Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells

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Abstract

Nitric oxide (NO) has been implicated in a number of important brain functions, such as long-term potentiation (LTP) and long-term depression (LTD), and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO production is increased by an inducible enzyme (iNOS), which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide (LPS). This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 and/or cyclooxygenase-2 (e.g., acetyl salicylic acid, SC 58125, L 745337), but is decreased by approx. 50% with PDTC, a scavenger of reactive oxygen intermediates (ROI) that inhibits nuclear factor κB (NF-κB) activation. Furthermore, inhibitors of protein kinase C (PKC) strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component (besides NF-κB) in the signaling pathway regulating iNOS expression in microglia.

Introduction

The function of nitric oxide (NO) in brain is still a matter of debate. It has been considered (along with CO) to belong to a new class of neurotransmitters (Bredt and Snyder, 1992; Dawson et al., 1992; Snyder, 1992), since it does not fulfil the classical criteria of those compounds, i.e., storage in and release from synaptic vesicles, binding to specific membrane receptors and inability to freely penetrate plasma membranes. The potential involvement of NO in both long-term potentiation (LTP) and long-term depression (LTD) (Bredt and Snyder, 1989; Choi, 1993) as well as its participation in damage of neurons after hypoxia (Choi, 1991) or in chronic neurodegenerative diseases has raised an extremely vigorous research interest in this substance. In most reports, cytotoxic effects of the NO radical have been studied (e.g., Dawson et al., 1991b; Zielasek et al., 1992). Cytoprotective effects described in some studies (Koh et al., 1986; Uemura et al., 1990; Lerner-Natoli et al., 1992; Wink et al., 1993) may be reconciled by its occurrence in at least two distinct ionic forms (Lipton et al., 1993). Three NO-synthesizing enzymes have been characterized and cloned: a constitutive NO-synthase (cNOS) located primarily in neurons (Bredt and Snyder, 1990; Bredt et al., 1991; Dawson et al., 1991a; Bredt et al., 1992), an endothelial NOS (Marsden et al., 1992), and an inducible enzyme (iNOS) found in various other cell types (Geller et al., 1993a; Nakane et al., 1993; Nunokawa et al., 1993) including macrophages (Stuehr et al., 1991; Lyons et al., 1992; Xie et al., 1992; Karupiah et al., 1993; Lorsbach et al., 1993). The inducible form of NO-synthase has raised increased scientific attention due to its involvement in inflammatory and degenerative events both in the periphery and in brain. Since the gene is only transcribed in these conditions, research has been focused on factors regulating iNOS mRNA expression. Cytokines, growth factors and neurotransmitters as well as components of second messenger pathways mediating expression of the iNOS gene are of primary importance for defining potential drug targets. Cytokines elevated mRNAiNOS in murine lung epithelial cells (Robbins et al., 1994) and, in combination with LPS, showed a synergistic effect on hepatocyte iNOS (Geller et al., 1993b). In 3T3 fibroblasts, iNOS mRNA was increased by PKC-activating, but also by cAMP-elevating substances. In murine macrophages, LPS synergized with interferon-γ (Lorsbach et al., 1993). In these cells, cAMP analogues were also able to trigger NO production (Park et al., 1997b). Similar results were obtained from vascular smooth muscle cells (Koide et al., 1993), where cAMP-elevating substances synergized with cytokines. Moreover, transforming growth factor (TGF)-β, which in other cellular systems inhibits iNOS mRNA expression, potentiated iNOS mRNA expression in 3T3 cells. Pyrrolidine dithiocarbamate (PDTC), however, an antioxidant that also inhibits superoxide-induced activation of the transcription factor NF-κB (Schreck et al., 1992), inhibited expression of iNOS mRNA and protein in these cells (Kleinert et al., 1996). Prostaglandin E2, which typically increases intracellular cAMP levels, has been shown to decrease nitrite formation and iNOS mRNA in rat hepatocytes (Harbrecht et al., 1996). Furthermore, the involvement of phosphatidylinositol-3 kinase (PI-3K) and related downstream elements has been reported recently in the colonic epithelial cell line HT-29 (Wright et al., 1997) and in murine peritoneal macrophages (Park et al., 1997a). N-acetyl cysteine, a potent antioxidant, also inhibited iNOS mRNA production in murine peritoneal macrophages, in the rat C6 glioma cell line, and in rat astrocytes (Pahan et al., 1998). The substance seems to stabilize the I-κB/NF-κB complex, which again underscores the crucial role of NF-κB. The tyrosine kinase pathway is an additional second messenger pathway leading to iNOS mRNA expression as shown in C6 glioma cells (Galea et al., 1995) and in human articular chondrocytes (Geng et al., 1995). The MAP kinase pathway has also been shown to induce iNOS expression in murine astrocytes (Kitamura et al., 1996; Dasilva et al., 1997).

Several groups have investigated NO production by astrocytes and microglial cells (Murphy et al., 1990; Chao et al., 1992; Galea et al., 1992; Simmons and Murphy, 1992; Zielasek et al., 1992; Feinstein et al., 1993a, Feinstein et al., 1993b, Feinstein et al., 1993c; Murphy et al., 1993). The major cellular elements in brain injury or brain diseases are microglial cells. They are a potent source of iNOS and NO upon stimulation with lipopolysaccharide (LPS) and/or interferon-γ. Lipopolysaccharide has often been used to induce both NO release and iNOS mRNA. In conditions of increased NO production, cytotoxic events are frequently observed both in culture (Dawson et al., 1994; Peterson et al., 1994) and in vivo (Tran et al., 1997; Garcion et al., 1998; Weldon et al., 1998). As shown recently, nitrite production and appearance of iNOS enzyme protein in cultured microglia are inhibited by cAMP-elevating substances such as 8-Br-cAMP, isobutyl methylxanthine (IBMX), or forskolin (Zuckerman et al., 1998), or isoproterenol and PGE2 (Minghetti et al., 1997). Surprisingly, in the latter report, inhibitors of prostaglandin synthesis such as salicylates or indometacin reduced both iNOS enzyme protein and NO production. Reports, however, on NO-synthase mRNA expression in microglia are sparse. This report includes an evaluation of the involvement of NF-κB, cAMP, and PKC in the regulation of iNOS mRNA expression, of iNOS protein occurrence and of nitrite formation in cultured rat microglial cells.

Section snippets

Materials

Dulbecco's modified Eagle's medium and fetal calf sera were from Gibco-BRL, Eggenstein, LPS (Salmonella typhimurium) from Sebak, Aidenbach. Cholera toxin, Gö 6976, GF 109203X, and PDTC were from Calbiochem, Bad Soden, and Pertussis toxin, dbcAMP, forskolin, PGE2 and aspirin from Sigma, Deisenhofen, Germany Specific, polyclonal iNOS antibodies were from Biomol, Hamburg, Germany L745337, SC 58125, and meloxicam were kindly provided by Drs. Pairet, Trummlitz and Engelhardt, Boehringer Ingelheim

Inhibition of NO synthesis by cAMP-elevating substances in cultured microglia

A number of cAMP-elevating substances were tested for their ability to influence NO production in microglia cultures. Cholera toxin, which activates the stimulatory G-protein subunit αs, was used at concentrations from 1–100 ng/ml. None of these concentrations caused a substantial effect on NO production in microglial cultures (Fig. 1). Significant inhibitory effects (15–20%) were observed, however, using dibutyryl cAMP (10−4 M; 84.2±2.8%) and forskolin (10−5 M; 79.0±2.4%). A comparable

Discussion

The present data provides more detailed insights into second messenger-involvement in LPS-mediated regulation of iNOS protein expression and NO production as well as some view on iNOS mRNA regulation in rat microglia. The appearance of substantial amounts of NO/nitrite in culture media after increased cellular protein and mRNA expression supports the notion of a tight correlation between these distinct metabolic levels. Although direct influences of second messenger-mediated events on stability

Acknowledgements

The authors are indebted to Prof. M. Berger for his generous support of microglial research. The skilful technical assistance of U. Götzinger-Berger and Sandra Hess is greatly acknowledged. Supported by DFG-grants Ge 486/9-1, Fi 683/1-1, Li 643/2-1 and SFB 505, Teilprojekt B1.

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