vFLIP protects PC-12 cells from apoptosis induced by Sindbis virus: implications for the role of TNF-alpha.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor alpha (TNF-alpha), and pre-treatment of the cells with an anti-TNF-alpha blocking antibody or with soluble TNF-alpha receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-alpha R1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-alpha plays an important role in the apoptotic response.

Protein kinase Calpha and protein kinase Cdelta play opposite roles in the proliferation and apoptosis of glioma cells.

Protein kinase C (PKC) has been implicated in the proliferation and apoptosis of glial tumors, but the role of specific PKC isoforms remains unresolved. Comparing brain tumors differing in degree of malignancy, we found that malignant gliomas expressed higher levels of PKCalpha and lower levels of PKCdelta as compared with low-grade astrocytomas. Consistent with a mechanistic role for these differences, overexpression of PKCalpha in the human U87 glioma cell line resulted in enhanced cell proliferation and decreased glial fibrillary acidic protein (GFAP) expression as compared with controls. Reciprocally, overexpression of PKCdelta inhibited cell proliferation and enhanced GFAP expression. Using PKC chimeras, we found that the regulatory domains of PKCalpha and PKCdelta mediated their effects on cell proliferation and GFAP expression. PKCalpha and delta have been implicated as potential signaling molecules in apoptosis. Therefore, we examined the role of these isoforms in the resistance of glioma cells to apoptotic stimuli. In U87 cells, manipulation of PKCalpha levels had little effect on apoptosis in response to etoposide. In contrast, overexpression of PKCdelta rendered the U87 cells more sensitive to the apoptotic effect of etoposide, and PKCdelta was cleaved in these cells by a caspase-dependent process. Furthermore, the glioma cell line U373, which expresses endogenous PKCdelta, underwent apoptosis in response to etoposide, and the apoptotic response was blocked by the PKCdelta inhibitor rottlerin. Our results suggest that PKCalpha and PKCdelta play opposite roles in the proliferation and apoptosis of glioma cells.

Nerve growth factor regulates TNF-alpha production in mouse macrophages via MAP kinase activation.

In this study, we examined the expression of nerve growth factor (NGF) and its receptors in mouse macrophages and the mechanisms involved in the effect of NGF on tumor necrosis factor (TNF)-alpha production. Macrophages expressed NGF and the NGF receptors TrkA and p75. Treatment of J744 cells or peritoneal macrophages with NGF induced a large increase in the production of TNF-alpha. In addition, NGF induced the secretion of nitric oxide in interferon-gamma-treated J774 cells or lipopolysaccharide-treated peritoneal macrophages. The induction of TNF-alpha production by NGF was blocked by K252a, an inhibitor of the TrkA receptor. NGF induced phosphorylation and activation of extracellular signal-regulated kinase, Erk1/Erk2 and c-Jun amino-terminal kinase, whereas it did not induce phosphorylation of p38 mitogen-activated protein kinase. Inhibition of the MAP kinase-Erk kinase pathway with PD 098059 decreased the secretion of TNF-alpha by NGF. Our results suggest that NGF has an important role in the activation of macrophages during inflammatory responses via activation of mitogen-activated protein kinases.

Macrophages express neurotrophins and neurotrophin receptors. Regulation of nitric oxide production by NT-3.

In this study we examined the expression of neurotrophins and their receptors in mouse macrophages and the effects of the neurotrophins on nitric oxide secretion. Macrophages expressed TrkB and TrkC but not BDNF, NT-3 or NT-4. LPS induced up-regulation of TrkB and TrkC and of BDNF and NT-3 expression. Treatment of macrophages with NT-3 increased the secretion of nitric oxide in LPS-treated macrophages and this increase was blocked by K252a, a Trk kinase inhibitor. In contrast, BDNF and NT-4 had no significant effects on the induction of nitric oxide. Our results suggest that NT-3 play important roles in the function of macrophages during inflammatory responses and in tissue repair.

Roles of BCL-2 and caspase 3 in the adenosine A3 receptor-induced apoptosis.

Selective A3 adenosine receptor agonists have been shown to induce apoptosis in a variety of cell types. In this study we examined the effects of adenosine receptor agonists selective for A1, A2A, or A3 receptors on the induction of apoptosis in primary cultures of rat astrocytes and in C6 glial cells. Treatment of the cells with the A3 receptor agonist Cl-IB-MECA (10 microM) induced apoptosis in both cell types. The effects of Cl-IB-MECA were partially antagonized by the A3 receptor-selective antagonist MRS 1191. In contrast, the A1 and A2A receptor agonists, CPA and CGS 21680, respectively, did not have significant effects on apoptosis in these cells. Cl-IB-MECA reduced the expression of endogenous Bcl-2, whereas it did not affect the expression of Bax. Overexpression of Bcl-2 in C6 cells abrogated the induction of apoptosis induced by the A3 agonist. Cl-IB-MECA also induced an increase in caspase 3 activity and caspase inhibitors decreased the apoptosis induced by the A3 agonist. These findings suggest that intense activation of the A3 receptor is pro-apoptotic in glial cells via bcl2 and caspase-3 dependent pathways.

Differential regulation of Bcl-2 and Bax expression in cells infected with virulent and nonvirulent strains of sindbis virus.

Sindbis virus is an alphavirus that infects cells in either lytic or persistent infection. In this study we examined the effects of Sindbis virus on cell apoptosis and on the expression of Bcl-2 and Bax. Of the two strains studied, SVA and SVNI, only the neurovirulent strain, SVNI, induced apoptosis of astrocytes and PC-12 cells. SVA, which infects cells in a persistent manner, induced up-regulation of bcl-2 mRNA and Bcl-2 protein, whereas SVNI induced an increase in Bax levels. Our results indicate a differential regulation of Bcl2 and Bax expression by SVA and SVNI, which may be associated with the apoptotic potential of the viruses.

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues regulates specific cellular functions.

Protein kinase Cdelta (PKCdelta) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKCdelta are involved in these two responses. Transfection of cells with PKCdelta mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKCdelta wild-type transfectant. Conversely, transfection with PKCdelta mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKCdelta wild-type transfectant. The tyrosine phosphorylation of PKCdelta and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKCdelta via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKCdelta induced by PDGF. We conclude that the tyrosine phosphorylation of PKCdelta and its association with tyrosine kinases may be an important point of divergence in PKC signaling.

Differential regulation of neurotrophin expression by mitogens and neurotransmitters in mouse lymphocytes.

In this study, we examined the expression of neurotrophins in mouse lymphocytes and the regulation of their expression by mitogens and neurotransmitters. We found that mixed splenocytes as well as T and B lymphocytes expressed mRNA for all the neurotrophins examined. Differential regulation of the neurotrophins was obtained upon stimulation of the cells. Thus, LPS increased the expression of NGF, BDNF and NT-3 in splenocytes and B cells, whereas Con-A increased the mRNA of NT-3 and NT-4 in T cells and NGF expression in splenocytes. The neurotransmitter substance P and the beta-adrenergic agonist, isoproterenol induced an increase in the expression of NGF. Our results suggest an important role for the different neurotrophins in the function of the immune system and point to a bi-directional interaction between neurotrophins and neurotransmitters in this system.

Functional IL-4 receptors on mouse astrocytes: IL-4 inhibits astrocyte activation and induces NGF secretion.

IL-4 is a Th2-derived cytokine which plays an important role in the function of various immunocompetent cells as well as in the pathophysiology of various CNS disorders. In this study we characterized the expression of IL-4R in cultured astrocytes and explored the effects of IL-4 on the function of these cells. We found that astrocytes express the mRNA of both the membrane-bound and the soluble forms of the IL-4R, whereas they do not secrete IL-4. IL-4 inhibited both NO production and iNOS expression induced by LPS stimulation and decreased the secretion of TNF-alpha and the expression of ICAM-1. In contrast, IL-4 induced the secretion of NGF by astrocytes and synergized with LPS and TNF-alpha in this effect. These results suggest an important role for IL-4 as an immunosuppressive and a neurotrophic factor in the CNS.

Regulation of GDNF expression in cultured astrocytes by inflammatory stimuli.

Astrocytes express increased levels of neurotrophic factors in response to pathological conditions in the CNS such as injury and inflammation. We have examined the effects of lipopolysaccharide (LPS) and inflammatory cytokines on the expression of GDNF by mouse astrocytes and by C6 glial cells. LPS and tumor necrosis factor-alpha (TNF-alpha) induced an increase in level of glial-derived neurotrophic factor (GDNF) mRNA in both cell types. Similarly, the synthesis of GDNF protein was increased by both treatments. Interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induced similar effects on GDNF production, whereas IL-2 and IL-6 had no significant effects. These results indicate that the expression of GDNF in astrocytes is regulated by inflammatory stimuli and therefore may provide neurotrophic support to injured neurons in inflammatory conditions in the CNS.

Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures.

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.

Staurosporine induces astrocytic phenotypes and differential expression of specific PKC isoforms in C6 glial cells.

In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-beta 2 and an increase in that of PKC-gamma. In addition, it induced translocation of PKC-epsilon from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.

The immunomodulator AS101 administered orally as a chemoprotective and radioprotective agent.

AS101 [ammonium-trichloro (0,0' dioxyethylene)tellurate] is a new immunomodulator shown previously to stimulate the production of various cytokines in vitro and in vivo, and to have minimal toxicity. In the present study we explore the possibility of oral administration of AS101 to mice via cannulation in lieu of interperitoneal or intravenous administration reported to date. Our studies show that oral administration of AS101 at a dose ranging between 50 and 100 micrograms/mouse promotes hemopoietic regeneration after treatment with sublethal doses of cyclophosphamide (CYP) and protects mice from the lethal effects of this compound. In addition, AS101 administered orally confers a strong radioprotective effect upon mice when given before irradiation.