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.

Resveratrol inhibits foam cell formation via NADPH oxidase 1- mediated reactive oxygen species and monocyte chemotactic protein-1.

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.

Involvement of glycogen synthase kinase-3beta in hydrogen peroxide-induced suppression of Tcf/Lef-dependent transcriptional activity.

Hydrogen peroxide (H(2)O(2)) mediates induction of cytotoxicity in various cell types. GSK-3beta has been found to participate in a number of signaling pathways, including cell proliferation and cell death. In the present study, we show that GSK-3beta is rapidly dephosphorylated and activated in response to H(2)O(2) treatment. H(2)O(2) also dephosphorylates Akt/PKB in a dose- and time-dependent manner. Overexpression of Akt/PKB attenuates H(2)O(2)-induced dephosphorylation of GSK-3beta. Ectopic expression of Dvl-1, a component of Wnt signaling, stimulates Akt/PKB and inhibits dephosphorylation of GSK-3beta by H(2)O(2). Furthermore, H(2)O(2) causes the reduction of beta-catenin level and LiCl-mediated activation of Tcf/Lef-dependent transcription activity. These findings suggest that GSK-3beta is involved in H(2)O(2)-mediated inhibition of Tcf/Lef-dependent transcriptional activity.

Methyl-beta-cyclodextrin inhibits cell growth and cell cycle arrest via a prostaglandin E(2) independent pathway.

Methyl-beta-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-beta-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-beta-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.

Apoptosis, proliferation and p12(doc-1) profiles in normal, dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model.

Disruption of the homeostatic balance between proliferation and apoptosis is widely believed to contribute to human oral carcinogenesis. Using the Syrian hamster oral cancer model, we examined normal, hyperplastic, dysplastic and malignant oral epithelium for the fraction of apoptotic, proliferating and p12(doc-1) expressing keratinocytes using the TUNEL assay, as well as PCNA and p12(doc-1) immunostaining, respectively. The percentage of TUNEL positive cells progressively increased from normal to dysplastic epithelium (P<0.0019), but returned to normal keratinocyte levels in the malignant epithelium (P<0.20). However, PCNA positive cells increased progressively through hamster oral malignant progression (P<0.0012). The overall ratio of apoptotic to proliferating keratinocytes remains similar until the transition between dysplastic and malignant epithelium, where the ratio is markedly reduced (P<0.05). p12(doc-1) labeling demonstrated a similar expression pattern (P<0.008). This study demonstrates that apoptosis, proliferation and the expression of p12(doc-1) reflects alterations reported during human oral carcinogenesis and supports the use of the Syrian hamster model for the further examination of these pathways.

Inflammatory granuloma caused by injectable soft tissue filler (Artecoll).

Artecoll (Artes Medical Inc., San Diego, CA, USA) has recently been developed as a permanent synthetic cosmetic filler. We experienced an inflammatory granuloma resulting from a previous injection of Artecoll at the upper lip, which was regarded as a rare side effect of this filler. A 50-year-old female patient complained of swelling, dull pain, and heat in the right upper nasolabial fold area, which had started one week before her visit to Kyungpook National University Hospital. The patient received topical steroid therapy at a local clinic, which was not effective. At the injection site, a hard nodule was palpated and erythema was observed with mild tenderness. Antibiotic treatment and subsequent incision and drainage did not result in complete cure of the facial swelling, and the facial swelling and pain persisted. Computed tomography showed a lesion approximately 1-cm in size without clear boundaries and relatively increased nodular thickening. Finally, a subdermal lesion was removed via an intraoral vestibular approach. The lesion was diagnosed as inflammatory granuloma by a permanent biopsy. The patient had healed at two months after the filler injection. Although the soft tissue filler is widely used for cosmetic purposes, there is potential for complication, such as the inflammatory granuloma should be considered before treatment.

Resveratrol inhibits inflammation induced by heat-killed Listeria monocytogenes.

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 heat-killed 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 3ß (GSK3ß) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The involvement of GSK3ß and ERK1/2 was tested using inhibitors. While the GSK3ß 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 GSK3ß and ERK1/2 pathways.

The JAK2-Akt-glycogen synthase kinase-3ß signaling pathway is involved in toll-like receptor 2-induced monocyte chemoattractant protein-1 regulation.

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-3ß (GSK3ß) 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 GSK3ß as well as Akt by Pam3CSK4 stimulation. While the inactivation of Akt by LY294002 suppressed TLR2-mediated MCP-1 induction, the inactivation of GSK3ß by LiCl potentiated TLR2-mediated MCP-1 induction. Furthermore, Akt inhibitor suppressed TLR2-mediated phosphorylation of GSK3ß. Taken together, these results suggest that a MyD88-independent pathway exists in TLR2 signaling; the JAK2-Akt-GSK3ß pathway is a novel MyD88-independent pathway for MCP-1 induction.

TRAIL is involved in CpG ODN-mediated anti-apoptotic signals.

Synthetic oligodeoxynucleotides (ODNs) with the CpG-motifs are recognized by toll-like receptor 9 (TLR9), which elicits an immune response. Serum starvation of Raw264.7 cells increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression. However, treatment with CpG ODN reduced TRAIL expression as well as apoptosis by serum starvation. In serum starved cells, TLR9 inhibitors recovered the decreasing TRAIL expression and sub-G1 accumulation by CpG ODN. CpG ODN-regulated anti-apoptotic signals which were dependent on the Akt-FoxO3a signaling pathway. CpG ODNs activated Akt and inactivated FoxO3a in serum starved cells. Knockdown of FoxO3a by siRNA decreased TRAIL expression and apoptosis in serum-starved cells. In contrast, FoxO3a overexpression increased apoptosis by serum starvation, and CpG ODNs blocked these effects through TRAIL expression. LY294002, a PI3K-Akt inhibitor, blocked the CpG ODN effect of TRAIL expression and the sub-G1 population in serum starved cells. In contrast, overexpression of wild-type Akt reduced additional sub-G1 cells both in non-CpG ODN- and CpG ODN-treated cells. Taken together, these results demonstrate the involvement of Akt-FoxO3a signaling in TLR9-mediated downregulation of TRAIL and anti-apoptotic signals.

Glycogen synthase kinase 3beta and beta-catenin pathway is involved in toll-like receptor 4-mediated NADPH oxidase 1 expression in macrophages.

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 beta-catenin accumulation by glycogen synthase kinase 3beta (GSK3beta) inactivation, and that beta-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 GSK3beta phosphorylation, induced beta-catenin accumulation and increased nuclear translocation. The GSK3beta inhibitor LiCl potentiated LPS-induced Nox1 expression in accordance with beta-catenin accumulation and nuclear translocation. Conversely, ectopic expression of a constitutively active GSK3beta mutant severely attenuated Nox1 expression. These findings identify a novel regulatory pathway controlling Nox1 expression by LPS-stimulated macrophages.

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.

Differential Sensitivity of Taxol-induced Apoptosis in U2OS and SaOS2 Osteogenic Sarcoma Cells.

PURPOSE: Taxol (Paclitaxel) is a new generation of chemotherapeutic drug proven to be effective in the treatment of many cancers. In this study, to further demonstrate the differential effect of the tumor suppressor gene, p53, on the Taxol-induced apoptosis in osteogenic sarcoma cell lines, we used p53-defected SaOS2 cells and wild type p53-expressed U2OS cells. MATERIALS AND METHODS: The cell viability was measured by the XTT assay. To examine whether the differential expressions of p53, in U2OS and SaOS2 cells, were associated with Taxol-induced apoptosis, DNA fragmentation assays were performed on both cytosolic and genomic DNA. Since the cleavage of poly (ADP-ribose) polymerase (PARP) is primarily responsible for apoptosis, the cleavage of PARP, and the expression of cyclin B1, polo-like kinase, Bax, Bcl-xL, Bcl-2 in U2OS and SaOS2 cells were compared by Western blot analyses. RESULTS: The cell viability of the p53-defected SaOS2 cells was markedly decreased with Taxol treatment. Whereas, the cell viabilities due to 6-mercaptopurine and adriamycin were no different between the U2OS and SaOS2 cells. Treatment with Taxol induced a ladder- like pattern of DNA fragments, which is a biochemical hallmark of apoptosis, consisting of multiples of approximately 180-200 base pairs, in a dose-dependent manner in the SaOS2 cells, but insignificantly with the U2OS cells. When the cells were treated with Taxol, the 89 kDa cleavage product of PARP clearly appeared as a function of time in the SaOS2 cells, but not in the U2OS cells. The Taxol-induced apoptosis in p53 defected-osteogenic sarcoma cells was associated with the PARP cleavage as a result of the increased activity of caspase 3, and the high expressions of cyclin B1 and PLK. Bax, as a proapoptotic factor, was increased in the SaOS2cells, but the Bcl-xL and Bcl-2 were decreased when the cells were exposed to 10miceoM Taxol. CONCLUSION: From these results, it was concluded that p53-defected SaOS2 cells are much more sensitive to Taxol-induced apoptosis than p53-expressed U2OS cells.