School of Health and Sports Science, Mukogawa Women's University, Nishinomiya, Japan.
Faculty of Human Care, Department of Nursing Science, Tohto College of Health Sciences, Saitama, Japan.
J Cell Physiol. 2017 Dec;232(12):3317-3336. doi: 10.1002/jcp.25776. Epub 2017 Apr 10.
Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.
Sox9 是软骨发育的主要调节因子，控制着从间充质细胞向软骨细胞分化的细胞命运决定。此外，Sox9 调节软骨细胞的增殖和分化，以及软骨特异性蛋白聚糖的产生。软骨发育中 Sox9 独立机制的存在仍有待确定。在这里，我们试图通过使用小鼠软骨细胞系 N1511 的微阵列分析来鉴定参与这些假定机制的基因。我们首先关注在 Bmp2 处理后表达上调但 Sox9 siRNA 后续处理不改变的转录因子。在这些转录因子中，我们选择了对软骨形成有正向调节作用的转录因子，并确定了同源框 3（Irx3）是候选基因之一。Irx3 的表达随着软骨细胞终末分化逐渐增加，与 Sox9 的表达呈相反趋势，并且在 Bmp2 处理时促进间充质细胞的软骨分化。此外，Irx3 通过上调 Sox9 表达下调时，部分挽救了由 Sox9 表达下调引起的软骨形成受损，从而上调了核心蛋白聚糖和聚糖蛋白的表达。最后，Irx3 与 Bmp2 信号协同作用激活 p38 MAPK 途径，从而以 Sox9 非依赖性方式刺激 Sox9 的表达以及核心蛋白聚糖和聚糖蛋白的表达。这些结果表明，Irx3 代表了间充质细胞的一种新型软骨形成因子，与 Bmp2 介导的信号协同作用，并调节软骨形成独立于 Sox9 介导的调节相关的转录机制。
