The inhibitory effect of the deubiquitinase cylindromatosis (CYLD) on inflammatory responses in human gingival fibroblasts

Abstract

Objective Experiments were performed to evaluate CYLD expression in human gingival tissue samples and to examine the effects of CYLD on inflammatory responses in lipopolysaccharide (LPS)‐ or TNF‐α‐stimulated human gingival fibroblasts (HGFs). Methods Immunohistochemistry for CYLD and p65 expression was performed with healthy and inflamed gingival tissue samples. siRNA was used to knock down the expression of CYLD in HGFs. Upon LPS or TNF‐α stimulation, NF‐κB activation was detected in control and CYLD‐knockdown HGFs. RT‐PCR was applied to determine gene expression. Western blot analyses were employed to assess protein expression. Immunofluorescence staining was carried out to evaluate the nuclear translocation of p65. Results Immunohistochemical staining showed the expression of CYLD in human gingival tissues. In addition, CYLD protein expression was reduced in inflamed gingival tissue samples compared with healthy tissue samples. CYLD knockdown greatly enhanced the mRNA expression of proinflammatory cytokines in LPS‐ or TNF‐α‐stimulated HGFs. Furthermore, knocking down CYLD expression increased LPS‐stimulated NF‐κB activation in HGFs. Unexpectedly, CYLD knockdown did not affect TNF‐α‐induced NF‐κB activation. Conclusions Our results suggest that CYLD participates in periodontal inflammatory responses by negatively regulating LPS‐induced NF‐κB signalling.

Expression of deubiquitinases in human gingiva and cultured human gingival fibroblasts

“…Similarly, western blotting data also showed that the protein level of CYLD had no obvious change in the presence of LPS or TNF-α. These ndings are in line with our previous study [4]. In contrast, several early reports indicated that CYLD expression was upregulated by LPS or TNF-α treatment [28][29][30].…”

“…Then these samples were processed to para n blocks and sectioned at 5 µm. Immunohistochemical study was undertaken as previously described using a commercial immunohistochemistry kit [4]. Anti-CYLD (1:400), anti-A20…”

“…Ubiquitin, a protein containing 76 amino acids, has an essential role in variety of biological processes [1][2][3][4]. Ubiquitination is a critical post-translational protein modi cation, performed by speci c ubiquitin enzymes [5,6].…”

Expression of deubiquitinases in human gingiva and cultured human gingival fibroblasts

“…Then these samples were processed to paraffin blocks and sectioned at 5 µm. Immunohistochemical study was undertaken as previously described using a commercial immunohistochemistry kit [4]. Anti-CYLD (1:400), anti-A20 (1:400) and anti-OTULIN (1:400,) antibodies were applied in this study.…”

“…Ubiquitin, a protein containing 76 amino acids, has an essential role in variety of biological processes [1-4]. Ubiquitination is a critical post-translational protein modification, performed by specific ubiquitin enzymes [5, 6].…”