Macrophage activation contributes to the pathogenesis of atherosclerosis. In the vascular system, the major source of reactive oxygen species is the NADPH oxidase (Nox) family. Nox1 is induced by lipopolysaccharide (LPS) in macrophages, but the expression mechanism is not fully understood. We found that LPS causes β‐catenin accumulation by glycogen synthase kinase 3β (GSK3β) inactivation, and that β‐catenin accumulation increases Nox1 expression. LPS induced Nox1 mRNA expression and reactive oxygen species generation in Raw264.7 cells. Using bone marrow‐derived macrophages from toll‐like receptor 4 mutant mice, we also tested whether LPS‐induced Nox1 expression is toll‐like receptor 4 dependent. LPS caused GSK3β phosphorylation, induced β‐catenin accumulation and increased nuclear translocation. The GSK3β inhibitor LiCl potentiated LPS‐induced Nox1 expression in accordance with β‐catenin accumulation and nuclear translocation. Conversely, ectopic expression of a constitutively active GSK3β mutant severely attenuated Nox1 expression. These findings identify a novel regulatory pathway controlling Nox1 expression by LPS‐stimulated macrophages.

This study examined the hypothesis that the control of NADPH oxidase-2 (Nox2)-mediated reactive oxygen species (ROS) regulates the expression of matrix metalloproteinases (MMPs) and the migration of macrophages. Lipopolysaccharide (LPS) stimulation of Raw264.7 cells and mice peritoneal macrophages increased the expression of MMP-9, 10, 12 and 13 mRNA, and also increased Raw264.7 cell migration. Treatment with an antioxidant (N-acetyl cysteine) or Nox inhibitors strongly inhibited the expression of MMPs by LPS and inhibited cell migration. LPS caused ROS production in macrophages and increased the mRNA expression of Nox isoforms Nox1 and Nox2 by 20-fold and two-fold, respectively. While Nox1 small interfering RNA (siRNA) did not inhibit LPS-mediated expression of MMPs, Nox2 siRNA inhibited the expressions of MMP-9, 10 and 12. Neither Nox1 nor Nox2 siRNA influenced the LPS-mediated expression of MMP-13. In addition, NAC or apocynin attenuated LPS-induced ROS production and MMP-9 expression. MMP-9 expression and cell migration were controlled by ERK1/2-ROS signaling. Collectively, these results suggest that LPS stimulates ROS production via ERK and induce various types of MMPs expression and cell migration.

Resveratrol is a polyphenolic compound in red wine that has antioxidant and cardioprotective effects in animal models. Listeria monocytogenes is a pathogen that mainly affects immunocompromised individuals and is initially detected at the cell surface or in phagosomes by toll-like receptor 2. Many antioxidants also exert anti-inflammatory activities; therefore, we evaluated the anti-inflammatory properties of resveratrol by studying the various inflammatory responses induced by heatkilled L. monocytogenes (HKLM). Resveratrol strongly blocked HKLM-induced NADPH oxidase-1 mRNA and reactive oxygen species production by macrophages. Resveratrol also suppressed monocyte chemotactic protein-1 expression, cyclooxygenase-2 expression, prostaglandin production, inducible nitric oxide (NO) synthase expression, and NO production induced by HKLM. We investigated the signaling pathway involved in the resveratrol effect. HKLM stimulated glycogen synthase kinase 3b (GSK3b) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The involvement of GSK3b and ERK1/2 was tested using inhibitors. While the GSK3b inhibitor LiCl potentiated the effect of HKLM, the MEK inhibitor U0126 blocked these responses. Additionally, pretreatment with resveratrol blocked phosphorylation of both kinases induced by HKLM. These results suggest that HKLM is strong inducer of inflammatory mediators, and that the inhibitory effect of resveratrol may be mediated by the GSK3b and ERK1/2 pathways.KEY WORDS: COX-2 HKLM iNOS MCP-1 NADPH oxidase 1 resveratrol

Monocyte chemoattractant protein-1 (MCP-1) is an essential cytokine for the migration of monocytes into vessels, and is also involved in the pathogenesis of atherosclerosis. In this study, we investigated the importance of janus kinase 2 (JAK2) and the function of the Akt and glycogen synthase kinase-3β (GSK3β) pathway in toll-like receptor (TLR2)-mediated MCP-1 expression. The TLR2 agonist, Pam3CSK4, induced MCP-1 expression in the Raw264.7 cell line. The induction of MCP-1 was seen in the bone marrow-derived macrophages of wild-type mice but not in TLR2 knockout mice. The TLR2-mediated MCP-1 induction was myeloid differentiation primary response gene 88 (MyD88)-independent. By contrast, the inactivation of JAK2 attenuated TLR2-mediated MCP-1 expression. The JAK inhibitor suppressed the phosphorylation of GSK3β as well as Akt by Pam3CSK4 stimulation. While the inactivation of Akt by LY294002 suppressed TLR2-mediated MCP-1 induction, the inactivation of GSK3β by LiCl potentiated TLR2-mediated MCP-1 induction. Furthermore, Akt inhibitor suppressed TLR2-mediated phosphorylation of GSK3β. Taken together, these results suggest that a MyD88-independent pathway exists in TLR2 signaling; the JAK2-Akt-GSK3β pathway is a novel MyD88-independent pathway for MCP-1 induction.

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