Expression Profiling and Functional Analysis of Candidate Col10a1 Regulators Identified by the TRAP Program

Abstract

Hypertrophic chondrocytes and their specific marker, the type X collagen gene (Col10a1), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse Col10a1 gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp Col10a1 cis-enhancer. However, the roles of these candidate TFs in Col10a1 expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp Col10a1 enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of Col10a1 expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, Tbx5 was downregulated upon Col10a1 upregulation, overexpression of Tbx5 decreased Col10a1 expression, and knock-down of Tbx5 increased Col10a1 expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of Col10a1. We further generated a stable Tbx5-overexpressing ATDC5 cell line and ColX-Tbx5 transgenic mice driven by Col10a1-specific enhancers and promoters. Tbx5 overexpression decreased Col10a1 expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate Col10a1 transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.

DLX5 promotes Col10a1 expression and chondrocyte hypertrophy and is involved in osteoarthritis progression

“…To investigate the underlying mechanism of Dlx5 's up-regulation of Col10a1 , we analyzed the binding sites within the 150-bp Col10a1 cis-enhancer using JASPAR software. 19 The result confirmed the existence of the DLX5 binding site as previously identified by the TRAP program. 19 Importantly, we demonstrate that such DLX5 binding site is functional as evidenced by the results of the dual-luciferase reporter and ChIP assays, as well as the results of the co-transfection studies.…”

“… 19 The result confirmed the existence of the DLX5 binding site as previously identified by the TRAP program. 19 Importantly, we demonstrate that such DLX5 binding site is functional as evidenced by the results of the dual-luciferase reporter and ChIP assays, as well as the results of the co-transfection studies. Not surprisingly, the functional DLX5 binding site is adjacent to the tandem-repeat RUNX2 binding sites within the Col10a1 enhancer as previously described.…”

“…This suggested that DLX5 may work with Runx2 together to drive the cell-specific Col10a1 expression. 19 , 25 Indeed, it has been reported that DLX5 and myocyte enhancer factor-2C (MEF2C) can directly bind to the 343-bp enhancer of Runx2 and play an important role in directing its expression to osteoblast lineage cells. 32 …”

“…For the past 2-to-3 decades, we and others have made significant progress toward understanding the type X collagen gene, especially murine Col10a1 gene regulation. These include the identification of multiple Col10a1 promoters and enhancer elements 19 as well as the characterization of many candidate Col10a1 regulators. 20 , 21 We have previously localized murine Col10a1 cis-enhancer to a 150-bp distal promoter and demonstrated that RUNX2 directly interacts with this enhancer and is required but not sufficient for hypertrophic chondrocyte-specific Col10a1 promoter activity in vivo .…”

RUNX2 stabilization by long non-coding RNAs contributes to hypertrophic changes in human chondrocytes

“…This study proved that COL10A1 is a direct target of RUNX2 during chondrogenesis and has multiple RUNX2 binding sites within the proximal promoters of humans, mice, and chicks. Since COL10A1 is a specific marker of chondrocyte hypertrophy 13 , 14 it has been shown that the regulation of COL10A1 expression by RUNX2 is a key regulatory axis of chondrocyte hypertrophy. Although the importance of RUNX2 in chondrocyte hypertrophy has been elucidated, studies on the regulation of RUNX2 activity during chondrocyte hypertrophy are lacking.…”