Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.
