Egf receptor inhibitor sc-120 disrupts Tgf-ß2 activity in sutures

Objective: Tgf-ß2 induces Egf expression prior to induction of suture fusion. However, Egf alone does not induce suture closure. Pretreatment of calvariae with Egf blocks Tgf-ß2-induced suture closure, whereas post treatment has no effect on Tgf-ß2-induced suture closure. This study was done to test if Egf receptor signaling modulates Tgf-ß2 activity in sutures. Methods: Twenty E17.5 mice were sacrificed, their calveriae dissected and cultured for 5 days in 1) serum free medium (SFM) alone, 2) SFM plus 50mM sc-120 (sc-120), 3) SFM and 3 ng/ml Tgf-ß2 (Tgf-ß2), with sc-120 added at 48 hours, 4) SFM and sc-120, with Tgf-ß2 added at 48 hours, and 5) SFM with sc-120 and Tgf-ß2 with n=4 per group. Tissues were then harvested and prepared for histological sectioning of coronal sutures and H&E staining. Sutures were viewed under a microscope and the shortest distances between bone fronts across the suture were measured using Metamorph®. Data was stored in an Excel file and averages and standard deviations calculated within each group and between the groups. Results: No significant differences in suture width were found within or between the groups, demonstrating that blocking Egf receptor activity either before or after Tgf-ß2 treatment blocked Tgf-ß2-induced suture closure. Conclusion: Since Egf alone does not induce suture closure, it was not unexpected that sc-120 alone had no effect on suture width. That blocking Egf activity after Tgf-ß2 treatment abrogates suture obliteration indicates Tgf-ß2 requires Egf signaling to induce suture closure. An unexpected finding was that blocking Egf receptor activity prior to Tgf-ß2 treatment also blocked Tgf-ß2 induced suture closure. However, since sc-120 was present for the duration of culture, it is likely that the presence of sc-120 after Tgf-ß2 treatment was responsible for blocking the Tgf-ß2 effect on sutures.