miR-214 is known to play a role in mammalian skeletal development through inhibition of osteogenesis and stimulation of osteoclastogenesis, but data regarding other vertebrates, as well as a possible role in chondrogenesis, remain unknown. Here, we show that miR-214 expression is detected in bone and cartilage of zebrafish skeleton, and is downregulated during murine ATDC5 chondrocyte differentiation. Additionally, we observed a conservation of the transcriptional regulation of miR-214 primary transcript Dnm3os in vertebrates, being regulated by Ets1 in ATDC5 chondrogenic cells. Moreover, overexpression of miR-214 in vitro and in vivo mitigated chondrocyte differentiation probably by targeting activating transcription factor 4 (Atf4). Indeed, miR-214 overexpression in vivo hampered cranial cartilage formation of zebrafish and coincided with downregulation of atf4 and of the key chondrogenic players sox9 and col2a1. We show that miR-214 overexpression exerts a negative role in chondrogenesis by impacting on chondrocyte differentiation possibly through conserved mechanisms.
Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short-and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85±0.40 versus 3.95± 0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems.
We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/ Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.
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