Dong Yu-Feng, Soung Do Y, Chang Yoon, Enomoto-Iwamoto Motomi, Paris Mark, O'Keefe Regis J, Schwarz Edward M, Drissi Hicham
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14642, USA.
Mol Endocrinol. 2007 Nov;21(11):2805-20. doi: 10.1210/me.2007-0199. Epub 2007 Aug 7.
We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.
我们研究了转化生长因子-β（TGF-β）和经典Wnt信号下游未成熟软骨细胞与成熟软骨细胞之间转变的分子机制。我们使用了两种发育上不同的软骨细胞模型，分别从胚胎期鸡胸骨的尾部或鸡生长板中分离得到。胸骨下部的软骨细胞表现出未成熟的表型特征，而从生长板中提取的细胞则呈现肥大表型。TGF-β在未成熟软骨细胞中显著诱导β-连环蛋白，而在成熟软骨细胞中则抑制其表达。TGF-β进一步增强了经典Wnt介导的下胸骨软骨细胞中Topflash报告基因表达的反式激活。然而，它在生长板软骨细胞中以时间依赖性方式抑制Topflash活性。我们的免疫沉淀实验表明，TGF-β在未成熟软骨细胞中诱导Sma和Mad相关蛋白3与T细胞因子4相互作用，而在成熟软骨细胞中则抑制这种相互作用。染色质免疫沉淀实验也观察到类似结果，表明TGF-β在下胸骨软骨细胞与生长板软骨细胞中对Runx2启动子上T细胞因子4的占据情况有不同影响。为了进一步确定未成熟软骨细胞与肥大软骨细胞之间的分子开关，我们在用TGF-β和Wnt3a处理两种细胞模型后，评估了Twist1和Runx2的表达及调控情况。我们发现Runx2和Twist1在软骨细胞成熟过程中受到不同调控。此外，TGF-β在成熟软骨细胞中诱导Twist1表达，却抑制这些细胞中Runx2的表达。Wnt3a处理则产生相反效果，且在这些细胞中其作用强于TGF-β。最后，在成熟软骨细胞中过表达鸡Twist1可显著抑制其肥大。总之，我们的研究结果表明，Twist1可能是软骨细胞响应TGF-β和经典Wnt信号向终末成熟发展过程中的重要调节因子。
