Peisheng Zhang

Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death.

Abstract : Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen‐activated protein kinase (MAPK) cascades in relation to oxidant‐induced cell death in an oligodendrocyte cell line, central glia‐4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet‐derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal‐regulated kinase (ERK), p38 MAPK, and c‐Jun N‐terminal kinase (JNK). Dose‐response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK‐activated protein kinase‐2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant‐induced cell death was indicated by morphological changes, decreased 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose‐dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant‐mediated cytotoxicity.

TNFα potentiates IFNγ‐induced cell death in oligodendrocyte progenitors

Oligodendrocytes in multiple sclerosis brain may be under a direct attack by proinflammatory cytokines, particularly tumor necrosis factor‐α (TNFα) and interferon‐γ (IFNγ). In this study, we have examined the in vitro cytotoxic effects of the two cytokines, individually and in combination, on oligodendrocyte lineage cells using morphological criteria, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide reduction assay (MTT), terminal deoxynucleotide transferase‐mediated dUTP nick end‐labeling (TUNEL), and agarose‐gel electrophoretic analysis of fragmented DNA. IFNγ exerted a dose‐dependent cytotoxic effect on cultured CG4 cells, an oligodendrocyte progenitor cell line, and in primary cultures of purified oligodendrocyte progenitors. TNFα, while by itself being only mildly toxic, greatly potentiated the cytotoxicity of IFNγ. The cytokine effects were developmentally modified in that their cytotoxic and cooperative effects became less evident in more differentiated cells. A cell‐permeable peptide inhibitor (i.e., z‐VAD.fmk) of caspases partially suppressed apoptotic changes elicited by the cytokine combination in CG4 cells but not in primary oligodendrocytes. Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis of mRNA prepared from cytokine‐treated cultures revealed an increased expression of the death receptor, Fas. The results suggest particular vulnerability of oligodendrocyte progenitors to a combination of TNFα and IFNγ involving an activation of the cell death program. J. Neurosci. Res. 54:574–583, 1998.

Activation of mitogen-activated protein kinases in oligodendrocytes.

Abstract: The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen‐activated protein (MAP) kinase [also called extracellular signal‐regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet‐derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an in‐gel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti‐MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C‐activating tumor promoter, phorbol 12‐myristate 13‐acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti‐phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42‐kDa phosphoprotein band cross‐reacting with anti‐MAP kinase antibodies. The phosphorylation of p42mapk in PDGF‐treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti‐pan‐ERK antibodies indicated the presence of ERK‐immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan‐ERK‐immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis.

Cytokine induction of inducible nitric oxide synthase in an oligodendrocyte cell line: role of p38 mitogen-activated protein kinase activation.

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen‐activated protein kinase (MAPK) activation and cytokine‐mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor‐α (TNFα), interleukin‐1 (IL‐1), and interferon‐γ (IFNγ), had either minimal (TNFα) or no (IL‐1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT‐PCR analysis, the expression of iNOS. TNFα and IL‐1 were able to activate p38 MAPK in a time‐ and dose‐dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL‐1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination‐treated cells in a dose‐dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine‐treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro‐L‐arginine methyl ester, had a minimal protective effect against cytokine‐mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.

ACTIVATION OF C-JUN N-TERMINAL KINASE/STRESS-ACTIVATED PROTEIN KINASE IN PRIMARY GLIAL CULTURES

Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuropathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress‐activated protein kinase (SAPK) or c‐Jun N‐terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti‐JNK antibodies along with GST c‐Jun (1‐79) as the substrate. Among the cytokines tested, TNF‐α had the strongest effect on JNK activation followed by TNF‐β in both the glial cell types while a substantial level of kinase activation was observed in response to IL‐1 in astrocytes. JNK activation by TNF‐α in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in‐gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF‐α. JNK was also activated by several other stress‐inducing factors including UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell‐permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF‐α. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes.

p38 MAP Kinase Regulation of Oligodendrocyte Differentiation with CREB as a Potential Target

Despite a substantial understanding of the factors regulating oligodendrocyte differentiation, the signaling mechanisms involved in this process are not well-understood. This study elaborates on the findings (Bhat NR, Zhang P (1997) FASEB J 11:A925; Baron W, Metz B, Bansal R, Hoekstra D, de Vries H (2000) Mol Cell Neurosci 15:314–329) of a role for p38 MAP kinase signaling in oligodendrocyte differentiation and myelin gene expression. When proliferating oligodendrocyte progenitors were switched to a growth factor-free differentiation medium, there was a rapid activation of p38 kinase that correlated with an increased phosphorylation of CREB, a down-stream target and a factor involved in oligodendrocyte differentiation. Addition of forskolin, a known inducer of intracellular c-AMP and of oligodendrocyte differentiation, also stimulated CREB phosphorylation in a p38 kinase dependent way. Pharmacological inhibition of p38 interfered with the morphological and antigenic changes associated with differentiating oligodendrocytes as well as with the developmental and forskolin-induced expression of myelin basic protein, thereby supporting an essential role for p38 MAPK pathway in oligodendrocyte differentiation.

Activation of JNK/SAPK in Primary Glial Cultures: II. Differential Activation of Kinase Isoforms Corresponds to Their Differential Expression

Recently, we reported on the activation of c-Jun N-terminal kinase (JNK) in primary glial cells noting certain differences in the patterns of kinase activation in astrocytes and oligodendrocytes (Zhang et al., J Neurosci Res 46:114–121;1996). In this extended study, we have examined the activation and expression levels of JNK1 and JNK2 isoforms in different glial cell types including the two in vitro-defined astroglial subtypes (type-1 and type-2), oligodendrocytes and microglia. An in-gel kinase assay of cell extracts and JNK-immunoprecipitates revealed the activation of both JNK1 and JNK2 in type-1 astrocytes in response to TNFα, and in microglia, in response to TNFα and bacterial lipopolysaccharide. The strong activation of the two JNK isoforms in type-1 astrocytes and microglia contrasted with a predominant activation of JNK1 over JNK2 in type-2 astrocytes and oligodendrocytes, the two glial subtypes sharing a common lineage. Immunoblot and immunocytochemical analyses using isoform-speciflc antibodies showed a differential expression of the two isoforms in different glial cells thereby accounting for their observed differential activation.

THE EXPRESSION OF MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE IN OLIGODENDROCYTES IS DEVELOPMENTALLY REGULATED

Myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of activated protein kinase C (PKC), is thought to be involved in PKC-mediated signal transduction events. In the present study, we have examined the expression of MARCKS in primary cultures of rat glial cells. Western blot analysis of different glial cell types (i.e., astrocytes, oligodendrocytes and microglia) revealed a relatively high level of MARCKS protein in oligodendrocytes. MARCKS protein and MARCKS mRNA levels in oligodendrocytes increased with time in culture, indicating a developmental regulation in MARCKS gene expression in differentiating oligodendrocytes. Immunocytochemical examination of developing oligodendrocytes indicated a strong labeling of MARCKS distributed both in the cell body and in the lacy network of processes. These findings, in concert with our previous observations on the role of PKC in oligodendrogenesis, strongly implicate a PKC-signaling system in oligodendrocyte development.

Hyaluronectin binds to laminin and blocks laminin-dependent process formation by astrocytes.

The interaction of hyaluronectin (HN), a hyaluronic acid-binding extracellular matrix (ECM) glycoprotein with two other ECM-associated molecules, laminin and fibronectin, was studied by ligand blot and solid phase ligand binding assays. Ligand blot analysis with biotin-labeled HN revealed a strong binding of HN to immobilized laminin and a weaker binding to fibronectin. Ligand binding studies indicated a concentration dependent, Ca(2+)-independent binding of HN to laminin. Binding of HN to laminin but not to fibronectin was resistant to increased salt concentrations indicating a non-electrostatic, protein-protein interaction of HN with laminin. The functional relevance of HN-laminin interaction was demonstrated by an inhibition of laminin-supported astroglial process formation by HN.