USP36 regulates the proliferation, survival, and differentiation of hFOB1.19 osteoblast

Abstract

BackgroundEffective 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.MethodsIn 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.ResultsUSP36 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.ConclusionsThese 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.