Abstract
The majority of human skeleton develops through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and enlarge into hypertrophic chondrocytes that eventually undergo apoptosis and are replaced by bone. Although at a terminal differentiation stage, hypertrophic chondrocytes have been implicated as the principal engine of bone growth. Abnormal chondrocyte hypertrophy has been seen in many skeletal dysplasia and osteoarthritis. Meanwhile, as a specific marker of hypertrophic chondrocytes, the type X collagen gene (COL10A1) is also critical for endochondral bone formation, as mutation and altered COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases. However, how the type X collagen gene is regulated during chondrocyte hypertrophy has not been fully elucidated. We have recently demonstrated that Runx2 interaction with a 150-bp mouse Col10a1 cis-enhancer is required but not sufficient for its hypertrophic chondrocyte-specific reporter expression in transgenic mice, suggesting requirement of additional Col10a1 regulators. In this study, we report in silico sequence analysis of this 150-bp enhancer and identification of its multiple binding factors, including AP1, MEF2, NFAT, Runx1 and TBX5. Using this enhancer as bait, we performed yeast one-hybrid assay and identified multiple candidate Col10a1-interacting genes, including cyclooxygenase 1 (Cox-1) and Cox-2. We have also performed mass spectrometry analysis and detected EF1-alpha, Fus, GdF7 and Runx3 as components of the specific complex formed by the cis-enhancer and nuclear extracts from hypertrophic MCT (mouse chondrocytes immortalized with large T antigen) cells that express Col10a1 abundantly. Notably, some of the candidate genes are differentially expressed in hypertrophic MCT cells and have been associated with chondrocyte hypertrophy and Runx2, an indispensible Col10a1 regulator. Intriguingly, we detected high-level Cox-2 expression in hypertrophic MCT cells. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed the interaction between Cox-2 and Col10a1 cis-enhancer, supporting its role as a candidate Col10a1 regulator. Together, our data support a Cox-2-containing, Runx2-centered Col10a1 regulatory mechanism, during chondrocyte hypertrophic differentiation.
Highlights
The type X collagen gene (COL10A1) is expressed by hypertrophic chondrocytes
Mutations and abnormal expression of COL10A1 are closely linked to abnormal chondrocyte hypertrophy that has been seen in multiple skeletal dysplasia and osteoarthritis.[6–15]
Schmid metaphyseal chondrodysplasia (SMCD) is an autosomal dominantly inherited skeletal disorder caused by human COL10A1 mutation
Summary
The type X collagen gene (COL10A1) is expressed by hypertrophic chondrocytes. Extensive studies have identified multiple TFs or signaling pathways that contribute to type X collagen gene regulation. These include TFs AP1, carbonic anhydrase (CA) IX, GADD45beta, HIF-2a, MEF-2C, PTH/ PTHrP, SOX9, SP3/SP1 and the Bmp/Ihh/Wnt signaling pathways.[13,27–35]. Most of these factors interact with the proximal promoter of type X collagen gene. We have been working on murine Col10a1 gene regulation and have demonstrated that Runx[2] contributes to hypertrophic chondrocyte-specific Col10a1/reporter expression in vivo through interaction with both the proximal promoter and the distal enhancer element (150-bp, − 4296 to − 4147).[36–38]. We report identification and characterization of these regulators by dissection of the 150-bp enhancer using comprehensive bioinformatics, proteomics and transgenic (TG) approaches
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have