Department of Orthopaedics, Xiamen Humanity Hospital, Xiamen, 361006, Fujian, China.
Department of ICU, Xiang'an Hospital of Xiamen University, Xiamen, 361100, Fujian, China.
J Orthop Surg Res. 2024 Aug 17;19(1):483. doi: 10.1186/s13018-024-04893-8.
Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive.
In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining.
USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells.
These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.
有效的骨形成依赖于成骨细胞分化，这是一个受复杂翻译后调控的过程。泛素特异性蛋白酶（USPs）抑制由泛素-蛋白酶体途径介导的蛋白质降解。已经有几种 USPs 被证明可以调节成骨细胞分化，但其他 USPs 是否参与这个过程仍然难以捉摸。
在这项研究中，我们对分化和未分化的 hFOB1.19 成骨细胞中的 48 种 USPs 进行了比较分析，确定了明显上调的 USPs。随后，我们生成了 USP 敲低的 hFOB1.19 细胞，并通过茜素红染色评估它们的成骨分化。我们还分别通过 MTT、7-氨基放线菌素 D 染色和 Annexin V/PI 染色测定细胞活力、细胞周期进程和细胞凋亡。定量 PCR 和 Western blot 用于测量成骨分化标记物的表达水平。此外，我们使用免疫共沉淀（co-IP）研究了 USP 与其靶蛋白之间的相互作用。此外，我们在 hFOB1.19 细胞中耗尽 USP，并用免疫沉淀（IP）和 Western blot 检测其对靶蛋白泛素化和稳定性的影响。最后，我们在 USP 缺陷的 hFOB1.19 细胞中转染靶蛋白，并通过茜素红染色评估其对成骨分化的影响。
USP36 是分化的 hFOB1.19 成骨细胞中上调最明显的 USP。USP36 敲低导致 hFOB1.19 细胞活力降低、细胞周期停滞、凋亡增加和成骨分化受损。USP36 与 WD 重复蛋白 5（WDR5）相互作用，USP36 敲低导致 WDR5 泛素化水平增加和 WDR5 降解加速。过量的 WDR5 改善了 USP36 缺陷的 hFOB1.19 细胞受损的成骨分化。
这些观察结果表明，USP36 可能是成骨细胞分化的关键调节因子，其调节机制可能与 WDR5 的稳定有关。
