Positive feedback regulation between USP15 and ERK2 inhibits osteoarthritis progression through TGF-\u03b2/SMAD2 signaling

Wenjuan Wang,Yanhui Zhu,Xiang Wang,Zhenyu Sun,Chen Jin

doi:10.1186/s13075-021-02456-4

Wenjuan Wang, Yanhui Zhu + Show 3 more

Open Access

https://doi.org/10.1186/s13075-021-02456-4

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Abstract

BackgroundThe transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). However, the specific mechanism of non-SMAD and classical SMAD signaling interactions is still unclear, which needs to be further explored.MethodsIn ATDC5 cells, USP15 overexpression and knockout were performed using the transfected lentivirus USP15 and Crispr/Cas9. Western blotting and immunofluorescence staining were used to test p-SMAD2 and cartilage phenotype-related molecular markers. In rat OA models, immunohistochemistry, hematoxylin and eosin (HE)/Safranin-O fast green staining, and histology were used to examine the regulatory activity of USP15 in TGF-β/SMAD2 signaling and the cartilage phenotype. Then, ERK2 overexpression and knockout were performed. The expressions of USP15, p-SMAD2, and the cartilage phenotype were evaluated in vitro and in vivo. To address whether USP15 is required for ERK2 and TGF-β/SMAD2 signaling, we performed rescue experiments in vitro and in vivo. Immunoprecipitation and deubiquitination assays were used to examine whether USP15 could bind to ERK2 and affect the deubiquitination of ERK2. Finally, whether USP15 regulates the level of p-ERK1/2 was evaluated by western blotting, immunofluorescence staining, and immunohistochemistry in vitro and in vivo.ResultsOur results indicated that USP15 stimulated TGF-β/SMAD2 signaling and the cartilage phenotype. Moreover, ERK2 required USP15 to influence TGF-β/SMAD2 signaling for regulating the cartilage phenotype in vivo and in vitro. And USP15 can form a complex with ERK2 to regulate ubiquitination of ERK2. Interestingly, USP15 did not regulate the stability of ERK2 but increased the level of p-ERK1/2 to further enhance the TGF-β/SMAD2 signaling pathway.ConclusionsTaken together, our study revealed positive feedback regulation between USP15 and ERK2, which played a critical role in TGF-β/SMAD2 signaling to inhibit OA progression. Therefore, this specific mechanism can guide the clinical treatment of OA.

Highlights

The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA)
Endogenous USP15 knocked out by Crispr/Cas9 in untreated and TGF-β1-treated ATDC5 cells decreased the level of phosphorylated SMAD2 (p-SMAD2) instead of decreasing SMAD2 and SMAD4 expression (Fig. 1c)
Immunofluorescence staining results further revealed that compared to ERK2, USP15 had more influence on maintaining the cartilage phenotype, such as increasing the expression of Col2a1 and Aggrecan; this was not the case for Col10a1 expression (Fig. 3b, c). These results confirmed that USP15 is required for ERK2 to influence the TGF-β/SMAD2 signaling for regulating the cartilage phenotype

Summary

Introduction

The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining tissue homeostasis through anabolic signaling for cartilage growth and repair [5]. The specific receptor-regulated (R)-SMAD proteins SMAD2 and SMAD3 form a complex with the coSMAD SMAD4 and are phosphorylated via the activated TGF-β type I receptor (TbRI). Knowledge of the ability of DUB USP15 to deubiquitinate and stabilize R-SMADs and TbRI has increased our understanding of TGF-β signaling activity in many diseases, such as glioblastoma and systemic sclerosis fibroblasts [8,9,10]; this knowledge has attracted our attention in terms of applying this to OA experiments

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