c-Jun N-terminal kinase and, to a lesser extent, p38 mitogen-activated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia

doi:10.1016/j.jneuroim.2003.10.034

Journal of Neuroimmunology

Volume 146, Issues 1–2, January 2004, Pages 50-62

https://doi.org/10.1016/j.jneuroim.2003.10.034 Get rights and content

Abstract

Lower molecular weight of hyaluronan (HA) fragments are capable of activating macrophages to express a number of inflammatory mediators through the interaction with the HA receptor CD44. Recent evidence has demonstrated that concomitant induction of CD44 and HA synthase 2 (HAS-2) mRNA in microglia of the ischemic brain. However, the influence of HA fragments on the activation of microglia is poorly understood. In this study, we demonstrated that HA fragments induced inducible NO synthase (iNOS) expression in BV-2 microglia in a dose-dependent manner and was synergized with interferon-γ (IFN-γ). Moreover, HA fragments could induce the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/ERK2), and c-Jun N-terminal kinase (JNK) in a time and dose-dependent fashion. The HA fragments-induced iNOS expression was suppressed by the selective inhibitors of JNK and, to a lesser extent, p38 MAPK. These results suggest that the induction of iNOS by HA fragments is significantly dependent on JNK than on p38 MAPK signaling pathways and support the hypothesis that HA fragments may be an important regulator in the activation of microglia at sites of ischemic brain.

Introduction

Microglia are cells of the monocyte/macrophage lineage that reside in the brain parenchyma and exist in two major states: nonactivated (ramified, or resting) and activated (ameboid). Microglia have been proposed to play a role in host defense and tissue repair in the CNS (Perry and Gordon, 1988). However, activation of microglia and consequent release of proinflammatory and/or cytotoxic factors, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen species (ROS), are believed to contribute to the pathophysiology of ischemia and several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease Dirnagl et al., 1999, Orr et al., 2002, Pocock et al., 2002

In the CNS, NO has been implicated as a mediator of neurotoxicity as well as a neuromodulator Chao et al., 1992, Moncada et al., 1991. The expression of inducible NO synthase (iNOS) has been predominantly attributed to glial cells in the CNS. iNOS is rapidly expressed in glial cells after stimulation with bacterial lipopolysaccharide (LPS), cytokines, amyloid β, glutamate and N-methyl-d-aspartate (NMDA) Galea et al., 1992, Tikka and Koistinaho, 2001, Tikka et al., 2001, Weldon et al., 1998. Various activators or inhibitors of signaling kinases, including protein kinase C (PKC), protein kinase A (PKA), protein tyrosine kinases (PTK) and mitogen-activated protein kinases (MAPK) have been shown to alter iNOS induction in cytokine and LPS-stimulated glial cells Bhat et al., 1998, Fiebich et al., 1998.

Hyaluronan (HA), a low m.w. fragments of the extracellular matrix (ECM) component, is a nonsulfated glycosaminoglycan polymer made up of repeating disaccharide units of (β,1-4)-d-glucuronic acid-(β,1-3)-N-acetyl-d-glucosamine. High molecular weight fragments of HA is believed to have many functions in healthy tissue (Laurent and Fraser, 1992). However, accumulation of low molecular mass of HA species at sites of inflammation and tissue injury has been shown to activate macrophages and dendritic cells to produce inflammatory mediators, including cytokines, chemokines, reactive nitrogen species, and growth factors McKee et al., 1996, McKee et al., 1997, Noble et al., 1993, Termeer et al., 2002. This effect is mediated, in part, by interaction with the HA receptor CD44 McKee et al., 1996, McKee et al., 1997, Noble et al., 1993, or Toll-like receptor-4 (TLR-4)(Termeer et al., 2002).

A recent report showed that the expression of CD44 gene and hyaluronan synthase 2 (HAS-2) mRNA (a HA biosynthesis enzyme) are concomitantly upregulated in the brain after ischemic stroke (Wang et al., 2001). In addition, the localization of CD44 and HAS-2 expression in microglia indicates that HA may, in part, participate in the inflammatory response of microglia after ischemic injury. The murine microglial cell line BV-2, generated by immortalizing primary murine microglial cells with a v-raf/v-myc oncogene carrying retrovirus, is shown to reproduce many microglial responses in culture and has been used as a model of microglial system in many studies Blasi et al., 1990, Jana et al., 2002, Petrova et al., 1999. Thus in this study, we investigate the effect of HA fragments on BV-2 microglia activation by using the iNOS expression as a model and characterize the molecular mechanisms of this induction.

Section snippets

Materials

HA fragments from human umbilical cords (M.W. 500–800 kDa) were obtained from Calbiochem (San Diego, CA, USA). LPS from Escherichia coli serotype 0111: B4 was obtained from Sigma (St. Louis, MO, USA). Murine recombinant interferon-γ (IFN-γ; sp. act. 4×10⁶ U/mg) was from R&D (Minneapolis, MN, USA). Cell culture ingredients were purchased from Life Technologies (Grand Island, NY, USA). Monoclonal antibody against the CR3 complement receptor (OX-42) was obtained from PharMingen (San Diego, CA,

HA fragments induce NO production in microglia

To investigate whether HA fragments induce microglia activation, we used the NO production in BV-2 microglia as a model. The cells were treated with various concentrations of HA fragments for 24 h, then the content of nitrite (the stable NO metabolite) in medium was measured. The results showed that incubation of BV-2 cells with HA fragments led to a dose-dependent accumulation of nitrite (Fig. 1A). This effect was unlikely to be due to LPS contamination because the LPS inhibitor polymixin B

Discussion

In the present study, we demonstrated that HA fragments induced the production of NO, iNOS mRNA and protein in BV-2 microglia. HA fragments were able to activate all three members of MAPK in a time and dose-dependent manner. However, the expression of iNOS induced by HA fragments was mediated via the activation of the JNK and, to a lesser extent, p38 MAPK signaling pathways. Furthermore, the induction of iNOS by HA fragments was enhanced synergistically with the treatment of IFN-γ, and

Acknowledgments

This study was supported by Grants TCVGH 927320D from Taichung Veterans General Hospital.

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c-Jun N-terminal kinase and, to a lesser extent, p38 mitogen-activated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia

Abstract

Introduction

Section snippets

Materials

HA fragments induce NO production in microglia

Discussion

Acknowledgments

Cytokine Growth Factor Rev.

J. Neuroimmunol.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Trends Cell Biol.

FEBS Lett.

Trends Neurosci.

J. Neuroimmunol.

Kidney Int.

J. Neuroimmunol.

J. Neuroimmunol.

J. Biol. Chem.

J. Neuroimmunol.

J. Biol. Chem.

Prog. Neurobiol.

Trends Neurosci.

J. Biol. Chem.

Cell. Immunol.

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase

Proc. Natl. Acad. Sci. U. S. A.

Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinase regulate inducible nitric oxide synthase and tumor necrosis factor-α gene expression in endotoxin-stimulated primary glial cultures

J. Neurosci.

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

J. Neurochem.

Inhibition of caspase 3 abrogates lipopolyssacharide-induced nitric oxide production by preventing activation of NF-κB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells

Infect. Immun.

Activated microglia mediate neuronal cell injury via a nitric oxide mechanism

J. Immunol.

Inhibition of caspase 3 abrogates lipopolyssacharide-induced nitric oxide production by preventing activation of NF-κB and c-Jun NH₂-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells