Deborah V. Novack

Mice lacking β3 integrins are osteosclerotic because of dysfunctional osteoclasts

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-α

Estrogen deficiency induces bone loss by upregulating osteoclastogenesis by mechanisms not completely defined. We found that ovariectomy-enhanced T-cell production of TNF-alpha, which, acting through the TNF-alpha receptor p55, augments macrophage colony-stimulating factor-induced (M-CSF-induced) and RANKL-induced osteoclastogenesis. Ovariectomy failed to induce bone loss, stimulate bone resorption, or increase M-CSF- and RANKL-dependent osteoclastogenesis in T-cell deficient mice, establishing T cells as essential mediators of the bone-wasting effects of estrogen deficiency in vivo. These findings demonstrate that the ability of estrogen to target T cells, suppressing their production of TNF-alpha, is a key mechanism by which estrogen prevents osteoclastic bone resorption and bone loss.

M-CSF mediates TNF-induced inflammatory osteolysis

TNF-alpha is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokines osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-alpha stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-kappaB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-alpha enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti-c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-alpha, anti-c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.

The IκB Function of NF-κB2 p100 Controls Stimulated Osteoclastogenesis

The prototranscription factor p100 represents an intersection of the NF-κB and IκB families, potentially serving as both the precursor for the active NF-κB subunit p52 and as an IκB capable of retaining NF-κB in the cytoplasm. NF-κB–inducing kinase (NIK) controls processing of p100 to generate p52, and thus NIK-deficient mice can be used to examine the biological effects of a failure in such processing. We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100. In the absence of NIK, p100 expression is increased by RANKL, but its conversion to p52 is blocked, leading to cytosolic accumulation of p100, which, acting as an IκB protein, binds NF-κB complexes and prevents their nuclear translocation. High levels of unprocessed p100 in osteoclast precursors from NIK−/− mice or a nonprocessable form of the protein in wild-type cells impair RANKL-mediated osteoclastogenesis. Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL. These data demonstrate a novel, biologically relevant means of regulating NF-κB signaling, with upstream control and kinetics distinct from the classical IκBα pathway.

The IB Function of NF- B2 p100 Controls Stimulated Osteoclastogenesis

The prototranscription factor p100 represents an intersection of the NF-κB and IκB families, potentially serving as both the precursor for the active NF-κB subunit p52 and as an IκB capable of retaining NF-κB in the cytoplasm. NF-κB–inducing kinase (NIK) controls processing of p100 to generate p52, and thus NIK-deficient mice can be used to examine the biological effects of a failure in such processing. We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100. In the absence of NIK, p100 expression is increased by RANKL, but its conversion to p52 is blocked, leading to cytosolic accumulation of p100, which, acting as an IκB protein, binds NF-κB complexes and prevents their nuclear translocation. High levels of unprocessed p100 in osteoclast precursors from NIK−/− mice or a nonprocessable form of the protein in wild-type cells impair RANKL-mediated osteoclastogenesis. Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL. These data demonstrate a novel, biologically relevant means of regulating NF-κB signaling, with upstream control and kinetics distinct from the classical IκBα pathway.

PLCγ2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2

Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cgamma (PLCgamma) isoforms are known modulators of T and B lymphocyte-mediated immune responses, we found that blockade of PLCgamma enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCgamma2, independent of PLCgamma1, was required for receptor activator of NF-kappaB ligand-induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator protein-1 (AP1), and NF-kappaB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCgamma2 downstream of Dap12/Fc receptor gamma (Dap12/FcRgamma) receptors and is blocked by the PLCgamma inhibitor U73122. In contrast, activation of JNK and NF-kappaB was not affected by U73122 or Dap12/FcRgamma deletion. Interestingly, we found that in osteoclasts, PLCgamma2 formed a complex with the regulatory adapter molecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCgamma2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy.

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by β3 integrin

The β3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony–stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the β integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various β3 integrins with cytoplasmic tail mutations in β3-deficient OC precursors. We find that S752 in the β3 cytoplasmic tail is required for growth factor–induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional β3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on β3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional β3. Instead, its activation is dependent upon intracellular calcium, and on the β2 integrin. Thus, the β3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

Parathyroid hormone stimulates osteoblastic expression of MCP-1 to recruit and increase the fusion of pre/osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-κB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-κB ligand stimulation to initiate greater bone remodeling.

Marrow Stromal Cells and Osteoclast Precursors Differentially Contribute to TNF-α-Induced Osteoclastogenesis In Vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-κB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-α-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-α. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-α treatment or whether the recipient animals possess TNF-α-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-α requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-α increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-α is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-α depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-α may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.

A Glanzmann’s mutation in β3 integrin specifically impairs osteoclast function

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the αvβ3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the β3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of β3–/– osteoclast function following expression of a full-length β3 integrin. In contrast, truncated β3, lacking a cytoplasmic domain (hβ3Δc), is completely ineffective in restoring function to β3–/– osteoclasts. To identify the components of the β3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate β integrin signaling. Of the six, only the S752P substitution, which also characterizes a form of the human bleeding disorder Glanzmann’s thrombasthenia, fails to rescue β3–/– osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y747F/Y759F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the β3 integrin regulates platelets and osteoclasts.