背景:有效的骨形成依赖于成骨细胞的分化,受复杂的翻译后调节的过程。泛素特异性蛋白酶(USP)抑制泛素-蛋白酶体途径介导的蛋白质降解。一些USP已经被证明可以调节成骨细胞的分化,但是其他USP是否参与此过程仍然难以捉摸。
方法:在本研究中,我们对分化和未分化的hFOB1.19成骨细胞中的48个USP进行了比较分析,确定显著上调的USP。随后,我们产生了USP敲低hFOB1.19细胞,并使用茜素红染色评估了它们的成骨分化。我们还评估了细胞活力,细胞周期进程,通过MTT和细胞凋亡,7-氨基放线菌素D染色,和膜联蛋白V/PI染色测定,分别。定量PCR和Western印迹用于测量成骨分化标志物的表达水平。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细胞中清除USP,以使用免疫沉淀(IP)和Western印迹检测其对靶蛋白泛素化和稳定性的影响.最后,我们在USP缺陷型hFOB1.19细胞中过表达靶蛋白,并使用茜素红染色评估其对成骨分化的影响.
结果:USP36是分化的hFOB1.19成骨细胞中最显著上调的USP。USP36的击倒导致生存能力降低,细胞周期停滞,细胞凋亡加剧,hFOB1.19细胞成骨分化受损。USP36与含WD重复序列的蛋白5(WDR5)相互作用,USP36的敲低导致WDR5泛素化水平升高并加速WDR5的降解。过量WDR5改善USP36缺陷型hFOB1.19细胞成骨分化受损。
结论:这些观察表明USP36可能是成骨细胞分化的关键调节因子,其调控机制可能与WDR5的稳定有关。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.