Abstract
Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.
Highlights
Skeletal loss that can occur with inflammation
Type 1 and Type 2 IL-4 receptors are formed by heterodimerization of IL-4 receptor ␣ (IL-4R␣) protein to either the ␥ common chain (␥c), a receptor component that is found in receptors for IL-2, IL-7, IL-9, IL-15, and IL-21, or to the IL-13 receptor ␣1 (IL-13R␣1) protein, respectively [21, 22]
Cells were stained for tartrate-resistant acid phosphatase (TRAP), and cells positive for TRAP and having three or more nuclei were considered osteoclasts, and the number of multinucleated osteoclasts counted. Osteoclasts formed in these cultures stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL were able to form pits when cultured on slices of bovine bone, and osteoclast formation was associated with increased mRNA expression of calcitonin receptor (CTR), TRAP, and cathepsin K
Summary
M-CSF, macrophage colony-stimulating factor; ALP, alkaline phosphatase; ␣-MEM, ␣-modification of minimum essential medium; BMM, bone marrow macrophages; CT, calcitonin; CTR, calcitonin receptor; DAP12, DNAX-activating protein 12; D3, 1,25(OH)2-vitamin D3; ERK, extracellular signal-regulated kinase; ␥c, common ␥ chain; FBS, fetal bovine serum; FcR␥, Fc receptor common ␥ subunit; IB, inhibitor of B; IL, interleukin; IL-4R␣, interleukin-4 receptor ␣; IL-13R␣, interleukin-13 receptor ␣; JNK, c-Jun NH2-terminal kinase; MAP kinase, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; NF-B, nuclear factor B; OPG, osteoprotegerin; OSM, oncostatin M; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; PBS, phosphate-buffered saline; RANK, receptor activator of nuclear factor B; RANKL, receptor activator of nuclear factor B ligand; RT, reverse transcriptase; STAT, signal transducers and activators of transcription; TAMRA, 6-carboxyltetramethylrhodamine; TNF, tumor necrosis factor; TRAF, tumor necrosis factor receptor-associated factor; TRAP, tartrate-resistant acid phosphatase; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. In inflammatory diseases, increased osteoclast activity can be stimulated by bone resorbing proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-11, IL-17, tumor necrosis factor-␣ (TNF-␣), and kinins released in the kallikrein-kinin system [15,16,17,18] Several of these compounds have been shown to increase RANKL expression [19, 20]. Several molecular mechanisms have been suggested for the inhibition of osteoclast formation by IL-4 These include decreased RANK stimulation of osteoclast progenitor cells by activation of peroxisome proliferator-activated receptor ␥1 [38], inhibition of inhibitor of B (IB) phosphorylation, and decreased nuclear factor B (NF-B) nuclear translocation [39] and inhibition of signaling through c-Jun NH2-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) [40]. The present study was undertaken to determine whether IL-4 and IL-13 might inhibit bone resorption by initiating responses in both stromal cells/osteoblasts and osteoclast progenitor cells that alter the RANKL/RANK/OPG system
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