We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti– IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry. IL-6 (5 ng/l to 10 μg/l), but not IL-11 (10 and 100 μg/l), reversed the inhibition of osteoclastic bone resorption induced by high extracellular Ca2+ (15 mM). The IL-6 effect was abrogated by excess soluble IL-6 receptor (500 μg/l). Additionally, IL-6 (5 pg/l to 10 μg/l) inhibited cytosolic Ca2+ signals triggered by high Ca2+ or Ni2+. In separate experiments, osteoclasts incubated in 10 mM Ca2+ or on bone released more IL-6 than those in 1.25 mM Ca2+. Furthermore, IL-6 mRNA histostaining was more intense in osteoclasts in 10 or 20 mM Ca2+ than cells in 1.25 mM Ca2+. Similarly, IL-6 receptor mRNA histostaining was increased in osteoclasts incubated in 5 or 10 mM Ca2+. Thus, while high Ca2+ enhances IL-6 secretion, the released IL-6 attenuates Ca2+ sensing and reverses inhibition of resorption by Ca2+. Such an autocrine–paracrine loop may sustain osteoclastic activity in the face of an inhibitory Ca2+ level generated locally during resorption.

The formation of multinucleated cells such as myotubes, macrophage-derived giant cells (MGC), and osteoclasts is the result of cell-cell fusion of mononuclear precursors. Meltrin-alpha, -beta, and -gamma are members of a recently discovered family of proteins that contain disintegrin and metalloprotease domains and are related to fertilin, a protein involved in egg-sperm fusion. Based on this and evidence implicating meltrin-alpha in myoblast formation, we have investigated the possibility that meltrins may also play a role in the formation of MGC and osteoclasts. Using in situ RT-PCR, we have determined that murine mononuclear alveolar macrophages cultured under basal conditions express the transcript for meltrin-beta, but not for meltrin-alpha. However, meltrin-alpha mRNA appeared in mononuclear cells before cell fusion after treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent inducer of giant cell and osteoclast formation. Moreover, addition of meltrin-alpha antisense oligonucleotides to the cultures caused a 50% inhibition of giant cell formation. Similarly, meltrin-alpha antisense oligonucleotides inhibited by 70% the formation of multinucleated osteoclast-like cells expressing tartrate-resistant acid phosphatase (TRAP) in co-cultures of bone marrow cells and osteoblastic cells (2107) in the presence of 1,25(OH)2D3. Mononucleated TRAP-positive cells, induced by 1,25(OH)2D3 in the co-cultures, also expressed meltrin-alpha mRNA, but their number was not changed in the presence of meltrin-alpha antisense oligonucleotide. In contrast to mononuclear macrophages and osteoclast-like cells, murine bone marrow stroma and calvaria derived-cell lines (+/+ LDA.11 and 2107), primary cultures of calvaria cells, and primary cultures of bone marrow cells expressed both meltrin-alpha and -beta mRNA under basal conditions; whereas embryonic fibroblasts (NIH3T3) expressed only the meltrin-beta transcript. Upregulation of meltrin-alpha protein expression during cell fusion in alveolar macrophages and expression in osteoblastic cell lines were confirmed by Western blot analysis. These observations demonstrate that meltrins play a role in MGC and osteoclast formation from mononuclear precursors, as in the case with myotubes.

Both estrogen and androgen exert their antiosteoporotic effects, at least in part, by inhibiting IL-6 production, thereby suppressing osteoclastogenesis. Several observations, however, suggest that besides increased IL-6 production, sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy. Based on this and evidence that the ligand-binding subunit of the IL-6 receptor (gp80) is a limiting factor for the actions of IL-6 on bone, we hypothesized that sex steroids regulate expression of the IL-6 receptor as well. We report that 17 ␤ -estradiol or dihydrotestosterone in vitro decreased the abundance of the gp80 mRNA as well as the mRNA of the signal-transducing subunit of the IL-6 receptor (gp130) in cells of the bone marrow stromal/osteoblastic lineage, and also decreased gp130 protein levels. These effects did not require new protein synthesis. In contrast to sex steroids, parathyroid hormone stimulated gp130 expression; this effect was opposed by sex steroids. Consistent with these findings, ovariectomy in mice caused an increase in expression of gp80, gp130, and IL-6 mRNAs in ex vivo bone marrow cell cultures as determined by quantitative reverse transcription (RT)-PCR, and confirmed on an individual cell basis using in situ RT-PCR. The demonstration of increased expression of the IL-6 receptor after loss of sex steroids provides an explanation for why IL-6 is important for skeletal homeostasis in the sex steroid-deficient, but not replete, state. (