TRAF4 positively regulates the osteogenic differentiation of mesenchymal stem cells by acting as an E3 ubiquitin ligase to degrade Smurf2

Abstract

TNF receptor-associated factor 4 (TRAF4), a member of the TRAF family, plays an important role in the embryogenesis and development of the bone system. Mesenchymal stem cells (MSCs), which are the primary origin of osteoblasts in vivo, are key cells in bone development; however, whether TRAF4 modulates the osteogenic capacity of MSCs has never been explored. In this study, we demonstrated that TRAF4 positively regulates the osteogenic process of MSCs both in vitro and in vivo. In addition, we further demonstrated that TRAF4 modulates the osteogenic process of MSCs by acting as an E3 ubiquitin ligase to mediate the K48-linked ubiquitination of Smurf2 at the K119 site and cause degradation. Furthermore, TRAF4 was abnormally decreased in bone sections of ovariectomized rat and osteoporosis patients. Taken together, our findings suggest that TRAF4 positively regulates the osteogenic differentiation of MSCs by acting as an E3 ubiquitin ligase to degrade Smurf2. These results emphasize the critical role of TRAF4 in bone formation and could not only improve the clinical use of MSCs in tissue engineering but also clarify the pathogenesis of bone metabolism disorders.

Introduction

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes [1, 2]. Due to their strong potential in osteogenic differentiation capacity, MSCs are considered to be the most promising cell types used in tissue engineering technology for bone regeneration and repair [3,4,5]. However, the molecular mechanism that governs the osteogenic differentiation of MSCs remains largely unknown and hampers the further development of MSC-based cell therapies for bone defects in the clinic. Therefore, to efficiently harness MSCs for therapeutic purposes, it is necessary to understand the molecular mechanisms underlying MSC osteogenic differentiation.

TNF receptor-associated factor 4 (TRAF4) is a unique member of the TRAF family [6]. Previous in vivo studies have clearly demonstrated that TRAF4 is involved in embryogenesis and that TRAF4 deficiency results in severe malformation of the skeleton system, which indicates that TRAF4 is indispensable in bone development [7]. However, further studies illustrating the concrete role of TRAF4 in the process of bone remodeling at the molecular biological level have not been reported. Osteoblasts are among the most important cells in regulating the bone remodeling process in vivo [8, 9], and MSCs are the major origin of osteoblasts [10, 11]. However, whether TRAF4 modulates the osteogenic capacity of MSCs has never been explored.

Smad ubiquitination-related factor 2 (Smurf2) is a member of the Hect domain family of E3 ubiquitin ligases [12, 13], and it interacts with and degrades various essential osteogenesis-related molecules including Smad1 and Runx2 to negatively regulate the osteogenic differentiation process [12, 14, 15]. Although the mechanism of Smurf2 in modulating the osteogenic differentiation process has been widely studied, the upper regulatory network to modulate the expression of Smurf2 during the osteogenic differentiation process of MSCs remains unclear.

Taken together, our findings suggest that TRAF4 positively regulates the osteogenic process of MSCs both in vitro and in vivo, can be a promising target not only to improve the efficiency of MSC-based tissue engineering therapy for bone regeneration and repair but also to treat the pathogenesis of bone metabolism disorders, such as osteoporosis.

Methods and materials

Isolation and culture of MSCs

This study was approved by the ethics committee of Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. Written informed consent was obtained from all subjects included in the study. MSCs were isolated and cultured as previously described [

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Acknowledgements

This study was financially supported by grants from the National Natural Science Foundation of China (81672097 and 31700884) and the Science and Technology Project of Guangdong Province (2015B020228001).

Author information

1. These authors contributed equally: **teng Li, Peng Wang, Zhongyu **e

Authors and Affiliations

1. Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-Sen University, Shenzhen, P.R. China

   **teng Li, Peng Wang, Zhongyu **e, Mengjun Ma & Huiyong Shen

2. Department of Orthopedics, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, P.R. China

   **teng Li, Peng Wang, Zhongyu **e, Shuizhong Cen, Ming Li, Wenjie Liu, Su’an Tang, Guiwen Ye, Guan Zheng, Mengjun Ma & Huiyong Shen

3. Center for Biotherapy, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, P.R. China

   Shan Wang, Hongjun Su, **aohua Wu & Yanfeng Wu

Contributions

JL, PW, ZX, SW, and SC performed the experiments. ML, WL, and ST collected the human samples. HS, MM, and XW isolated and cultured the MSCs. JL, ZX, GY, and GZ wrote the initial draft of the paper. HS, YW, PW, JL, and ZX contributed to the conceptualization and experimental design of the study and edited the paper.

Corresponding authors

Correspondence to Yanfeng Wu or Huiyong Shen.

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Li, J., Wang, P., **e, Z. et al. TRAF4 positively regulates the osteogenic differentiation of mesenchymal stem cells by acting as an E3 ubiquitin ligase to degrade Smurf2. Cell Death Differ 26, 2652–2666 (2019). https://doi.org/10.1038/s41418-019-0328-3

• Received: 04 October 2018

• Revised: 13 March 2019

• Accepted: 22 March 2019

• Published: 10 May 2019

• Issue Date: December 2019

• DOI: https://doi.org/10.1038/s41418-019-0328-3

• Springer Nature Limited

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