optic neuropathy. The mechanism by which astrocytes in the optic nerve head (ONH) respond to changes in IOP is under study. Gene transcription by ONH astrocytes exposed either to 60 mmHg hydrostatic pressure (HP) or control ambient pressure (CP) for 6, 24, and 48 h was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Data were normalized across arrays within each gene. A linear regression model applied to test effect of time and HP on changes in expression level identified 596 genes affected by HP over time. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5-fold at 6, 24, or 48 h. Expression of selected genes was confirmed by real-time RT-PCR; protein levels were detected by Western blot. Among the genes highly responsive to HP were those involved in signal transduction, such as Rho nucleotide exchange factors, Ras p21 protein activator, tyrosine kinases and serine threonine kinases, and genes involved in transcriptional regulation, such as c-Fos, Egr2, and Smad3. Other genes that increased expression included ATP-binding cassettes, solute carriers, and genes associated with lipid metabolism. Among the genes that decreased expression under HP were genes encoding for dual activity phosphatases, transcription factors, and enzymes involved in protein degradation. These HP-responsive genes may be important in the establishment and maintenance of the ONH astrocyte phenotype under conditions of elevated IOP in glaucoma.

The scope and activation mechanisms of chemokine gene expression in primary astrocytes following infection with Theiler's virus

Intracerebral infection with Theiler's virus induces a demyelinating disease that resembles human MS. In order to delineate the early events in virus-induced inflammatory disease, we have analyzed chemokine gene activation following Theiler's murine encephalomyelitis virus (TMEV) infection. Infection of primary astrocyte cultures results in activation of various chemokine genes (GRO-1, MCP-1, MCP-5, MIP-1α, MIP-1β, MIP-2, RANTES, IP-10 and MCP-3) that are important in the initiation of an inflammatory response. As early as 1–3 h after TMEV infection, chemokine gene expression is strongly activated. In addition, proinflammatory cytokines do not interfere with TMEV-induced chemokine gene expression and some cytokines may function synergistically for virus-induced upregulation of chemokine gene expression. Chemokine gene activation by TMEV appears to be largely independent of the IFNαβ pathway and partly dependent on dsRNA-dependent protein kinase (PKR) and MAP kinase pathways. However, TMEV-induced chemokine gene expression is completely dependent on the NFκB pathway. These results strongly suggest that the expression of select chemokine genes upon TMEV infection is activated via the NFκB pathway, similar to that of proinflammatory cytokine genes, and these cellular gene products appear to synergistically promote inflammatory responses in the CNS.

Suppression of proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha in astrocytes by a V1 vasopressin receptor agonist: a cAMP response element-binding protein-dependent mechanism.

Previous research from our laboratory has demonstrated that V1 vasopressin receptor agonist (V1 agonist) induces a complex intracellular Ca2+-signaling cascade in cortical astrocytes that is initiated by G-protein-coupled V1a vasopressin receptor-mediated cytoplasmic and nuclear Ca2+ rise and converges during activation of the nuclear transcription factor cAMP response element-binding protein (CREB). In the current study, we pursued the downstream functional consequences of V1 agonist-induced Ca2+-signaling cascade for gene expression. Because astrocytes can exert immune effects analogous to immune cells in the periphery, we investigated V1 agonist regulation of cytokine gene expression in astrocytes. Results from gene array studies indicated that V1 agonist dramatically decreased the mRNA level of five cytokines. Two prominent proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), were selected for detailed analysis, and their expression was also confirmed with reverse transcriptase-PCR. Furthermore, ELISA analyses demonstrated that the peptide level of IL-1beta and TNF-alpha in the astrocyte medium was also decreased in response to V1 agonist. Using CREB antisense to determine the causal relationship between V1 agonist-induced CREB activation and suppression of IL-1beta and TNF-alpha, we demonstrated that decreased IL-1beta and TNF-alpha gene expression was dependent on upstream CREB activation. V1 agonist-induced decrease of cytokine release from cortical astrocytes was also shown to be neuroprotective in cortical neurons. To our knowledge, this is the first documentation of V1 agonist modulation of cytokine gene expression in any cell type. Implications for vasopressin as an antipyretic agent and the role of vasopressin in neurodegeneration, autoimmune diseases, stress, and neuropsychiatric behaviors are discussed.

THE EFFECT OF STIMULATED MICROGLIA CONDITIONED MEDIA ON BDNF GENE EXPRESSION OF STRIATAL ASTROCYTES: QUANTIFICATION BY REAL-TIME PCR

It is well accepted that there is mutual relation between glia-glia and glia-neuron in the central nervous system. In the present study, the authors aimed to evaluate the effect of microglia conditioned medium (MCM) on brain derived neurotrophic factor (BDNF) gene expression of astrocytes by quantitative real-time polymerase chain reaction (PCR). Real-time PCR is one of the most recent techniques for determination of gene expression. It is the first choice when sensitivity, specifity and cost effectiveness are concerned. The authors present, for the first time, the settings of real-time PCR for quantification of BDNF gene expression of rat strital astrocytes. Astrocytes that cultured­ from the striatum were incubated with conditioned medium of either Zymosan A stiumulated or unstimulated microglia which were cultured from striatum and cortex of the rat pups. Our results have shown that incubation with stimulated striatal MCM induced BDNF gene expression of striatal astrocytes (1.33 fold) when compared to astrocytes treated with regular medium or unstimu­lated striatal MCM. We have also seen the similar effect with cortical MCM implying that effect of MCM does not change with regionally different microglia.

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis.

Neurodegeneration and dementia caused by human immunodeficiency virus type 1 (HIV-1) infection of the brain are common complications of acquired immunodeficiency syndrome (AIDS). Introduction of highly active antiretroviral therapy (HAART) reduced the incidence of HIV-1-associated dementia, but so far had no effect on the high frequency of milder neurological disorders caused by HIV-1. This indicates that some neuropathogenic processes persist during limited HIV-1 replication in the central nervous system (CNS). The authors are evaluating the hypothesis that interaction of HIV-1 with astrocytes, which bind HIV-1 but support limited productive HIV-1 infection, may contribute to these processes by disrupting astrocyte functions that are important for neuronal activity or survival. Using laser-capture microdissection on brain tissue samples from HIV-1-infected individuals, we found that HIV-1 DNA can be detected in up to 1% of cortical and basal ganglia astrocytes, thus confirming HIV-1 infection in astrocytes from symptomatic patients. Using rapid subtraction hybridization, the authors cloned and identified 25 messenger RNAs in primary human fetal astrocytes either up-regulated or down-regulated by native HIV-1 infection or exposure to gp120 in vitro. Extending this approach to gene microarray analysis using Affymetrix U133A/B gene chips, the authors determined that HIV-1 alters globally and significantly the overall program of gene expression in astrocytes, including changes in transcripts coding for cytokines, G-coupled protein receptors, transcription factors, and others. Focusing on a specific astrocyte function relevant to neuropathogenesis, the authors showed that exposure of astrocytes to HIV-1 or gp120 in vitro impairs the ability of the cells to transport L-glutamate and the authors related this defect to transcriptional inhibition of the EAAT2 glutamate transporter gene. These findings define new pathways through which HIV-1 may contribute to neuropathogenesis under conditions of limited virus replication in the brain.

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

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JC virus-induced changes in cellular gene expression in primary human astrocytes.

Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor beta receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.

755 Stroma activation and anglogenesis in tumour progression

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9–11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.

Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.

An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis.

Cloning and characterization of the 3'-untranslated region of the human excitatory amino acid transporter 2 transcript.

The 3'-untranslated region (UTR) of the human excitatory amino acid transporter 2 (EAAT2) transcript was cloned and characterized. The full-length EAAT2 cDNA of 11 692 bp was found to contain 283 bp of 5' UTR, a 1725-bp open reading frame and an unusually long 3'-UTR of 9684 bp. The 3'-UTR of EAAT2 cDNA was well conserved among mammals, and human, macaque, rat and mouse cDNA had nearly identical 3' ends. The human EAAT2 transcripts were detected in brain, spinal cord, liver, adrenal gland, placenta and pancreas by northern hybridization, and many ESTs homologous to the human EAAT2 cDNA were found in numerous tissues. To investigate the role of human EAAT2 3'-UTR in gene expression, we constructed luciferase expression vectors containing 3'-UTR fragments spanning the entire length of the region. The individual fragments varied in their effects on reporter gene expression in human astrocytes by a factor of eight to ten suggesting a complex role of the 3'-UTR in post-transcriptional regulation of EAAT2 gene expression.

Vasopressin-Induced Cytoplasmic and Nuclear Calcium Signaling in Embryonic Cortical Astrocytes: Dynamics of Calcium and Calcium-Dependent Kinase Translocation

The present study sought to determine the downstream consequences of V1a vasopressin receptor (V1aR) activation of Ca2+ signaling in cortical astrocytes. Results of these analyses demonstrated that V1aR activation led to a marked increase in both cytoplasmic and nuclear Ca2+. We also investigated V1aR activation of Ca2+-activated signaling kinases, protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the mitogen-activated protein (MAP) kinases [MAPK and extracellular signal-regulated kinases 1 and 2 (ERK1/2)], their localization within cytoplasmic and nuclear compartments, and activation of their downstream nuclear target, the transcription factor cAMP response element-binding protein (CREB). Results of these analyses demonstrated that V1aR activation led to a significant rise in PKC, CaMKII, and ERK1/2 activation, with CaMKII and ERK1/2 demonstrating dynamic transport between cytoplasmic and nuclear compartments. Although no evidence of PKC translocation was apparent, PKC and CaMKs were required for activation and nuclear translocation of ERK1/2. Subsequent to CaMKII and ERK1/2 translocation to the nucleus, CREB activation occurred and was found to be dependent on upstream activation of ERK1/2 and CaMKs. These data provide the first systematic analysis of the V1aR-induced Ca2+ signaling cascade in cortical astrocytes. In addition, results of this study introduce a heretofore unknown effect of vasopressin, dynamic Ca2+ signaling between the cytoplasm and nucleus that leads to comparable dynamics of kinase activation and shuttling between cytoplasmic and nuclear compartments. Implications for development and regeneration induced by V1aR activation of CREB-regulated gene expression in cortical astrocytes are discussed.

Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes

Brain injury is often associated with proliferation and hypertrophic response of glial cells (reactive gliosis). We have previously reported immunosuppressant effects on survival of glioma cells and adult reactive astrocytes. In the present study, we demonstrate growth-inhibitory effect of FK506 on cortical astrocytes from newborn rats. FK506 inhibits Erk and PI-3K/Akt signaling, two crucial pro-survival pathways. The levels of phosphorylated Akt and p42/44 Erk decline in few hours after FK506 addition. Furthermore, in FK506-treated astrocyte cultures the levels of mRNA encoding PDGF, bFGF, and CNTF decreased. Downregulation of growth factor expression by FK506 may play a role in the inhibition of mitogenic/hypertrophic responses. FasL mRNA level was elevated and interaction of FasL with Fas receptor expressed in astrocytes may trigger cell death. Interestingly, expression of BDNF increased in a dose-dependent manner in FK506-treated astrocytes. Upregulation of BDNF mRNA and protein level in astrocytes exposed to FK506 may underlie neuroprotective action of FK506.