Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that expression was markedly induced at early time points during chondrogenesis. We examined the expression of in human cartilage development, identifying significant enrichment of in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

中文翻译：

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SERPINA3 是软骨分化的标志物，对于早期软骨形成过程中细胞外基质基因的表达至关重要


丝氨酸蛋白酶抑制剂 (serpin) 是一个结构相似的蛋白质家族，可调节从凝血到细胞外基质 (ECM) 重塑的许多不同的生物过程。软骨形成涉及间充质干细胞（MSC）在早期发育过程中浓缩和分化为软骨细胞。在这里，我们首次证明了一种丝氨酸蛋白酶抑制剂 SERPINA3（基因名蛋白质，也称为 α-1 抗胰凝乳蛋白酶）在软骨分化中发挥着关键作用。我们观察到表达在软骨形成过程的早期时间点被显着诱导。我们检查了人类软骨发育中的表达，发现与整个肢体相比，发育中的软骨中的显着富集，这与众所周知的软骨分化标志物相关。当使用 siRNA 沉默时，通过二甲基亚甲蓝测定 (DMMB) 和番红-O 染色测定，软骨沉淀更小并且含有更低的蛋白聚糖。与此一致的是，RNA 测序揭示了与软骨 ECM 形成相关的基因显着下调，从而扰乱了软骨形成。相反，沉默对成骨过程中基因表达谱的影响可以忽略不计，这表明 SERPINA3 的作用是软骨细胞分化所特有的。对软骨形成的整体影响促使我们研究沉默对软骨形成的主要转录调节因子 SOX9 的影响。事实上，我们观察到 SOX9 蛋白水平在早期时间点显着降低，表明 SERPINA3 在调节 SOX9 表达和活性中发挥作用。总之，我们的数据支持 SERPINA3 在通过调节 SOX9 水平促进软骨形成中发挥非冗余作用。