Annexin A5 involvement in bone overgrowth at the enthesis

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5‐deficient (Anxa5–/–) mice, the sizes of bone ridge outgrowths at the entheses of the tibiae and femur were increased after 7 weeks of age. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP‐expressing cells were observed in the fibrocartilage layer in Anxa5–/– mice than in wild‐type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5–/– mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5–/– mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail‐suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5–/– and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of AnxaA5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. AnxaA5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in AnxaA5 KO mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators.