Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-␣, interleukin-1␤, interleukin-6, and the production of nitric oxide, prostaglandin E 2 , which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.

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We have found previously that phosphatidic acid (PA) can induce inflammatory mediators such as cytokines, which implies that PA plays a role in inflammatory response. In the present study, we provide evidence of the PA-mediated activation of the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which results in the production of interleukin (IL)-1␤ and IL-6. PA elicited the rapid phosphorylations of JAK2 and STAT1/3, and the subsequent nuclear translocation. Macrophages that had been transiently transfected with a luciferase reporter construct containing eight consecutive ␥-interferon activating sequence (GAS) elements, a known STAT binding site, exhibited enhanced reporter gene activity in response to PA stimulation, which further supports the involvement of JAK-STAT activation in the PA-induced signaling pathway. Of the inflammatory cytokines, IL-1␤, IL-6, and tumor necrosis factor (TNF)-␣ were detected in media from macrophages stimulated with PA.Moreover, the JAK2 inhibitor ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) abolished PA-induced IL-1␤ and IL-6 release but not TNF-␣ production, which is consistent with the notion that IL-1␤ and IL-6 but not TNF-␣ contain a STAT binding element in their promoter region. The knockdown of JAK2 in macrophages by small interfering RNA significantly attenuated PA-induced IL-1␤ and IL-6 production. In addition, JAK2 inhibitor suppressed PA-induced Akt phosphorylation, and the Akt inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocked GAS activation (GAS contains a promoter that responds to PA), suggesting that PA-mediated JAK2 activation leads to phosphatidylinositol 3-kinase/Akt phosphorylation and STAT activation, and the subsequent translocation of STAT to the nucleus. Together, our data demonstrate that PA-activated macrophages produce IL-1␤ and IL-6 and that these processes require the activation of the JAK2-STAT1/3 or JAK2-Akt-STAT signaling pathways.Phosphatidic acid (PA) is an important metabolite that is involved in phospholipid biosynthesis and membrane remodeling (Lim et al., 2003). PA can be generated by several cellular processes, such as the hydrolysis of phosphatidylcholine by phospholipase D, the phosphorylation of DAG by DAG kinase, and the acylation of lyso-PA by lyso-PA acyltransferase (Koch et al., 2004;van Baal et al., 2005), and can be metabolized to other bioactive lipids, such as lyso-PA and DAG (Nanjundan and Possmayer, 2003).It has been suggested that PA may play a crucial role in the regulation of various biological events. For example, PA is involved in the phosphorylations of many proteins Avila-Flores et al., 2005), activation because of oxidative stress (de Jong et al., 2004), modulation of membrane trafficking (Kooijman et al

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-κB activation and NF-κB is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-κB activation.

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.