Abstract 8

Identification and Functional Characterization of an Osteoclast-Derived Osteoblastic Factor (ODOF)

Tony C.A. Phan, Renzhi Han, Tamara Davey, Verity J. Smuts, Ming H Zheng, Jiake Xu*

Molecular Orthopaedic Laboratory, School of Surgery and Pathology, The University of Western Australia, Nedlands, WA, 6009, Australia

Bone is a living tissue and is maintained by the coordinate action of osteoblasts and osteoclasts. Intercellular communication between osteoblasts and osteoclasts is the quintessential mechanism in bone remodelling. Here we report the identification and functional characterization of an Osteoclast-Derived Osteoblastic Factor (ODOF), which is expressed by osteoclasts, binds specifically to osteoblasts and elevates cytosolic calcium ([Ca²⁺]i) resulting in the proliferation of osteoblastic cells. The ODOF gene was identified in RAW264.7 cell-derived osteoclasts utilising a PCR-selected subtractive hybridisation screening process. Further investigations using reverse transcriptase PCR, revealed that ODOF mRNA was up-regulated during RANKL-induced osteoclastogenesis but was not expressed in osteoblasts or osteoblast-like cells. Recombinant His-tagged ODOF was subsequently produced and labelled with ¹²⁵I to ascertain its binding profile. The protein exhibits highly specific binding to primary calvarial osteoblasts with a binding affinity of 1.7±0.4nM and 2.7x10⁴±306 receptors per cell but not with osteoclasts and their precursor cells. Functional studies demonstrated that ODOF stimulates an increase in the growth and proliferation of osteoblastic cells. In contrast, the protein did not promote osteoclastogenesis, osteoclast survival or bone resorption. Mechanistic analysis revealed that ODOF, alone, elevates intracellular [Ca²⁺]i through the PLC-IP3 induced depletion of calcium stores. Moreover, western blot analysis reveals that ODOF induces the activation of the PI3K-Akt and ERK pathways in osteoblasts. Taken together, our results provide evidence for a novel cross-talk mechanism between the osteoclast and osteoblast and indicate that osteoclasts play a role in regulating osteoblastic growth and proliferation.

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