Effect of transmembrane proteoglycans in human periodontal ligament fibroblasts

碱性成纤维细胞生长因子(碱性成纤维细胞生长因子、成纤维细胞生长因子-2)是牙周膜细胞的重要有丝分裂原之一。然而，碱性成纤维细胞生长因子对PDL细胞基质金属蛋白酶-3表达的影响尚不清楚。本研究旨在探讨碱性成纤维细胞生长因子对人视网膜色素上皮细胞中基质金属蛋白酶-3表达的影响及其机制。碱性成纤维细胞生长因子呈浓度依赖性地促进[~3H]-胸腺嘧啶核苷掺入，抑制蛋白多糖合成。然而，相似浓度范围的碱性成纤维细胞生长因子增加了细胞相关蛋白多糖的释放。此外，逆转录聚合酶链式反应检测到，bFGF呈浓度依赖性地增加了基质金属蛋白酶-3的mRNA水平。碱性成纤维细胞生长因子刺激后12h即可观察到基质金属蛋白酶-3的表达，2 4h达高峰，以后逐渐下降，直至72h，放线菌酮阻断了碱性成纤维细胞生长因子对基质金属蛋白酶-3的诱导，提示这种刺激需要从头合成蛋白质。此外，丝裂原活化蛋白(MAP)激酶通路中MEK1/2和ERK1/2的特异性抑制剂U0126可抑制碱性成纤维细胞生长因子诱导的基质金属蛋白酶-3的表达，而MEK1的特异性抑制剂PD98059不能抑制该作用。此外，Western blotting显示，bFGF在5min内上调ERK1/2的磷酸化水平，在20min时达到最大值，而U0126抑制bFGFF诱导的ERK1/2的磷酸化。这些结果表明，bFGF通过激活MAP激酶通路中的MEK2而诱导PDL细胞表达MMP3。碱性成纤维细胞生长因子对基质金属蛋白酶-3合成的刺激可能参与了牙周再生和降解过程中细胞相关蛋白多糖的调控。

Basic fibroblast growth factor (bFGF, FGF-2) is one of the potent mitogens for periodontal ligament (PDL) cells. However, the role of bFGF on the matrix metalloproteinase (MMP) -3 expression in PDL cells is unknown. In this study, the effect of bFGF on MMP-3 expression in PDL cells and the mechanism of this process were examined.Human PDL cells were exposed to bFGF at various concentrations (0.01 - 10 ng/ml) in monolayer cultures. bFGF increased [3H]-thymidine incorporation and suppressed proteoglycan synthesis concentration-dependently. However, similar concentration ranges of bFGF increased the release of the cell-associated proteoglycans into the medium. Furthermore, bFGF increased MMP-3 mRNA levels concentration-dependently as examined by reverse transcription-polymerase chain reaction. Induction of MMP-3 after the stimulation with bFGF was observed as early as 12 h with maximal at 24 h. Thereafter the MMP-3 mRNA level gradually decreased until 72 h. Cycloheximide blocked the induction of MMP-3 by bFGF, indicating the requirement of de novo protein synthesis for this stimulation. Furthermore, MMP-3 expression induced by bFGF was abrogated by U0126, a specific inhibitor of MEK1/2 and ERK1/2 in mitogen-activated protein (MAP) kinase pathway, not by PD98059, a specific inhibitor of MEK1. In addition, bFGF up-regulated the phosphorylated ERK1/2 in 5 min with the maximal at 20 min as examined by Western blotting, and U0126 inhibited the ERK1/2 phosphorylation induced by bFGF.These findings suggest that bFGF induces MMP-3 expression in PDL cells through the activation of the MEK2 in MAP kinase pathway. bFGF stimulation on MMP-3 synthesis may be involved in the control of the cell-associated proteoglycans in PDL cells during periodontal regeneration and degradation.