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Activation and mitogen-activated protein kinase regulation of transcription factors Ets and NF-kappaB in Mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function.

ABSTRACT: Previous studies have shown that primary murine macrophages infected with Mycobacterium avium produced lower levels of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase 2 (NOS2) compared to cells infected with nonpathogenic Mycobacterium smegmatis. TNF-alpha and NOS2 levels correlated with and were dependent on the activation of mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). To define the macrophage transcriptional responses dependent on ERK1/2 activation following a mycobacterial infection, we used RAW 264.7 cells transfected with a TNF-alpha or NOS2 promoter vector. We determined that macrophages infected with M. avium compared to M. smegmatis showed diminished TNF-alpha and NOS2 promoter activity. A more pronounced difference in promoter activity was observed when only the consensus ETS and NF-kappaB binding sites were used as promoters. Mutational analysis of the ETS and NF-kappaB binding sites present on the TNF-alpha and NOS2 promoters, respectively, showed that these sites were essential for a functional promoter. Moreover, the Ets/Elk but not the NF-kappaB transcriptional response was dependent on ERK1/2. This correlated with the requirement for ERK1/2 in TNF-alpha but not NOS2 promoter activity. Our data indicate that the increased Ets/Elk and NF-kappaB promoter activities associated with M. smegmatis-infected macrophages are responsible, at least in part, for the increased TNF-alpha and NOS2 production observed in these infected cells and that ERK1/2 is required for Ets/Elk activity and full TNF-alpha production.

SUBMITTER: Lee SB

PROVIDER: S-EPMC1230939 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Activation and mitogen-activated protein kinase regulation of transcription factors Ets and NF-kappaB in Mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function.

Lee Seong-Beom SB Schorey Jeffrey S JS

Infection and immunity 20051001 10

Previous studies have shown that primary murine macrophages infected with Mycobacterium avium produced lower levels of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase 2 (NOS2) compared to cells infected with nonpathogenic Mycobacterium smegmatis. TNF-alpha and NOS2 levels correlated with and were dependent on the activation of mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). To define the macrophage transcriptional ...[more]

PMID: 16177323

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Project description:We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.

Dataset Information

Activation and mitogen-activated protein kinase regulation of transcription factors Ets and NF-kappaB in Mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function.

Publications

Activation and mitogen-activated protein kinase regulation of transcription factors Ets and NF-kappaB in Mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function.

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