Karen Fuller

A crucial role for thiol antioxidants in estrogen-deficiency bone loss.

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-beta estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while l-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-beta estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-kappaB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-alpha, a target for NF-kappaB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-beta estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption.

TNFα Potently Activates Osteoclasts, through a Direct Action Independent of and Strongly Synergistic with RANKL

TNFalpha is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFalpha promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFalpha promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFalpha was equipotent with RANKL for this action. Activation by TNFalpha was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFalpha promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFalpha strongly synergized with RANKL, such that miniscule concentrations of TNFalpha were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFalpha suggests that the most sensitive osteolytic response of bone to TNFalpha is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.

Calcitonin gene-related peptide inhibits osteoclastic bone resorption: a comparative study.

SummaryBesides the calcitonin (CT) precursor, the calcitonin gene also encodes another peptide—calcitonin gene-related peptide (CGRP). We have previously reported that CGRP lowers plasma calcium in the rat. In the present study we have evaluated the effect of CGRP on resorption of bone by isolated rat osteoclasts and have compared these effects to those produced by calcitonins from three species (salmon, pig, and human calcitonins). There was a significant inhibition of bone resorption with rat calcitonin gene-related peptide (rCGRP) at a 1000-fold higher dose than that used for human CT. This effect well explains the CT-like effect of CGRP seen in thein vivo rat CT bioassay. Our results suggest that though CGRP may not be involved in the hormonal control of plasma calcium, the peptide may be an important local regulator of bone cell function.

CCL9/MIP‐1γ and its receptor CCR1 are the major chemokine ligand/receptor species expressed by osteoclasts

Although much has been learned recently of the mechanisms by which the differentiation of osteoclasts is induced, less is known of the factors that regulate their migration and localization, and their interactions with other bone cells. In related cell types, chemokines play a major role in these processes. We therefore systematically tested the expression of RNA for chemokines and their receptors by osteoclasts. Because bone is the natural substrate for osteoclasts and may influence osteoclast behavior, we also tested expression on bone slices. Quantitative RT‐PCR using real‐time analysis with SYBR Green was therefore performed on RNA isolated from bone marrow cells after incubation with macrophage‐colony stimulating factor (M‐CSF) with/without receptor‐activator of NFκB ligand (RANKL), on plastic or bone. We found that RANKL induced expression of CCL9/MIP‐1γ to levels comparable to that of tartrate‐resistant acid phosphatase (TRAP), a major specialized product of osteoclasts. CCL22/MDC, CXCL13/BLC/BCA‐1, and CCL25/TECK were also induced. The dominant chemokine receptor expressed by osteoclasts was CCR1, followed by CCR3 and CX3CR1. Several receptors expressed on macrophages and associated with inflammatory responses, including CCR2 and CCR5, were down‐regulated by RANKL. CCL9, which acts through CCR1, stimulated cytoplasmic motility and polarization in osteoclasts, identical to that previously observed in response to CCL3/MIP‐1α, which also acts through CCR1 and is chemotactic for osteoclasts. These results identify CCL9 and its receptor CCR1 as the major chemokine and receptor species expressed by osteoclasts, and suggest a crucial role for CCL9 in the regulation of bone resorption. J. Cell. Biochem. 87: 386–393, 2002.

Secretion of tartrate‐resistant acid phosphatase by osteoclasts correlates with resorptive behavior

There have been dramatic advances recently in our understanding of the regulation of osteoclastic differentiation. However, much less is known of the mechanisms responsible for the induction and modulation of resorptive behavior. We have developed a strategy whereby osteoclasts can be generated in vitro and released into suspension in a fully‐functional state. We now exploit this approach to show that tartrate‐resistant acid phosphatase (TRAP) is released by osteoclasts during bone resorption. TRAP release was inhibited by the secretion‐inhibitor Brefeldin A, and was not accompanied by LDH release. This suggests that TRAP release is due to secretion, rather than cell death. Consistent with this, TRAP secretion was stimulated by resorbogenic cytokines, was inhibited by the resorption‐inhibitor calcitonin, and correlated with excavation of the bone surface. We found that, in contrast to incubation on bone, incubation on plastic, glass, or vitronectin‐coated plastic substrates did not induce secretion of TRAP. This suggests that the induction of resorptive behavior in osteoclasts depends upon stimulation by bone matrix of a putative osteoclastic “mineral receptor.” Release of TRAP by osteoclasts thus represents not only a productive approach to the analysis of the mechanisms that modulate the rate of resorptive activity, but also a system whereby the mechanism through which bone substrates induce resorptive behavior can be identified. J. Cell. Biochem. 98: 1085–1094, 2006.

TGF-β1 and IFN-γ Direct Macrophage Activation by TNF-α to Osteoclastic or Cytocidal Phenotype

TNF-related activation-induced cytokine (TRANCE; also called receptor activator of NF-κB ligand (RANKL), osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and TNFSF11) induces the differentiation of progenitors of the mononuclear phagocyte lineage into osteoclasts in the presence of M-CSF. Surprisingly, in view of its potent ability to induce inflammation and activate macrophage cytocidal function, TNF-α has also been found to induce osteoclast-like cells in vitro under similar conditions. This raises questions concerning both the nature of osteoclasts and the mechanism of lineage choice in mononuclear phagocytes. We found that, as with TRANCE, the macrophage deactivator TGF-β1 strongly promoted TNF-α-induced osteoclast-like cell formation from immature bone marrow macrophages. This was abolished by IFN-γ. However, TRANCE did not share the ability of TNF-α to activate NO production or heighten respiratory burst potential by macrophages, or induce inflammation on s.c. injection into mice. This suggests that TGF-β1 promotes osteoclast formation not only by inhibiting cytocidal behavior, but also by actively directing TNF-α activation of precursors toward osteoclasts. The osteoclast appears to be an equivalent, alternative destiny for precursors to that of cytocidal macrophage, and may represent an activated variant of scavenger macrophage.

Osteoclast resorption — stimulating activity is associated with the osteoblast cell surface and/or the extracellular matrix

Osteoblasts mediate much of the hormonal responsiveness of osteoclasts. We and others have found that one mechanism through which this regulation is effected is by release of osteoclast resorption-stimulating activity (ORSA) into culture supernatants. However, although hormonal responsiveness is regularly observed in co-cultures, ORSA is not always detectable in conditioned media. We show here that one explanation for this finding is that ORSA may be retained by heparin-like glycosaminoglycans (GAGs) of the cell surface or extracellular matrix of osteoblasts. We found that protease-sensitive ORSA could be extracted from monolayers of the osteoblastic cell line UMR 106 with 2M NaCl or collagenase. Production of this activity was increased in response to 1,25(OH)2D3. The presence of the GAG heparin was required to reveal ORSA. Immobilisation of ORSA by GAGs may assist osteoblastic cells in the regulation of the complex patterns of osteoclastic activity observed during skeletal morphogenesis and restructuring.

Osteoclast Lineage Commitment of Bone Marrow Precursors Through Expression of Membrane-bound TRANCE

Osteoclast formation from hemopoietic precursors is induced by TRANCE (also called RANKL, ODF, and OPGL), a membrane-bound ligand expressed by bone marrow stromal cells. Because soluble recombinant TRANCE is a suboptimal osteoclastogenic stimulus, and to eliminate the need for such dependence on stromal cells, membrane-bound TRANCE was expressed in hematopoietic precursors using retroviral gene transfer. Four TRANCE-expressing osteoclast cell lines were established that continuously generate large numbers of multinucleated cells and express tartrate-resistant acid phosphatase and calcitonin receptors. The multinuclear cells are long-lived and either fuse continuously with each other and with mononuclear cells to form enormous syncytia, or separate to form daughter multinuclear cells. When formed on bone, but not on plastic, the majority of multinuclear cells develop actin rings on bone, and resorb bone, suggesting that bone matrix may provide additional signals that facilitate osteoclastic functional maturation. Surprisingly, multinuclear cells originate from fusion of proliferating mononuclear cells that strongly express the mature macrophage markers F4/80 and Fc receptor, which are not expressed by osteoclasts. These results indicate that osteoclasts can be derived from F4/80-positive and Fc receptor-positive cells, and that TRANCE induces osteoclastic differentiation partly by suppressing the macrophage phenotype.

Osteoclast activation: Potent inhibition by the bisphosphonate alendronate through a nonresorptive mechanism

Alendronate, an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its mechanism of action is unknown. Because it localizes to bone surfaces, we compared the sensitivity of components of the resorptive process to incubation on alendronate‐coated bone surfaces. We found that bone resorption by osteoclasts isolated from neonatal rat bone was unaffected by alendronate (10‐4 M). Osteoclast production in bone marrow cultures, as assessed by the production of calcitonin‐receptor positive cells, was observed even at 10‐4 M, but bone resorption in these cultures was almost completely abolished by 10‐6 M alendronate. The greater sensitivity of osteoclast activation to inhibition by alendronate that these results suggest was supported by similar inhibition of osteoblast‐mediated activation of osteoclasts from neonatal rat bone. Thus, activation of osteoclasts by osteoblastic/stromal cells is apparently the most sensitive component of the pathway whereby bone resorption is affected. Moreover, the ability of alendronate to suppress osteoclastic activation does not depend on resorption‐mediated release of alendronate from bone surfaces. This ability extends the range of cell types and processes that might be affected by alendronate, beyond those in the immediate vicinity of resorbing cells, to include any cell that comes into contact with alendronate‐coated bone surfaces. J. Cell. Physiol. 172:79–86, 1997.

Activation of osteoclasts by interleukin-1: divergent responsiveness in osteoclasts formed in vivo and in vitro.

Recently, it has been found that osteoclasts are induced and activated by osteoblastic cells through expression of receptor activator NF‐kB ligand (RANKL), and that soluble recombinant RANKL, with M‐CSF, can replace the need for osteoblastic cells in osteoclast formation. We exploited this opportunity to compare the responsiveness of osteoclast‐like cells (OCL) formed in vitro in the absence of osteoblasts, with that of osteoclasts ex vivo. We found that while OCL responded to several hormones and cytokines like ex vivo osteoclasts, their responsiveness to interleukin‐1 (IL‐1) was fundamentally different: IL1 directly stimulated actin ring formation in OCL, but had no effect on actin rings or survival in osteoclasts ex vivo unless osteoblastic cells were present. This difference could not be attributed to the use of plastic culture substrates for OCL formation, nor to osteoblastic contamination, and did not seem to be mediated by the macrophages that form in OCL cultures. To understand the mechanisms by which IL‐1 induces bone loss, it will need to be determined whether or not IL‐1‐responsive OCLs have a counterpart in vivo. Whichever is the case, our data suggest that the behavior of osteoclasts formed in culture will not always predict that of osteoclasts in vivo. J. Cell. Physiol. 184:334–340, 2000.