Immunohistochemical localization of fibroblast growth factors FGF-1 and FGF-2, and receptors FGFR2 and FGFR3 in the epithelium of human odontogenic cysts and tumors

Acidic (FGF-1) and basic (FGF-2) fibroblast growth factors are members of a family of growth factors that function in growth, differentiation and regeneration of a variety of tissues. Their presence in human odontogenic cysts and tumors has not been previously investigated. This study was designed to detect immunohistochemically the presence of these factors and two fibroblast growth factor receptors (FGFR2 and FGFR3) in a cross section of odontogenic cysts and tumors, to determine if they may be involved in the differentiation of odontogenic epithelium or, more specifically, in the development of particular cysts or tumors. Archival formalin-fixed paraffin-embedded tissues were used. With some exceptions, FGF-2 and the receptor FGFR2, were found in the cytoplasm and occasionally in the nuclei of cells of odontogenic epithelium, while FGF-1 and the receptor FGFR3, were absent or only focally or weakly detected, using standard immunohistochemical techniques. The data are similar to those published for normal murine odontogenesis, suggesting that these factors are associated with odontogenic differentiation rather than pathogenesis. The presence of significant nuclear staining in odontogenic epithelium associated with embryonic mesenchyme in ameloblastic fibromas and ameloblastic fibro-odontomas suggests that FGF-2 may be involved in directing nuclear activity at the histodifferentiation stage of odontogenesis.     

FGF-2对体外培养人牙髓细胞FGFR和OPN表达活性的影响

目的:研究成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)对体外培养的人牙髓细胞表面成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)及骨桥蛋白(osteopontin,OPN)表达的影响。方法:以改良组织块培养法体外获得人牙髓细胞,采用Western blotting法检测不同浓度FGF-2作用下牙髓细胞FGFR的表达情况;应用Real Time-PCR法,检测不同浓度FGF-2作用下牙髓细胞OPN mRNA的表达。结果:与对照组相比,FGF-2在1～50 ng/mL浓度下均可促进牙髓细胞FGFR的表达(P<0.05),最佳显效浓度为10 ng/mL。FGF-2在1～50 ng/mL浓度下均可诱导牙髓细胞OPN mRNA表达(P<0.05),OPN mRNA的表达在10 ng/mL的FGF-2作用下达到高峰。结论:FGF-2可促进体外培养人牙髓细胞FGFR和OPN的表达,在牙髓牙本质复合体修复中可能发挥重要作用。     

Immunohistochemical localization of fibroblast growth factor-1 (FGF-l)and FGF-2 in cultured human ameloblastoma epithelial cells and ameloblastoma tissues

Fibroblast growth factor-1 (FGF-1) and FGF-2 are mitogenic polypeptides that may contribute to neoplastic cell proliferation. In the present study, we established a serum-free culture system for ameloblastoma cells and demonstrated that the addition of FGF-1 and FGF-2 enhanced cell growth in a dose-dependent manner. Immunoperoxidase staining of cultured cells demonstrated strong expression of FGF-1 and FGF-2. In tissue specimens, FGF-1 was localized in epithelial cell components of ameloblastomas, whereas FGF-2 was mainly found in the basement membranes with only moderate staining in epithelium. These data suggest that both FGF-1 and FGF-2 may contribute to the growth and development of ameloblastomas.     

Immunocytochemical localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in oral squamous cell carcinoma (SCC)

The localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in human oral squamous cell carcinoma (SCC) was examined by immunohistochemical techniques using anli-FGF-1 and anti-FGF-2 monoclonal antibodies. Immunofluorescence staining of two oral SCC cell lines revealed that growing cancel-cells were intensely positive for both FGF-1 and FGF-2, but confluent cells showed a faint immunostaining. In addition, two molecular mass species of FGF-1(16 and 18 kDa) and one of FGF-2 (18 kDa) were identified by Western blot in cell extracts derived from growing SCC cells, but not from confluent SCC cells. The growing cell extracts significantly stimulated the proliferation of human umbilical vein endothelial cells. Immunoperoxidase staining of 13 oral SCC cases showed that both well-differentiated and poorly-differentiated cancer cells were positive for FGF-1 and FGF-2 with high frequency and intensity as compared to normal oral epithelium. These results indicate that SCC cells express high levels of endogenous FGF-1 and FGF-2, and suggest that these growth factors may contribute to cancer cell growth.     

Immunohistochemical localization of fibroblast growth factor-1 (FGF-1), FGF-2 and fibroblast growth factor receptor-1 (FGfR-1) in pleomorphic adenoma of the salivary glands

Fibroblast growth faclor-1 (FGF-l) and FGF-2 are heparin-binding polypeplides that are potent mitogens for neoplastic cells. In this study, fibroblast growth factor-1 (FGF-l), FGF-2, and fibroblast growth factor receptor-1 (FGfR-1) were immunohistochemically analyzed in 10 patients with pleomorphic adenoma of the salivary gland by using specific monoclonal antibodies. The tumor tissues were histopathologically classified as: tubular, solid, myxoid or chondroid. Both FGF-1 and FGF-2 were immunohistochemically identified in the tumor cells of all histological types. In addition, immunoreactive FGF-2 was also found in the basement membrane of tubular type tumor cells. Conversely. FGfR-1-positive tumor cells were essentially confined to the tubular and solid areas of tumors. Tumor cells in the myxoid and chondroid areas were FGfR-1 immunonegative. These results suggest that the co-expression of FGF and its receptor appears to be related to the proliferative activity of tumor cells in the tubular and solid areas, whereas loss of FGF receptor expression may be associated with the differentiation of tumor cells into myxoid and chondroid tissue types.     

白细胞介素-6对人牙周膜细胞MMP-3 mRNA及蛋白表达的影响

目的 观察白细胞介素—6(IL—6)对人牙周膜细胞MMP—3蛋白及mRNA表达的影响，探讨IL—6介导MMPs对牙周组织ECM代谢影响的机制。方法 在体外培养条件下，观察不同浓度IL—6作用前后是否对人牙周膜细胞MMP—3表达的情况产生影响。采用原位杂交、免疫组化技术对HPDLC比表达MMP—3的情况进行检测。结果 当IL—6的浓度为10U／ml及100U／ml时，可显著促进人牙周膜细胞MMP—3 mRNA及蛋白的表达，且具有明显的浓度依赖效应。结论 IL—6的刺激作用，可使HPLCD比表达MMP—3阳性信号增强，表明IL—6介导MMP—3参与调节牙周膜细胞外基质代谢。     

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

FGF2基因转染对人牙周膜细胞生物学性状的影响

目的:探讨碱性成纤维细胞生长因子(Fibroblast Growth Factor 2,FGF2)基因转染对人牙周膜细胞(Periodontalligament cells,PDLCs)生物学性状的影响.方法:将FGF2基因转入PDLCs,通过免疫组化SABC及免疫印记法检测其稳定表达,并检测转基因细胞增殖活力及碱性磷酸酶合成情况.结果:免疫细胞化学证实转染与未转染细胞均在胞浆内表达FGF2,但转染FGF2细胞较未转染组染色深,免疫印迹杂交结果显示转染前后均表达17KD FGF2,但转染后表达量增高.FGF2基因转染可增强细胞增殖能力,但碱性磷酸酶活性未见明显变化(P＞0.05).结论:FGF2基因能被转入PDLCs并得到稳定表达,转基因细胞增殖与合成明显增强,但转染FGF2基因不能促进细胞分化.     

Specific inhibition of cyclooxygenase-2 results in inhibition of proliferation of oral cancer cell lines via suppression of prostaglandin E2 production

Prostaglandins (PGs) are known to play important roles in the proliferation of various types of cancer cells. PGs are produced by the action of cyclooxygenase (COX) enzymes, and two forms of COX, COX-1 and COX-2, have been described. Previous studies have demonstrated that overexpression of COX-2 is associated with colon carcinogenesis, tumor invasion and metastatic potential of colon cancer. In this study, the role of COX-2 on proliferation of squamous cell carcinoma cell lines was investigated. NS-398, a selective COX-2 inhibitor, inhibited proliferation of NA cells, a squamous cell caricinoma cell line that constitutively expresses COX-2 mRNA. NS-398 suppressed the spontaneous production of PGE2 by NA cells, and the antiproliferative effect of NS-398 was abolished by addition of PGE2. Similar results were obtained from experiments using COX-2 antisense oligonucleotide. These results suggest that specific inhibition of COX-2 inhibits proliferation of cancer cells expressing COX-2 mRNA via suppression of PGE2 production.     

The inhibition of DNA synthesis by prostaglandin E2 in human gingival fibroblasts is independent of the cyclic AMP-protein kinase A signal transduction pathway

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE₂ on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE₂ upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE₂-induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE₂ on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE₂ on DNA synthesis in Gin-1. These results suggest that in Gin-I, PGE₂-induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE₂, and that PGE₂ does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.     

Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-1β release from rat periosteal and human gingival fibroblasts in vitro

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease-digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum-free medium with 0.1, 1.0 and 10.0μg/ml LPS. The levels of prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE₂ (24.5 ± 1.5 (SD) ng/ml) and IL-1β (0.34 ± 0.29 ng/ml). LPS stimulated statistically significant dose-related increases hi PGE₂ and IL-1β at the concentrations of LPS tested. At 10.0μg/ml, LPS-stimulated fibroblasts produced 363.5 ± 40.3 ng/ml PGE, and 1.81±0.1 ng/ml IL-1β in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE, and IL-1β release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.     

骨形成蛋白-2和碱性成纤维细胞生长因子对牙周膜细胞碱性磷酸酶活性的联合效应

目的 :了解重组人骨形成蛋白 - 2 (rhBMP - 2 )和碱性成纤维细胞生长因子 (bFGF)单独和联合作用对人牙周膜细胞 (PDLC)碱性磷酸酶 (ALP)活性的影响。方法 :体外培养人PDLC ,分别用不同浓度的rhBMP- 2和bFGF单独或联合作用 ,用酶动力学方法检测PDLC的ALP活性。结果 :5 0～ 2 0 0 μg/L浓度的rhBMP - 2可显著增强人PDLC的ALP活性 (P <0 .0 1) ,而 10 μg/L浓度的bFGF可显著抑制人PDLC的ALP活性(P <0 .0 1) ,rhBMP - 2和bFGF联合作用仍可较明显地增强人PDLC的ALP活性 (P <0 .0 5 )。结论 :rhBMP - 2和bFGF联合应用可增强人PDLC的ALP活性     

Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model

OBJECTIVE: The aim of this study was to evaluate the influence of Emdogain (EMD) on cultured gingival fibroblasts, periodontal ligament fibroblasts and dermal fibroblasts, using an in vitro model of wound healing. BACKGROUND: Enamel matrix derivative has been demonstrated to promote periodontal regeneration. However, the precise mechanisms by which this agent acts are still unclear. METHODS: The effect of EMD on proliferation of the cells was studied using subconfluent cultures of gingival fibroblasts and periodontal ligament fibroblasts. The cells were made quiescent overnight and then stimulated with various concentrations of EMD (10, 50, 100 and 150 microg/ml) for 24 h. Negative and positive controls were cells cultured in media containing 0.2% and 10% fetal calf serum (FCS). The DNA synthesis was measured by the cellular uptake of [3H]thymidine. For in vitro wounding the cells were cultured, wounded and stimulated with 0.2% FCS, 10% FCS and EMD at a concentration of 20 microg/ml. The percentage of wound fill after treatment was measured after d 1, 4, 6, 12 and 16. The proliferation of cells was also calculated by the extent of incorporation of crystal violet. RESULTS: The results demonstrated that cells in vitro fill an empty space by a combination of proliferation and cell migration. The most rapid closure of a wound area occurred where both proliferation and migration can occur as was seen when wounded cultures were maintained in 10% FCS or at a concentration of 20 microg/ml EMD which promoted proliferation. CONCLUSIONS: Therefore, EMD appears to exert an influence on cells that is compatible with improved wound healing.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [¹²⁵I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [¹²⁵I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4°C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-β₁. Scatchard analysis revealed the presence of approximately 1.0 × 10⁵ FGF-2 binding sites per cell with an apparent Kd of 1.2 × 10⁻¹⁰ M. Interestingly, the binding of [¹²⁵I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.     

Differential effects of prostaglandin E2 and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes

Weinberg E, Topaz M, Dard M, Lyngstadaas P, Nemcovsky C, Weinreb M. Differential effects of prostaglandin E ₂ and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes. J Periodont Res 2010; 45: 731–740. © 2010 John Wiley & Sons A/S Background and Objective: Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E₂ (PGE₂) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. Material and Methods: Cells from healthy human gingiva or skin were treated with PGE₂ and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. Results: In hGFs, PGE₂ (1 μm ) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE₂‐induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal‐regulated kinase (p‐ERK), PGE₂ reduced it (by 70–80%) and EMD restored it when present with PGE₂. Prostaglandin E₂ stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE₂ was milder than with hGFs. When present together, EMD abolished the PGE₂‐induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE₂ had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP₂ receptor mRNA than hGFs, while hGKs contained none. Conclusion: Prostaglandin E₂ inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE₂ can be explained by the levels of EP₂ expression.     

碱性成纤维细胞生长因子对体外培养人牙髓细胞迁移和碱性磷酸酶活性的影响

目的:研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对体外培养人牙髓细胞迁移和碱性磷酸酶(ALP)活性的影响。方法:以组织块培养法体外获得人牙髓细胞,采用Transwell培养法和ALP活性检测法,观察10 ng/mL bFGF对体外培养人牙髓细胞迁移和分化能力的影响。结果:bFGF可显著诱导体外培养牙髓细胞迁移,与对照组相比差异有统计学意义(P<0.05),并能抑制细胞ALP活性(P<0.05),随着培养时间延长,该抑制作用更加显著(P<0.05)。结论:bFGF能促进牙髓细胞迁移,抑制ALP活性,在牙本质牙髓复合体修复中可能发挥重要作用。     

Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-1β release from rat periosteal and human gingival fibroblasts in vitro

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease-digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum-free medium with 0.1, 1.0 and 10.0 micrograms/ml LPS. The levels of prostaglandin E2 (PGE2) and interleukin-1 beta (IL-1 beta) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE2 (24.5 +/- 1.5 (SD) ng/ml) and IL-1 beta (0.34 +/- 0.29 ng/ml). LPS stimulated statistically significant dose-related increases in PGE2 and IL-1 beta at the concentrations of LPS tested. At 10.0 micrograms/ml, LPS-stimulated fibroblasts produced 363.5 +/- 40.3 ng/ml PGE2 and 1.81 +/- 0.1 ng/ml IL-1 beta in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE2 and IL-1 beta release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.