IL-6 In Human Gingival Fibroblasts Research Articles

MiR-200b attenuates IL-6 production through IKK\u03b2 and ZEB1 in human gingival fibroblasts

ObjectiveMicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Materials and methodsTotal RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.ResultsIL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.ConclusionsThese results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4 Signaling in Human Gingival Fibroblasts.

Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-κB, and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862-1871, 2017. © 2016 Wiley Periodicals, Inc.

Effects of miR-223 on expression of IL-1β and IL-6 in human gingival fibroblasts.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional expression by translational inhibition or mRNA degradation. miRNAs bind to target mRNAs through partial complementarity, and can regulate many genes. In the present study, we investigated the effects of miR-223 on the expression of inflammatory cytokines in human gingival fibroblasts (HGF). To determine the effects of miR-223 on the expressions of interleukin-1β (IL-1β) and IL-6, HGF were stimulated by IL-1β (1 ng/mL) or tumor necrosis factor-α (TNF-α; 10 ng/mL) and transfected with a miR-223 expression plasmid. Levels of mRNA for IL-1β, IL-6, inhibitor of kappa-B kinase α (IKKα) and mitogen-activated protein kinase phosphatase-5 (MKP-5) were measured by real-time PCR, and levels IL-1β, IL-6 and IKKα protein were determined by enzyme-linked immunosorbent assay and Western blotting. Expression of IL-1β and IL-6 mRNAs was induced by IL-1β and TNF-α and further increased by miR-223 overexpression. IL-1β and TNF-α induced the expression of IL-1β and IL-6 mRNAs, and this was reduced by miR-223 inhibitor. Overexpression of miR-223 decreased the levels of IKKα protein and MKP-5 mRNA in HGF. These findings indicate that miR-223 might control the inflammatory response via IKKα and MKP-5 in periodontal tissue. (J Oral Sci 58, 101-108, 2016).

Heterogeneous Expression of Toll-like Receptor 4 and Downregulation of Toll-like Receptor 4 Expression on Human Gingival Fibroblasts by Porphyromonas gingivalis Lipopolysaccharide

Porphyromonas gingivalis (P. gingivalis) is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. P. gingivalis or its components such as lipopolysaccharide (LPS) upregulate the production of various inflammatory cytokines including interleukin (IL)-1 and IL-6 in HGFs. Recently, we demonstrated that the binding of P. gingivalis LPS to Toll-like receptor 4 (TLR4) on HGFs activates various second messenger systems (Biochem. Biophys. Res. Commun. 273, 1161–1167, 2000). In the present study, we examined the level of TLR4 expression on HGFs by flow cytometric analysis (FACS), and studied the levels of IL-1 and IL-6 in the culture medium upon LPS stimulation of HGFs by enzyme-linked immunosorbent assay (ELISA). Upon stimulation by P. gingivalis LPS for 24 h, HGFs that expressed a high level of TLR4 secreted significantly higher levels of IL-1 and IL-6 than HGFs that expressed a low level of TLR4. On the other hand, after stimulation with P. gingivalis LPS for 24 h, the level of TLR4 on the surface of HGFs decreased. These results suggest that the level of TLR4 expression on HGFs reflects the extent of inflammation in the gingival tissue, and that P. gingivalis LPS downregulates TLR4 expression on HGFs. These findings may be used to control inflammatory and immune responses in periodontal disease.