Simon W. Fox

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.

Role of Nitric Oxide and Prostaglandins in Mechanically Induced Bone Formation

We have previously shown that prostaglandins (PG) and nitric oxide (NO) are required in the induction of bone formation by mechanical stimulation. We therefore tested the ability of NO donors, S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP), and S‐nitroso‐glutathione (GSNO) to mimic or augment the osteogenic response of bone to a minimal mechanical stimulus. In rats administered vehicle or the vasodilator hydralazine, stimulation of the 8th caudal vertebra increased bone formation. In animals treated with SNAP or GSNO, there was significant potentiation of this osteogenic response. The bone formation rate in nonloaded vertebrae was unaffected by administration of the NO donors. We also found that while inhibition of either PG or NO production at the time of loading caused a partial suppression of c‐fos mRNA expression in the loaded vertebrae, administration of indomethacin and NG‐monomethyl‐L‐arginine together markedly suppressed c‐fos expression. This suggests that although both PG and NO are required in mechanically induced osteogenesis, they appear to be generated largely independently of each other. Moreover, while exogenous NO potentiates the stimulatory effect of mechanical loading on bone formation, the lack of effect in nonloaded vertebrae suggests that NO is necessary but not sufficient for induction of bone formation.

Interleukin-10 inhibits osteoclastogenesis by reducing NFATc1 expression and preventing its translocation to the nucleus

BackgroundIL-10 has a potent inhibitory effect on osteoclastogenesis. In vitro and in vivo studies confirm the importance of this cytokine in bone metabolism, for instance IL-10-deficient mice develop the hallmarks of osteoporosis. Although it is known that IL-10 directly inhibits osteoclastogenesis at an early stage, preventing differentiation of osteoclast progenitors to preosteoclasts, the precise mechanism of its action is not yet clear. Several major pathways regulate osteoclastogenesis, with key signalling genes such as p38, TRAF6, NF-κB and NFATc1 well established as playing vital roles. We have looked at gene expression in eleven of these genes using real-time quantitative PCR on RNA extracted from RANKL-treated RAW264.7 monocytes.ResultsThere was no downregulation by IL-10 of DAP12, FcγRIIB, c-jun, RANK, TRAF6, p38, NF-κB, Gab2, Pim-1, or c-Fos at the mRNA level. However, we found that IL-10 significantly reduces RANKL-induced NFATc1 expression. NFATc1 is transcribed from two alternative promoters in Mus musculus and, interestingly, only the variant transcribed from promoter P1 and beginning with exon 1 was downregulated by IL-10 (isoform 1). In addition, immunofluorescence studies showed that IL-10 reduces NFATc1 levels in RANKL-treated precursors and suppresses nuclear translocation. The inhibitory effect of IL-10 on tartrate-resistant acid phosphatase-positive cell number and NFATc1 mRNA expression was reversed by the protein kinase C agonist phorbol myristate acetate, providing evidence that interleukin-10 disrupts NFATc1 activity through its effect on Ca2+ mobilisation.ConclusionIL-10 acts directly on mononuclear precursors to inhibit NFATc1 expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of Ca2+ mobilisation. We detected downregulation only of the NFATc1 isoform 1 transcribed from promoter P1. This is the first report indicating that one of the ways in which IL-10 directly inhibits osteoclastogenesis is by suppressing NFATc1 activity.

Nitric oxide synthase expression in bone cells.

We have localized the expression of the three main nitric oxide synthases (eNOS, bNOS, and iNOS) in bone cells of rats and humans using immunohistochemistry. The predominant isoform expressed in normal adult bone was the constitutive isoform, eNOS, mainly in cells of osteoblastic lineage. In adult bone, the osteoblast lineage cells exhibiting eNOS expression were flat bone lining cells and osteocytes, but cuboidal osteoblasts were consistently negative. Expression for bNOS was not detected in any bone cells. iNOS expression was not detected in any cells of osteoblastic lineage in normal adult rat or human bone, but was observed in cuboidal osteoblasts of adult rats with experimental colitis, in which the suppression in bone formation may be cytokine mediated. Osteoclasts in normal rat tissue showed expression for both eNOS and iNOS, but these were patchy. As for cells of the osteoblast lineage, osteoclasts were negative for bNOS. Thus, our findings support evidence, from in vitro studies and from animal experiments, that nitric oxide may play an important role in the physiology of bone.

Role for parathyroid hormone in mechanical responsiveness of rat bone

We investigated the relationship between parathyroid hormone (PTH) and mechanical stimulation in mechanically induced osteogenesis. In normal rats, mechanical stimulation of the eighth caudal vertebra induced an osteogenic response. This was augmented by a single injection of human PTH-(1-34) 30-45 min before loading. No osteogenic response was seen in thyroparathyroidectomized (TPTX) rats; the osteogenic response was restored by a single injection of PTH before stimulation, suggesting that physiological levels of PTH are necessary for the mechanical responsiveness of bone. c- fosexpression was detected only in the osteocytes of those rats that were both mechanically stimulated and given PTH. This suggests that PTH supports mechanically induced osteogenesis by sensitizing either the strain-sensing mechanism itself or early responses of bone to strain-generated signals. The osteogenic response was not augmented by two further daily injections of PTH and was not seen in TPTX rats in which PTH administration was started 3 days after loading. These results reveal a major role for PTH in the mechanical responsiveness of rat bone.

Mechanical loading stimulates bone formation by reactivation of bone lining cells in 13-week-old rats.

The bone formation that occurs in response to mechanical stimulation is generally considered to be a means by which bone adapts to changes in its mechanical environment. We have previously shown that the expression of genes for bone matrix proteins is maximal 72 h after a single 5‐minute episode of loading of tail vertebrae of 13‐week‐old female rats, that the predominant increase in mineralization occurs after 3 days, and that the osteogenic response to mechanical stimulation is not dependent on prior bone resorption. We have now investigated the cellular correlates of this osteogenic response. No proliferation was detected, by pulse or flash labeling, in the trabecular bone surface cells of animals killed 1 h to 10 days after the loading episode. Ultrastructural examination revealed that most of the cells covering the trabecular bone surface of control vertebrae were flat bone lining cells. After mechanical stimulation, the trabecular bone surface cells developed ultrastructural features of osteoblastic differentiation and activity, with acquisition of an increasingly cuboidal shape, rounded nuclei, and abundant rough endoplasmic reticulum. Morphometric analysis of the mean cell area, mean nuclear area, and cell and nuclear height showed that they were all maximal 48 h after loading. By 120 h after loading, the appearances of bone surface cells had reverted to those of control vertebrae. Thus, mechanical loading appears to activate lining cells, with a temporal sequence that correlates with bone matrix production.

Current insights into the role of transforming growth factor-β in bone resorption

Transforming growth factor-beta (TGF-beta) elicits a variety of effects on cellular proliferation and differentiation. The major repository for TGF-beta is bone, where it possesses separate facilitative and suppressive actions on osteoclast differentiation and bone resorption. Without a direct enabling stimulus from TGF-beta monocytes cannot form osteoclasts but instead follow macrophage differentiation pathways. This facilitative action depends on an ability to promote a state in which precursors are resistant to anti-osteoclastic inflammatory signals. Following the initiation of resorption TGF-beta is released from bone matrix. This acts on osteoblasts to reduce the availability of the osteoclast differentiation factor, RANKL (receptor activator of NFkappaB ligand) and thereby indirectly limits further osteoclast formation. Thus TGF-beta has a fundamental role in the control of bone resorption having actions that first allow monocytes to develop into osteoclasts then subsequently limiting the extent and duration of resorption after its release from the bone matrix.

The Possible Role of TGF-β-Induced Suppressors of Cytokine Signaling Expression in Osteoclast/Macrophage Lineage Commitment In Vitro

Osteoclast formation is dependent on the ability of TGF-β to enable receptor activator of NF-κB ligand (RANKL)-induced commitment of hemopoietic precursors to the osteoclastic lineage. The mechanism by which TGF-β enables formation is unknown. One possibility is that TGF-β opposes Janus kinase (JAK)/STAT signals generated by inhibitory cytokines such as IFN-β. The JAK/STAT pathway is activated by cytokines that induce resistance to osteoclast formation, such as IFN-γ and M-CSF, and the effect of these is opposed by TGF-β. Recently, a group of STAT-induced factors, termed suppressors of cytokine signaling (SOCS), has been identified that inhibit JAK/STAT signals. Therefore, we tested the ability of TGF-β to induce SOCS expression in osteoclast precursors and examined the effect of SOCS expression on osteoclast/macrophage lineage commitment. We found that while SOCS mRNA is undetectable in macrophages, osteoclasts express SOCS-3, and TGF-β up-regulates this expression. Furthermore, TGF-β rapidly induces sustained SOCS-3 expression in macrophage/osteoclast precursors. To determine whether SOCS-3 plays a role in osteoclast differentiation we expressed SOCS-3 in precursors using a retroviral system. We found that osteoclast differentiation was significantly enhanced in SOCS-3-infected precursors, and SOCS-3 expression enables formation in the presence of anti-TGF-β Ab. On the other hand, antisense knockdown of SOCS-3 strongly suppressed osteoclast formation and significantly blunted the response to TGF-β. Moreover, like TGF-β, SOCS-3 expression opposed the inhibitory effect of IFN-β. These data suggest that TGF-β-induced expression of SOCS-3 may represent a mechanism by which TGF-β suppresses inhibitory cytokine signaling, priming precursors for a role in bone resorption.

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.

A Longitudinal Study of the Effect of Subcutaneous Estrogen Replacement on Bone in Young Women With Turner's Syndrome

It is desirable that young women with primary ovarian failure achieve normal peak bone mass to reduce the subsequent risk of osteoporosis, and that there are management strategies to replace bone that is already lost. While estrogen (E2) is generally considered to prevent bone loss by suppressing bone resorption, it is now recognized that estrogen also exerts an anabolic effect on the human skeleton. In this study, we tested whether estrogen could increase bone mass in women with primary ovarian failure. We studied the mechanism underlying this by analyzing biochemical markers of bone turnover and iliac crest biopsy specimens obtained before and 3 years after E2 replacement. Twenty‐one women with Turners syndrome, aged 20‐40 years, were studied. The T scores of bone mineral density at lumbar spine and proximal femur at baseline were −1.4 and ‐1.1, respectively. Hormone replacement was given as subcutaneous E2 implants (50 mg every 6 months) with oral medroxy progesterone. Serum E2 levels increased incrementally from 87.5 pM at baseline to 323, 506, 647, and 713 pM after 6 months and 1, 2, and 3 years of hormone replacement therapy (HRT), respectively. The bone mineral density at the lumbar spine and proximal femur increased after 3 years to T scores of ‐0.2 and ‐0.4, respectively. The cancellous bone volume increased significantly from 13.4% to 18.8%. There was a decrease in activation frequency, but the active formation period was increased by HRT. There was a significant increase in the wall thickness from 33.4 μm at baseline to 40.9 μm after 3 years of HRT, reflecting an increase in bone formed at individual remodeling units. Although there was an early increase in biochemical markers of bone formation, these declined thereafter. Our results show that estrogen is capable of exerting an anabolic effect in the skeleton of young women with Turners syndrome and low bone mass.