Bone Abstracts

Skeletogenesis is an intricate process controlled by numerous transcriptional factors, hormones and signalling pathways. Recently, microRNAs emerged as important players in skeletogenesis but, only few were identified and most of their targets remain unknown. Previous works showed that miR-199a-2/214 cluster is essential for skeletal development and that miR-214 inhibits bone formation in mammals. However, data regarding its skeletal role in other vertebrates is scarce and its role in chondrogenesis is still unknown. Here, we aimed to uncover the potential role of miR-214 in chondrogenesis, by analysing its transcriptional regulators and mechanisms of action, using an in vivo accepted model for vertebrate skeletogenesis, the zebrafish, and an in vitro chondrocyte-like cell model, the ATDC5 cell line.

First, we characterized miR-214 expression throughout zebrafish development, by in situ hybridization, and identified an association with skeletal formation, since miR-214 is particularly expressed in zebrafish mineralizing cartilaginous structures. Moreover, reporter gene assays and chromatin immunoprecipitation studies led us to conclude that both human and zebrafish miR-199a-2/214 promoters are active and similarly regulated in chondrocyte cells and that Ets1 regulates miR-214 transcription in ATDC5 cells. Importantly, overexpression of miR-214 in ATDC5 cells mitigated chondrocyte differentiation probably by targeting Atf4. Two key skeletal markers, Mgp and Osteocalcin, were simultaneously decreased upon miR-214 overexpression in ATDC5 cells, suggesting that mineralization, the late stage of chondrocyte differentiation, is compromised. Interestingly, like Osteocalcin, we show that Mgp transcription could be controlled by the complex Atf4-Runx2-Satb2. Our data indicates that miR-214 exerts a key role in skeletal development, not only by inhibiting osteogenesis but also by affecting chondrogenesis. In this process, we hypothesize that miR-214 affects the expression of proteins relevant for bone and cartilage formation (Mgp and Osteocalcin) by targeting a transcriptional activator of both molecules, Atf4.