Michael Soo Ho Kim

MCP-1 Is Induced by Receptor Activator of Nuclear Factor-κB Ligand, Promotes Human Osteoclast Fusion, and Rescues Granulocyte Macrophage Colony-stimulating Factor Suppression of Osteoclast Formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-κB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP‐1 and RANTES

Chemokines MCP‐1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP‐1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1α is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1α was able to overcome the potent inhibition of GM‐CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6‐ and 49‐fold, P = 4.0 × 10−7 and 4.0 × 10−8, respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP‐1, RANTES and MIP1α. Unexpectedly, treatment with MCP‐1 in the absence of RANKL resulted in 458‐fold induction of CCR4 (P = 1.0 × 10−10), while RANTES treatment resulted in twofold repression (P = 1.0 × 10−4). Since CCR2b and CCR4 are MCP‐1 receptors, these data support the existence of an MCP‐1 autocrine loop in human osteoclasts differentiated using RANKL. J. Cell. Biochem.

Gene array identification of osteoclast genes: Differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) stimulates the formation of multinucleate osteoclast‐like cells. Treatment with M‐CSF alone results in the formation of macrophage‐like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R∝), transcription factors (nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPα)), and ribosomal proteins (60S L17 and 40S S20). Real‐time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c‐Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate‐resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30‐fold. Constant exposure of M‐CSF‐ and RANKL‐treated cells to GM‐CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.

NFAT expression in human osteoclasts

Nuclear factor of activated T‐cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T‐cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage‐like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium‐calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T‐cells but are similar to those seen in naïve T‐cells.