Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis

Abstract

Gingival overgrowth is a side effect of certain medications and occurs in non-drug-induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin; the least fibrotic lesions are caused by cyclosporin A; and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. The present study has investigated the hypothesis that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies showed that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real-time polymerase chain reaction (PCR) analyses of RNA extracted from drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analysed for basal and transforming growth factor beta1 (TGF-beta1) or lysophosphatidic acid-stimulated CTGF/CCN2 expression at protein and RNA levels. These data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues; that cultured gingival epithelial cells express CTGF/CCN2; and that lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.

Conflict of interest statement

The authors have no conflicts of interest.

Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Figures

Figure 1. CTGF/CCN2 protein expression in gingival biopsies from fibrotic and normal control tissues

Representative immunohistochemical analysis for CTGF/CCN2 protein expression (A) shows the extracellular (asterisk) and intracellular (arrows) CTGF in deep connective tissue (DCT) in phenytoin (PHE) and human gingival fibromatosis (HGF) samples and not in controls. Epithelium (Ep) of fibrotic lesions stained for CTGF/CCN2 in PHE and HGF samples. Both gingival fibrosis lesions exhibited elongated rete pegs. Non-immune IgG stained serial sections showed no CTGF/CCN2 staining. Measure bars represent 200 μm at a magnification of 200×. (B); quantitative analyses of fibrosis, inflammation, and CTGF expression in connective tissue stroma are means +/− SE, (*, p

Figure 2. Expression of CTGF/CCN2 mRNA in gingival fibrosis

(A) Representative in situ hybridization sections of cyclosporin induced gingival overgrowth samples (CsA) and phenytoin (PHE) shows connective tissue and epithelial cell expression in PHE and not in CsA or no overgrowth controls. Measure bars are 400 μm, 200 μm, and 100 μm at 100×, 200×, and 400×-magnifications, respectively. (B) Real-time PCR quantification of CTGF/CCN2 mRNA expression isolated from PHE and CsA tissues normalized to GAPDH compared to RNA isolated from a non-inflamed no gingival overgrowth control tissue, expressed as fold induction compared to the same control sample +/− SD. PHE tissues (n=3) expressed significantly higher levels of CTGF/CCN2 mRNA (~7-fold) compared to CsA tissues (n=3) where CTGF/CCN2 expression was close to the no overgrowth controls (1.2-fold); *, p

Figure 3. CTGF/CCN2 mRNA expression and regulation in primary gingival epithelial cell and fibroblast cultures

Real time PCR analyses of CTGF/CCN2 RNA expression of primary human gingival epithelial cells (A) and human gingival fibroblasts (B). Cells were cultured and treated with or without TGF-β1 or LPA as described in “Materials and Methods”. No treatment control RNA’s were isolated from cultures not treated with vehicle; fold changes were relative to vehicle treated controls at each time point +/− SD; all values are normalized to GAPDH internal controls; *, p

Figure 4. CTGF/CCN2 protein expression in primary gingival epithelial and fibroblast cultures determined by Western blotting

Western blots of cell layer extracts of primary human gingival epithelial cells (A) and human gingival fibroblasts (B) cultured and treated as described in “Methods and Materials” with 5 ng/ml TGF-β1, 10 μM LPA, or no treatment control. Blots analyzed with non-immune IgG gave no background signal. β-actin antibody was used to verify equal loading of gels. Blots are representative of four experiments with consistent results.