Tgf-ß2 regulates suture patency via phosphorylation of Erk1/2

Objectives: Premature suture obliteration is associated with disruptions of the Fgf signaling pathway due to mutations in Fgf receptors (Fgfr). Blocking the Erk1/2 signaling pathway downstream of Fgfr rescues sutures from obliteration. Tgf-β2 is known to induce premature suture fusion both in vitro and in vivo. Tgf-ßs act via Smad signaling pathways, but may also interact with the Erk1/2 pathway. To test the hypothesis that Erk1/2 signaling is required for Tgf-ß2-induced suture closure, embryonic mouse calvariae were cultured in the presence of Tgf-ß2 with or without Erk1/2 inhibitor PD98059 (PD). Methods: Fetal day 17.5 mouse calvaria were cultured for 3 days in the presence of 0 and 3 ng/ml Tgf-ß2 and 0 and 50 µM PD98059. Calvariae were photographed daily and the width of the posterior frontal sutures (PFS) measured using Metamorph® software. In a second set of experiments, calvariae were treated as before, and harvested at 0, 15 and 30 minutes after addition of Tgf-ß2, and prepared for western blotting. Results: Tgf-ß2 induced narrowing of the sutures after 72 hours, an effect inhibited by treatment with PD. Erk1/2 but not Smad2/3 protein was upregulated by Tgf-ß2 in calvarial tissues at 72 hours. PD inhibited endogenous and Tgf-ß2 stimulated Erk1/2 protein, but potentiated Smad2/3 protein expression. Tgf-ß2 induced Erk1/2 phosphorylation after 15 minutes, an effect abrogated by the addition of PD. Summary and Conclusion: Tgf-ß2 stimulated Erk1/2 phosphorylation, and induced Erk1/2 expression, associated with suture closure after 72 hours. Blocking Erk1/2 activity with PD inhibited these effects, but potentiated Smad2/3 expression. We postulate that Tgf-ß2 regulates suture closure directly via phosphorylation of Erk1/2, and indirectly by upregulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration.