[Show abstract][Hide abstract] ABSTRACT: We undertook genetic and nongenetic approaches to investigate the relationship between telomere maintenance and osteoblast differentiation, as well as to uncover a possible link between a known mediator of cellular aging and senile bone loss. Using mouse models of disrupted telomere maintenance molecules, including mutants in the Werner helicase (Wrn(-/-) ), telomerase (Terc(-/-) ), and Wrn(-/-) Terc(-/-) double mutants predisposed to accelerated bone loss, we measured telomere dysfunction-induced foci (TIFs) and markers of osteoblast differentiation in mesenchymal progenitor cells (MPCs). We found that telomere maintenance is directly and significantly related to osteoblast differentiation, with dysfunctional telomeres associated with impaired differentiation independent of proliferation state. Telomere-mediated defects in osteoblast differentiation are associated with increased p53/p21 expression and concomitant reduction in RUNX2. Conversely, MPCs from p53(-/-) mice do not have substantial telomere dysfunction and spontaneously differentiate into osteoblasts. These results suggest that critical telomere dysfunction may be a prominent mechanism for age-related osteoporosis and limits MPC differentiation into bone-forming cells via the p53/p21 pathway.
[Show abstract][Hide abstract] ABSTRACT: The regulation of NFATc1 expression is important for osteoclast differentiation and function. Herein, we demonstrate that macrophage-colony-stimulating factor induces NFATc1 degradation via Cbl proteins in a Src kinase-dependent manner. NFATc1 proteins are ubiquitinated and rapidly degraded during late stage osteoclastogenesis, and this degradation is mediated by Cbl-b and c-Cbl ubiquitin ligases in a Src-dependent manner. In addition, NFATc1 interacts endogenously with c-Src, c-Cbl, and Cbl-b in osteoclasts. Overexpression of c-Src induces down-regulation of NFATc1, and depletion of Cbl proteins blocks NFATc1 degradation during late stage osteoclastogenesis. Taken together, our data provide a negative regulatory mechanism by which macrophage-colony-stimulating factor activates Src family kinases and Cbl proteins, and subsequently, induces NFATc1 degradation during osteoclast differentiation.
Journal of Biological Chemistry 02/2010; 285(8):5224-31. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Involvement of Treg in transplant tolerance has been demonstrated in multiple models. During the active process of graft rejection, these regulatory cells are themselves regulated and inactivated, a process termed counter-regulation. We hypothesize that ligation of the costimulatory molecule glucocorticoid-induced TNF receptor-related protein (GITR) on Treg inhibits their ability to promote graft survival, and by blocking GITR ligation graft survival can be prolonged. To this aim, we have designed a soluble GITR fusion protein (GITR-Fc), which binds GITR ligand and inhibits activation of GITR. Here, we show that GITR-Fc prolonged mouse skin graft survival, and this prolongation is dependent on Treg. In a full MHC-mismatched skin graft setting, GITR-Fc significantly improved graft survival when used in combination with MR1, anti-CD40L, while GITR-Fc alone did not demonstrate graft prolongation. These results demonstrate that disruption of binding of GITR with GITR ligand may be an important strategy in prolonging allograft survival.
European Journal of Immunology 02/2010; 40(5):1369-74. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many models of transplant tolerance have been found to depend on the induction of regulatory T cells (Tregs). Innate immune signals are known to suppress Tregs thereby augmenting immunity by abrogating Treg function. Such signals may also provide a barrier to transplantation tolerance mediated by Tregs. A number of cell surface molecules expressed by Tregs have been found to inhibit Treg activity, the best characterized of which is the glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein.
By using an adoptive transfer model of allograft rejection, we can study the effects of inflammation and antigen-specific Tregs on graft survival. Inflammation resulting from the transplant procedure counter-regulates the suppressor activity of Tregs. To assess whether Treg activity could be enhanced by blocking GITR signaling, we compared the capacity of Tregs to prolong the survival of grafts in the presence or absence of activation-inducible TNF receptor (AITRL)-Fc, a novel construct that binds GITR.
We report that interruption of GITR-GITR ligand (GITRL) binding by AITRL-Fc resulted in long-term Treg-dependent acceptance of skin grafts in the setting of innate immune signals that otherwise interfere with Treg activity.
Inflammation and other innate immune signals may activate antigen presenting cells to upregulate GITRL. GITR-GITRL interaction is one pathway by which antigen presenting cells may enhance the adaptive response to foreign antigen by counter-regulating Tregs and by costimulating effector T cells. By blocking this interaction with AITRL-Fc, one can sustain the benefit conferred by graft-protective Tregs.
[Show abstract][Hide abstract] ABSTRACT: It has become clear that complex interactions underlie the relationship between the skeletal and immune systems. This is particularly true for the development of immune cells in the bone marrow as well as the functions of bone cells in skeletal homeostasis and pathologies. Because these two disciplines developed independently, investigators with an interest in either often do not fully appreciate the influence of the other system on the functions of the tissue that they are studying. With these issues in mind, this review will focus on several key areas that are mediated by crosstalk between the bone and immune systems. A more complete appreciation of the interactions between immune and bone cells should lead to better therapeutic strategies for diseases that affect either or both systems.
[Show abstract][Hide abstract] ABSTRACT: NFATc1 has been characterized as a master regulator of nuclear factor kappaB ligand-induced osteoclast differentiation. Herein, we demonstrate a novel role for NFATc1 as a positive regulator of nuclear factor kappaB ligand-mediated osteoclast fusion as well as other fusion-inducing factors such as TNF-alpha. Exogenous overexpression of a constitutively active form of NFATc1 in bone marrow-derived monocyte/macrophage cells (BMMs) induces formation of multinucleated osteoclasts as well as the expression of fusion-mediating molecules such as the d2 isoform of vacuolar ATPase V(o) domain (Atp6v0d2) and the dendritic cell-specific transmembrane protein (DC-STAMP). Moreover, inactivation of NFATc1 by cyclosporin A treatment attenuates expression of Atp6v0d2 and DC-STAMP and subsequent fusion process of osteoclasts. We show that NFATc1 binds to the promoter regions of Atp6v0d2 and DC-STAMP in osteoclasts and directly induces their expression. Furthermore, overexpression of Atp6v0d2 and DC-STAMP rescues cell-cell fusion of preosteoclasts despite reduced NFATc1 activity. Our data indicate for the first time that the NFATc1/Atp6v0d2 and DC-STAMP signaling axis plays a key role in the osteoclast multinucleation process, which is essential for efficient bone resorption.
[Show abstract][Hide abstract] ABSTRACT: Osteoporosis and the associated risk of fracture are major clinical challenges in the elderly. Telomeres shorten with age in most human tissues, including bone, and because telomere shortening is a cause of cellular replicative senescence or apoptosis in cultured cells, including mesenchymal stem cells (MSCs) and osteoblasts, it is hypothesized that telomere shortening contributes to the aging of bone. Osteoporosis is common in the Werner (Wrn) and dyskeratosis congenita premature aging syndromes, which are characterized by telomere dysfunction. One of the targets of the Wrn helicase is telomeric DNA, but the long telomeres and abundant telomerase in mice minimize the need for Wrn at telomeres, and thus Wrn knockout mice are relatively healthy. In a model of accelerated aging that combines the Wrn mutation with the shortened telomeres of telomerase (Terc) knockout mice, synthetic defects in proliferative tissues result. Here, we demonstrate that deficiencies in Wrn-/- Terc-/- mutant mice cause a low bone mass phenotype, and that age-related osteoporosis is the result of impaired osteoblast differentiation in the context of intact osteoclast differentiation. Further, MSCs from single and Wrn-/- Terc-/- double mutant mice have a reduced in vitro lifespan and display impaired osteogenic potential concomitant with characteristics of premature senescence. These data provide evidence that replicative aging of osteoblast precursors is an important mechanism of senile osteoporosis.
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts play an important role in bone metabolism by resorbing the bone matrix. These cells originate from hematopoietic
precursors and share a common progenitor with macrophages and dendritic cells (DCs). Two essential cytokines, macrophage colony-stimulating
factor (M-CSF) and TRANCE (also called RANKL, OPGL, and ODF), enable osteoclast differentiation from their monocyte/macrophage
lineage precursors (Suda, Takahashi, Udagawa, et al. 1999; Yasuda, Shima, Nakagawa et al. 1998; Lacey, Timms, Tan, et al.
TRANCE, a TNF family member, supports osteoclast differentiation, survival, and activation. Binding of TRANCE to its receptor,
receptor activator of nuclear factor κB (RANK), activates multiple signaling pathways mediated by TNF receptor-associated
factors (TRAFs), including NF-κB, c-Jun N-terminal kinase (JNK), p38 MAP kinase, extracellular signal-related kinase (ERK),
and AKT (Lee and Kim 2003; Boyle, Simonet and Lacey 2003).
It has been shown that TRANCE induces activation and/or induction of transcription factors such as Mitf, PU.1, and NFATc1
(Boyle, Simonet and Lacey 2003; Teitelbaum 2000; Teitelbaum and Ross 2003). Mitf is known to be important for osteoclastogenesis
in vitro and in vivo (Holtrop, Cox, Eilon, et al. 1981; Thesingh and Scherft 1985; Luchin, Purdom, Murphy, et al. 2000).TRANCE activates Mitf
via the MKK6/p38 signaling cascade. Subsequently, activated Mitf induces the expression of target genes, including TRAP, cathepsin
K, and OSCAR, which are important for osteoclast differentiation or function (Luchin, Purdom, Murphy, et al. 2000; Motyckova,
Weilbaecher, Horstmann, et al. 2001; Mansky, Sankar, Han, et al. 2002; So, Rho, Jeong, et al. 2003; Kim, Takami, Rho, et al.
2002), by binding to the canonical E-box sequence in the promoter region of those genes.
Advances in Experimental Medicine and Biology 02/2007; 602:23-31. · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H(+)) ATPase (v-ATPase) V(0) domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.
Nature Medicine 01/2007; 12(12):1403-9. · 28.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) induces osteoclast formation from monocyte/macrophage lineage cells via various transcription factors, including the Mi transcription factor (Mitf). Here, we show that inhibitors of differentiation/DNA binding (Ids), helix-loop-helix (HLH) transcription factors, negatively regulate TRANCE-induced osteoclast differentiation. Expression levels of Id1, Id2, and Id3 genes are significantly reduced by TRANCE during osteoclastogenesis. Interestingly, overexpression of the 3 Id genes in bone marrow-derived monocyte/macrophage lineage cells (BMMs) inhibits the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts, but it does not alter the ability of BMMs to either phagocytose or differentiate into dendritic cells (DCs). Overexpression of Id2 in BMMs attenuates the gene induction of nuclear factor of activated T cells c1 (NFATc1) and osteoclast-associated receptor (OSCAR) during TRANCE-mediated osteoclastogenesis. Furthermore, Id proteins interact with Mitf, a basic HLH (bHLH) transcription factor, and inhibit its transactivation of OSCAR, which is a costimulatory receptor expressed by osteoclast precursors, by attenuating the DNA binding ability of Mitf to the E-box site of the OSCAR promoter. Taken together, our results reveal both a new facet of negative regulation, mediated by Id proteins, as well as the mechanism whereby TRANCE signaling overcomes it, allowing osteoclastogenesis to proceed.
[Show abstract][Hide abstract] ABSTRACT: Osteoclast differentiation from hematopoietic precursors is controlled by the tumor necrosis factor family member tumor necrosis factor-related activation-induced cytokine (TRANCE) via induction of various transcription factors, including nuclear factor of activated T cells (NFAT) c1. During osteoclast differentiation, NFATc1 is further activated via calcium signaling when costimulatory receptors expressed on osteoclast precursors, such as osteoclast-associated receptor (OSCAR), are stimulated. Here we show that NFATc1 expression precedes that of OSCAR during TRANCE-mediated osteoclastogenesis and that inhibition of NFATc1 by cyclosporin A abolishes TRANCE-induced OSCAR expression and subsequent osteoclast differentiation. Moreover, we show that the 1.0-kb promoter region of the OSCAR gene contains three potential NFATc1-binding sites. Induction of an OSCAR promoter-luciferase reporter is significantly increased when transiently transfected into 293T cells in combination with NFATc1 expression plasmid. Deletion and site-directed mutant constructs confirmed that NFATc1-binding sites are both functional and NFATc1-specific. Furthermore, NFATc1 synergistically activates an OSCAR reporter construct together with microphthalmia transcription factor and PU.1, transcription factors previously shown to be critical for osteoclast differentiation. In addition, a plasmid expressing constitutively active MAP kinase kinase 6 enhances the transactivation activity of NFATc1/microphthalmia transcription factor/PU.1 on the OSCAR promoter. Taken together, our results indicate that NFATc1 is an important transcription factor in the induction of OSCAR during osteoclastogenesis. Elucidation of NFATc1 as a transcription factor for OSCAR expression implies the presence of a positive feedback circuit of TRANCE-induced activation of NFATc1, involving NFATc1-mediated OSCAR expression and its subsequent activation of NFATc1, necessary for efficient differentiation of osteoclasts.
Journal of Biological Chemistry 11/2005; 280(42):35209-16. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.
Journal of Experimental Medicine 10/2005; 202(5):589-95. · 13.91 Impact Factor