Retinoic acid enhances osteogenesis in cranial suture-derived mesenchymal cells: potential mechanisms of retinoid-induced craniosynostosis.
BACKGROUND
In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion.


METHODS
Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed.


RESULTS
Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands.


CONCLUSIONS
Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.
James
Aaron W
AW
Stanford and San Francisco, Calif. From the Hagey Pediatric Regenerative Research Laboratory, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, and the University of California, San Francisco, School of Medicine.
Levi
Benjamin
B
Xu
Yue
Y
Carre
Antoine L
AL
Longaker
Michael T
MT
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003393:Cranial Sutures / Q000166:cytology*; D003398:Craniosynostoses / Q000196:embryology*; D015870:Gene Expression; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects*; D012176:Retinoids / Q000009:adverse effects*; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology*
D019485:Bone Morphogenetic Proteins; D012176:Retinoids; D014212:Tretinoin; D000469:Alkaline Phosphatase