Erk1/2 signaling is required for Tgf‐β2–induced suture closure

Abstract

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.

TGF-β Family Signaling in Connective Tissue and Skeletal Diseases

“…TGF-b1 and TGF-b2 are expressed in sutures undergoing fusion, whereas TGF-b3 expression is restricted to patent sutures, possibly suggesting that TGF-b1 and TGF-b2 promote, whereas TGF-b3 prevents, closure (Opperman et al 1997;Most et al 1998;Chong et al 2003). In accordance with these observations, TGF-b2 has been shown to induce suture closure in an Erk1 and Erk2 MAPK-dependent manner in an ex vivo murine calvaria model (Opperman et al 2006). In addition to their role in calvaria fusion, BMP and TGF-b signaling are also essential for development and fusion of the palatal shelves (Bush and Jiang 2012).…”

Ephrin‐B stimulation of calvarial bone formation

“…Thus, the unclustered protein acts as a competitive inhibitor of Eph signaling. We tested the ability of clustered ephrin-B2/Fc to stimulate bone growth in ex vivo cultured embryonic mouse calvariae (Opperman et al, 2006). After 5 days of culture, ephrin-B2 treated skulls had double the bone volume of those treated with IgG-Fc control protein ( Fig.…”

“…Calvariae (containing the frontal, parietal, and part of the occipital bones) were harvested from day-17.5 CD-1 mouse embryos and cultured as previously described (Opperman et al, 2006), except that the tissues were cultured for 5 days in alpha-MEM with 10% fetal bovine serum. The medium was changed on day 3.…”

Insights into the development of molecular therapies for craniosynostosis

“…33 The TGFb2 molecule seems to promote suture fusion, whereas TGFb3 promotes patency. 31 Capitalizing on the therapeutic potential of TGFb, this group demonstrated that application of antibodies to TGFb2 prevented normal molecular signaling and resulted in suture patency. 30,32 Other studies have revealed that treatment of suturectomy sites in rabbits with anti-TGFb2 antibodies can maintain suture patency.…”