h-89 and Glioma

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.

Topics: Animals; Astrocytes; Brain Neoplasms; Bucladesine; Cell Differentiation; Cell Line, Tumor; Glioma; Immunity, Innate; Interleukin-6; Isoquinolines; Mannose-Binding Protein-Associated Serine Proteases; Phosphorylation; Protein Kinase Inhibitors; Rats; Recombinant Proteins; RNA, Messenger; STAT3 Transcription Factor; Sulfonamides

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.

Topics: Animals; Apoptosis Regulatory Proteins; Astrocytes; Cell Differentiation; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioma; Humans; Isoquinolines; Membrane Proteins; Rats; S100 Proteins; Sulfonamides

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt(2)AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt(2)AMP/theophylline. Taken together, these results indicated that Bt(2)AMP/theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.

Topics: Animals; Astrocytes; Blotting, Western; Brain Neoplasms; Bucladesine; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Genes, Reporter; Glial Fibrillary Acidic Protein; Glioma; Interleukin-6; Isoquinolines; Microscopy, Fluorescence; Models, Biological; Phosphorylation; Plasmids; Promoter Regions, Genetic; Rats; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Theophylline; Time Factors; Trans-Activators; Transcription, Genetic

This study has demonstrated the mechanism of protein kinase A (PKA)-dependent inhibition of astrocytic nitric oxide production and inducible NO synthase mRNA expression induced by lipopolysaccharide. In C6 glioma cells, the stimulation with lipopolysaccharide (LPS; 1 microg/ml) evoked increases of nitric oxide (NO) production, NO synthase (iNOS) mRNA expression, phosphorylation of p38 mitogen activated protein kinase (p-p38), and the activation of NF kappa B. LPS-induced NO production and iNOS mRNA expression were inhibited by the pretreatment with forskolin (FSK; 5 microM) in a dose-dependent manner, and which were reversed by PKA inhibition by compound H89. Furthermore, LPS-induced increases of p-p38, but not activation of NF kappa B, were also reduced by FSK and H89 reversed the FSK-induced inhibition response. The dose-dependent inhibition of NO production and iNOS mRNA expression by compound SB203580 (p38 inhibitor) suggests the participation of p38 in PKA-dependent inhibition of LPS-induced NO production and iNOS mRNA expression. However, the activation of NF kappa B by LPS also not affected by SB203580. Therefore, our results suggest that, in C6 glioma cells, LPS-induced NO production and iNOS gene expression may be regulated by PKA pathway through the reduction of activity of p38 kinase. This inhibitory role of PKA may not involve the activation of NF kappa B.

Topics: Animals; Colforsin; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Enzymologic; Glioma; Imidazoles; Isoquinolines; Lipopolysaccharides; Mitogen-Activated Protein Kinases; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; RNA, Messenger; Sulfonamides; Transcription, Genetic; Tumor Cells, Cultured

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Androstadienes; Animals; Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Chromones; Cyclic AMP; Enzyme Inhibitors; Flavonoids; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Indoles; Isoproterenol; Isoquinolines; Lactams, Macrocyclic; Maleimides; Morpholines; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Sirolimus; Sulfonamides; Tumor Cells, Cultured; Wortmannin

To investigate the possible mechanisms involved in forskolin-induced c-jun mRNA decrease in rat C6 glioma cells, we examined effects of a PKA inhibitor (H-89), a L-type Ca2+ channel blocker (nimodipine), a calmodulin activation inhibitor (calmidazolium chloride) and a Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) on forskolin-induced c-jun mRNA down-regulation. H-89 caused a reversal of forskolin-induced c-jun mRNA decrease. Furthermore, nimodipine, KN-62 and calmidazolium chloride partially blocked forskolin-induced c-jun mRNA down-regulation. Our results suggest that activation of adenylate cyclase appears to be involved in a down-regulation of c-jun mRNA expression through a PKA pathway. In addition, L-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II may be partially involved in c-jun mRNA down-regulation induced by forskolin.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenylyl Cyclases; Animals; Calcium Channel Blockers; Calmodulin; Colforsin; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Glioma; Imidazoles; Isoquinolines; Nimodipine; Proto-Oncogene Proteins c-jun; Rats; RNA, Messenger; Sulfonamides; Tumor Cells, Cultured

The effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bucladesine; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Extracellular Space; Glial Fibrillary Acidic Protein; Glioma; Isoquinolines; Kinetics; Rats; RNA, Messenger; Sulfonamides; Superoxide Dismutase; Transcription, Genetic

The brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925-1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14-fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA i) is due to increased transcription; ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy-demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP-mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Brain; Brain Neoplasms; Cloning, Molecular; Colforsin; Creatine Kinase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Energy Metabolism; Enzyme Activation; Enzyme Induction; Glioblastoma; Glioma; HeLa Cells; Humans; Isoenzymes; Isoquinolines; Myelin Sheath; Nerve Tissue Proteins; Neuroglia; Protein Synthesis Inhibitors; Rats; Recombinant Fusion Proteins; RNA, Antisense; Sulfonamides; Transcription, Genetic; Transfection; Tumor Cells, Cultured