T.J. Chambers

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.

Regulation of the differentiation and function of osteoclasts

The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation‐induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane‐inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor‐β (TGF‐β), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF‐α can induce osteoclast formation in vitro from bone marrow‐derived mononuclear phagocytes, especially in the presence of TGF‐β. Whether or not TNF‐α does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF‐β, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics. Copyright

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.

Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation.

Estrogen is generally considered to maintain bone mass through suppression of bone resorption. We have previously demonstrated that administration of pharmacologic doses of estrogen increases bone formation in ovary-intact rats. To assess the effects of physiological concentrations of estrogen on bone formation, estrogen was administered to ovariectomized rats in which bone resorption was suppressed by the bisphosphonate 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP). Animals receiving exogenous 17 beta-estradiol (E2) (1, 10, and 100 micrograms/kg daily for 17 d) showed a dose-dependent increase in trabecular bone volume of 1.9, 25.8, and 43.6%, respectively, compared with those rats treated with AHPrBP alone. The increase in bone volume was associated with an increase in bone formation in E2-treated animals, in which bone resorption had been almost completely suppressed by AHPrBP. Neither ovariectomy, AHPrBP, nor E2 treatment had a significant effect on the volume or rate of formation of cortical bone. Thus, the increased bone resorption, which is a consequence of estrogen-deficiency, entrains increased bone formation, which masks a simultaneous reduction in estrogen-dependent bone formation. Therefore, in addition to the nonspecific effect of estrogen to depress formation via coupling, we have identified a specific effect of estrogen to increase formation independent of coupling. Thus it appears that estrogen maintains bone volume not only through inhibition of bone resorption, but also through stimulation of bone formation.

Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain

The nature of the stimulus sensed by bone cells during mechanical usage has not yet been determined. Because nitric oxide (NO) and prostaglandin (PG) production appear to be essential early responses to mechanical stimulation in vivo, we used their production to compare the responsiveness of bone cells to strain and fluid flow in vitro. Cells were incubated on polystyrene film and subjected to unidirectional linear strains in the range 500-5,000 microstrain (με). We found no increase in NO or PGE2 production after loading of rat calvarial or long bone cells, MC3T3-E1, UMR-106-01, or ROS 17/2.8 cells. In contrast, exposure of osteoblastic cells to increased fluid flow induced both PGE2 and NO production. Production was rapidly induced by wall-shear stresses of 148 dyn/cm2 and was observed in all the osteoblastic populations used but not in rat skin fibroblasts. Fluid flow appeared to act through an increase in wall-shear stress. These data suggest that mechanical loading of bone is sensed by osteoblastic cells through fluid flow-mediated wall-shear stress rather than by mechanical strain.

Dichloromethylenebisphosphonate (Cl2MBP) inhibits bone resorption through injury to osteoclasts that resorb Cl2MBP-coated bone.

Dichloromethylenebisphosphonate (Cl2MBP, formerly Cl2MDP) inhibits bone resorption in vivo and in vitro. The mechanism by which it inhibits osteoclastic bone resorption has not been established. To investigate this, osteoclasts were isolated from rat long bone and incubated with Cl2MBP (10(-9)-10(-5) M) on bone slices. Bone resorption was assessed as plan area resorbed after 6 and 24 h by scanning electron microscopy. Although Cl2MBP inhibited bone resorption in the first 6 h of culture, inhibition was more marked in the incubation period between 6 and 24 h. This pattern of accelerating inhibition is unlike the pattern we have observed using other resorption-inhibitors, and suggested resorption-mediated osteoclast injury. In keeping with this, we found reduced numbers of osteoclasts, and morphological features of cell injury and degeneration of osteoclasts, after incubation with Cl2MBP on bone slices. Bone seemed to be an essential component in Cl2MBP-mediated injury, since osteoclast numbers and morphology on plastic coverslips were unaffected by the bisphosphonate. Moreover, bone slices preincubated with Cl2MBP showed similar effects on resorption and morphology to cultures in in which osteoclasts on bone were continuously immersed in Cl2MBP. Neither non-resorptive cells (macrophages, UMR 106 cells), nor osteoclasts prevented from resorption by calcitonin, showed evidence of cytotoxicity after incubation on bone slices with Cl2MBP. These results suggest that even relatively high concentrations of Cl2MBP in the fluid phase do not affect osteoclasts, nor does contact with Cl2MBP-coated bone surfaces, but that injury to osteoclasts, and a consequent reduction in bone resorption, occurs when osteoclasts excavate bone surfaces upon which Cl2MBP is adsorbed.

Inhibition of bone resorption by bisphosphonates: Interactions between bisphosphonates, osteoclasts, and bone

SummaryBisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to the cells that resorb Cl2MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorptionin vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl2MBP. The greater sensitivity of resorption to AHPrBPin vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormonesin vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl2MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generationin vivo in AHPrBP-treated rats. These results suggest that the comparisons of potencyin vitro do not include all the factors responsible for determining bisphosphonate potencyin vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10−3 M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl2MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influencedin vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.

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.

Induction of bone formation in rat tail vertebrae by mechanical loading.

We have developed an experimental model in which pins, inserted into the seventh and ninth caudal vertebrae of 13-week-old rats, are used to load the eighth caudal vertebra in compression. Four groups of animals were used in the study: unpinned; animals with pins inserted, but non-loaded; animals loaded once, for 360 cycles at 0.5 Hz; and animals subjected to daily loading for 36 cycles at 0.5 Hz. Pins were immobilised by clamps when not undergoing loading. The animals were killed 9 days after pinning, and the eighth caudal vertebra was subjected to histomorphometric and histodynamic analysis. We found that vertebrae subjected to 36 daily loading cycles showed a 30-fold increase in bone formation compared to non-loaded controls. A single loading regime of 360 cycles was sufficient to increase bone formation 4-fold. Bone formation on trabecular surfaces was of lamellar rather than woven bone and was accompanied by a decrease in indices of bone resorption. Loaded vertebrae also showed substantial periosteal woven bone formation, although a minor degree of periosteal woven bone formation was also seen in one non-loaded pinned control vertebra. Our results suggest that in the rat, as in avian species, short loading regimes are capable of inducing bone formation. The model may assist an analysis of the interactions between bone resorption, bone formation and mechanical stimuli, and may enable identification of the molecular signals that mediate induction of lamellar bone formation on trabecular surfaces.

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.