RNA测序揭示软骨钙化的关键因素：对骨关节炎发病机制的潜在影响

RNA sequencing uncovers key players of cartilage calcification: potential implications for osteoarthritis pathogenesis.

作者信息

Bernabei Ilaria, Faure Elodie, Wegrzyn Julien, Bertheaume Nicolas, Falgayrac Guillaume, Hugle Thomas, Nasi Sonia, Busso Nathalie

机构信息

Service of Rheumatology, Department of Musculoskeletal Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

Service of Orthopedics, Department of Musculoskeletal Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

出版信息

Rheumatology (Oxford). 2025 May 1;64(5):3151-3159. doi: 10.1093/rheumatology/keae587.

DOI:10.1093/rheumatology/keae587

PMID:39432678

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12048056/

Abstract

OBJECTIVE

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 analysed by Bulk RNA sequencing. Differentially expressed (DE) genes were identified [cutoff: abs(LogFC)>1 and adjusted P-value < 0.05], and top 50 DE genes were cross-referenced with human OA datasets from previous studies (i.e. 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 five 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.

摘要

目的

骨关节炎（OA）是一种与病理性软骨钙化相关的关节疾病，由软骨细胞沉积含钙晶体所致。尽管其具有临床意义，但驱动钙化的确切机制仍不清楚。本研究旨在确定软骨钙化中的关键因素，为未来针对OA的靶向干预提供见解。

方法

用继发性钙蛋白颗粒（CPP2）刺激原代小鼠软骨细胞6小时，或不进行处理（NT）。通过茜素红染色评估钙化情况。通过批量RNA测序分析RNA。鉴定差异表达（DE）基因[截断值：|LogFC|>1且校正P值<0.05]，并将前50个DE基因与先前研究中的人类OA数据集（即健康软骨与OA软骨，或未受损软骨与受损软骨）进行交叉参照。用来自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刺激的前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，有些在钙化和/或OA中已有描述（Errfi1、Ngf、Inhba、Col9a1）。另外两个基因（Rcan1、Tnfrsf12a）在钙化和OA背景下显得新颖且有趣。我们在来自5名患者的钙化人软骨细胞中验证了这6个基因的调节情况。最终，我们揭示了受CPP2调节的两个不同基因家族：第一个家族包括细胞骨架基因（Actb、Tpm1、Cfl1、Tagln2、Lmna），而第二个家族包括细胞外基质基因（Fmod、Sparc、Col9a1、Cnmd）。