Transforming growth factors ␤ (Tgf-␤s) act by means of Smad signaling pathways and may also interact with the mitogen-activated protein kinase pathway. The hypothesis was tested that Erk1/2 signaling is required for Tgf-␤2-induced suture closure, by culturing embryonic mouse calvariae in the presence of Tgf-␤2 with or without Erk1/2 inhibitor PD98059 (PD). Suture widths were measured daily, and microdissected sutures and bones were homogenized and protein analyzed by Western blots. Tgf-␤2 induced narrowing of the sutures after 72 hr, an effect inhibited by treatment with PD. Erk1/2 and Egf but not Smad2/3 protein expression was up-regulated by Tgf-␤2 calvarial tissues at 72 hr. PD inhibited endogenous and Tgf-␤2-stimulated Erk1/2 protein as well as Tgf-␤2-stimulated Egf, but increased Smad2/3 protein expression. In tissues harvested 0, 15, and 30 min after exposure to Tgf-␤2, Erk1/2 phosphorylation was up-regulated after 15 min, an effect abrogated by the simultaneous addition of PD. In summary, Tgf-␤2 stimulated Erk1/2 phosphorylation and induced Egf and Erk1/2 expression, associated with suture closure after 72 hr. Blocking Erk1/2 activity with PD inhibited these effects but increased Smad2/3 expression. We postulate that Tgf-␤2 regulates suture closure directly by means of phosphorylation of Erk1/2 and indirectly by up-regulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration.

No abstract

Introduction: Ephrin-B2 on osteoclasts was reported to promote bone formation as part of homeostasis by activating the EphB4 tyrosine kinase receptor on osteoblasts. Little is known about the role of ephrin-B signaling to EphBs in developmental bone formation. Results: We observed expression of an ephrin-B2 LacZ chimeric allele in the periosteum, sutural bone fronts, and dura mater of embryonic and neonatal mice. Expression in the adult skull was confined to sutures, but was heavily upregulated at sites of bone injury. Culture of embryonic calvariae with soluble recombinant ephrin-B2/Fc doubled their bone content without altering suture width or overall skull morphology. Ephrin-B2/Fc also stimulated osteoblast marker gene expression in cultured MC3T3 preosteoblastic cells without the need for type 1 collageninduced differentiation. EphB4 was absent in embryonic and adult skulls. However, EphB1 and EphB2, both physiological receptors for ephrin-Bs, were expressed at sites of osteogenesis, and EphB1 knockout mice displayed a reduction in calvarial bone content compared to controls. Conclusions: These data support a role for ephrin-B2 in the development and healing of bone through activation of osteoblastspecific gene expression. EphB1 and EphB2 are likely candidates receptors for the ephrin-B2 in bone.

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.

The Eph receptor tyrosine kinases and their ligands, the ephrins, control boundary formation during cranial suture development. They also regulate mineral homeostasis in the long bones. We hypothesized that ephrins regulate bone growth in the calvarium separate from pattern formation, and that exogenous activation of Eph signaling can be used to promote bone synthesis. We found strong expression of beta‐galactosidase in the periosteum and the dura mater of late embryonic ephrin‐B2/LacZ and EphB2/LacZ mice (in which a lacZ reporter gene was knocked in to the ephrin‐B2 or EphB2 locus, respectively) after suture patterning was complete, but during the period of vigorous bone growth. EphB2 was particularly concentrated in the osteogenic fronts of the frontal bones. Treatment of e17.5 mouse calvariae for five days with recombinant ephrin‐B2 ectodomain fused to human Fc dramatically increased bone volume without changing bone density or interfrontal/sagittal suture widths. We conclude that Eph signaling stimulates bone synthesis apart from its role in cranial patterning. Thus, recombinant ephrin may be useful for the repair of cranial defects. Ongoing studies are concentrating on the mechanism of ephrin action in osteoblast differentiation and gene expression.