The ROLE of RANK-RANKL-OPG AXIS IN Cranial Suture Homeostasis

Background: Craniosynostosis is a significant disorder affecting 1 in 2500 live births worldwide. While a large body of work has focused on dural regulation and the contributions of molecular mediators such as FGF, BMP, and TGFβ, minimal attention has been directed towards osteoclast function in cranial suture biology. Receptor activator of NF-κB (RANK) is an essential mediator of osteoclastogenesis and osteoclast activation. We now report differential expression of RANK in fused and patent cranial sutures as well as the necessity of RANK expression for maintenance of suture patency. Methods: Murine posterior frontal, coronal, and sagittal sutures were harvested at 2 weeks, 5 weeks, 7 weeks, and >12 weeks of age and analyzed for RANK mRNA using real time reverse transcriptase PCR and RANK protein with immunohistochemistry. Human suture samples from four nonsyndromic craniosynostotic patients undergoing cranial vault reconstruction were obtained and analyzed for RANK and RANKL expression via immunohistochemistry. Murine calvarial organ cultures and suture strip cultures were infected with adenoviral siRANK and suture densities were analyzed using serial micro-CT scans at 0, 1, 2, 3 and 4 weeks post infection. Results: Physiologic fusion of posterior frontal sutures in murine development correlated with decreasing protein expression of RANK in immunohistochemistry in comparison to age-matched coronal and sagittal sutures. However, RANK mRNA did not exhibit a similar pattern suggesting that RANK is post-translationally regulated. Fused cranial sutures in nonsyndromic craniosynostotic children also showed decreased levels of RANK staining in immunohistochemistry in comparison to patent sutures. Immunohistochemistry with a RANKL antibody did not show differences in fused or patent sutures. Moreover, siRANK infected calvarial and strip suture cultures displayed increased bone density specifically in the suture line following infection. Conclusions: Cranial suture biology, similar to bone biology in general, likely depends on a complex interplay between osteoblasts and osteoclasts. In this study, the temporospatial correlation between RANK expression and suture morphology suggests that osteoclast activity is important in maintenance of cranial suture patency in normal physiology and disease. Furthermore, RANK downregulation promoted suture fusion establishing a causal relationship between the presence of RANK and patency. Future studies focusing on upstream regulatory signaling molecules and RANK protein stability are required.