The effect of gonadal status on body composition and bone mineral density in transfusion-dependent thalassemia.

UNLABELLED: Patients with transfusion-dependent thalassemia have abnormal growth, hormonal deficits, and increased bone loss. We investigated the relationship between skeletal muscle mass, fat mass, and bone mineral density in adult subjects with transfusion-dependent thalassemia based on their gonadal status. Our findings show that hypogonadism attenuates the strength of the muscle-bone relationship in males but strengthens the positive correlation of skeletal muscle mass and fat mass in female subjects. INTRODUCTION: Transfusion-dependent thalassemia is associated with a high prevalence of fractures. Multiple hormonal complications, in particular hypogonadism, can lead to changes in body composition and bone mineral density (BMD). We investigated for the first time the relationship between skeletal muscle mass (SMM), fat mass, and BMD in adult subjects with transfusion-dependent thalassemia based on their gonadal status. METHODS: A retrospective cohort study of 186 adults with transfusion-dependent thalassemia was analyzed. Body composition and BMD were measured using dual energy X-ray absorptiometry. The association between skeletal muscle, fat, and BMD was investigated through uni-, multi-, and stepwise regression analyses after adjusting for multicollinearity. SMM was derived using the formula, SMM = 1.19 × ALST-1.65, where ALST is equivalent to the sum of both arm and leg lean tissue mass. RESULTS: There were 186 subjects, males (43.5 %) and females (56.5 %), with a median age of 36.5. Hypogonadism was reported in 44.4 % of males and 44.7 % of females. SMM and BMD were positively correlated and strongest in eugonadal males (0.36 ≤ R (2) ≤ 0.59), but the association was attenuated in hypogonadal males. SMM (0.27 ≤ R (2) ≤ 0.69) and total fat mass (0.26 ≤ R (2) ≤ 0.55) were positively correlated with BMD in hypogonadal females, but the correlation was less pronounced in eugonadal females. Leg lean tissue mass and arm lean tissue mass in males and females, respectively, were most highly correlated to BMD in the stepwise regression analysis. CONCLUSION: Hypogonadism attenuates the strength of the muscle-bone relationship in males but strengthens the positive correlation of skeletal muscle mass and fat mass in female subjects. This study supports the notion that exercise is important for maintaining BMD and the need to optimize treatment of hypogonadism in patients with transfusion-dependent thalassemia.

Prolonged hypocalcemia following denosumab therapy in metastatic hormone refractory prostate cancer.

Prostate cancer is a leading cause of cancer death, frequently associated with widespread bone metastases. We report two cases of hypocalcemia following the first dose of denosumab in metastatic hormone refractory prostate cancer, the first case requiring 26 days of intravenous calcium therapy. This is the first report of prolonged hypocalcemia following denosumab in a patient with normal renal function.

Thalassemia bone disease: the association between nephrolithiasis, bone mineral density and fractures.

UNLABELLED: Thalassemia bone disease is well described, but the prevalence of nephrolithiasis has not been characterized. The association between nephrolithiasis, reduced bone density, and increased fractures has been demonstrated through this retrospective study of 166 participants with transfusion-dependent thalassemia. The findings support the need for increased vigilance of kidney and bone disease in this cohort. INTRODUCTION: Previous studies have revealed that thalassemia is associated with reduced bone mineral density (BMD) and fractures. Many causes are implicated including hypogonadism, growth hormone deficiency, marrow expansion, and iron overload. Nephrolithiasis is associated with reduced BMD and increased fractures in the general population. However, the prevalence of nephrolithiasis and its association with bone density and fractures have not been characterized in thalassemia. METHODS: We have addressed this question by performing a retrospective cohort study of 166 participants with transfusion-dependent thalassemia who had undergone dual-energy X-ray absorptiometry between 2009 and 2011. Logistic regression modeling was used to adjust for potential confounders. RESULTS: We found a high prevalence of kidney stones (18.1 %) which was greater in males compared to females (28.7 vs 9.7 %, respectively). Renal stones were associated with reduced femoral neck Z-score and fractures in men after adjusting for potential confounders. These results indicate that nephrolithiasis is highly prevalent in patients with transfusion-dependent thalassemia and is significantly associated with reduced BMD and increased fractures. CONCLUSIONS: The findings from this study strongly support the need for ongoing surveillance of BMD, fractures, and nephrolithiasis in the management of transfusion-dependent thalassemia.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

Communication between ephrinB2 and EphB4 within the osteoblast lineage.

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Mechanisms involved in skeletal anabolic therapies.

Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.

Effects of parathyroid hormone on Wnt signaling pathway in bone.

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

Myeloma cells can directly contribute to the pool of RANKL in bone bypassing the classic stromal and osteoblast pathway of osteoclast stimulation.

Summary The ratio of osteoprotegerin [OPG, tumour necrosis factor receptor superfamily, member 11b (TNFRSF11B)] to receptor activator of nuclear factor kappaB ligand [RANKL, tumour necrosis factor (ligand) superfamily, member 11 (TNFSF11)] in bone is critical for the regulation of bone remodelling. Myeloma cells can home to bone, triggering increased RANKL and decreased OPG expression by stromal cells, leading to osteolysis. Whether myeloma cells contribute directly to the pool of RANKL or OPG in bone has been contentious. Here we provide evidence of RANKL expression by reverse transcription polymerase chain reaction and in situ hybridization, demonstrating transcripts encoding both the membrane-bound and secreted forms of RANKL in five human multiple myeloma cell lines (LP-1, NCI-H929, OPM-2, RPMI8226, U266) and myeloma cells purified from bone marrow aspirates of myeloma patients. We demonstrated that RANKL encoding mRNAs are translated to protein by antibody detection of RANKL. In vitro assays showed that myeloma cells induced bone marrow derived mononuclear cells to differentiate into adherent tartrate-resistant acid phosphatase positive multinucleated cells, indicative of the formation of functional osteoclasts. This differentiation could also be achieved with passaged myeloma media alone, implicating secreted products. Finally, we provide evidence that the differentiation observed is at least in part the result of myeloma cell expression of RANKL. We therefore conclude that myeloma cells can directly contribute to the pool of RANKL in bone.

Interleukin-18 is a novel mitogen of osteogenic and chondrogenic cells.

IL-18 was identified due to its ability to induce interferon-gamma (IFNgamma) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFNgamma. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFNgamma production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes. The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFNgamma or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFNgamma production by primary osteoblasts, but, of greater significance, IFNgamma had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFNgamma or GM-CSF.

Parathyroid hormone-related protein (PTHrP)-(1-139) isoform is efficiently secreted in vitro and enhances breast cancer metastasis to bone in vivo.

Parathyroid hormone-related peptide (PTHrP) is a mediator of local osteolysis due to breast cancer. Three isoforms of PTHrP, (1-139), (1-141), and (1-173), are products of alternative splicing in humans, but the specific contribution of each of these isoforms to osteolytic metastasis caused by breast cancer has not been evaluated. To determine the role of PTHrP isoforms in breast cancer metastasis to bone, the human breast cancer cell line MDA-MB-231 (MDA-231) was stably transfected with cDNAs for human prepro PTHrP-(1-139), -(1-141), or -(1-173). Stable MDA/PTHrP-(1-139) clones expressed more PTHrP mRNA and secreted more PTHrP protein, compared with MDA/PTHrP-(1-141), -(1-173), or parental MDA-231. Parental MDA-231 cells and clones expressing each isoform had similar growth rates in vitro. In a mouse model of bone metastases, the osteolytic lesion area of radiographs was greatest in mice bearing MDA/PTHrP-(1-139) compared with those bearing MDA/PTHrP-(1-141), -(1-173), or parental MDA-231. Ca(++) and plasma PTHrP concentrations were significantly higher in the MDA/PTHrP-(1-139) compared with the MDA/PTHrP-(1-141), -(1-173), or parental MDA-231 groups. These data demonstrate that the PTHrP-(1-139) isoform was produced to a greater extent than PTHrP-(1-141) or -(1-173), and in vivo enhanced osteolysis with increased plasma PTHrP concentrations and hypercalcemia compared with overexpression of PTHrP-(1-141) or -(1-173).

Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis.

Bone loss represents a major unsolved problem in rheumatoid arthritis (RA). The skeletal complications of RA consist of focal bone erosions and periarticular osteoporosis at sites of active inflammation, and generalized bone loss with reduced bone mass. New evidence indicates that osteoclasts are key mediators of all forms of bone loss in RA. TNF-alpha is one of the most potent osteoclastogenic cytokines produced in inflammation and is pivotal in the pathogenesis of RA. Production of tumor necrosis factor-alpha (TNF-alpha) and other proinflammatory cytokines in RA is largely CD4(+) T-cell dependent and mostly a result of interferon-gamma (IFN-gamma) secretion. Synovial T cells contribute to synovitis by secreting IFN-gamma and interleukin (IL)-17 as well as directly interacting with macrophages and fibroblasts through cell-to-cell contact mechanisms. Activated synovial T cells express both membrane-bound and soluble forms of receptor activator of NF-kappaB ligand (RANKL). In rheumatoid synovium, fibroblasts also provide an abundant source of RANKL. Furthermore, TNF-alpha and IL-1 target stromal-osteoblastic cells to increase IL-6, IL-11, and parathyroid hormone-related protein (PTHrP) production as well as expression of RANKL. In the presence of permissive levels of RANKL, TNF-alpha acts directly to stimulate osteoclast differentiation of macrophages and myeloid progenitor cells. In addition, TNF-alpha induces IL-1 release by synovial fibroblasts and macrophages, and IL-1, together with RANKL, is a major survival and activation signal for nascent osteoclasts. Consequently, TNF-alpha and IL-1, acting in concert with RANKL, can powerfully promote osteoclast recruitment, activation, and osteolysis in RA. The most convincing support for this hypothesis has come from in vivo studies of animal models. Protection of bone in the presence of continued inflammation in arthritic rats treated with osteoprotegerin (OPG) supports the concept that osteoclasts mediate bone loss, providing further evidence that OPG protects bone integrity by downregulating osteoclastogenesis and promoting osteoclast apoptosis. Modulation of the RANKL/OPG equilibrium in arthritis may provide additional skeletal benefits, such as chondroprotection. The nexus between T-cell activation, TNF-alpha overproduction, and the RANKL/OPG/RANK ligand-receptor system points to a unifying paradigm for the entire spectrum of skeletal pathology in RA. Strategies that address osteoclastic bone resorption will represent an important new facet of therapy for RA.

Increased expansion of the lung stimulates calmodulin 2 expression in fetal sheep.

Obstruction of the fetal trachea causes the lungs to expand with accumulated liquid. Although this is a potent stimulus for lung growth, the mechanisms involved are unknown. Our aim was to identify genes that are differentially expressed as a result of increased fetal lung expansion. Using differential display RT-PCR, we isolated a cDNA fragment partially encoding calmodulin 2 (CALM2) and identified the remainder of the coding region by 5'-rapid amplification of cDNA ends. Differential expression of CALM2 was confirmed by Northern blot analysis; CALM2 mRNA levels were increased to 161 +/- 5% of control at 2 days of increased lung expansion, induced by tracheal obstruction (TO), and had returned to control levels at days 4 and 10. Using in situ hybridization analysis, we found that the proportion of CALM2-labeled cells increased from 10.3 +/- 1.0% to 21.4 +/- 6.8% by 2 days of TO. This increase in CALM2 expression was reflected by a tendency for calmodulin protein levels to increase from 122.7 +/- 17.3 to 156.5 +/- 17.7 at 2 days of TO. Thus increases in fetal lung expansion result in time-dependent changes in CALM2 mRNA levels, which closely parallels the changes in lung DNA synthesis rates. As calmodulin is essential for cell proliferation, increased CALM2 mRNA levels may reflect an important role for calmodulin in expansion-induced fetal lung growth.

The generation of highly enriched osteoclast-lineage cell populations.

Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.

Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein.

Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin beta1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 -10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.

Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions.

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

Molecular dissection of the importin beta1-recognized nuclear targeting signal of parathyroid hormone-related protein.

Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin beta1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin beta using a series of alanine mutated PTHrP peptides and truncated human importin beta1 derivatives. Our results indicate that PTHrP amino acids 83-93 (KTPGKKKKGK) are absolutely essential for importin beta1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of importin beta1 are required for the interaction. Binding of importin beta1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin beta is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin beta1-recognized nuclear targeting signal.

IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro.

IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.

A novel osteoblast-derived C-type lectin that inhibits osteoclast formation.

We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.