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Expression profile of human gingival fibroblasts induced by interleukin-1beta reveals central role of nuclear factor-kappa B in stabilizing human gingival fibroblasts during inflammation.

ABSTRACT: Interleukin (IL)-1beta is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1beta on the gene expression using expression arrays containing approximately 40,000 genes, and tested the role of nuclear factor-kappa B (NF-kappaB) in maintaining an activated HGF population.Total RNA was isolated from IL-1beta-induced and mock-induced control cells. Gene expression analyses were performed using expression arrays and confirmed by quantitative real-time polymerase chain reaction. Western blot analysis to show inhibitor of kappa B-alpha (IkappaBalpha) phosphorylation and immunostaining of cells for NF-kappaB nuclear translocation were performed. Apoptosis was confirmed by assay of poly ADP-ribose polymerase (PARP) cleavage.A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1beta-induced cells (P <0.001). A total of 215 genes were upregulated, and 39 genes were downregulated. Most notable NF-kappaB pathway members (NFkappaB1, NFkappaB2, IkappaBalpha, IkappaBepsilon, IkappaBzeta, REL, RELB, and TA-NFKBH) were upregulated. IkappaBalpha was phosphorylated, and NF-kappaB accumulated in the nucleus. An IL-1beta-induced set of 27 genes was downregulated by an NF-kappaB inhibitor, leading to a decreased number of viable cells and suggesting an antiapoptotic role for NF-kappaB.IL-1beta leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and especially NF-kappaB-dependent antiapoptotic genes. NF-kappaB activation blocks apoptosis, thereby stabilizing the HGF population in inflammation.

SUBMITTER: Vardar-Sengul S

PROVIDER: S-EPMC4150685 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Expression profile of human gingival fibroblasts induced by interleukin-1beta reveals central role of nuclear factor-kappa B in stabilizing human gingival fibroblasts during inflammation.

Vardar-Sengul Saynur S Arora Shilpi S Baylas Haluk H Mercola Dan D

Journal of periodontology 20090501 5

<h4>Background</h4>Interleukin (IL)-1beta is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1beta on the gene expression using expression arrays containing approximately 40,000 genes, and tested the role of nuclear factor-kappa B (NF-kappaB) in maintaining an activated HGF population.<h4>Methods</h4>Total RNA was isolated from IL-1beta-i ...[more]

PMID: 19405838

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Project description:ObjectivesTo investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression.MethodsThe presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry.ResultsERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2.ConclusionsOur data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

Dataset Information

Expression profile of human gingival fibroblasts induced by interleukin-1beta reveals central role of nuclear factor-kappa B in stabilizing human gingival fibroblasts during inflammation.

Publications

Expression profile of human gingival fibroblasts induced by interleukin-1beta reveals central role of nuclear factor-kappa B in stabilizing human gingival fibroblasts during inflammation.

Similar Datasets

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