Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFß (transforming growth factor ß) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity.

Membrane-bound receptor activator of NFκB ligand (RANKL) activity displayed by osteoblasts is differentially regulated by osteolytic factors.

Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFκB ligand (RANKL) on osteoblasts. Osteolytic hormones induce osteoblast RANKL expression, and reduce production of RANKL decoy receptor osteoprotegerin (OPG). However, rather than RANKL and OPG mRNA or protein levels, to measure hormonally-induced osteoclastogenic stimuli the net RANKL activity at the osteoblast surface needs to be determined. To estimate this we developed a cell reporter approach employing pre-osteoclast RAW264.7 cells transfected with luciferase reporter constructs controlled by NFκB (NFκB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24h. When NFκB-RAW cells were co-cultured on osteoblastic cells (primary osteoblasts or Kusa O cells) stimulated by osteolytic factors 1,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)), a strong dose dependent signal in NFκB-RAW cells was induced. These signals were completely blocked by soluble recombinant RANKL receptor, RANK.Fc. This osteoblastic RANKL activity was sustained for 3 days in Kusa O cells; with 1,25(OH)(2)D(3) withdrawal, RANKL-induced signal was still detectable 24 h later. However, conditioned medium from stimulated osteoblasts induced no signal. TGFß treatment inhibited osteoclast formation supported by 1,25(OH)(2)D(3)-treated Kusa O cells, and likewise blocked RANKL-dependent signals in NFAT-RAW co-cultured with these cells. These data indicate net RANKL stimulus at the osteoblast surface is increased by 1,25(OH)(2)D(3) and PGE(2), and suppressed by TGFß, in line with their effects on RANKL mRNA levels. These results demonstrate the utility of this simple co-culture-based reporter assay for osteoblast RANKL activity.