Proteinase expression during differentiation of human osteoclasts in vitro

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acid ic extracellular space. This differentiation includes expression of protein ases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high l evels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate -resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied us ing human apheresis macrophages cocultured with MG63 osteosarcoma cells, wh ich produce cytokines including RANKL and CSF-1 that mediate efficient oste oclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsi n K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusi on or bone attachment, and TRAP activity reached much higher levels in oste oclasts attached to bone fragments. Two proteinases that occur in the precu rsor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, a nd an unrelated lysosomal aspartate proteinase, cathepsin D, were also stud ied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two-to threefold in parallel with cat hepsin K. Cathepsin D activity did not change with differentiation, and sec reted activity was not significant. In situ acid transport measurements sho wed increased acid accumulation after 7 days either in cells on osteosarcom a matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attac hment. However, major increases in acid secretion and proteinases expressio n continue during cell fusion and bone attachment from 7 to 14 days. J. Cel l. Biochem. 78:627-637, 2000. (C) 2000 Wiley-Liss, Inc.