Matsuzaki Etsuko, Takahashi-Yanaga Fumi, Miwa Yoshikazu, Hirata Masato, Watanabe Yutaka, Sato Noriharu, Morimoto Sachio, Hirofuji Takao, Maeda Katsumasa, Sasaguri Toshiyuki
Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
J Bone Miner Res. 2006 Aug;21(8):1307-16. doi: 10.1359/jbmr.060512.
Because DIF-1 has been shown to affect Wnt/beta-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site.
Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3beta, suggesting that this chemical could affect the Wnt/beta-catenin signaling pathway. This pathway has been shown to be involved in bone biology.
We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/beta-catenin signaling pathway.
DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor alpha1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. We found that DIF-1 suppressed the expression of beta-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (-1023/-1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site.
Our data suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (-1023/-1017 bp) of the ALP gene promoter in osteoblast-like cell lines.
由于已证明DIF-1会影响Wnt/β-连环蛋白信号通路，因此研究了DIF-1对成骨细胞样细胞系SaOS-2和MC3T3-E1的影响。我们发现DIF-1抑制了该信号通路，通过TCF/LEF结合位点导致碱性磷酸酶（ALP）启动子活性受到抑制。
分化诱导因子-1（DIF-1）是盘基网柄菌的一种形态发生素，可抑制多种哺乳动物细胞的增殖并诱导其分化。先前的研究表明DIF-1可激活糖原合酶激酶-3β，提示这种化学物质可能会影响Wnt/β-连环蛋白信号通路。该信号通路已被证明与骨生物学有关。
我们分别研究了DIF-1对SaOS-2和MC3T3-E1的影响，SaOS-2是一种广泛用作成骨细胞模型系统的骨肉瘤细胞系，MC3T3-E1是小鼠成骨细胞样细胞系。还进行了报告基因检测以研究DIF-1对Wnt/β-连环蛋白信号通路的影响。
DIF-1以浓度和时间依赖性方式抑制SaOS-2增殖并降低碱性磷酸酶（ALP）活性。DIF-1处理在蛋白质和mRNA水平上均显著抑制了ALP的表达。DIF-1还在蛋白质和mRNA水平上抑制了其他成骨细胞分化标志物的表达，包括核心结合因子α1、I型胶原蛋白和骨钙素，并抑制了成骨细胞介导的矿化。随后，我们研究了DIF-1对Wnt/β-连环蛋白信号通路的影响。我们发现DIF-1抑制了β-连环蛋白的表达以及含有T细胞因子/淋巴增强子结合因子（TCF/LEF）共有结合位点的报告基因的活性。我们研究了DIF-1对由人ALP启动子驱动的报告基因的影响，发现DIF-1通过TCF/LEF结合位点（-1023/-1017 bp）显著降低了ALP报告基因的活性。此外，还研究了DIF-1对小鼠成骨细胞样细胞系MC3T3-E1的影响，发现DIF-1通过TCF/LEF结合位点降低ALP报告基因活性，从而抑制了ALP mRNA的表达。
我们的数据表明DIF-1抑制Wnt/β-连环蛋白信号通路，导致ALP启动子活性受到抑制。据我们所知，这是第一份分析ALP基因启动子的TCF/LEF结合位点（-1023/-1017 bp）在成骨细胞样细胞系中作用的报告。
