Abstract
ABSTRACTOsteoclasts are responsible for bone destruction in degenerative, inflammatory and metastatic bone disorders. Although osteoclastogenesis has been well-characterized in mouse models, many questions remain regarding the regulation of osteoclast formation in human diseases. We examined the regulation of human precursors induced to differentiate and fuse into multinucleated osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL). High-content single cell microscopy enabled the time-resolved quantification of both the population of monocytic precursors and the emerging osteoclasts. We observed that prior to induction of osteoclast fusion, RANKL stimulated precursor proliferation, acting in part through an autocrine mediator. Cytokines secreted during osteoclastogenesis were resolved using multiplexed quantification combined with a Partial Least Squares Regression model to identify the relative importance of specific cytokines for the osteoclastogenesis outcome. Interleukin 8 (IL-8) was identified as one of RANKL-induced cytokines and validated for its role in osteoclast formation using inhibitors of the IL-8 cognate receptors CXCR1 and CXCR2 or an IL-8 blocking antibody. These insights demonstrate that autocrine signaling induced by RANKL represents a key regulatory component of human osteoclastogenesis.
Highlights
Osteoclasts are responsible for the physiological resorption of calcified tissues during skeletal development and bone remodeling, and for bone destruction in osteoporosis, periodontitis and cancer metastases to bone (Burr, 2002; Honore et al, 2000; Kong et al, 1999; Pearse et al, 2001; Ritchlin et al, 2003; Teng et al, 2000)
While no osteoclasts were formed when the cultures were treated with macrophage colonystimulating factor (MCSF) only (Fig. 1B), when receptor activator of nuclear factor kB ligand (RANKL) and MCSF were added we observed that large, multinucleated osteoclasts that stain positive for tartrateresistant acid phosphatase (TRAP) (Fig. 1B) emerged after 4 days of culture (Fig. 1C)
In the presence of RANKL, the expression of TRAP, MMP9 and cathepsin K was strongly up-regulated at day 3 after induction of differentiation, prior to active osteoclast fusion (Fig. 1G), and the expression of calcitonin receptor (CTR) increased at day 5
Summary
Osteoclasts are responsible for the physiological resorption of calcified tissues during skeletal development and bone remodeling, and for bone destruction in osteoporosis, periodontitis and cancer metastases to bone (Burr, 2002; Honore et al, 2000; Kong et al, 1999; Pearse et al, 2001; Ritchlin et al, 2003; Teng et al, 2000). Osteoclasts attach to the bone matrix, lower the extracellular pH to dissolve hydroxyapatite, and secrete proteolytic enzymes, cathepsin K and. Receptor activator of nuclear factor kB ligand (RANKL) and macrophage colony stimulating factor (MSCF), were identified as the key regulators of osteoclast formation and function. In particular calcium oscillations observed in osteoclast precursors, stimulate phosphatase calcineurin, which dephosphorylates NFATc1 allowing its nuclear translocation (Hwang and Putney, 2011; Takayanagi et al, 2002). Many aspects of osteoclastogenesis remain unclear and accelerated remodeling, especially associated with inflammatory conditions, such as rheumatoid arthritis and periodontitis, remains poorly tractable
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