Ddx5 participates in regulation of Col10a1 expression and chondrocyte hypertrophic differentiation in vitro

Tiaotiao Han; Tianxiang Zhu; Huiqin Bian; Jinnan Chen; Yaojuan Lu; Junxia Gu; Tong-Chuan He; Longwei Qiao; Qiping Zheng

doi:10.62347/ZDBO3541

Ddx5 participates in regulation of Col10a1 expression and chondrocyte hypertrophic differentiation in vitro

Am J Transl Res. 2024 Apr 15;16(4):1454-1467. doi: 10.62347/ZDBO3541. eCollection 2024.

Authors

Tiaotiao Han^{1

2}, Tianxiang Zhu^{1

2}, Huiqin Bian^{1

2}, Jinnan Chen^{1

2}, Yaojuan Lu^{1

2

3}, Junxia Gu^{1

2}, Tong-Chuan He⁴, Longwei Qiao⁵, Qiping Zheng^{1

2

3

4}

Affiliations

¹ Department of Laboratory Medicine, School of Medicine, Jiangsu University Zhenjiang 212013, Jiangsu, China.
² Department of Hematological Laboratory Science, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University Zhenjiang 212013, Jiangsu, China.
³ Shenzhen Walgenron Bio-Pharm Co., Ltd. Shenzhen 518118, Guangdong, China.
⁴ The Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center Chicago, IL 60637, USA.
⁵ The Affiliated Suzhou Hospital of Nanjing Medical University Suzhou 215008, Jiangsu, China.

Abstract

Background and aims: The type X collagen gene (Col10a1), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. Col10a1 expression is known to be influenced by many regulators. In this study, we aim to investigate how DEAD-box helicase 5 (DDX5), a potential binding factor for Col10a1 enhancer, may play a role in Col10a1 expression and chondrocyte hypertrophic differentiation in vitro.

Methods: The potential binding factors of the 150-bp Col10a1 cis-enhancer were identified with the hTFtarget database. The expression of DDX5 and COL10A1 was detected by quantitative real-time PCR (qRT-PCR) and Western blot in chondrogenic ATDC5 and MCT cell models with or without Ddx5 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the Col10a1 enhancer. The effect and mechanism of DDX5 on chondrocyte differentiation and maturation was evaluated by alcian blue, alkaline phosphatase (ALP), and alizarin red staining in ATDC5 cell lines with stable knockdown of Ddx5.

Results: DDX5 was identified as a potential binding factor for the Col10a1 enhancer. The expression of DDX5 in hypertrophic chondrocytes was higher than that in proliferative chondrocytes. Knockdown of Ddx5 decreased, while overexpression of Ddx5 slightly increased COL10A1 expression. DDX5 promotes the enhancer activity of Col10a1 as demonstrated by dual-luciferase reporter assay, and the ChIP experiment suggests a direct interaction between DDX5 and the Col10a1 enhancer. Compared to the control (NC) group, we observed weaker alcian blue and ALP staining intensity in the Ddx5 knockdown group of ATDC5 cells cultured both for 7 and 14 days. Whereas weaker alizarin red staining intensity was only found in the Ddx5 knockdown group of cells cultured for 7 days. Meanwhile, knockdown of Ddx5 significantly reduced the level of runt-related transcription factor 2 (RUNX2) in related ATDC5 cells examined.

Conclusions: Our results suggest that DDX5 acts as a positive regulator for Col10a1 expression and may cooperate with RUNX2 together to control Col10a1 expression and promote the proliferation and maturation of chondrocytes.

Keywords: ATDC5 cells; Chondrocyte differentiation and hypertrophy; Col10a1; Ddx5; Runx2.