Stage-Specific Secretion of HMGB1 in Cartilage Regulates Endochondral Ossification

Abstract

High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1^−/− mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1^−/− mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.

SUPPLEMENTAL MATERIAL

We are grateful to Yasuhiko Kawakami and Thiennu Vu for helpful discussions; Kim Henriksen, Noriyuki Namba, and Chisa Shukunami for technical advice; Yasuhiko Yamamoto and Hiroshi Yamamoto for providing Rage^−/− mice; and Shingo Yamada for providing the neutralizing anti-HMGB1 IgY and ELISA kit. We especially thank Lilo Creighton for her excellent immunohistochemical technique.

This study was supported by grants from the NIH (AR47360, AR50631, and AG07996), NIBI (ID05-24), Arthritis Foundation, JST SORST, Genome Network Project (MEXT), and Grants-in Aid for Scientific Research (MEXT) (H.A.) and a postdoctoral fellowship of the Japan Research Foundation for Clinical Pharmacology and Research Grant for Rheumatology Disease, Japan Rheumatism Foundation (N.T.).