Role of transglutaminase 2 in promoting biglycan synthesis in idiopathic gingival fibromatosis

Abstract

Background: This study aimed to elucidate the pathogenesis of idiopathic gingival fibromatosis (IGF).

Methods: Human gingival fibroblasts (hGFs) were isolated from patients with IGF and periodontitis. Differential gene expression in the hGFs was analyzed using RNA sequencing. Extracellular matrix-related gene expression in the hGFs was analyzed. The effect of specific protein (SP)1 inhibitor or recombinant human transglutaminase 2 (rh-TGM2) on biglycan (BGN) expression in hGFs was also determined.

Results: RNA sequencing showed that TGM2 expression was downregulated and BGN mRNA expression was upregulated in patients with IGF relative to periodontitis. rh-TGM2 stimulation of hGFs in patients with IGF significantly reduced BGN expression. SP1 inhibitors downregulated BGN expression in the hGFs.

Conclusion: BGN upregulation via SP1 causes TGM2 downregulation in gingival fibroblasts in IGF.

Keywords: Biglycan; Human gingival fibroblast; Idiopathic gingival fibromatosis; Periodontitis; Specific protein 1; Transglutaminase 2.

Fig. 1

(a) Oral photograph of periodontal disease (PD) and idiopathic gingival fibromatosis (IGF). (b) Images of gingival fibroblasts isolated from patients with PD and IGF. The bar indicates 1 μm. (c) Cell proliferation was analyzed using the MTT assay. The bar graph shows the mean MTT absorbance and standard deviation (SD), **p < 0.01, n = 16

Fig. 2

(a) Gene Ontology enrichment analysis of the differentially expressed genes; cellular component. (b) Volcano map of differentially expressed genes in the human gingival fibroblasts (hGFs) of patients with idiopathic gingival fibromatosis (IGF)1/2 and periodontal disease (PD). Arrows indicate TGM2. (c) mRNA expression of TGM2 in the hGFs of a patient with PD and IGF. The bar graph shows the mean mRNA expression and standard deviation (SD), **p < 0.01, n = 3. (d) Protein levels of TGM2. The bar graph shows the protein level and standard deviation (SD), **p < 0.01, n = 3. (e) Immunofluorescence of hGFs isolated from a patient with PD and IGF1. Green, TGM2; red; actin; blue, nuclear. The bar indicates 75 mm

Fig. 3

Expression of mRNA related to the extracellular matrix, COL1A1 (a), ACTA2 (b), and FN1 (c). The bar graph shows the mean level of mRNA expression and standard deviation (SD), n = 3. To investigate the mRNA expression related to the extracellular matrix stimulated with transforming growth factor (TGF)-β (10 ng/mL, 24 h). (d) TGM2. (e) COL1A1. (f) ACTA2. (g) FN1. The bar graph shows the mean mRNA expression and standard deviation (SD), *p < 0.05, **p < 0.01, n = 3

Fig. 4

(a) The mRNA expression of POSTN and biglycan (BGN) related to the extracellular matrix stimulated with transforming growth factor (TGF)-β (10 ng/mL, 24 h). The mean mRNA expression and standard deviation (SD) are shown, *p < 0.05, **p < 0.01, n = 3. (b) Immunofluorescence images of gingival tissue in patients with drug-induced gingival enlargement (DIGE) and idiopathic gingival fibromatosis (IGF). BGN (green), COL1A1 (red), and nuclear (blue). The bar indicates 150 μm. (c) The effect of rhTGM2 on BGN mRNA expression in human gingival fibroblasts (hGF) isolated from a patient with IGF. TGM2 at the indicated dose (ng/mL) was introduced for 24 h. The mean mRNA expression and SD are shown, *p < 0.05, n = 3. (d) Protein levels of SP1 in hGFs isolated from a patient with periodontal disease (PD) and patients with IGF. The mean protein level and SD are shown, **p < 0.01, n = 3. (d) The effect of plicamycin on BGN mRNA expression. Plicamycin was applied at the indicated concentration (mM) for 24 h. The mean mRNA expression and SD are shown, **p < 0.01, n = 3. (f) Schema of the mechanisms underlying IGF development, showing that biglycan upregulation via SP1 causes TGM2 downregulation in gingival fibroblasts in IGF