Expression of metalloproteinases and their tissue inhibitors in gingiva affected by hereditary gingival fibromatosis: analysis of three cases within a family.

BACKGROUND AND OBJECTIVE: Hereditary gingival fibromatosis (HGF) is a benign disorder manifested by fibrous enlargement of keratinized gingiva. Evidence exists concerning the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in mediating normal and pathological processes, including HGF. Nevertheless, there are few and contradictory results on the analysis of MMPs and TIMPs transcripts in this pathology. MATERIAL AND METHODS: We studied the expression of the transcripts encoding MMP-1, -2 and -9 and TIMP-1 and -2 in gingival biopsies, obtained from three cases of HGF within a family, by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. Samples were also processed for gelatin zymography. RESULTS: Except for MMP-9, most transcripts presented a higher level of expression in biopsies from HGF patients compared with control subjects. Accordingly, MMP-9 gelatinase activity was detected at low and similar levels among samples. Moreover, MMP-2 enzymatic activity was not detected at all. CONCLUSION: The mRNA expression of MMP-1 and -2 and TIMP-1 and -2 does not explain the gingival overgrowth presented in these cases. In addition, it is suggested that the gene expression of those molecules in the course of HGF is regulated at the translational or post-translational level.

Increased expression of collagen prolyl 4-hydroxylases in Chinese patients with hereditary gingival fibromatosis.

OBJECTIVES: Hereditary gingival fibromatosis (HGF) is characterized by excess accumulation of interstitial collagen. However, until now, there has been controversy about the mechanism of collagen accumulation in HGF gingivae. The present study aimed to clarify the pathogenic mechanisms potentially involved. DESIGN: Gingival fibroblasts from three Chinese HGF patients and three healthy subjects were cultured. Cell proliferation was assessed by MTT assay. The mRNA levels of type I collagen, MMP-1, MMP-3, TIMP-1, prolyl 4-hydroxylase (P4H)alpha(I), alpha(II), alpha(III) and P4Hbeta were analyzed in gingival fibroblasts by RT-PCR. The protein production of type I collagen and P4H was examined respectively by ELISA and Western blot. RESULTS: In culture, HGF gingival fibroblasts showed similar growth characteristics to fibroblasts isolated from control gingivae. The mRNA and protein levels of type I collagen and P4Halpha in HGF fibroblasts were higher than those in controls. There were no detected differences in mRNA expression levels of MMP-1, MMP-3, TIMP-1, P4Halpha(II), alpha(III) and P4Hbeta between HGF and control fibroblasts. CONCLUSIONS: These data suggest that increased collagen post-translational modification by P4H may be one mechanism by which increased collagen accumulation occurs in some forms of HGF.

Morphological and molecular analysis of idiopathic gingival fibromatosis: a case report.

AIM: We analyse a case of idiopathic gingival overgrowth using morphological and molecular methods. As this overgrowth involves collagen accumulation in the gingival connective tissue, we measured the collagen turnover to clarify the pathogenic mechanisms potentially involved. MATERIALS AND METHODS: The patient was a 29-year-old Italian woman with enlargement of the gingivae throughout the entire mandible and maxilla. Morphological analyses were carried out on haematoxylin-eosin and Sirius red-stained paraffin-embedded gingival sections. mRNA levels of collagen type I and III, matrix metalloproteinase (MMP)-1, transforming growth factor-beta1 and lysyl hydroxylase (LH)2b were determined by RT-PCR on cultured gingival fibroblasts and compared with healthy control fibroblasts. Interstitial collagen and MMP-1 content in the supernatants were assessed, respectively, by dot blot and SDS zymography. RESULTS AND CONCLUSIONS: In Sirius red-stained sections of the patient's overgrown gingivae, interstitial collagen content was 29% higher than controls. Her gingival fibroblasts had higher collagen type I, MMP-1 and LH2b gene expression and unmodified interstitial collagen, type I protein levels in the supernatants. These findings would seem to suggest that in this case collagen accumulation in the gingival connective tissue was not associated with increased synthesis and decreased degradation.

Proliferation of fibroblasts cultured from normal gingiva and hereditary gingival fibromatosis is dependent on fatty acid synthase activity.

BACKGROUND: Fatty acid synthase (FAS) is the enzyme that synthesizes palmitate from malonyl-CoA and acetyl-CoA. Recent studies have shown that FAS is overexpressed in human cancers and that its activity is necessary for cell proliferation. Hereditary gingival fibromatosis (HGF) is a genetic disease manifested as a progressive enlargement of the gingiva. The pathogenesis of this condition is not understood; however, a proliferative advantage of HGF fibroblasts in comparison with cells from normal gingiva (NG) has been described. The aim of this study was to investigate the role of FAS in NG and HGF fibroblast proliferation. METHODS: NG and HGF fibroblasts had their proliferative potential assessed by automated cell counting and immunocytochemistry against Ki-67 or proliferating cell nuclear antigen (PCNA). The production of FAS, androgen receptor (AR), and ErbB2 was analyzed by Western blot and the pattern of FAS expression studied by immunocytochemistry. FAS activity was blocked by the specific inhibitor cerulenin. RESULTS: Higher proliferation rates were found in fibroblasts isolated from HGF than from NG. HGF fibroblasts with greater proliferative potential produced more FAS and AR than the cell lines with lower growth rates, and all studied cell lines produced similar amounts of the ErbB2 protein. In addition, the FAS inhibitor cerulenin was able to significantly reduce the proliferation of both NG and HGF cells. CONCLUSIONS: These results show that FAS is expressed by gingival fibroblasts and that highly proliferative HGF cells produced more FAS and AR than the other fibroblast cell lines. Moreover, FAS inhibition significantly reduced both NG and HGF fibroblast growth, suggesting a role for the androgen-driven fatty acid biosynthesis in their proliferation.

Alteration in expression of MMP-1 and MMP-2 but not TIMP-1 and TIMP-2 in hereditary gingival fibromatosis is mediated by TGF-beta 1 autocrine stimulation.

Hereditary gingival fibromatosis (HGF) is characterized by an excess accumulation of extracellular matrix (ECM) resulting in a generalized and fibrotic enlargement of the gingiva. To investigate some of the regulatory features of this condition, gingival fibroblasts from normal gingiva (NG) and HGF were examined for the expression and production of matrix metalloproteinases (MMPs) and their inhibitors, tissue matrix metalloproteinases inhibitor (TIMPs). Our results, obtained from 2 different assays, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography, clearly demonstrated that the expression and production of MMP-1 and MMP-2 was significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher to those from HGF. Addition of antibodies against transforming growth factor-beta 1 (TGF-beta 1), which is produced in greater amounts by HGF fibroblasts, resulted in a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas TIMP-1 and TIMP-2 expressions were unaffected. These patterns of expression and production suggest that enhanced TGF-beta 1 production reduce the proteolytic activities of HGF fibroblasts, which favor the accumulation of ECM.

DNA content and alkaline phosphatase expression in cells of different gingival overgrowths.

The present study compared the alkaline phosphatase (ALPase) expression and DNA content at specific periods in cultured cells derived from non-inflamed enlarged gingivae of idiopathic gingivofibromatosis (IGF) and phenytoin-induced hyperplasia (PHG). Cultured cells from healthy gingiva or periodontal ligament (PDL) were used as controls. The DNA assay, ALPase assay and cytochemical staining for ALPase in cultured cells were performed at four, seven, and nine days. The presence of intense ALPase activity was a prominent feature in cultured IGF cells, whereas very low ALPase activity was detected in PHG cells. The cell lines tested showed no significant differences in DNA content. The expression of ALPase in these cells was population density-dependent. The observation that cells isolated from both types of gingival overgrowth exhibited a different ALPase profile at variance with normal gingival fibroblasts suggested that a distinct pathogenic mechanism may be involved in each type of gingival overgrowth.

Creatine kinase and lactate dehydrogenase isoenzyme patterns in cultured normal and pathological gingival fibroblasts.

The isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) were examined in cultured fibroblasts derived from the gingival tissue of a healthy person and patients with periodontitis and gingival fibromatosis. Human foreskin fibroblasts were also included for comparison. Cytosol fractions were prepared by sonication and centrifugation. Gingival fibroblasts from normal subjects (A), and from patients with periodontitis (B) or fibromatosis (C), exhibited CK-MM (muscle form) as a predominant isoenzyme and CK-BB (brain form) as a minor fraction. There was no significant difference in specific activities of CK-BB fraction in all 3 types of fibroblasts. However, B fibroblasts contained a higher proportion of MM type isoenzyme than A fibroblasts. C fibroblasts showed the lowest amount of CK-MM isoenzyme as compared to either A or B fibroblasts. In contrast, fibroblasts derived from human foreskin (D) contained only a small amount of CK-BB isoenzyme, but CK-MM was not present. In the studies of LDH isoenzymes, A, B, and C fibroblasts possessed only 3 isoenzymes, namely, LDH-3, LDH-4 and LDH-5. In terms of the relative abundance of M vs H subunits, A, B, and C fibroblasts were shown to have a preponderance of M subunits (85-87% M vs 13-15% H subunits). In comparison, D fibroblasts contained 4 isoenzymes (LDH-2, LDH-3, LDH4, LDH-5) with 73% M and 27% H subunits. The ratio of LDH-5 to LDH-4 was calculated for all fibroblasts tested, and the values were 1.11 in A, 1.39 in B, 1.16 in C and 0.64 in D.(ABSTRACT TRUNCATED AT 250 WORDS)