All-Trans-Retinoid Acid (ATRA) Activates Notch Signaling to Inhibit Hind Limb Chondrogenesis by Suppressing Differentiation of Chondrogenic Cells.

All-trans retinoic acid (ATRA) plays an important role in chondrogenesis, this study is performed to dissect the correlated factors of inhibiting chondrogenesis by ATRA in rat embryonic hind limb bud mesenchymal cells (rEHBMCs), and investigate the underlying mechanisms. In this study, the proliferation of rEHBMCs was tested by CCK8 assays. Cartilage formation was assessed by Alcian blue staining, HE staining and immunohistochemistry. RT-PCR and western blotting were performed to examine the effects of ATRA on cartilage-specific molecules, SOX9 and COL2A1, and the expression of Notch1 genes and Hes-1 of the Notch signaling pathway in rEHBMCs. It was found that the proliferation and differentiation of chondrocytes were delayed in limb cartilage of ATRA-treated embryos in utero. ATRA markedly inhibited the proliferation and differentiation of rEHBMCs, as well as reduced the cartilage nodules formation of rEHBMCs in vitro. Furthermore, ATRA decreased the mRNA levels of SOX9 and COL2A1, but contrary on Notch1 and Hes-1. Consistently, ATRA increased Notch1 protein expression but had reverse effect on SOX9 and COL2A1. Moreover, compared to rEHBMCs treated with ATRA alone, DAPT, the Notch inhibitor, reversed the downregulated expression of SOX9 and COL2A1, and increased chondrocyte differentiation. In conclusion, ATRA reduced chondrogenesis of rEHBMCs by downregulating SOX9 and COL2A1 via the Notch1-Hes-1 signaling pathway. [ABSTRACT FROM AUTHOR]