Abstract：
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 SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing foetal cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 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, SERPINA3 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 SERPINA3 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.
摘要：
丝氨酸蛋白酶抑制剂（serpin）是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）在早期发育过程中发生的凝结和分化为软骨细胞。这里,第一次，我们证明了一个Serpin，SERPINA3（基因名称SERPINA3，蛋白质也称为α-1抗胰凝乳蛋白酶），在软骨分化中起关键作用。我们观察到SERPINA3表达在体外软骨形成的早期时间点被显著诱导。我们检测了SERPINA3在人软骨发育中的表达，与全肢相比，在发育中胎儿软骨中SERPINA3的显着富集，与良好描述的软骨分化标志物相关。当SERPINA3使用siRNA沉默时，通过二甲基亚甲蓝测定法（DMMB）和藏红蛋白-O染色测定，软骨颗粒较小，含有较低的蛋白聚糖。与此一致，RNA测序揭示了与软骨ECM形成相关的基因的显着下调，扰乱了软骨形成。相反，SERPINA3沉默对成骨过程中基因表达谱的影响可忽略不计，表明SERPINA3的作用对软骨细胞分化具有特异性。对软骨形成的整体影响使我们研究了SERPINA3沉默对软骨形成的主转录调节因子的影响，SOX9的确,我们观察到SOX9蛋白水平在早期时间点显著降低,提示SERPINA3在调节SOX9表达和活性中的作用.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的非冗余作用.
