COUP-TFII induction by FGF2 priming leads to a reduction in osteodifferentiation potential.
(A, B) Pre-exposure to FGF2 prior to differentiation stimuli inhibits osteoblast differentiation of C3H10T1/2 cells. (A) Cells were treated with 10 ng/mL of FGF2 every other day for 4 days. After removing FGF2-containing media, cells were incubated with osteogenic media (OM) (50 μg/mL of ascorbic acid, 5 mM of β-glycerophosphate, and 100 ng/mL of BMP2). Cells were stained for alkaline phosphatase activity after 5 days of differentiation (left, ALP staining), and were subjected to alizarin red staining after 10 days of differentiation (right, AR staining). The bar graph shows the relative intensity of AR staining. Cells stained with AR were incubated in 10% cetylpyridinium chloride, and staining was quantified at 562 nm. The ratio of OM/GM was determined. (B) After cells were prepared as in panel A, cells were harvested on day 5 for COUP-TFII, ALP, and Osterix, and on day 10 after differentiation for BSP and osteocalcin (Oc). Total RNA was isolated and subjected to real-time RT-PCR. (C-F) Blocking of COUP-TFII induction abolished the anti-osteogenic effect of FGF2 priming. (C) After COUP-TFII–silenced cells were pretreated with FGF2 as in panel A, osteogenic differentiation was induced for 4 days. Alkaline phosphatase activity in the differentiated cells was analyzed by ALP staining. Control, non-FGF2 treated and control siRNAs-transfected cells; FGF2-primed control, FGF2-treated and control siRNA-transfected cell. (D) Osteogenic differentiated cells were harvested and subjected to real-time RT-PCR to analyze expression levels of Osterix (on day 4), ALP (on day 2), BSP and Oc (on day 10). The relative expression levels of COUP-TFII and Runx2 were analyzed on day 0 (that is, before the onset of differentiation). (E) Cells were pretreated with FGF2 as in panel A in the presence or absence of U0126, and the cells then underwent osteogenic differentiation for 4 days. Alkaline phosphatase activity was determined by ALP staining, and magnified images of the differentiated cells are representative of the relevant wells (left). (F) Before the onset of differentiation (day 0), the cells were harvested for analysis of COUP-TFII levels. The relative expression levels of ALP (on day 2), Osterix (on day 4), and Oc (on day 10) were determined. Representative data from three independent experiments are shown. Values for the relative expression of the indicated genes are expressed as the mean ± SEM of triplicate reactions in one representative experiment. Statistical analysis was performed by ANOVA followed by the Tukey post hoc test. * p<0.05; ** p<0.01; *** p<0.001. (G) Working model for the role of overexpressed COUP-TFII in the FGF2-primed mesenchymal cells. FGF2 priming in uncommitted mesenchymal cells induces COUP-TFII expression via the MEK1/2 pathway and it might bring about low osteogenic potential and high pluripotency.