MicroRNA-142-3p regulates TGF-β3-mediated region-dependent chondrogenesis by regulating ADAM9

Position-dependent chondrogenesis is regulated by processes that are both common to and differ among all limb types and limb skeletal elements. Despite intrinsic differences between wing and leg bud mesenchyme, the exact regulatory molecules and mechanisms involved in these processes have not been elucidated. Here, we show the limb type-specific role of TGF-β3 during chondrogenic differentiation of chick limb mesenchymal cells. Exposure of wing cells to TGF-β3 stimulated chondrogenic differentiation, whereas in leg bud mesenchymal cells, TGF-β3 induced apoptotic cell death via G2M arrest. Consistent with a limb type-specific effect of TGF-β3 on chondrogenic differentiation, we found different levels of miR-142-3p induction. Inhibition of miR-142-3p via PNA-based antisense oligonucleotides (ASOs) markedly promoted cell migration and precartilage condensation, while exogenous induction of miR-142-3p reduced cell survival and increased cell death. Overexpression of ADAM9 significantly reduced chondrogenic differentiation via downregulation of cell migration and cell survival and upregulation of apoptotic cell death. Limb type-specific expression levels of ADAM9 induced by TGF-β3 were observed. Collectively, this study demonstrates that differential induction of miR-142-3p is involved in the limb type-specific effect of TGF-β3 on wing vs. leg mesenchymal cells through direct modulation of ADAM9 transcription.