IL-33 is an important inflammatory mediator in allergy, asthma, and joint inflammation, acting via its receptor, ST2L, to elicit Th₂ cell cytokine secretion. IL-33 is related to IL-1 and IL-18, which both influence bone metabolism, IL-18 in particular inhibiting osteoclast formation and contributing to PTH bone anabolic actions. We found IL-33 immunostaining in osteoblasts in mouse bone and IL-33 mRNA expression in cultured calvarial osteoblasts, which was elevated by treatment with the bone anabolic factors oncostatin M and PTH. IL-33 treatment strongly inhibited osteoclast formation in bone marrow and spleen cell cultures but had no effect on osteoclast formation in receptor activator of nuclear factor-κB ligand/macrophage colony-stimulating factor-treated bone marrow macrophage (BMM) or RAW264.7 cultures, suggesting a lack of direct action on immature osteoclast progenitors. However, osteoclast formation from BMM was inhibited by IL-33 in the presence of osteoblasts, T cells, or mature macrophages, suggesting these cell types may mediate some actions of IL-33. In bone marrow cultures, IL-33 induced mRNA expression of granulocyte macrophage colony-stimulating factor, IL-4, IL-13, and IL-10; osteoclast inhibitory actions of IL-33 were rescued only by combined antibody ablation of these factors. In contrast to osteoclasts, IL-33 promoted matrix mineral deposition by long-term ascorbate treated primary osteoblasts and reduced sclerostin mRNA levels in such cultures after 6 and 24 h of treatment; sclerostin mRNA was also suppressed in IL-33-treated calvarial organ cultures. In summary, IL-33 stimulates osteoblastic function in vitro but inhibits osteoclast formation through at least three separate mechanisms. Autocrine and paracrine actions of osteoblast IL-33 may thus influence bone metabolism.

In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC) generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors) induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate) these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF) to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL) to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM) greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM), and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2) actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation) or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells, including osteoclasts but not classically activated macrophages, can strongly drive MSC-osteoblastic commitment in OSM-dependent manner. This supports the notion that eliciting gp130-dependent signals in human MSC would be a useful approach to increase bone formation.

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Breast cancer cells metastasize to bone where they cause bone destruction. Here, cancer cells promote bone loss through the recruitment of osteoclasts, specialised macrophage-like cells that resorb bone. Bone resorption releases bone matrix factors that increase tumor growth, resulting in vicious cycle of bone resorption and tumor progression. Previously we found 17-AAG, an Hsp90 inhibitor and anti-cancer therapeutic significantly increases MDA-MB-231 breast cancer cell growth in bone following intracardiac inoculation in nude mice. 17-AAG also increases osteoclast formation in vitro and in vivo even though Hsp90 client proteins are important in osteoclast formation. In addition to degradation of Hsp90 client proteins, inhibition by 17-AAG also causes cell stress responses through the activation of Heat Shock Factor 1 (HSF1). Therefore we sought to characterize 17-AAG and new HSP90 inhibitors, to examine the role of HSF1 in their effects and to determine the molecular mechanism of 17-AAG. 17-AAG and new, but structurally unrelated, Hsp90 blockers CCT018159 and NVP-AUY922 dose-dependently enhanced osteoclast formation from mouse bone marrow and RAW264.7 cells. Ablating HSF1 action by shRNA transfection and by pharmacological inhibition using KNK437 inhibited 17-AAG effects upon osteoclast formation. Osteoclast formation from HSF1 knockout mouse bone marrow were unresponsive to 17-AAG, but wild type cells did respond. To determine the mechanism of how 17-AAG is increasing osteoclast formation, the effect of 17-AAG upon critical transcription factors in the osteoclast formation pathway was observed. 17-AAG treatment did not affect transcriptional activity of NFκB, AP-1 or NFATc1. Moreover 17-AAG did not increase NFATc1 or cFos protein expression over time. In contrast, 17-AAG treatment increased protein expression of MITF,in a dose and time dependent manner. RAW264.7 cells were then transiently transfected with a luciferase reporter construct containing the promoter region of a MITF target gene v-ATPase-d2. 17-AAG treatment increased the transcriptional activity of this promoter. RANKL and 17-AAG combination treatment increased reporter activity of the v-ATPase-d2 promoter in an additive manner. The stress induced kinase p38 is known to be important in osteoclast formation. Our results show inhibiting p38 with SB203580 decreases MITF protein expression. These data indicate that Hsp90 inhibition causes an increase in osteoclast formation in an HSF1-dependent manner. The data provides a mechanism of action, whereby 17-AAG is increasing osteoclast numbers, through the increased protein expression of Mitf. Citation Format: A. Gabrielle J. van der Kraan, Ryan R. Chai, Michelle M. Kouspou, Ben J. Lang, Preetinder P. Singh, Jiake Xu, Damien Eeles, Matthew T. Gillespie, Julian M. Quinn, John T. Price. HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2013-2933