Abstract
MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.
Highlights
Inhibition of Jun N-terminal kinase (JNK) Signaling Suppresses Precartilage Condensation and Chondrogenesis—Numerous protein kinases and pathways have been implicated in the chondrogenic process, including protein kinase A (PKA) [30], protein kinase C (PKC) [31], Rho kinase I and -II (ROCK I and -II) [32], and the Sma- and Mad-related protein (Smad) 15/8 [33] and -2/3 [34] pathways
We found that the JNK inhibitor-induced decreases in peanut agglutinin (PNA) and Alcian blue staining intensity were recovered in cells that had been co-treated with the miR-221 inhibitor (Fig. 3B)
The level of ubiquitinated Slug was increased (Fig. 4G, top), but no change was observed in the mRNA expression level of slug (Fig. 4G, bottom), indicating that the protein was increasingly tagged for proteasomal degradation. These findings suggest that the JNK inhibitor-mediated inhibition of Mdm2 via miR-221 suppresses the ubiquitination and subsequent degradation of Slug, leading to up-regulation of the Slug protein in these cells
Summary
To verify the effect of JNK signaling during chondrogenesis in our model system, we treated chick wing bud mesenchymal cells with a 5 M concentration of a JNK inhibitor and used PNA and Alcian blue staining to analyze precartilage condensation and chondrogenic differentiation, respectively. To examine the possible involvement of JNK signaling in the development, the highest induction of miR-221 was detected control of cell motility, we used a wound-healing assay to test in the limb buds of chick embryos at Hamburger-Hamilton the effects of the JNK inhibitor on the motility of chick wing stages 32 and 35 (Fig. 2C) and at later periods of culture
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