A number of bone diseases characterised by excessive osteolysis (e.g. osteoporosis and Paget's disease) exhibit a marked gender difference in prevalence and are more common in the elderly population. Bone resorption is carried out by osteoclasts, which are formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. In this study, we have determined whether there are gender- and age-related differences in osteoclast formation from circulating precursors. Peripheral blood mononuclear cells (PBMCs) were co-cultured with UMR106 osteoblast-like cells in the presence of macrophage-colony stimulating factor (M-CSF) and 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) or cultured alone in the presence of sRANKL (soluble receptor activator of nuclear factor kappa B ligand) and M-CSF. As assessed by the formation of tartrate resistant acid phosphatase (TRAP)-positive (TRAP(+)) and vitronectin receptor-positive (VNR(+)) multinucleated cells (MNCs), there was no difference in the number of circulating osteoclast precursors in males and females. Lacunar resorption carried out by osteoclasts formed from these precursors was generally increased in males compared with females (P=0.03). An increase in the number of TRAP(+) and VNR(+) MNCs formed from male PBMCs was noted in response to 1,25(OH)(2)D(3) (P<0.005). An increase in lacunar resorption in cultures of PBMCs (10(5) per well) from males was also noted in response to 10(-9) M 1,25(OH)(2)D(3) (P<0.05) and sRANKL (P=0.05), but not M-CSF. The addition of dexamethasone resulted in a marked increase in osteoclast formation and lacunar resorption in both males and females. Post-menopausal females and males of comparable age showed similar levels of osteoclastogenesis. Pre-menopausal women showed similar levels of osteoclastogenesis but less resorption (P=0.01) compared with males of comparable age. These results show that there are specific gender/age-related differences in osteoclast formation and bone resorption and have implications for evaluating osteoclastogenesis in skeletal diseases such as primary osteoporosis and Paget's disease.
M Jevon, A Sabokbar, Y Fujikawa, T Hirayama, SD Neale, J Wass and NA Athanasou
CH Kim, SL Cheng and GS Kim
It is well documented that glucocorticoid excess causes bone loss, but the mechanisms of these effects remain poorly defined. To understand further the mechanisms of glucocorticoid-induced osteoporosis, we investigated the effects of glucocorticoids on bone formation and bone resorption by examining the proliferation, functional activities, and cytokine secretion of cultured human bone marrow stromal cells (hBMSC). Treatment with dexamethasone for 24 h at the concentration of 10(-8) M significantly suppressed [(3)H]thymidine incorporation and further inhibition was observed with longer treatment (8 days) or higher concentration (10(-7) M). Alkaline phosphatase activity of hBMSC was markedly stimulated with addition of dexamethasone (10(-8) M), to 191 +/- 22% (after 4 days) and 317 +/- 46% (after 7 days) of control. Dexamethasone (10(-8) M) treatment for 48 h decreased the incorporation of [(3)H]proline into collagenase-digestible protein (CDP; 43.7+/-7.9% of control) and non-collagen protein (65.2+/-8.4% of control), with a greater effect on CDP. Northern blot analysis indicated that alpha1(I)-collagen mRNA level was decreased by dexamethasone to 27.6 +/- 9.0% of the control value after 1 day of exposure, and to 55.2 +/- 6.2% after 7 days. Dexamethasone markedly suppressed basal production of interleukin (IL)-6 and IL-11 and that stimulated by parathyroid hormone (PTH), IL-1alpha, or tumour necrosis factor-alpha in a dose-dependent manner. These results suggest that the glucocorticoid-induced bone loss is derived at least in part via inhibition of bone formation, which includes the suppression of osteoblast proliferation and collagen synthesis. As both basal and PTH-stimulated production of IL-6 and IL-11 are decreased by dexamethasone, the increased bone resorption observed in glucocorticoid-induced osteopenia does not appear to be mediated by IL-6 or IL-11.
K Fuller, JM Owens and TJ Chambers
It is believed that parathyroid hormone (PTH) increases the resorptive activity of pre-existing osteoclasts through a primary interaction with cells of the osteoblastic lineage. Much less is known, however, of the mechanisms by which PTH induces osteoclast formation. It is known that osteoclast formation occurs through a contact-dependent interaction between stromal cells and haemopoietic precursors, but it is not known whether PTH acts on stromal cells or precursors to induce osteoclast formation. To address this issue, we compared the ability of haemopoietic cultures to generate osteoclasts, identified as calcitonin receptor positive (CTRP) cells, and to resorb bone in response to PTH and 1,25(OH)2 vitamin D3 (1,25(OH)2D3). We found that when murine haemopoietic tissues were incubated at densities sufficiently high to support haemopoiesis, both PTH and 1,25(OH)2D3 induced bone resorption in bone marrow cells, but in cultures of haemopoietic spleen only 1,25(OH)2D3 induced CTRP cells, and neither hormone induced bone resorption. To determine whether these differences were attributable to differences in stromal cells or haemopoietic precursors, lower densities of haemopoietic spleen cells were incubated on osteoblastic (UMR 106), splenic or bone marrow stromal cells. We found that the behaviour of the cocultures reflected the characteristics and origin of the stromal cells. Thus, the ability of both osteoblastic and splenic stromal cells to induce CTRP cells with 1,25(OH)2D3, while only osteoblastic cells induced osteoclasts with PTH, from the same precursors, suggests that the ability of PTH to induce osteoclastic differentiation cannot be attributed to a hormonal action on osteoclast precursors, but depends on a response in stromal cells.
V Sibilia, AE Rigamonti, F Pagani, N Lattuada, F Guidobono, WB Wehrenberg, EE Muller and C Netti
The effects of neonatal passive immunization against GHRH on bone was examined in male and female rats. Pups were treated subcutaneously with GHRH-antiserum (GHRH-Ab) from day 1 to day 10 of age. Bone mineral content (BMC) and bone mineral density (BMD) were evaluated at monthly intervals until 7 months. Markers of bone resorption (urinary lysylpyridinoline, LP), bone formation (serum osteocalcin, OC) and serum IGF-I were measured at 2, 3 and 7 months. In male rats, GHRH-Ab did not modify BMC and BMD when compared with controls. In contrast, female rats demonstrated lower whole body and femoral BMC and BMD from 2 to 7 months of age. Reduced bone growth in the females was associated with lower IGF-I levels than controls at 2 and 3 months of age, whereas in males IGF-I titers did not change during the period of the study. LP excretion was higher in GHRH-Ab-treated rats at 2 and 3 months in both sexes. In females, no difference in OC values was recorded, whereas in GHRH-Ab-treated males, there was an increase in OC levels at 2 and 3 months. These data indicate that transient GHRH deprivation induces an osteopenic effect in female rats which is not evident in male rats.
J. H. Tobias, A. Gallagher and T. J. Chambers
We have previously found that administration of oestradiol-17β (OE2) to intact adult female rats of 19 days stimulates cancellous bone formation. However, this effect is not observed following longer periods of OE2 treatment, suggesting that the responsiveness of the skeleton to oestrogen's anabolic action is reduced after prolonged administration. A possible explanation for this is that oestrogen also suppresses bone resorption, which is an important stimulus for bone formation. We therefore investigated the effect of omitting OE2 for short periods, on the proximal tibial metaphysis of intact female rats. We found that, unlike continuous treatment with OE2 (40 pg/kg) for 56 days, omission of OE2 for 4 days out of every 20 was associated with a significant increase in cancellous bone volume. Although continuous and intermittent OE2 were both associated with a reduction in osteoclast surface, a decrease in the proportion of double fluorochrome-labelled surface was only seen after continuous OE2 treatment. We then studied the effects of longer periods of OE2 omission by giving OE2 (40 pg/kg) for three repeated cycles of: (1) OE2 for 16 days/vehicle for 4 days, (2) OE2 for 12 days/vehicle for 8 days, (3) OE2 for 8 days/vehicle for 12 days, or (4) OE2 for 4 days/vehicle for 16 days. We found a significant increase in cancellous bone volume when OE2 was stopped for either 4 or 8 days at a time. However, longer periods of OE2 omission did not affect bone volume, possibly because these were associated with an increase in bone resorption and/or a reduction in bone formation during the OE2-free period. In conclusion, we observed an increase in cancellous bone volume after prolonged treatment with oestrogen only if OE2 was omitted for short periods. This may be due, at least in part, to bone formation being maintained at a higher rate by such treatment than by either continuous OE2 administration or by intermittent administration where OE2 is discontinued for longer periods.
Journal of Endocrinology (1993) 139, 267–273
K Ono, T Akatsu, T Murakami, M Nishikawa, M Yamamoto, N Kugai, K Motoyoshi and N Nagata
Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.
D Swolin-Eide and C Ohlsson
High levels of glucocorticoids are believed to alter bone remodeling by decreasing bone formation and increasing bone resorption. It has been suggested that different cytokines, like interleukin-6 (IL-6) and interleukin-1 (IL-1), are involved in bone resorption by activating immature osteoclasts, and some studies indicate that IL-6 promotes bone formation by a mitogenic effect on osteoblasts. The aim of the present investigation was to study whether cortisol regulates the expression of IL-6 and IL-1 beta in human osteoblast-like cells. A high dose of cortisol (10(-7)M) decreased, as expected, the C-terminal propeptide of type I collagen released into the culture medium. The IL-6 mRNA levels and IL-6 protein released into the culture medium were also decreased by cortisol in a dose-dependent manner. The maximum effect was seen at 1 microM cortisol (mRNA 23.1 +/- 7.9% of control culture; protein 28.2 +/- 8.3% of control culture). The decrease in IL-6 mRNA levels was apparent 4 h after the addition of cortisol and was still present 20 h later. The decrease in IL-6 protein released into the culture medium was seen 20 h later than the decrease in IL-6 mRNA levels. The production of IL-1 beta protein released into the culture medium was decreased in a dose-dependent manner after the addition of cortisol with a maximum effect at 1 microM. The effect of cortisol on IL-1 beta protein released into the culture medium was seen 16 h after the addition of cortisol. To summarize, cortisol decreases the expression of IL-6 as well as IL-1 beta in human osteoblast-like cells.
C E Evans, C Ward and I P Braidman
Bone metastases in breast cancer may be osteolytic, osteosclerotic, or a mixture of the two. Although stimulation of bone resorption by breast cancer cells has attracted some interest, the formation of osteosclerotic secondary tumours and the influence of human mammary carcinoma cells on osteoblasts (bone forming cells), both important in understanding breast cancer - bone interactions, have been largely neglected. We therefore examined the effects of conditioned medium (CM) from two cultured human breast cancer cell lines (MCF7 and ZR-75) and from primary cultures of breast carcinomas from two patients, on osteoblasts and recruitment of bone-resorbing cells (osteoclasts) in vitro. Osteoblast-like cells (BDC) were cultured from human trabecular bone explants. Osteoclast maturation was studied in fetal rat calvaria cultured on collagen gels. CM from the MCF-7 line and cells derived from one patient each inhibited BDC DNA synthesis, but stimulated osteoclast recruitment. In contrast, CM from the second patient's cells or ZR-75 enhanced DNA synthesis in BDC, but blocked osteoclast maturation. This suggests that human breast carcinomas secrete soluble factors which influence both osteoclasts and osteoblasts. A further unexpected implication is that mammary carcinoma cells may cause local osteosclerosis by directly stimulating osteoblasts, rather than through raised bone turnover in metastases.
J. P. MANNING, B. G. STEINETZ, MARGARET C. BUTLER and SARA PRIESTER
The histochemical changes in acid phosphatase activity of the pubic joint of ovariectomized mice treated with various hormones have been studied. Oestrogen administration resulted in marked increase in acid phosphatase activity of the osteoclasts, osteocytes, chondroclasts, young chondrocytes and chondroblasts in addition to bone resorption and transformation of the hyaline cartilage caps of the pubic bones into fibrocartilage. Relaxin, progesterone and cortisone acetate failed to influence the quantity or distribution of the enzyme in the pubic joint of mice primed with oestrogen. Progesterone and cortisone inhibited ligament formation produced by relaxin in oestrogen-treated animals. The results are discussed in relation to the activities of the hormones on the symphysis pubis of the mouse.
H Inoue, T Tsujisawa, T Fukuizumi, S Kawagishi and C Uchiyama
1,25 Dihydroxy vitamin D3 (1,25(OH)2D3), prostaglandin (PG) E2 and parathyroid hormone (PTH) induce osteoclast formation in cell cultures. Previously, we have shown that SC-19220, an antagonist of the EP1 subtype of PGE receptors, inhibited tartrate-resistant acid phosphatase (TRAP)-positive cell formation by PGE2 and PTH in adherent cell cultures taken from neonatal rats. Since 1,25(OH)2D3 has been shown to induce osteoclast formation through PGE2 synthesis, in this study we have examined the effect of SC-19220 on osteoclast formation induced by 1,25(OH)2D3 in cell cultures by measuring bone resorption as well as TRAP-positive cell formation. SC-19220 inhibited osteoclast formation by 1,25(OH)2D3 as well as by PGE2 in cell cultures. The addition of SC-19220 to the later half but not to the earlier half of the culture inhibited 1,25(OH)2D3-induced formation. In the culture in which hydroxyurea was added in the later half period, SC-19220 inhibited osteoclast formation by 1, 25(OH)2D3. Under these conditions, 17-phenyl PGE1, an EP1 agonist, induced osteoclast formation. Thus, SC-19220 inhibits certain reactions in the later processes of osteoclast formation induced by 1,25(OH)2D3. In addition, SC-19220 also inhibited osteoclast formation induced by interleukin (IL)-11 and IL-6 as well as by PTH. It is suggested that the SC-19220 inhibiting reactions are shared by all the inducers including 1,25(OH)2D3 and are essential for osteoclast formation.