IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages

The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.     

Interferons inhibit tumor necrosis factor-alpha-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with NF-kappa B

Enhanced expression of matrix metalloproteinase-9 (MMP-9) correlates with invasion during tumor progression. Interferons (IFNs) inhibit MMP-9 activation in response to tumor necrosis factor-alpha (TNF-alpha), and the latter activates the MMP-9 gene through NF-kappaB. Understanding the molecular basis for MMP-9 inhibition may provide tools to control cell invasion. The data reported here show the critical role of interferon regulatory factor-1 (IRF1) in the inhibition of MMP-9. (i) IFN treatment suppresses TNF-alpha-induced MMP-9 reporter activity in STAT1(+/+) cells but not in STAT1(-/-) cells. (ii) IRF1 transfection blocks TNF-alpha-mediated MMP-9 activation. (iii) IFNs phosphorylate STAT1 and induce IRF1 but do not affect Ikappa-B degradation nor NF-kappaB nuclear translocation. (iv) Nuclear NF-kappaB (p50/p65) and IRF1, but not STAT1, bind to the MMP-9 promoter region containing an IFN-responsive-like element overlapping the NF-kappaB-binding site. (v) Recombinant IRF1, although unable to bind to an NF-kappaB consensus sequence, competes with NF-kappaB proteins for binding to the MMP-9 promoter. (vi) Conversely recombinant p50/p65 proteins reduce IRF1-DNA binding. (vii) In cells cotransfected with IRF1 and/or p65 expression vectors, an excess of IRF1 reduces MMP-9 reporter activity, whereas an excess of p65 blocks the inhibitory effect of IFN-gamma. Thus, in contrast to the known synergism between IRF1 and NF-kappaB, our data identify a novel role for IRF1 as a competitive inhibitor of NF-kappaB binding to the particular MMP-9 promoter context.     

Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.     

Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis

Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.