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.

Traditionally, binding proteins are known to regulate the activity of ligands by prolonging their half-life, and insulin-like growth factor (IGF)-binding proteins (IGFBPs) are no exception to this. The IGFBP family contains six high-affinity members with variable functions and mechanisms of actions. In addition to functioning as simple carrier proteins, IGFBPs in serum function to regulate the endocrine actions of IGFs by regulating the amount of IGF available to bind to signaling IGF-I receptors, whereas locally produced IGFBPs act as autocrine/paracrine regulators of IGF action. Furthermore, recent in vitro and in vivo findings that IGFBPs function independently of the IGFs as growth modulators are particularly exciting. Regarding the role of IGFBPs as ligand-independent growth modulators, our recent data that IGFBP-5 stimulates markers of bone formation in osteoblasts lacking functional IGFs provide evidence that IGFBP-5 itself is a growth factor that can act independently of IGFs to regulate bone formation. In terms of the mechanism by which certain IGFBPs mediate their effects in a ligand-independent manner, the binding of IGFBP to its putative receptor on the cell membrane may stimulate the signaling pathway independent of an IGF receptor, to mediate the effects of IGFBPs in certain target cell types. IGFBPs may also exert IGF-independent effects by transcriptional activation of genes by IGFBPs transported into the nucleus via their nuclear localization signal. In conclusion, IGFBPs are unusually pleotrophic molecules with functions ranging from the traditional role of prolonging the half-life of the IGFs to functioning as growth factors independent of the IGFs. In this regard, it was surprising to find that the human genome contains only about 35 000 genes. One mechanism to account for such complexity with a relatively small number of genes is strikingly illustrated by the multifunctional IGFBP class of proteins.

It was previously reported that the expression of cyclo-oxigenase-2 (COX-2) is induced by prostaglandin E(2) (PGE(2)) in vitro in an osteogenic cell line and organ culture, suggesting an autoamplification mechanism. In this study, we first tested whether this phenomenon also occurs in bone tissue in vivo and found that a single anabolic dose of PGE(2) (5 mg/kg) induced (between 30 and 120 min) in rat tibiae, an increase in the mRNA level of COX-2 (2.5- to 9-fold) but not that of COX-1. Secondly, to test whether COX-2 activity in generating endogenous prostaglandins (PGs) is required for the in vivo anabolic properties of PGE(2), young male rats were injected daily with either vehicle (8% ethanol) or 5 mg/kg PGE(2) for 21 days. PGE(2)-injected rats received, 45 min prior to PGE(2), either dimethyl sulphoxide (as vehicle) or one of two doses of NS-398, a selective COX-2 inhibitor: a low dose (3 mg/kg) or a high dose (10 mg/kg). PGE(2) increased bone formation (measured as cancellous mineralizing surface, mineral apposition rate and bone formation rate) and bone mass (measured as cancellous bone area and surface and cortical width). None of these increases was suppressed by pre-administration of NS-398. In contrast, the high dose of NS-398 effectively suppressed an increase in rat hind-paw volume induced by a local carrageenan injection. Furthermore, since COX-2 inactivation may affect PG receptor expression, we found that pre-administration of NS-398 did not abolish the induction in EP(4) receptor mRNA levels, caused by PGE(2) in rat bone tissue. For in vitro testing, rat femoral bone marrow stromal cell cultures were initiated and were incubated in the absence or presence of PGE(2) at 100 nM (as an inducer) and with increasing concentrations of NS-398 (10(-8) M to 10(-5) M) for 21 days, after which time mineralized (Von-Kossa positive) nodules were counted. PGE(2) increased nodule formation as previously reported; however, NS-398 reduced nodule formation in both control and PGE(2)-treated cultures to the same extent. We conclude that while the level of COX-2 mRNA is increased in vivo by administration of PGE(2), inhibition of its activity (i.e. generation of endogenous PGs) does not abolish the anabolic effect of PGE(2).

Abstract

Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells.

Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10⁻⁶ mol/l) decreased IGF-I mRNA levels.

In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I.

Journal of Endocrinology (1996) 149, 397–403

ABSTRACT

Insulin-like growth factor-I (IGF-I) is a potent stimulator of bone formation. Whether this growth factor also induces bone resorption has not been studied in detail. We used two organ culture systems to examine the direct effect of IGF-I on bone resorption. Fetal mouse radii/ulnae, containing mature osteoclasts, showed no response to IGF-I, indicating that osteoclastic activity is not influenced by IGF-I. Fetal mouse metacarpals/metatarsals, containing just osteoclast precursors and progenitors, showed an increase in resorption in response to IGF-I, indicating that IGF-I stimulates the formulation of osteoclast precursors/progenitors and thereby increases the number of osteoclasts.

Interleukin-6 (IL-6) has been hypothesized to be a mediator of bone resorptive agents such as parathyroid hormone (PTH). Both radii/ulnae and metacarpals/metatarsals reacted to IGF-I with an increase in IL-6 production. IL-6 production by UMR-106 osteogenic osteosarcoma cells was positively modulated by IGF-I, indicating that osteoblasts are likely to be the cells responsible for increased IL-6 production by the bones, and that IL-6 might be a mediatory of IGF-I-stimulated bone resorption.

Journal of Endocrinology (1992) 132, 433–438

ABSTRACT

Oestrogens play an important role in bone metabolism; they preserve bone mass after the menopause. Their action in bone has recently been shown to be, partly, a direct one, as oestrogen receptors and their effects have been demonstrated in bone cells. The role of progestogens in bone metabolism is less clear. In this study it has been shown that 17β-oestradiol exerts only a small, although not significant, stimulatory action with regard to SaOS-2 human osteosarcoma cell proliferation. A pure progestogen (Org 2058) has no effect when added alone. In combination with 17β-oestradiol, however, it has a highly synergistic action on SaOS-2 cell proliferation. The same effect was observed in primary rat osteoblasts, showing that this synergism is a general phenomenon in osteoblastic cells. High numbers of oestrogen and progestogen receptors have been demonstrated in SaOS-2 cells, indicating that the effects of these steroids are mediated via the normal route of steroid receptors. These data provide a cellular basis for the clinically recognized positive effect of oestrogen/progestogen combinations on bone formation.

ABSTRACT

When growth cartilage from rachitic chicks was cultured in the presence of the calcium-regulating hormone 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), collagen resorption was increased and collagen synthesis decreased compared to control cultures containing no hormone. The minimum concentration of the hormone that caused a statistically significant inhibition of collagen synthesis was 10 ⁻⁸ mol/l. Collagen synthesis by growth cartilage from normal chicks was also reduced by 1,25-(OH)₂D₃, showing that it was not an abnormal response of vitamin D-depleted tissue. 25-Hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ also inhibited collagen synthesis by cultures of growth cartilage but only at higher metabolite concentrations. 1,25-Dihydroxyvitamin D₃ (10⁻⁷ mol/l) did not significantly inhibit collagen synthesis by cultures of articular fibrocartilage and of sternal cartilage, tissues that do not calcify physiologically. The minimum concentration of 1,25-(OH)₂D₃ (10⁻⁹ mol/l) necessary to cause decreased collagen synthesis by embryonic chick calvaria was lower than the value obtained with growth cartilage; this suggests that bone cells may be more sensitive to the hormone in this respect than are growth cartilage chondrocytes. These findings provide evidence of a direct role of 1,25-(OH)₂D₃ in the control of endochondral bone formation which is consistent with its primary role in the maintenance of plasma calcium homeostasis.

J. Endocr. (1985) 105, 79–85

SUMMARY

The effect of glucagon on bone was studied in rats. Urinary hydroxyproline excretion and incorporation of [³H]proline into bone hydroxyproline were used as indices of bone collagen breakdown and formation respectively. Parathyroid extract (15 USP units/rat/h, i.v.), infused into thyroparathyroidectomized animals, increased urinary hydroxyproline excretion. This increase was nullified by simultaneous administration of glucagon (50 μg/rat/h, i.v.). Rats treated with glucagon for 12 days (30 μg/100 g/day, s.c.) excreted slightly less hydroxyproline in their urine than controls. In both intact and thyroparathyroidectomized rats, glucagon (10 μg/100 g/h, s.c.) decreased incorporation of [³H]proline into bone. Similar results were obtained in nephrectomized rats, evidence that changes produced by glucagon were not solely due to alterations in proline pool size caused by increased renal excretion. From these data it is concluded that: (1) glucagon can inhibit bone resorption (the effect being slight in normal rats, but easily demonstrable in parathyroid hormone-treated thyroparathyroidectomized rats), (2) release of endogenous calcitonin is not required to produce this effect, (3) parathyroid hormone and glucagon may act on the same target cell in bone, (4) inhibition of skeletal resorption may contribute to glucagon-induced hypocalcaemia, and (5) the hormone possibly decreases bone formation. Since pharmacological doses of glucagon were used in our studies, the relationship of the observations made to the physiological role of glucagon is unknown.

Gross deformities appeared in Xenopus laevis maintained for about 2 yr. under laboratory conditions on a diet containing no live food. Radiographs of the affected animals revealed defective calcification of the skeleton. All animals bred in the colony were defective, and only the original adults taken from the wild had normal bones. A description of the different types of skeleton found, and later produced experimentally, is given. An analysis of the conditions under which the defects became manifest showed X. laevis to be very susceptible to lack of vitamin D. Calcification in this species is greatly affected by the nature of the basal food. Normal bones are formed when the toads are fed on a diet of rabbit liver (or ox liver) supplemented with cod-liver oil and calcium. Horse liver is toxic, causing a depression of growth and a failure of calcification even with the supplements. With guinea-pig muscle a far larger supplement of vitamin D is required to prevent the development of general calcium deficiency and to permit normal bone formation. The failure of calcification is identified as rickets with osteoporosis and its relation to these diseases in other species is discussed.

SUMMARY

Isolated rat liver, when perfused with medium containing bovine growth homone produced somatomedin-like activity (liver somatomedin).

Liver somatomedin is useful in studies of the hormonal control of the cartilage plate in vitro, since unlike serum it is not contaminated with other hormones or growth factors (apart from growth hormone). Chondrocytes isolated from various regions of the growth cartilage responded differently to liver somatomedin; proliferative chondrocytes, like those isolated from the articular cartilage, showed increased [³H]thymidine uptake in response to liver somatomedin, whereas hypertrophic chondrocytes did not respond. It is suggested that there is a reduction in the response to somatomedin by growth plate chondrocytes as they pass from the proliferative to the hypertrophic state.

Thyroxine, thought to be involved in the processes of hypertrophy and new bone formation, did not directly affect [³H]thymidine uptake by proliferative chondrocytes, but inhibited stimulation of their activity by liver somatomedin.

Measurement of [³H]thymidine uptake by isolated articular chondrocytes may provide a useful assay for both liver and serum somatomedin. The graded response of chondrocytes to increasing concentrations of liver somatomedin paralleled the response to increasing levels of serum somatomedin.