Genetic regulation of osteoclast development and function

Osteoclasts are the principal, if not exclusive, bone-resorbing cells, and their activity has a profound impact on skeletal health. So, disorders of skeletal insufficiency, such as osteoporosis, typically represent enhanced osteoclastic bone resorption relative to bone formation. Prevention of pathological bone loss therefore depends on an appreciation of the mechanisms by which osteoclasts differentiate from their precursors and degrade the skeleton. The past five years have witnessed important insights into osteoclast formation and function. Many of these discoveries have been made through genetic experiments that involved the rare hereditary disorder osteopetrosis.     

Cell adhesion is a prerequisite for osteoclast survival

The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF-kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival.     

MIP-1 gamma promotes receptor-activator-of-NF-kappa-B-ligand-induced osteoclast formation and survival

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes, and have also been reported to modulate osteoclast differentiation from hemopoietic precursor cells of the monocyte-macrophage lineage. In this study, we examined the effect of MIP-1 gamma, a C-C chemokine family member, on receptor activator of NF-kappa B ligand (RANKL)-stimulated osteoclast differentiation, survival, and activation. RANKL induced osteoclasts to dramatically increase production of MIP-1 gamma and to also express the MIP-1 gamma receptor CCR1, but had only minor effects on the related C-C chemokines MIP-1 alpha and RANTES. Neutralization of MIP-1 gamma with specific Ab reduced RANKL-stimulated osteoclast differentiation by 60-70%. Mature osteoclasts underwent apoptosis within 24 h after removal of RANKL, as shown by increased caspase 3 activity and DNA fragmentation. Apoptosis was reduced by the addition of exogenous MIP-1 gamma or RANKL, both of which increased NF-kappa B activation in osteoclasts. Neutralization studies showed that the prosurvival effect of RANKL was in part dependent on its ability to induce MIP-1 gamma. Finally, osteoclast activation for bone resorption was stimulated by MIP-1 gamma. Taken together, these results demonstrate that MIP-1 gamma plays an important role in the differentiation and survival of osteoclasts, most likely via an autocrine pathway.     

Cytosolic free calcium dependent regulation of osteoclast bone resorbing activity

Osteoclasts are sensitive to KCl-induced depolarization and to increased extracellular calcium concentration, and respond to these treatments with cytosolic calcium increase. In this study we evaluated the possibility that these experimental conditions could affect osteoclast bone resorption. We found that, incubating osteoclasts with 3H-proline previously labeled bone particles the resorbing activity was inhibited by both depolarization and extracellular calcium concentration increase. The released radioactivity was, in fact, 48% and 52% respectively compared to the untreated cultures. These data demonstrated that cytosolic calcium increase is one of the messengers of the pathway that inhibits, in this condition, bone resorption. Furthermore, as in parathyroid cells, extracellular calcium acts with a negative direct feedback mechanism that controls osteoclast activity.     

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

Background  

Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin α_vβ₃ increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.     

Pycnodysostosis: role and regulation of cathepsin K in osteoclast function and human disease

Patients with pycnodysostosis, a rare skeletal dysplasia, present with bone abnormalities such as short stature, acroosteolysis of distal phalanges, and skull deformities. The disease is caused by a deficiency of the cysteine protease cathepsin K which is responsible for degradation of collagen type I and other bone proteins. Osteoclasts, bone cells of hematopoietic origin responsible for bone mineral as well as protein matrix degradation, are dysfunctional in patients with pycnodysostosis due to mutations in the cathepsin K gene. Cathepsin K deficient osteoclasts can demineralize bone but cannot degrade the protein matrix. Mutations in the cathepsin K gene disrupting wild type cathepsin K activity have been described in patients with pycnodysostosis. Animal models of cathepsin K deficiency have been created and provide a valuable tool to study osteoclast function and treatment for cathepsin K deficiency. Understanding the regulation and role of cathepsin K in osteoclast function is important for designing future therapies for pycnodysostosis. Cathepsin K inhibitors will be useful in pathological processes involving excess osteoclast activation and bone resorption such as osteoporosis, bone metastasis and multiple myeloma. This review will discuss the bone remodeling cycle, the human disease pycnodysostosis caused by cathepsin K deficiency and cathepsin K activity and regulation.     

Osteoblastic cells: differentiation and trans-differentiation

The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Involvement of iNOS-dependent NO production in the stimulation of osteoclast survival by TNF-alpha

Osteoclasts, cells primarily responsible for bone resorption, differentiate from hematopoietic progenitor cells under the influence of various hormones, cytokines, and differentiation factors. Once fully differentiated, osteoclasts rapidly die in the absence of any survival factor. We have previously shown that tumor necrosis factor alpha (TNF-alpha) promotes the survival of differentiated osteoclasts. The expression of inducible nitric oxide synthase (iNOS) and consequent NO production is often stimulated under inflammatory conditions. In this study, we found that TNF-alpha, but not receptor activator of nuclear factor kappa B ligand and interleukin 1, increased the expression of iNOS both at the mRNA and protein levels. Subsequently, an enhanced NO level was detected both inside the cells and the culture medium of TNF-alpha-stimulated osteoclasts. Blocking NOS activity with L-NAME prevented TNF-alpha-induced NO generation by osteoclasts and the osteoclast survival stimulated by TNF-alpha. The iNOS selective inhibitor L-NIL also suppressed TNF-alpha-induced osteoclast survival, whereas low concentrations of NO releaser NOC-18 were sufficient to promote osteoclast survival. Furthermore, antiapoptotic and caspase suppressive effects of TNF-alpha on osteoclasts were abolished by L-NAME. Our findings indicate that iNOS-dependent NO generation contributes to the survival-promoting function of TNF-alpha in osteoclasts.     

A transmembrane osteoclastic protein-tyrosine phosphatase regulates osteoclast activity in part by promoting osteoclast survival through c-Src-dependent activation of NFkappaB and JNK2

This study evaluated the effects of overexpression of wild-type (WT) or phosphatase-deficient (PD) mutant of an osteoclastic protein-tyrosine phosphatase (PTP-oc) in RAW/C4 cells. Osteoclast-like cells derived from WT-PTP-oc overexpressing clones increased, while those derived from PD-PTP-oc expressing clones decreased, their resorption activity. WT-PTP-oc clones had lower apoptosis, lower caspase 3/7 activity, reduced c-Src tyr-527 phosphorylation (PY527) and IkappaBalpha cellular levels, and increased NFkappaB activation and JNK phosphorylation. Overexpression of PD-PTP-oc or PTP-oc siRNA treatment increased apoptosis, caspase 3/7 activity, PY527 and IkappaBalpha levels, and decreased NFkappaB and JNK2 activation. Inhibition of the c-Src kinase blocked the PTP-oc-mediated NFkappaB and JNK2 activation. Blocking the NFkappaB activation had no effect on the JNK2 activation. Inhibiting the NFkappaB and/or JNK2 pathway prevented the PTP-oc-mediated reduction in apoptosis. In conclusion, PTP-oc activates osteoclast activity in part by promoting osteoclast survival through the PTP-oc-mediated c-Src-dependent activation of NFkappaB and JNK2.     

Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.     

Role of monocytes in the inhibitory effect of calcitriol on PHA-stimulated lymphocytes

The possible role played by monocytes in the inhibitory effect of calcitriol on phytohemagglutinin (PHA)-stimulated lymphocyte proliferation was assessed by testing the effect of this sterol under different cell culture conditions. Calcitriol had a dose-dependent inhibitory effect on lymphocyte proliferation in concentrations ranging from 10(-10) up to 10(-8) M. The effect of 10(-9) M calcitriol was almost completely abolished by: a) monocyte depletion, b) inhibition of prostaglandin (PG) synthesis by indomethacin, and c) addition of exogenous interleukin-2 (IL-2). These results suggested that the inhibitory effect of calcitriol was mediated through monocytes. This possibility was substantiated by the following observations: a) the calcitriol inhibitory effect was restored when autologous adherent cells were added to monocyte-depleted PBM cells; b) the supernatant of adherent cells cultured for 24 hours in the presence of calcitriol exerted a marked inhibitory effect on lymphocyte proliferation; and c) this effect was not longer evident when adherent cells were cultured in the presence of calcitriol plus indomethacin. These data support the hypothesis that calcitriol acts, at least partially, through the monocytes, inducing an increased release of PG, with subsequent inhibition of IL-2 synthesis, then resulting in a decreased lymphocyte proliferation.     

Aging and calcitriol regulation of IP3 production in rat skeletal muscle and intestine.

We previously reported that calcitriol [1,25(OH)2-vitamin D3] in rat skeletal muscle and duodenum stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG), and that this mechanism is altered in old animals. As previously reported in muscle, we show in the present study that GTPgammaS (100 microM, 15 s), the non-hydrolyzable analogue of GTP, increased IP3 release from young rats duodenum to the same extent as 1 nM calcitriol (+ 100%), while GDPbetaS (100 microM) suppressed hormone-dependent IP3 production. Similarly to calcitriol, GTPgammaS response was diminished in old rats. Contrary to muscle, pretreatment with Bordetella pertussis toxin did not modify calcitriol-dependent IP3 in duodenum. The antibody, anti-G alpha q/11 (1:200) and anti-G alpha i (1:200) blocked calcitriol-dependent IP3 release in muscle from young rats, indicating that the hormone activates an isoform of PLC coupled to the alpha subunit of Gq/11 and possibly the betagamma subunits of Gi. The aged muscle was insensitive to anti G alpha i. In rat duodenum the hormone effects were suppressed by anti-Gq/11 both in young and aged animals. In 24-month-old rats, Gq/11 and Gi protein levels were greatly reduced both in muscle and duodenum, suggesting that a deficiency in G protein expression with aging may have important consequences for correct receptor/effector coupling and could explain age-related declines in the function of second messenger systems linked to G-proteins.     

The effect of calcitriol on natural killer cell activity in hemodialyzed patients

We report the effect of calcitriol on natural killer (NK) cell activity in patients with chronic renal failure undergoing long-term hemodialysis. Natural killer cytotoxicity was significantly decreased in these patients when compared to healthy control subjects (13.1 +/- 1.3 vs 38.8 +/- 2.4%, P less than 0.001). These patients also have decreased levels of calcitriol (17 +/- 3 vs 36 +/- 3 pg/ml, P less than 0.001). After 14 days of oral treatment with calcitriol at a dose of 0.5 micrograms per day, a significant increase in NK activity was observed (20.2 +/- 1.6%, P less than 0.001). This increase was maintained after 28 days of treatment (21.1 +/- 2%, P less than 0.001). These results suggest that the decreased serum calcitriol might contribute to the diminished NK activity found in hemodialyzed patients, and suggests a new potential therapeutical utility of calcitriol as modulator of the immune function in these patients.