Magnolol suppresses NF-κB activation and NF-κB regulated gene expression through inhibition of IkappaB kinase activation

Abstract

The mis-regulation of nuclear factor-kappa B (NF-κB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-κB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1α, TNF-α. Pre-treatment of magnolol blocked TNF-α-induced NF-κB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-α-stimulated phosphorylation and degradation of the cytosolic NF-κB inhibitor IκBα and the effects were dose-dependent. Mechanistically, a non-radioactive IκB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-α-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IκBα. The involvement of IKK was further verified in a HeLa cell NF-κB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-α and by the transient transfection and expression of NIK (NF-κB-inducing kinase), wild type IKKβ, constitutively active IKKα and IKKβ, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-κB. In line with the observation that NF-κB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-α-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.

Introduction

The bark of Magnolia officinalis (Cortex Magnoliae Officinalis) is widely used as a folk remedy for gastrointestinal disorders, cough, anxiety and allergic diseases. Magnolol, a low molecular weight lignan originally isolated from the Chinese medicinal plant (Wang et al., 2004), shows a number of diverse pharmacological effects including inducing apoptosis (Lin et al., 2001, Ikeda and Nagase, 2002, Yang et al., 2003, Zhong et al., 2003), differentiation (Fong et al., 2005), calcium mobilization (Teng et al., 1990, Wang and Chen, 1998, Zhai et al., 2003), and anti-platelet aggregation (Teng et al., 1988, Pyo et al., 2002). It has also been suggested to have anxiolytic (Maruyama et al., 1998), anti-oxidative (Lo et al., 1994, Shen et al., 1998), anti-fungal and anti-bacterial (Chang et al., 1998, Bang et al., 2000, Ho et al., 2001, Park et al., 2004), anti-viral and anti-carcinogenic (Konoshima et al., 1991) and anti-metastatic activities (Nagase et al., 2001, Ikeda et al., 2003).

Magnolol has a board spectrum anti-inflammatory effect. It suppresses the expression of the inducible nitric oxide synthase (iNOS) in macrophages (Son et al., 2000, Matsuda et al., 2001), the production of inflammatory cytokines interleukin-8 and tumor necrosis factor α (TNF-α) in THP-1 cells (Park et al., 2004, Lee et al., 2005), the formation of prostaglandin E2 (Wang et al., 1995, Lee et al., 2000), and the artherosclerosis mediators monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) (Chen et al., 2001, Chen et al., 2002, Chen et al., 2006). Moreover, magnolol was also reported as a cyclooxygenase (COX) inhibitor (Hsu et al., 2004, Lee et al., 2005).

Nuclear factor-κB (NF-κB) is an ubiquitous nuclear transcription factor regulating dozens of genes involved in inflammation, and also in growth regulation, apoptosis, cancer invasion/metastasis, tumor promotion, carcinogenesis (reviewed in Aggarwal, 2004). NF-κB consists of a family of transcription factors including p65 (RelA), p105/p50, p100/p52, RelB and c-Rel. The classic form of NF-κB is the p65/p50 heterodimer that contains the transcriptional activation domain and is sequestered in the cytoplasm as an inactive complex by IκB (Baldwin, 1996). Acute stimuli such as TNF-α, LPS or PMA led to the activation of IκB kinases (IKK) which in turn phosphorylate Ser32 and Ser36 within the N-terminal response domain of IκB (Karin and Ben-Neriah, 2000). Phosphorylated IκB would undergo ubiquitination-dependent proteolysis and the release of IκB unmasks the nuclear localization signal and results in the translocation of NF-κB to the nucleus, followed by the activation of specific target genes (Karin and Ben-Neriah, 2000).

We investigated whether the attenuation of NF-κB activity may account for the anti-inflammatory and pharmacological effects of magnolol. In a previous study magnolol was shown to reduce the nuclear NF-κB content in TNF-α-stimulated endothelial cells (Chen et al., 2002). However, the action mechanisms are poorly understood. We first established the effects of magnolol on NF-κB-regulated gene expression induced by a number of inflammatory agents and carcinogens. Then, we demonstrated that magnolol inhibited TNF-α stimulated activation of NF-κB in different cells. We further showed that magnolol suppressed IKK activity, stabilized cytoplasmic IκBα and subsequently reduced the nuclear translocation and phosphorylation of the p65 subunit of NF-κB. Magnolol also inhibited NF-κB-dependent reporter gene expression induced by TNF-α, over-expression of NIK, IKK and p65 subunit and enhanced TNF-α-mediated apoptosis.