participates in regulation of expression and chondrocyte hypertrophic differentiation .

Background And Aims: The type X collagen gene (), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. 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 enhancer, may play a role in expression and chondrocyte hypertrophic differentiation .

Methods: The potential binding factors of the 150-bp 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 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the 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 .

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