Sox9 determines translational capacity during early chondrogenic differentiation of ATDC5 cells by regulating expression of ribosome biogenesis factors and ribosomal proteins

Introduction: In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation. Materials: Sox9 expression in ATDC5 cells was knocked-down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 hours, and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes with polysome profiling and ribosome modus with bicistronic reporter assays. Results: Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown. Conclusion: Here we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix-production of chondroprogenitors in the growth plate in vivo.

引言：除了学界公认的与软骨细胞外基质（cartilage extracellular matrix）相关的Sox9表达外，本研究证实，祖细胞（progenitor cells）的软骨生成分化（chondrogenic differentiation）受Sox9的明确双相表达调控：即刻早期相与晚期（与细胞外基质相关）相表达。本研究旨在探究在软骨生成分化的早期阶段，Sox9驱动了哪些生物学过程。

材料与方法：在软骨生成分化前1天或分化第6天，通过siRNA转染（siRNA transfection）敲低ATDC5细胞中的Sox9表达。分别在分化诱导2小时及7天时收集样本。采用通路与网络分析方法，对比两组样本的转录组（采用RNA-seq技术）与蛋白质组（采用无标记蛋白质组学（Label-free proteomics）技术）。通过SuNSET实验评估总蛋白质翻译能力，采用多聚核糖体谱分析（polysome profiling）检测活性核糖体水平，通过双顺反子报告基因检测（bicistronic reporter assays）分析核糖体调控模式。

结果：早期Sox9敲低会在数周后显著抑制软骨生成分化。ATDC5细胞软骨生成分化的即刻早期阶段，Sox9表达可调控核糖体生物发生因子（ribosome biogenesis factors）与核糖体蛋白亚基（ribosomal protein subunits）的表达。伴随Sox9敲低的是翻译能力的下降，这与活性单核糖体及多聚核糖体数量减少相关。此外，Sox9敲低会改变帽依赖型与内部核糖体进入位点（IRES）介导的翻译模式。Sox9过表达可产生与Sox9敲低相反的效应。

结论：本研究揭示了Sox9在软骨生成分化早期阶段的一项全新关键功能。Sox9在调控核糖体数量、活性及/或组成方面的作用，可能对于体内生长板中软骨祖细胞应对增殖阶段的高代谢需求，以及后续软骨细胞外基质合成都至关重要。