Abstract
The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFβ1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFβ1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFβ1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFβ1 signaling.
Highlights
The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types
Expression of Foxo1 started to increase on day 4 in the same manner as sexdetermining region Y box 9 (Sox9) over the course of chondrogenic differentiation, whereas expression of Foxo3 and Foxo4 was nearly unchanged (Fig. 1B)
We revealed the effects of FOXOs on chondrogenic differentiation
Summary
We confirmed the gene expression patterns of Sox, Col2a1, and Acan (as chondrogenic differentiation markers) and collagen type X ␣1 (Col10a1) (as a hypertrophic differentiation marker) during chondrogenic differentiation of ATDC5 cells, an in vitro model of chondrogenic differentiation [35, 36]. Gene expression of Sox, Col2a1, and Acan on day 5 were significantly decreased by AS1842856 treatment (Fig. 3F). TGF1 treatment continued to promote FOXO1 protein expression up to 21 days, whereas the absence of TGF1 did not increase FOXO1 at any point in time (Fig. 5K) These results indicate that TGF1/SMAD signaling is essential for promoting the expression and activity of FOXO1 during chondrogenic differentiation. To investigate the effects of FOXO1 on the gene expression of chondrogenic differentiation markers following TGF1 treatment, ATDC5 cells transfected with siRNA targeting Foxo or treated with AS1842856 were stimulated with TGF1. Expression of Col10a1 decreased on days 7 and 14 (Fig. 8F) These results indicate that p21 is necessary for expression of COL2 and ACAN during chondrogenic differentiation in ATDC5
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