BMP Signaling Regulates Cranial Suture Fusion Caused by Retinoic Acid

Objectives: Craniosynostosis, premature and pathological fusion of cranial sutures, is a common developmental disorder. Excess exogenous retinoic acid has been documented to have teratogenic effects on craniofacial skeleton including craniosynostosis. In the present study, we examined the in vitro and in vivo effects of all-trans retinoic acid(atRA) on the development of postero-frontal suture and probed the potential mechanisms of this teratogen on suture development.
Methods: atRA was injected in postero-frontal suture subcutaneously to induce craniosynostosis. Suture morphology was analyzed by microCT and HE staining. Suture-derived mesenchymal cells isolated from the postero-frontal suture were grown in osteogenic medium with or without atRA. CCK-8 assay, alkaline phosphatase activities assay and alizarin red staining was performed to evaluate biological effects of atRA on these cells. Markers associated with osteogenic differentiation and BMP signaling were assayed by RT-PCR and WB. Ad-BMPR-2 was used to block BMP signaling in vivo and in vitro. All quantitative data were analyzed by Student’s t-test or one-way ANOVA by Graphpad Prism(P<0.05).
Results: Postero-frontal suture fused prematurely in atRA subcutaneous injection mice model. atRA was found to repress the proliferation of suture-derived mesenchymal cells but enhanced osteogenic differentiation of these cells, which were confirmed by enhancing ALP activities on day 7 and 14, alizarin red staining on day 21. BMPR-2, BMP4 and Smad9 mRNA levels and osteoblastic gene expression increased significantly in cells cultured in osteogenic differentiation medium with atRA, as well as the protein level of BMPR-2 and pSmad1/5/9. Blocking of the BMPR-2 expression using adenovirus shRNA failed to rescue the enhancement of atRA on osteogenesis either in vitro or in vivo.
Conclusions: Our results demonstrated that atRA induced craniosynostosis in mice by enhancing osteogenic differentiation of cranial suture-derived mesenchymal cells through partially regulating BMP signaling pathway.