骨关节炎软骨中 GEM 的表达下调通过 Wnt/β-catenin 信号通路调节软骨分化。
Decreased expression of GEM in osteoarthritis cartilage regulates chondrogenic differentiation via Wnt/β-catenin signaling.
机构信息
Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China.
Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangzhou, 510515, Guangdong, China.
出版信息
J Orthop Surg Res. 2023 Oct 4;18(1):751. doi: 10.1186/s13018-023-04236-z.
BACKGROUND
GEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored.
METHODS
Cartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis.
RESULTS
A decreased expression of GEM in OA patients' cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total β-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells.
CONCLUSION
These findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/β-catenin signaling.
背景
GEM（骨骼肌中过表达的 GTP 结合蛋白）是最近被鉴定为细胞分化调节剂的非典型小 GTPase 亚家族成员之一。异常的软骨形成伴随着软骨基质形成的周转率失衡与骨关节炎（OA）的发生和进展高度相关。然而，GEM 如何调节软骨细胞分化仍未被探索。
方法
从 OA 患者中获取软骨组织，并根据 ORASI 和 ICRS 分级系统进行分级。在模拟 OA 的培养体系中，检测到与 Grade 0 相比，Grade 4 软骨中 GEM 的表达发生改变，并对其进行了验证。接下来，为了研究 GEM 在这些过程中的特定功能，我们通过转染靶向 Gem 的 siRNA 构建了 Gem 敲低（Gem-Kd）系统，并在 ATDC5 细胞中进行了检测。在诱导过程中检测 Gem-Kd ATDC5 细胞的 Acan、Col2a1、Sox9 和 Wnt 靶基因。通过实时 PCR 和免疫印迹分析验证 Wnt 信号通路。最后，通过用 Wnt 信号激动剂处理 Gem-KD ATDC5 细胞来建立挽救模型，以验证通过 RNA 测序分析确定的机制。
结果
在 OA 患者的软骨组织和模拟 OA 的软骨细胞中观察到 GEM 的表达降低。而在软骨细胞分化和软骨基质形成过程中，GEM 的表达增加。Gem 沉默抑制了软骨细胞分化以及 Acan、Col2a1 和 Sox9 的表达。RNA 测序分析显示，Gem-Kd 细胞中的 Wnt 信号通路下调。观察到 Wnt 信号相关基因的表达下调以及核和细胞质中总 β-CATENIN 的表达下调。外源性 Wnt 激活对 Gem 功能丧失细胞表现出相反的作用。
结论
这些发现共同验证了 GEM 通过 Wnt/β-catenin 信号作为一种新型调节因子，调节软骨细胞分化和软骨基质形成。
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