CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-α release from lipopolysaccharide-treated cells and mice

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Abstract

CEP-11004, a mixed lineage kinase (MLK) inhibitor, was examined for its effects on tumor necrosis factor-alpha (TNF-α) production in human THP-1 monocytes, mouse BV-2 microglia, and C57Bl/6 mice. CEP-11004 inhibited TNF-α secretion up to 90% in THP-1 cells incubated with 3 μg/ml lipopolysaccharide, with an IC50 of 137 ± 14 nM. CEP-11004 also inhibited TNF-α production in lipopolysaccharide-stimulated microglial cells, but did not inhibit the initial increase in TNF-α mRNA expression as measured by real-time polymerase chain reaction (PCR). The mitogen-activated protein kinases (MAPKs) phospho-c-jun N-terminal kinase (JNK), phospho-p38, and phospho-MAPK kinase 4 (MKK4) levels were increased in THP-1 cells following lipopolysaccharide treatment, and were reduced by CEP-11004 treatment. For in vivo studies, CEP-11004 was injected 2 h prior to lipopolysaccharide (20 mg/kg) administration. CEP-11004 significantly inhibited TNF-α production at doses of 1–10 mg/kg as measured by enzyme-linked immunosorbent assay (ELISA). These results suggest that MLK blockade may be useful in inhibiting pro-inflammatory cytokine production in a wide range of diseases.

Introduction

CEP-11004 (Fig. 1) is an indolocarbazole analogue derived from the natural product K252a and is characterized by its ability to inhibit mixed lineage kinases (MLKs). It inhibits MLK1, MLK2, and MLK3 with IC50 values of 45, 31, and 89 nM, respectively (Murakata et al., 2002). CEP-11004 was shown to inhibit activation of the mitogen-activated protein kinase (MAPK) c-jun N-terminal kinase (JNK) and to exhibit dose-dependent rescue of rat PC12 cells, human SH-SY5Y cells, and primary rat cortical neurons treated with apoptosis-inducing reagents in vitro. In NGF-deprived sympathetic neurons, CEP-11004 inhibited activation of the JNK pathway (Wang et al., 2005). In vivo studies indicated that administration of CEP-11004 inhibited the 1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated increase in phosphorylated MAPK kinase 4 (MKK4) and attenuated the loss of striatal dopaminergic terminals in mice (Murakata et al., 2002).

The MLKs are a family of serine/threonine kinases that are part of the upstream signaling cascade that activates the MAPKs JNK and p38. The MLKs phosphorylate and activate MKKs, which then activate JNK and p38 (for review, see Gallo and Johnson, 2002). JNK, also called stress-activated protein kinase (SAPK), represents a family of proteins that are key effectors in signal transduction pathways associated with cellular stress and inflammation (Dreskin et al., 2001, Kyriakis and Avruch, 2001, Johnson and Lapadat, 2002). The two primary kinases that activate JNK are MKK4 and MKK7, while MKK3 and MKK6 phosphorylate p38. Activated JNK then phosphorylates and activates the transcription factor c-jun, which is involved in expression of a number of pro-inflammatory cytokine genes, including tumor necrosis factor-alpha (TNF-α) (Yao et al., 1997). Gene and/or protein expression of TNF-α through the JNK pathway may be an important mechanism in sustaining an inflammatory response. Therefore, blockade of this pathway through JNK inhibition may reduce overexpression of TNF-α.

Cells of monocyte/macrophage lineage are particularly responsive to lipopolysaccharide and have been shown to express and activate JNK in vitro (Dreskin et al., 2001), which can be reversed by treatment with CEP-11004 (Hidding et al., 2002). The downstream results of this acute response include activation of transcription factors that regulate immediate early genes, expression of adhesion molecules, and secretion of various pro-inflammatory cytokines including interleukin-1 beta (IL-1β) and TNF-α (Caroff et al., 2002, Yates et al., 2000, Liu et al., 2001). Thus, misregulation of, or chronic exposure to, TNF-α may exacerbate a pro-inflammatory response and lead to tissue damage and cell loss.

The current study evaluated the effects of CEP-11004 on lipopolysaccharide-mediated MAPK activation and TNF-α production in human monocytic and mouse microglial cell lines and in mice. These models were chosen to assess the ability of CEP-11004 to alter MAPK activation and TNF-α production during a biological stress response in vitro and in vivo. Phospho-JNK, phospho-p38, and TNF-α were increased after lipopolysaccharide treatment and the MLK inhibitor CEP-11004 inhibited these reactions, thereby providing a potential anti-inflammatory therapeutic.

Section snippets

Animals

C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA) and housed under controlled conditions and a 12-h day/night cycle. All studies conformed carefully to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the U.S. Department of Health and Human Services and were approved by the Cephalon, Inc., Animal Care and Use Committee.

Cells and reagents

THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI with 10%

Inhibition of TNF-α secretion by CEP-11004 in lipopolysaccharide-stimulated THP-1 human monocytes and mouse microglia

Fig. 2 shows that in THP-1 cells, CEP-11004 caused a concentration-dependent decrease in TNF-α secretion, with an IC50 of 137.1 ± 14.4 nM. Background levels in untreated cells were undetectable, whereas a significant increase in TNF-α levels was observed in lipopolysaccharide-treated samples (data not shown). To verify that CEP-11004 was not toxic to the THP-1 cultures, an MTS toxicity assay was performed to determine the viability of cells after treatment with CEP-11004 for 3 or 24 h.

Discussion

This report demonstrates that CEP-11004 is a potent inhibitor of TNF-α production both in vitro and in vivo. The drug acts by inhibiting lipopolysaccharide-mediated activation of MAPK signaling pathways. Although lipopolysaccharide-induced TNF-α secretion is inhibited by CEP-11004, early gene expression is not altered, suggesting that the attenuation in secretion is not attributable to inhibition of mRNA expression. This indicates that different signaling pathways can be utilized for these

Acknowledgments

The authors wish to thank Tom Connors, Beth Ann McKenna, Andreas Rassow, and Dr. Joanne Mathiasen for technical expertise in characterizing CEP-11004 and establishing in vitro assays, and Beth Ann Thomas and Jeff Thomas for assistance with real-time PCR techniques.

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