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.     

Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway

In the present study, we sought to investigate the signal transduction pathways of expression of cytokines in the ethanol-stimulated human mast cell line, HMC-1. Ethanol significantly increased the intracellular calcium level in HMC-1. Ethanol also significantly enhanced IL-6, TNF-alpha, and TGF-beta1 production compared with media control, but did not significantly affect the IL-1beta production. After 8 h of stimulation, ethanol increased mRNA and protein expression levels of TNF-alpha and TGF-beta1 in HMC-1. The increased cytokine level was significantly inhibited by BAPTA-AM, PD98059, and SB203580. These inhibitors also inhibited ethanol-induced ERK and p38 MAPK phosphorylation. Ethanol resulted in a great increase in protein levels and promoter activity driving luciferase expression of HIF-1alpha and NF-kappaB in HMC-1 cells, but it did not affect on HIF-1alpha mRNA expression. Our observations show that calcium, MAPK activation, HIF-1alpha, and NF-kappaB are necessary for ethanol-induced TNF-alpha and TGF-beta1 expression. These results may have important implications for the study of alcohol-related diseases.     

Human macrophage inflammatory protein-3alpha/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-alpha via a non-standard NF-kappaB site.

The 5'-flanking sequences of the human macrophage inflammatory protein-3alpha/CCL20 gene were cloned and transfected into G-361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor-alpha (TNF-alpha) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF-alpha inducibility is conferred by a region between nt -111 and -77, which contains a non-standard nuclear factor-kappaB (NF-kappaB) binding site. The requirement for NF-kappaB was demonstrated as follows: (i) mutations in this NF-kappaB site abrogated TNF-alpha responsiveness; (ii) TNF-alpha activated a construct containing two copies of the CCL20 NF-kappaB binding site; (iii) overexpression of NF-kappaB p65 activated the CCL20 promoter; (iv) NF-kappaB from nuclear extracts of TNF-alpha-stimulated cells bound specifically to this NF-kappaB site.     

Hypoxia-inducible factor mediates hypoxic and tumor necrosis factor alpha-induced increases in tumor necrosis factor-alpha converting enzyme/ADAM17 expression by synovial cells

Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor alpha (TNFalpha), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNFalpha itself. We report that low oxygen concentrations and TNFalpha enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5' promoter region. This is associated with elevated TACE activity as shown by the increase in TNFalpha shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-kappaB activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNFalpha also requires NF-kappaB activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast- and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNFalpha. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.