Jin-Gu Lee

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation.

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFkappaB activity, and a potent inhibitor of NFkappaB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFkappaB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.

Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression

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-κ B activation and NF-κ B 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-κ B activation.

Role of NADPH oxidase-2 in lipopolysaccharide-induced matrix metalloproteinase expression and cell migration

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.

Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-α signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll‐like receptor (TLR)‐9 by CpG ODN can regulate the expression of matrix metalloproteinase‐9 (MMP‐9). CpG ODN induced the release of tumor necrosis factor (TNF)‐α and the expression of TNF receptor (TNFR)‐II, but not of TNFR‐I, in a time‐dependent manner and stimulated significant, though delayed, MMP‐9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN‐induced TNF‐α, TNFR‐II, and MMP‐9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN‐induced TNF‐α, TNFR‐II, and MMP‐9 expressions. Moreover, neutralizing TNF‐α antibody significantly suppressed CpG ODN‐induced MMP‐9 expression, suggesting the involvement of TNF‐α. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP‐9 via a TLR‐9/Akt/TNF‐α‐dependent signaling pathway.

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A(2) (cPLA(2)) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA(2)-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA(2) activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA(2) protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA(2) activity via p38 or Akt pathways and mediates iNOS expression.

Protein kinase C-η and phospholipase D2 pathway regulates foam cell formation via regulator of G protein signaling 2

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for Gαq, which is involved in regulating various vascular functions. To understand how RGS2 regulates foam cell formation, the present study identified signaling pathways controlled by lipopolysaccharide (LPS) and discovered new mechanisms whereby protein kinase C (PKC)-η and phospholipase D (PLD) 2 regulate RGS2 expression. The toll-like receptor (TLR) 4 agonist LPS caused foam cell formation of Raw264.7 macrophages and dramatically decreased RGS2 mRNA expression. RGS2 down-regulation by LPS was partially recovered by TLR4 small interfering RNA (siRNA). Peritoneal macrophages were separated from wild-type and TLR4 mutant mice, and treatment with LPS showed RGS2 expression decrease in wild-type macrophages but no change in TLR4 mutant macrophages. RGS2 overexpression was suppressed, whereas RGS2 down-regulation accelerated foam cell formation by LPS. Treatment of PKC-η pseudosubstrate weakened foam cell formation and recovered RGS2 down-regulation by LPS. In addition, LPS or phorbol 12-myristate 13-acetate stimulated PLD activity, and the pretreatment of PLD inhibitor weakened foam cell formation and recovered RGS2 down-regulation. Inhibition of PLD2 expression by siRNA also weakened foam cell formation and partially recovered LPS-mediated RGS2 down-regulation. On the other hand, PLD2 overexpression intensified RGS2 down-regulation and foam cell formation by LPS. These results suggest that LPS causes foam cell formation by increasing PKC-η and PLD2 activity by down-regulating RGS2 expression via TLR4 dependently.

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-α signaling pathway

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase‐9 (MMP‐9) expression in macrophages through tumor necrosis factor (TNF)‐α signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF‐α, but not of TNF receptor (TNFR)1 and TNFR2 in a time‐dependent manner and stimulated significant, though delayed, MMP‐9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c‐jun amino‐terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA‐induced TNF‐α production and MMP‐9 expression. Neutralizing TNF‐α, TNFR1 or TNFR2 antibodies significantly suppressed PA‐induced MMP‐9 expression, suggesting that the production of TNF‐α in response to PA preceded the expression of MMP‐9. Moreover, lipopolysaccharide‐induced PA also led to TNF‐α release and resulted in MMP‐9 expression. Taken together, these observations suggest that PA may play a role in MMP‐9 regulation through ERKs/TNF‐α/TNFRs‐dependent signaling pathway.