Expression of typical osteoclast markers by PBMCs after PEG-induced fusion as a model for studying osteoclast differentiation

摘要

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin beta 3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.