J. M. Lean

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.

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.

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.

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 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.

The anabolic action of 17β-estradiol (E2) on rat trabecular bone is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP)

We have previously found that high doses of 17 beta-estradiol (E2), similar to those seen in late pregnancy, stimulate bone formation in adult rats. In this communication we tested the effects of a combination of E2 and (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) on bone formation and bone volume in rat bone. E2 (4 mg/kg/day subcutaneously for 17 days) stimulated the bone formation rate to 6 times that of control rats. This was reduced by a single administration of AHPrBP (0.3 mg/kg subcutaneously) to 3 times control levels, and by similar daily injections of AHPrBP to levels not significantly different from those of untreated rats. Suppression of bone formation was effected predominantly through a reduction in the percentage of double-labelled surfaces, consistent with reduced osteoblast recruitment. We found only relatively minor effects of AHPrBP on the mineral apposition rate, suggesting that AHPrBP affected osteoblast function less than osteoblast recruitment. Suppression of histodynamic parameters of bone formation by AHPrBP was associated with suppression of the increase in bone volume otherwise induced by E2. The suppression by AHPrBP of the effect of E2 on bone formation contrasted with its lack of effect on other target tissues for E2, since AHPrBP did not affect the E2-induced changes in longitudinal bone growth or uterine weight. These results suggest that AHPrBP inhibits the anabolic effect of estrogen on rat trabecular bone.

The Role of Prostaglandins and Nitric Oxide in the Response of Bone to Mechanical Stimulation

The primary function for which bone has evolved is to act as mechanical support. Thus, the shape of bones is determined by the genetic programme, upon which is superimposed adaptation to the mechanical environment. The distinct contribution of these two influences is most clearly seen in limb bones, in which mechanical usage causes a substantial change in shape, and an increase in the quantity of bone, compared to that determined genetically1. Such mechanically adapted bones show a remarkably consistent strain response to mechanical usage: peak strains of 2,000–3,000 microstrain (μe) are observed over the cortical surface of bones in a wide variety of species during physiological activitysee 2.

The progesterone antagonist, RU486 does not affect basal or estrogen-stimulated cancellous bone formation in the rat.

Although it has been suggested that progesterone may have a role in preventing postmenopausal bone loss, a number of studies have shown that progesterone has no additive effect on estrogen therapy. We have recently found that high-dose estrogen stimulates bone formation in rats. The effect of progesterone on this anabolic action of estrogen has not been tested. We therefore investigated the role of progesterone in combination with endogenous or exogenous estrogen on bone formation in rats using RU486, which has been shown to be a potent progesterone antagonist without detectable agonist effects. Three-month-old Wistar female rats were treated for 17 days with RU486, and histomorphometric indices of bone formation were measured at the proximal tibial metaphysis after administration of double fluorochrome labels. Animals treated with RU486 (10 mg/kg) showed no change in either bone formation rate or double-labelled bone surfaces compared to vehicle-treated controls. Estrogen (17 beta-estradiol, 4 mg/kg) increased both indices by more than double compared with controls. RU486 did not affect the indices of increased bone formation in estrogen-treated rats. Estrogen also exhibited inhibition of longitudinal bone growth, while RU486 had no effect either in normal or estrogen-treated animals. These results show that the progesterone antagonist affects neither the stimulatory effect on formation nor the inhibitory effect on longitudinal bone growth by estrogen. These results suggest that progesterone does not play a significant role in either bone formation or bone growth in the rat.

Estrogen induces bone formation on non-resorptive surfaces in the rat

We have recently found that 17 beta-estradiol (E2) stimulates bone formation in rat cancellous bone, and that this bone formation is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP). To analyse the relationship between bone resorption and bone formation in the action of E2, we injected 13-week-old female rats sequentially with three fluorochromes (calcein, tetracycline and xylenol orange) at 7-day intervals. E2 (40 micrograms/kg) or vehicle was injected daily for 15 days, starting 24 hrs after the first fluorochrome. A third group was injected with AHPrBP (0.3 mg/kg) 24 hrs after the first two fluorochromes. The rats were killed 48 hrs after the third fluorochrome. We found that the perimeter of all three fluorochrome labels was increased by E2. The entire perimeter of the first label was non-crenated. Since the first label was given before E2-administration, this suggests that label that would otherwise have been eluted from the bone surface had been fixed in bone by E2-induced bone formation, which might have occurred either through prolongation of pre-existing bone formation, or induction of bone formation on quiescent surfaces. In either case, our results suggest that resorption did not precede formation at the site of bone formation. Since induction of bone formation by E2 is suppressed by inhibition of bone resorption, this suggests that the coupling of E2-induced formation to resorption in the rat does not necessarily require that formation occurs at the same site as bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)