Functional role of TGF-ß2/3 and FGF2 cross-talk in palate development.

Abstract

Introduction: The cross-talk between transforming growth factor-beta (TGF-β)2/3 and fibroblast growth factor (FGF)2 during secondary palatal fusion is not fully understood. This study aimed to elucidate the details of cross-talk signaling of TGF-β2/3 and FGF2 during secondary palatal fusion using the small interfering RNA (siRNA) technique.

Methods: Secondary palates of embryonic day 13 ICR mice were harvested, and the palates were cultured in the medium containing control siRNA, siTGF-β2, siTGF-β3, and siFGF2 (single or double knockdown). The expression levels of TGF-β2, TGF-β3, FGF2, and downstream genes were observed by western blotting and real-time RT-PCR.

Results: Transfection of siRNA targeting the target gene resulted in a statistically significant decrease in expression levels. When the expression of either TGF-β3 or FGF2 was suppressed, a tendency for the expression of the other to increase was observed. Although Smad2 phosphorylation was not affected by siFGF2 transfection, siFGF2 treatment reduced MAPK phosphorylation levels of ERK, p38, and JNK. Following siFGF2 treatment, TGF-β3 expression increased, and suppression of its expression by siTGF-β3 also significantly increased FGF2 expression levels. At E13 + 72 h of culture, the siFGF2 treatment resulted in incomplete palatal fusion. In the TGF-β2/FGF2 and TGF-β3/FGF2 double knockdown groups at the stage, fusion defects were observed, particularly in the anterior region of the secondary palate. In the double knockdown of TGF-β/FGF2, the downstream gene expressions were also reduced.

Conclusion: The results suggests that the TGF-β3 and FGF2 genes interact as a result of crosstalk, complement each other, and influence signal transduction to downstream genes.