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. 2021 Jun 6;21(1):290.
doi: 10.1186/s12903-021-01655-4.

Expression of deubiquitinases in human gingiva and cultured human gingival fibroblasts

Yong-Wei Fu  1 , Hong-Zhi Xu  2
Affiliations

Expression of deubiquitinases in human gingiva and cultured human gingival fibroblasts

Yong-Wei Fu et al. BMC Oral Health. .

Abstract

Background: Although deubiquitinating enzymes (DUBs) such as CYLD, A20 and OTULIN are expressed in multiple tissues and thought to be linked with inflammatory diseases, their expression in periodontal tissues remains to be determined. This research was designed to assess the expression of CYLD, A20 and OTULIN in human gingiva, and to evaluate the regulation of these DUBs in human gingival fibroblasts (HGFs) upon different stimuli.

Methods: Immunohistochemistry assay was conducted to determine the expression of CYLD, A20 and OTULIN in human gingiva. Immunofluorescence assay was employed to observe the protein expression of CYLD, A20 and OTULIN in HGFs. RT-PCR and western blots were carried out to assess gene and protein expression changes of these DUBs in HGFs upon LPS or TNF-α.

Results: CYLD, A20 and OTULIN were found to be expressed in human gingiva and HGFs. The expression of CYLD, A20 and OTULIN was lower in the inflamed gingival tissue samples compared with the healthy gingival tissue samples. Further, the expression of CYLD, A20 and OTULIN in HGFs exhibited distinct regulation by different stimuli. TNF-α treatment markedly increased NF-κB activation in HGFs CONCLUSIONS: Our findings suggest that CYLD, A20 and OTULIN might play a role in the progression of periodontitis.

Keywords: A20; CYLD; Fibroblasts; Gingiva; OTULIN.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Immunohistochemistry for CYLD, A20 and OTULIN on sections from gingival tissue. A CYLD staining in healthy gingival tissue. B CYLD staining in inflamed gingival tissue. C A20 staining in healthy gingival tissue. D A20 staining in inflamed gingival tissue. E OTULIN staining in healthy gingival tissue. F OTULIN staining in inflamed gingival tissue. Arrows show positive staining for CYLD (A, B), A20 (C, D) and OTULIN (E, F). Scale bar, 50 μm
Fig. 2
Fig. 2
Immunofluorescence staining for CYLD (A), A20 (B) and OTULIN (C) in HGFs. Scale bar, 50 μm
Fig. 3
Fig. 3
mRNA levels of CYLD, A20 and OTULIN in HGFs pretreated with LPS (A–C) or TNF-ɑ (D–F) for the indicated periods of time. *P < 0.05, **P < 0.01, ***P < 0.001, 0 h versus 1 h, 2 h, 3 h, 4 h or 5 h, one-way ANOVA with Dunnett’s multiple comparison test. Data presented are from three independent experiments
Fig. 4
Fig. 4
Protein expression of CYLD, A20 and OTULIN in HGFs pretreated with LPS (A) or TNF-α (B) for the indicated periods of time. Below, quantification of the band intensity results, presented relative to Tubulin. *P < 0.05, **P < 0.01, ***P < 0.001, 0 h versus 2 h or 6 h, one-way ANOVA with Dunnett’s multiple comparison test. Data presented are from three independent experiments. The grouping of blots was cropped from the same gel for each protein. Full-length blots are presented in the Additional file 1: Fig. S1
Fig. 5
Fig. 5
NF-κB activation in HGFs pretreated with LPS (A) or TNF-α (B) for the indicated periods of time. The grouping of blots was cropped from the same gel for each protein. Full-length blots are presented in the Additional file 1: Fig. S2
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