TGF-β isoforms and TGF-β receptors in drug-induced and hereditary gingival overgrowth

Drug therapy and hereditary factors are two of the main causes of gingival overgrowth (GO). Both of these forms of GO are associated with increased extracellular matrix production by fibroblasts. Transforming growth factor beta (TGF-beta) is an important mediator of wound healing and tissue regeneration, which stimulates fibroblasts to produce extracellular matrix materials. The aim of this immunohistochemical study was to determine whether there is any altered expression of TGF-beta isoforms or its receptors in tissue from patients with drug-induced GO (DIGO; n=10) and hereditary gingival fibromatosis (n=10) when compared to non-overgrowth tissue (n=10). Compared to control tissues, significantly more fibroblasts expressed TGF-beta1 in both DIGO and hereditary gingival fibromatosis tissues (P<0.03). Cells expressing TGF-beta2 were present at control levels in DIGO but were significantly reduced in hereditary gingival fibromatosis (P<0.02). By contrast, the number of TGF-beta3-positive cells was the same in overgrowth tissues and controls. However, because of differences in total fibroblast densities between groups, there was a proportional increase in TGF-beta3 as well as TGF-beta1 expressing cells within both overgrowth populations (P<0.0001). Furthermore, representation of the TGF-beta2-positive phenotype was reduced in hereditary gingival fibromatosis (P<0.01) but increased in DIGO (P<0.005) compared to controls. Absorbance measurements of the positive cell populations showed that the level of expression was significantly higher for TGF-beta1 in hereditary gingival fibromatosis (P<0.002) and significantly lower for TGF-beta3 in DIGO (P<0.03). No significant differences in the numbers of TGF-betaRI- or RII-positive cells were detected between overgrowth tissues and controls. However, there were increases in the proportion of receptor-positive cells in the total cell population analysed in overgrowth tissues (P<0.0001). These results indicate qualitative and quantitative differences in TGF-beta isoform and receptor expression by fibroblasts in gingival overgrowth that may contribute to disease pathogenesis.     

Human periodontal ligament and gingival fibroblast response to TGF-β1 stimulation

Abstract. The purpose of this study was to measure the time-sequence response of RNA and protein synthesis to transforming growth factor-β1 (TGF-β1) by human periodontal ligament (HPDLF) and gingival (HGF) fibroblasts in culture. HPDLF and HGF were cultured from explants of healthy gingival tissue and freshly extracted teeth. Cultures of 8×10⁴ cells/ml were exposed to medium containing ³H-uridine and ³⁵S-methionine with TGF-β1 at concentrations from 10^-9 M to 10^-21 M, or control medium, for up to 60 hours in order to assess RNA and protein synthesis. Protein concentrations of comparable cultures were also assayed colorimetrically. Results were reported as specific activity (CPM/μMg protein). The results indicate that 10^-9 M TGF-β1 treated cultures showed a significant increase in RNA synthesis by HPDLF and HGF over time, as compared to the control cultures. HPDLF showed a significant increase in protein synthesis over time while that by HGF was not significant as compared to the control cultures. Lower concentrations of TGF-β1 demonstraled no significant differences from control. Results suggest that the effects of TGF-β1 on HPDLF and HGF are both time and dose dependent, with 10^-9 M TGF-β1 providing the best response of those concentrations tested. These findings support the concept that TGF-β1 may play a role in periodontal regeneration due to its ability to promote fibroblast RNA and protein synthesis. The results also demonstrate that although these two cells types appear morphologically similar, they exhibit distinct biological responses to growth factors such as TGF-β1.     

遗传性牙龈纤维瘤病的临床研究

目的探讨遗传性牙龈纤维瘤病(hereditary gingival fibromatosis,HGF)的临床特征。方法回顾分析我院2001-2007年收治的3例HGF病例,对其遗传特点及相关的综合征,临床表现,X线片特征,组织病理,诊断分型及治疗预后进行分析。结果3例HGF患者1例为综合征型HGF,2例为非综合征型HGF,其遗传特性、发病年龄、临床表征各有所不同,但均具有典型全口牙龈增生及牙槽骨吸收。结论3例HGF不同个体表现不尽相同,具有一定的异质性。     

白细胞介素-1β对遗传性牙龈纤维瘤和正常牙龈上皮细胞β-防御素的影响

目的:比较白细胞介素1β(IL-1β)刺激体外培养的正常牙龈、遗传性牙龈纤维瘤(HGF)上皮细胞β-防御素(HBD)表达的差异,探讨该差异与遗传性牙龈纤维瘤发病机制的可能相关性。方法:体外培养3例遗传性牙龈纤维瘤病人和6例正常人牙龈上皮细胞,经0,0.01,0.1,1,10,100 ng/mL的IL-1β分别刺激12、24、36、48 h,提取细胞总RNA,逆转录后,采用HBD-1、2、3特异性引物经PCR扩增,以β-肌动蛋白为内参,计算HBD-1、2、3与内参扩增产物相对值,对HBD-1、2、3进行半定量分析,采用SPSS软件单向方差统计分析法分析结果。结果:HBD-1 mRNA在正常牙龈和遗传性牙龈纤维瘤上皮细胞中呈固有表达,不受IL-1β刺激的影响。IL-1β可上调两种牙龈上皮细胞HBD-2、3的表达,在浓度为0.1～10 ng/mL时,作用时间大于12 h,受刺激组与未受刺激组差异有显著性(P<0.01)。相同浓度IL-1β作用不同时间时,正常牙龈上皮细胞中HBD-2、3的表达水平显著高于遗传性牙龈纤维瘤上皮细胞中表达水平(P<0.05,P<0.01)。且随作用时间延长差异更加显著。结论:遗传性牙龈纤维瘤病变组织和正常牙龈组织中β-防御素的表达有差异,这种差异可能与遗传性牙龈纤维瘤上皮细胞的分化及发病机制相关。     

lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.     

Nonsyndromic hereditary gingival fibromatosis: Characterization of a family and review of genetic etiology

Our aim is to describe a family with a nonsyndromic form of hereditary gingival fibromatosis (HGF) and discuss genetic characteristics of this rare disease by reviewing reported cases. A mother and three descendants were diagnosed with HGF. There was marked variable expressivity: from severe generalized gingival overgrowth in a 16-year-old boy (the proband) to minimal manifestations in the mother. The proband was submitted to gingivectomy and gingivoplasty. In younger siblings, the disease remained stable for 5 years, suggesting that clinical surveillance is a good option. The diagnosis was supported by histopathological examination. Analysis of this family and literature-reported cases supports that HGF most frequently shows an autosomal dominant inheritance with high penetrance and variable expressivity. Neomutations and gonadal mosaicism do not seem to be a rare event. Although five loci have been mapped by linkage analysis, only two genes, SOS1 and REST, were identified in four families.     

Differential proliferation of fibroblasts cultured from hereditary gingival fibromatosis and normal gingiva

Hereditary gingival fibromatosis (HGF) is an oral condition characterized by the enlargement of the gingiva of both the maxilla and mandible. To study the cell proliferation index of fibroblasts from HGF and normal gingiva (NG), cell cultures from 4 members of the same family with HGF and from 4 healthy patients were established. Our results obtained from 6 different cell proliferation assays clearly showed that the cell proliferation rate was significantly higher in fibroblasts from HGF than from normal gingiva. HGF and control fibroblasts in subconfluent culture densities were typically spindle, but in saturation density HGF cells were shorter than control cells. These data suggest that the higher proliferative index of HGF fibroblasts possibly has a role in the pathogenesis of gingival outgrowth in HGF patients.     

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.     

Transforming growth factor-β response and expression in junctional and oral gingival epithelial cells

The junctional (JE) and oral gingival (OGE) epithelium show distinct morphological phenotypes and express different cell surface and keratin markers. Transforming growth factor-β (TGF-β) has been shown to stimulate extracellular matrix formation and inhibit proteolytic matrix degradation in periodontal wound healing. To elucidate potential roles of TGF-β in gingival epithelial regeneration and reattachement, the present study examined the effects of TGF-β on JE and OGE cell growth and determined the patterns of expression of mRNAs for the TGF-β isotypes β1, β2 and β3 and TGF-β receptor types I, II and III. Primary cell cultures were initiated from JE and OGE and the cell phenotypes confirmed using monoclonal antibodies to specific keratins. TGF-β induced a significant growth inhibition in OGE cells derived from 6 different patients with a mean inhibition of 46% and a range of 16–70% (p=0.031). Although responses varied between patients, in general maximum inhibition occurred at 10 ng/ml TGF-β. JE cells from 5 patients showed no significant growth inhibition by TGF-β (p=0.125). Greater expression of TGF-β2 and receptor type I mRNA was found in OGE than JE cells and thus appeared to be associated with differentiating epithelial cells. JE cells expressed more TGF-β type II receptor specific mRNA than did OGE cells, but TGF-β1 mRNA expression was similar in JE and OGE cells. JE or OGE cultures derived from 2 of 3 patients showed expression of mRNA for the TGF-β type III receptor. TGF-β3 mRNA was not detected in any of the JE or OGE samples examined. The greater sensitivity of OGE than JE to the growth inhibiting effects of TGF-β correlated with higher expression of receptor type I mRNA which, together with the type II receptor, is required for sensitivity to growth inhibition by TGF-β. The results suggest that, in addition to structural differences, the development of functional differences in the responses of JE and OGE to TGF-β may be associated with the formation of JE from OGE cells and the reformation of attachement after periodontal surgery.     

遗传性牙龈纤维瘤病的临床特点及致病基因检测

目的检测分析遗传性牙龈纤维瘤病（hereditary gingval fibromatosis,HGF）患者的临床特点及致病基因。方法收集HGF家系1个,采用先证者查证法对其家庭成员进行全身健康状况及口腔专科检查。收集患者及健康牙龈组织作HE和Masson染色进行组织学检查。抽取患者及正常家族成员的静脉血,提取基因组DNA,PCR扩增SOSI基因并测序,同源性比较分析（basiclocalalignmentsearchtool,BLAST）。结果患者牙龈组织HE染色显示典型牙龈纤维瘤病的组织病理表现,Masson染色显示胶原纤维较正常人丰富。SOSl基因突变检测未发现突变点。结论HGF具有高度遗传异质性,目前被成功克隆与鉴定的致病基因SOSl不是唯一的致病基因。     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1β

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1β (IL-β). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1β, IL-6 and IL-8. IL-1β activation of GF cells showed that IL-1β not only induces the expression of IL-6, IL-8 and TNF-α, but also acts in an autocrine manner on GF cells and induces IL-1βexpression. Furthermore, the continuous presence of IL-1β in GF cell cultures did not down regulate the response of GF cells to IL-1β. Pretreatment of GF cells with IL-lβ resulted in the enhanced synthesis of TNF-α in response to additional IL-lβ. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

Effect of phenytoin on interleukin-1β production in human gingival fibroblasts challenged to tumor necrosis factor αin vitro

Effects and interaction of tumor necrosis factor α (TNFα) and the antiepileptic drug phenytoin (PHT) on interleukin-1β (IL-1β) production as well as on prostaglandin E₂ (PGE₂) formation were studied in gingival fibroblasts in vitro. TNFα, in contrast to PHT, dose-dependently stimulated the production of cell-associated IL-1β. The stimulatory effect of TNFα on IL-1β production was accompanied by enhanced PGE₂ formation. When PHT and TNFα were added simultaneously, the drug potentiated the stimulatory effect of TNFα on both IL-Iβ production and PGE₂ formation. The major PHT metabolite, p-HPPH, did not affect IL-1β production, either alone or in combination with TNFα. The production of IL-1β induced by TNFα and the combination of TNFα and PHT was further enhanced in the presence of the prostaglandin endoperoxide (PGH) synthase inhibitors, indomethacin and flurbiprofen. The PHT-mediated enhancement of TNFα-induced IL-1β production and PGE₂ formation in gingival fibroblasts may be an important link in the pathogenesis of gingival overgrowth induced by PHT.