Fernández-Lloris R, Viñals F, López-Rovira T, Harley V, Bartrons R, Rosa J L, Ventura F
Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, 08907 L' Hospitalet de Llobregat, Spain.
Mol Endocrinol. 2003 Jul;17(7):1332-43. doi: 10.1210/me.2002-0254. Epub 2003 Apr 3.
Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.
软骨形成导致成熟软骨的形成，并产生作为软骨内骨形成模板的初始骨骼元素。骨形态发生蛋白（BMPs）参与多个发育和器官发生过程，并已被确定为软骨形成的关键调节因子。在本研究中，我们试图确定促成BMP - 2诱导软骨生成标志物的转录机制。用BMP - 2刺激C3H10T1/2细胞的时间进程研究显示，阿尔新蓝阳性物质呈剂量依赖性出现，且II型胶原mRNA表达上调。后一种效应需要新的蛋白质合成，因为添加环己酰亚胺完全阻断了II型胶原mRNA的诱导。包含软骨细胞特异性增强子的区域，定位于II型胶原α1链（Col2a1）基因的内含子I中，足以赋予BMP - 2依赖性的II型胶原基因表达的转录诱导。对软骨生成的Sry型高迁移率族（HMG）盒蛋白（SOX）转录因子表达水平的分析表明，BMP - 2可时间依赖性诱导Sox6表达，这与结合到软骨细胞特异性增强子上的BMP - 2诱导蛋白复合物的出现相关。用SOX6和SOX9抗体对核提取物进行预孵育可显著降低这些条带的强度。SOX6的强制表达模拟了BMP - 2的效应，而SOX9的共表达促进了两种因子在软骨细胞特异性增强子转录中的协同相互作用。此外，缺乏其高迁移率族结构域的SOX6突变体的过表达足以阻止BMP - 2对软骨细胞特异性增强子的转录诱导。综上所述，这些结果表明SOX6是软骨形成过程中BMP - 2信号的重要下游介质。
