Objective Osteoarthritis (OA) is a joint disease linked with pathologic cartilage calcification, caused by the deposition of calcium-containing crystals by chondrocytes. Despite its clinical significance, the precise mechanisms driving calcification remain elusive. This study aimed to identify crucial players in cartilage calcification, offering insights for future targeted interventions against OA. Methods Primary murine chondrocytes were stimulated with secondary calciprotein particles (CPP2) or left untreated (NT) for 6 h. Calcification was assessed by alizarin red staining. RNA was analyzed by Bulk RNA sequencing. Differentially expressed (DE) genes were identified (cutoff: abs(LogFC)>1 and adj.p-val < 0.05), and top 50 DE genes were cross-referenced with human OA datasets from previous studies (ie healthy vs OA cartilage, or undamaged vs damaged cartilage). RNA from NT and CPP2-stimulated primary human OA chondrocytes were used to validate genes by qPCR. Results CPP2 induced crystal formation by chondrocytes and significantly modulated 1466 genes. Out of the top 50 DE genes in CPP2, 27 were confirmed in published OA cartilage datasets. Of those genes, some are described in calcification and/or OA (Errfi1, Ngf, Inhba, Col9a1). Two additional ones (Rcan1, Tnfrsf12a) appear novel and interesting in the context of calcification and OA. We validated modulation of these six genes in calcifying human chondrocytes from 5 patients. Ultimately, we unveiled two distinct gene families modulated by CPP2: the first comprised cytoskeletal genes (Actb, Tpm1, Cfl1, Tagln2, Lmna), while the second encompassed extracellular matrix genes (Fmod, Sparc, Col9a1, Cnmd). Conclusion CPP2 modulates genes in chondrocytes that could represent new targets for therapeutic interventions in OA.

中文翻译：

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RNA 测序揭示了软骨钙化的关键因素：对骨关节炎发病机制的潜在影响


目的 骨关节炎 （OA） 是一种与病理性软骨钙化有关的关节疾病，由软骨细胞沉积含钙晶体引起。尽管具有临床意义，但驱动钙化的确切机制仍然难以捉摸。本研究旨在确定软骨钙化的关键参与者，为未来针对 OA 的针对性干预措施提供见解。方法 用次生钙蛋白颗粒 （CPP2） 刺激原代小鼠软骨细胞或不处理 （NT） 6 h。通过茜素红染色评估钙化。通过 Bulk RNA 测序分析 RNA。鉴定了差异表达 （DE） 基因 （截断值：abs（LogFC）>1 和 adj.p-val < 0.05），并将前 50 个 DE 基因与先前研究的人类 OA 数据集（即健康与 OA 软骨，或未受损与受损软骨）交叉引用。使用 NT 和 CPP2 刺激的原代人 OA 软骨细胞的 RNA 通过 qPCR 验证基因。结果 CPP2 诱导软骨细胞形成晶体并显著调节 1466 个基因。在 CPP2 的前 50 个 DE 基因中，有 27 个在已发表的 OA 软骨数据集中得到证实。在这些基因中，一些以钙化和/或 OA （Errfi1、Ngf、Inhba、Col9a1） 的形式描述。另外两个 （Rcan1， Tnfrsf12a） 在钙化和 OA 的背景下显得新颖有趣。我们验证了这 6 个基因在 5 例患者钙化人软骨细胞中的调节。最终，我们揭示了由 CPP2 调节的两个不同的基因家族：第一个包含细胞骨架基因 （Actb、Tpm1、Cfl1、Tagln2、Lmna），而第二个包含细胞外基质基因 （Fmod、Sparc、Col9a1、Cnmd）。结论 CPP2 调节软骨细胞中的基因，这些基因可能代表 OA 治疗干预的新靶点。