The mouse NKR-P1B:Clr-b recognition system is a negative regulator of innate immune responses.

NKR-P1B is a homodimeric type II transmembrane C-type lectinlike receptor that inhibits natural killer (NK) cell function upon interaction with its cognate C-type lectin-related ligand, Clr-b. The NKR-P1B:Clr-b interaction represents a major histocompatibility complex class I (MHC-I)-independent missing-self recognition system that monitors cellular Clr-b levels. We have generated NKR-P1B(B6)-deficient (Nkrp1b(-/-)) mice to study the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by Clr-b is abolished in Nkrp1b(-/-) mice, confirming the inhibitory nature of NKR-P1B(B6). Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation; hence, NK cells expressing NKR-P1B(B6) and Ly49C/I display augmented responsiveness to activating signals vs NK cells expressing either or none of the receptors. In addition, Nkrp1b(-/-) mice are defective in rejecting cells lacking Clr-b, supporting a role for NKR-P1B(B6) in MHC-I-independent missing-self recognition of Clr-b in vivo. In contrast, MHC-I-dependent missing-self recognition is preserved in Nkrp1b(-/-) mice. Interestingly, spontaneous myc-induced B lymphoma cells may selectively use NKR-P1B:Clr-b interactions to escape immune surveillance by wild-type, but not Nkrp1b(-/-), NK cells. These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self recognition.

Genetic investigation of MHC-independent missing-self recognition by mouse NK cells using an in vivo bone marrow transplantation model.

MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Urolithiasis is prevalent and associated with reduced bone mineral density in ß-thalassaemia major.

Asymptomatic urolithiasis is common and of mixed composition in patients with ß-thalassaemia major. Twenty-seven subjects were imaged using dual-energy computer tomography to determine the presence and composition of urolithiasis. The prevalence of urolithiasis was 59% and affected patients generally had multiple stones, often with more than one component: struvite (33%), calcium oxalate (31%) and cystine (22%). Hypercalciuria was present in 78% of subjects and calcium-containing urolithiasis was associated with reduced femoral neck Z scores.

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.

Zinc finger protein 467 is a novel regulator of osteoblast and adipocyte commitment.

Osteoblasts and adipocytes are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. cDNA microarrays and quantitative real-time PCR (Q-PCR) were carried out in a differentiating mouse stromal osteoblastic cell line, Kusa 4b10, to identify gene targets of factors that stimulate osteoblast differentiation including parathyroid hormone (PTH) and gp130-binding cytokines, oncostatin M (OSM) and cardiotrophin-1 (CT-1). Zinc finger protein 467 (Zfp467) was rapidly down-regulated by PTH, OSM, and CT-1. Retroviral overexpression and RNA interference for Zfp467 in mouse stromal cells showed that this factor stimulated adipocyte formation and inhibited osteoblast commitment compared with controls. Regulation of adipocyte markers, including peroxisome proliferator-activated receptor (PPAR) γ, C/EBPα, adiponectin, and resistin, and late osteoblast/osteocyte markers (osteocalcin and sclerostin) by Zfp467 was confirmed by Q-PCR. Intra-tibial injection of calvarial cells transduced with retroviral Zfp467 doubled the number of marrow adipocytes in C57Bl/6 mice compared with vector control-transduced cells, providing in vivo confirmation of a pro-adipogenic role of Zfp467. Furthermore, Zfp467 transactivated a PPAR-response element reporter construct and recruited a histone deacetylase complex. Thus Zfp467 is a novel co-factor that promotes adipocyte differentiation and suppresses osteoblast differentiation. This has relevance to therapeutic interventions in osteoporosis, including PTH-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

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.

Germline deletion of AMP-activated protein kinase beta subunits reduces bone mass without altering osteoclast differentiation or function.

Since AMP-activated protein kinase (AMPK) plays important roles in modulating metabolism in response to diet and exercise, both of which influence bone mass, we examined the influence of AMPK on bone mass in mice. AMPK is an alphabetagamma heterotrimer where the beta subunit anchors the alpha catalytic and gamma regulatory subunits. Germline deletion of either AMPK beta1 or beta2 subunit isoforms resulted in reduced trabecular bone density and mass, but without effects on osteoclast (OC) or osteoblast (OB) numbers, as compared to wild-type littermate controls. We tested whether activating AMPK in vivo would enhance bone density but found AICA-riboside treatment caused a profound loss of trabecular bone volume (49.5%) and density and associated increased OC numbers. Consistent with this, AICA-riboside strongly stimulated OC differentiation in vitro, in an adenosine kinase-dependent manner. OCs and macrophages (unlike OBs) lacked AMPK beta2 subunit expression, and when generated from AMPK beta1(-/-) mice displayed no detectable AMPK activity. Nevertheless, AICA-riboside was equally effective at stimulating OC differentiation from wild-type or beta1(-/-) progenitors, indicating that AMPK is not essential for OC differentiation or the stimulatory action of AICA-riboside. These results show that AMPK is required to maintain normal bone density, but not through bone cell differentiation, and does not mediate powerful osteolytic effects of AICA-riboside.

IL-23 inhibits osteoclastogenesis indirectly through lymphocytes and is required for the maintenance of bone mass in mice.

IL-23 stimulates the differentiation and function of the Th17 subset of CD4(+) T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4(+) T lymphocyte-dependent manner. When sufficiently enriched, gammadelta T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19(-/-)) had approximately 30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19(-/-) mice also had reduced BV/TV. IL-23p19(-/-) mice had no detectable osteoclast defect in trabecular bone but IL-23p19(-/-) had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.

Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells.

Bone morphogenetic protein-2 (BMP-2) is strongly involved in the induction of osteoblast differentiation from mesenchymal cell precursors, as well as in enhancing bone matrix production by osteoblastic cells. Likewise, the osteoporotic phenotype of PTHrP deficient mice makes clear the importance of this paracrine regulator in bone physiology. Here, we report that BMP-2 rapidly down-regulated PTHrP gene expression through a transcriptional mechanism in pluripotent mesenchymal C2C12 cells, whereas BMP-2 increased expression of PTHrP receptor. PTHrP did not significantly alter the BMP-dependent Smad transcriptional pathway. Similarly, PTHrP did not significantly modify the BMP-regulated expression of RANKL or OPG, cytokines involved in osteoclastogenesis. More importantly, addition of PTHrP, through the PKA signaling pathway, partially prevented the BMP-dependent induction of some osteogenic markers such as Runx2 and Osterix in C2C12 cells. Our data suggest that BMP-2 down-regulation of PTHrP could facilitate terminal differentiation of osteoblasts.

Osteoprotegerin overexpression by breast cancer cells enhances orthotopic and osseous tumor growth and contrasts with that delivered therapeutically.

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of NF-kappaB ligand (RANKL), which is a pivotal molecule required for osteoclast formation. In vitro OPG inhibits osteoclast formation and in vivo (administered as Fc-OPG) it reduces hypercalcemia and the establishment of osteolytic lesions in mouse models of tumor cell growth in bone. Osteolysis can be induced by parathyroid hormone-related protein (PTHrP) produced by breast cancer cells that results in an increased osteoblastic RANKL/OPG ratio. We examined the effect of local tumor production of OPG on the ability of breast cancer cells to establish and grow in bone and mammary fat pad. MCF-7 cells or MCF-7 cells overexpressing PTHrP were transfected with full-length OPG and inoculated into the proximal tibiae of athymic nude mice. Mice injected with cells overexpressing PTHrP and OPG showed enhanced tumor growth, increased osteolysis (2-fold compared with MCF-7 cells overexpressing PTHrP), and altered histology that was reflective of a less differentiated (more aggressive) phenotype compared with MCF-7 cells. In contrast, administration of recombinant Fc-OPG reduced tumor growth and limited osteolysis even in mice inoculated with OPG overexpressing cells. Similarly, OPG overexpression by breast cancer cells enhanced tumor growth following orthotopic inoculation. These results indicate that OPG overexpression by breast cancer cells increases tumor growth in vivo and that there are strikingly different responses between therapeutically administered Fc-OPG and full-length OPG produced by tumor cells.

Glycoprotein 130 regulates bone turnover and bone size by distinct downstream signaling pathways.

The gp130-dependent cytokines, which signal through at least two intracellular pathways, regulate osteoclast and osteoblast formation. To define their roles in regulating bone mass, we analyzed mice in which gp130 signaling via either the signal transducer and activator of transcription (STAT) 1/3 (gp130(DeltaSTAT/DeltaSTAT)) or SHP2/ras/MAPK (gp130(Y757F/Y757F)) pathway was attenuated. In gp130(DeltaSTAT/DeltaSTAT) mice, trabecular bone volume (BV/TV) and turnover were normal, but bone length was reduced by premature growth plate closure, indicating an essential role for gp130-STAT1/3 signaling in chondrocyte differentiation. In contrast, while bone size was normal in gp130(Y757F/Y757F) mice, BV/TV was reduced due to high bone turnover, indicated by high osteoclast surface/bone surface (OcS/BS) and osteoblast surface/bone surface (ObS/BS). Furthermore, generation of functional osteoclasts from bone marrow of gp130(Y757F/Y757F) mice was elevated, revealing that while gp130 family cytokines stimulate osteoclastogenesis through the osteoblast lineage, gp130, via SHP2/Ras/MAPK, inhibits osteoclastogenesis in a cell lineage-autonomous manner. Genetic ablation of IL-6 in gp130(Y757F/Y757F) mice exacerbated this osteopenia by reducing ObS/BS without affecting OcS/BS. Thus, while IL-6 is critical for high bone formation in gp130(Y757F/Y757F) mice, it is not involved in the increased osteoclastogenesis. In conclusion, gp130 is essential for normal bone growth and trabecular bone mass, with balanced regulation depending on selective activation of STAT1/3 and SHP2/ras/MAPK, respectively. Furthermore, the latter pathway can directly inhibit osteoclastogenesis in vivo.

Osteoclast inhibitory lectin (OCIL) inhibits osteoblast differentiation and function in vitro.

Osteoclast inhibitory lectin (OCIL) is a type II C-type lectin and binds NK cell-associated receptor Nkrp1d and sulfated glycosaminoglycans. OCIL is expressed by several cell types found in bone and inhibits osteoclast differentiation. To determine whether OCIL may have wider effects on bone metabolism, we examined the effects of recombinant soluble OCIL on cultured osteoblasts and pre-osteoblastic KUSA O cells. Although OCIL did not affect osteoblast proliferation or apoptosis, or the formation of alkaline phosphatase positive colonies in cultured bone marrow, OCIL profoundly inhibited mineralization by primary osteoblasts and KUSA O cells in vitro. Analysis of ascorbate-treated KUSA O cells showed that addition of OCIL reduced bone sialoprotein (BSP), osterix and osteocalcin mRNA expression, as well as alkaline phosphatase activity while, in contrast, expression of markers associated with the earlier stages of osteoblast maturation or the transcription factors Runx2, ATF4 and c-fos were not affected by OCIL treatment. Indeed, osteocalcin expression was strongly inhibited within 3 days in a dose-dependent manner, although after subsequent removal of OCIL, osteocalcin mRNA levels recovered within 4 days. OCIL treatment also reduced osteocalcin expression in BMP-2 stimulated C2C12 cells. In support of a role for OCIL in mineralization, OCIL anti-sense oligonucleotide treatment of KUSA O cells increased mineralization and osteocalcin expression. In addition, insulin-, dexamethasone- and IBMX-stimulated KUSA O cells undergo adipocyte differentiation and OCIL treatment greatly suppressed this process. Consistent with this, OCIL also reduced adiponectin and resistin mRNA expression in these cells. Our data indicate that OCIL reduces osteoblastic function in vitro and this may be due to an inhibitory effect on osteoblast maturation. In addition, the reduction of adipocyte formation in KUSA O cells by OCIL indicates that OCIL may have wider effects on the mesenchymal lineage that may be important for both bone metabolism and other connective tissue functions.

Generation and analysis of Siah2 mutant mice.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

The heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line.

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence caused by 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Melphalan modifies the bone microenvironment by enhancing osteoclast formation.

Melphalan is a cytotoxic chemotherapy used to treat patients with multiple myeloma (MM). Bone resorption by osteoclasts, by remodeling the bone surface, can reactivate dormant MM cells held in the endosteal niche to promote tumor development. Dormant MM cells can be reactivated after melphalan treatment; however, it is unclear whether melphalan treatment increases osteoclast formation to modify the endosteal niche. Melphalan treatment of mice for 14 days decreased bone volume and the endosteal bone surface, and this was associated with increases in osteoclast numbers. Bone marrow cells (BMC) from melphalan-treated mice formed more osteoclasts than BMCs from vehicle-treated mice, suggesting that osteoclast progenitors were increased. Melphalan also increased osteoclast formation in BMCs and RAW264.7 cells in vitro, which was prevented with the cell stress response (CSR) inhibitor KNK437. Melphalan also increased expression of the osteoclast regulator the microphthalmia-associated transcription factor (MITF), but not nuclear factor of activated T cells 1 (NFATc1). Melphalan increased expression of MITF-dependent cell fusion factors, dendritic cell-specific transmembrane protein (Dc-stamp) and osteoclast-stimulatory transmembrane protein (Oc-stamp) and increased cell fusion. Expression of osteoclast stimulator receptor activator of NFκB ligand (RANKL) was unaffected by melphalan treatment. These data suggest that melphalan stimulates osteoclast formation by increasing osteoclast progenitor recruitment and differentiation in a CSR-dependent manner. Melphalan-induced osteoclast formation is associated with bone loss and reduced endosteal bone surface. As well as affecting bone structure this may contribute to dormant tumor cell activation, which has implications for how melphalan is used to treat patients with MM.

Inflammation-induced bone loss: can it be prevented?

Inflammatory synovitis induces profound bone loss and OCLs are the instrument of this destruction. TNF blockers have an established role in the prevention of inflammatory bone loss in RA; however, not all patients respond to anti-TNF therapy and side effects may prevent long-term treatment in others. The B-cell--depleting antibody rituximab and the T-cell costimulation blocker abatacept are emerging as major treatment options for patients who are resistant to anti-TNF [96,97]. Proof-of-concept studies demonstrate that targeting RANK-mediated osteoclastogenesis prevents inflammatory bone loss and clinical application has only just begun. The efficacy of RANKL inhibition has been witnessed in trials of Denosumab, and RANKL-neutralizing antibodies are likely to become the treatment of choice for blocking RANKL in RA [77,78]. A major limitation of RANKL antagonism is that it does not treat synovitis. Therefore, anti-RANKL therapy most likely will be used in the context of MTX therapy. There is uncertainty about the possible extraskeletal adverse effects of long-term effects of long-term RANKL blockade. In particular, anti-RANKL therapy could jeopardize dendritic cell function or survival. The demonstrable role of OCLs in inflammation-induced bone loss also invites a reconsideration of the new BPs for bone protection [98]. Studies of ZA in preclinical models indicate that bone protection is comparable to that afforded by OPG. One possible caveat is that intravenous BPs are linked to jaw osteonecrosis [99], although the incidence is confined mainly to intensive treatment in the oncology setting. Although pulsed PTH stimulated bone formation in arthritic models, it has yet to be proven clinically in the context of powerful OCL inhibition with TNF or RANKL antagonists. With strategies that normalize OCL numbers, clinicians are poised to accomplish effective prevention of inflammation-induced bone loss.

Bone Disease in Thalassemia: A Molecular and Clinical Overview.

Thalassemia bone disease is a common and severe complication of thalassemia-an inherited blood disorder due to mutations in the α or ß hemoglobin gene. In its more severe form, severe anemia is present, and treatment with frequent red blood cell transfusion is necessary. Because the body has limited capacity to excrete iron, concomitant iron chelation is required to prevent the complications of iron overload. The effects of chronic anemia and iron overload can lead to multiple end-organ complications such as cardiomyopathy, increased risks of blood-borne diseases, and liver, pituitary, and bone disease. However, our understanding of thalassemia bone disease is incomplete and is composed of a complex piecemeal of risk factors that include genetic factors, hormonal deficiency, marrow expansion, skeletal dysmorphism, iron toxicity, chelators, and increased bone turnover. The high prevalence of bone disease in transfusion-dependent thalassemia is seen in both younger and older patients as life expectancy continues to improve. Indeed, hypogonadism and GH deficiency contribute to a failure to achieve peak bone mass in this group. The contribution of kidney dysfunction to bone disease in thalassemia is a new and significant complication. This coincides with studies confirming an increase in kidney stones and associated accelerated bone loss in the thalassemia cohort. However, multiple factors are also associated with reduced bone mineral density and include marrow expansion, iron toxicity, iron chelators, increased bone turnover, GH deficiency, and hypogonadism. Thalassemia bone disease is a composite of not only multiple hormonal deficiencies but also multiorgan diseases. This review will address the molecular mechanisms and clinical risk factors associated with thalassemia bone disease and the clinical implications for monitoring and treating this disorder.

Deferasirox at therapeutic doses is associated with dose-dependent hypercalciuria.

Deferasirox is an oral iron chelator used widely in the treatment of thalassemia major and other transfusion-dependent hemoglobinopathies. Whilst initial long-term studies established the renal safety of deferasirox, there are now increasing reports of hypercalciuria and renal tubular dysfunction. In addition, urolithiasis with rapid loss of bone density in patients with ß thalassemia major has been reported. We conducted a cross-sectional cohort study enrolling 152 adult patients comprising of ß thalassemia major (81.5%), sickle cell disease (8%), thalassemia intermedia (2%), HbH disease (6.5%) and E/ß thalassemia (2%). Cases were matched with normal control subjects on age, gender and serum creatinine. Iron chelator use was documented and urine calcium to creatinine ratios measured. At the time of analysis, 88.8% of patients were receiving deferasirox and 11.2% were on deferoxamine. Hypercalciuria was present in 91.9% of subjects on deferasirox in a positive dose-dependent relationship. This was not seen with subjects receiving deferoxamine. At a mean dose of 30.2±8.8mg/kg/day, deferasirox was associated with an almost 4 fold increase in urine calcium to creatinine ratio (UCa/Cr). Hypercalciuria was present at therapeutic doses of deferasirox in a dose-dependent manner and warrants further investigation and vigilance for osteoporosis, urolithiasis and other markers of renal dysfunction.