ABSTRACT

While the osteopenia associated with oestrogen deficiency is thought to arise from a relative defect in bone formation with respect to resorption, oestrogen administration itself leads to a decrease, rather than an increase, in bone formation. This decrease in bone formation, which arises from oestrogen's inhibitory effect on bone turnover, presumably masks any underlying tendency of oestrogen treatment towards stimulation of bone formation. To investigate this further, we have examined the early effect of discontinuing the administration of oestradiol-17β (OE₂; 40 μg/kg on bone formation indices in ovariectomized 13-week-old rats, before the turnover-induced increase in formation occurs. Histomorphometric indices were assessed at the proximal tibial metaphysis 0, 7, 10, 13 and 16 days following discontinuation of OE₂ treatment. Measurements of body weight, uterine weight and longitudinal growth rate confirmed that there were rapid effects of OE₂ deficiency on these parameters.

We could detect no significant increase in bone resorption, as measured by osteoclast surface and number, until 16 days after ending treatment with OE₂; this was coincidental with a reduction in bone volume. Shorter periods of OE₂ deficiency were associated with a marked decrease in bone formation, as assessed by dynamic histomorphometric indices. This inhibition of bone formation was largely due to a reduction in double fluorochrome-labelled trabecular surfaces, which were decreased by approximately 70%. We conclude that ending OE₂ administration in ovariectomized rats caused a striking decrease in trabecular bone formation, if such indices are assessed prior to the subsequent turnover-induced increase in formation. This suggests that oestrogen treatment in ovariectomized rats is associated with a stimulatory effect on bone formation, in addition to its recognized anti-resorptive action.

Journal of Endocrinology (1993) 137, 497–503

Abstract

During bone resorption, osteoclasts are closely associated with endothelial cells. The latter are able to produce several agents that regulate bone resorption. In view of the increasing evidence that angiotensin II, which can be generated by endothelial cells, has actions outside the traditional renin-angiotensin system, we tested the effect of angiotensin II on bone resorption. Angiotensin II showed no effect either on osteoclast formation or on bone resorption by isolated osteoclasts. However, in co-cultures of osteoclasts with calvarial or MC3T3-E1 osteoblastic cells, and in osteoclastic cultures co-cultured with other bone cells obtained by prolonged sedimentation, angiotensin II stimulated bone resorption to a similar degree to that observed with 1,25(OH)₂ vitamin D₃. Stimulation of resorption was noted at concentrations of 10⁻⁷ m and above. We found that angiotensin I also stimulated bone resorption in co-cultures of osteoclasts with osteoblastic cells, and that this action was inhibited by inhibitors of angiotensin-converting enzyme. These results identify angiotensin I and II as potent stimulators of osteoclastic bone resorption, and raise the possibility that bone might contain a tissue-renin-angiotensin system that might play a role in the regulation of bone resorption.

Journal of Endocrinology (1997) 152, 5–10

Abstract

We have recently found that administration of oestradiol-17β (OE₂) to rats stimulates trabecular bone formation. It is not known, however, whether oestrogen has a similar action on bone formation rate under physiological circumstances. Oestrogen is known to suppress bone resorption, and oestrogen-deficient states in the rat, as in humans, are associated with an increase in bone resorption that entrains an increase in bone formation. To see if the latter masks a relative reduction in bone formation, due to oestrogen deficiency, we measured bone formation very early after ovariectomy, before the resorption-induced increase in bone formation becomes established. To do this, rats were administered fluorochrome labels before and after ovariectomy, spaced at weekly intervals in the first, and 3-day intervals in the second experiment.

In both experiments there was a decrease in indices of bone formation in the labelling interval immediately following ovariectomy such that, using the shorter fluorochrome intervals, the mineral apposition rate fell to 69%, the double-labelled surface to 45%, and the bone formation rate to 36% of sham-ovariectomized levels. The reduction was not sustained in the subsequent label intervals, presumably masked by the increase in bone formation attributable to increased resorption. These results suggest that if bone formation is assessed before this resorption-entrained increase in bone formation occurs, oestrogen deficiency is associated with a reduction in dynamic indices of bone formation. Thus, these experiments suggest that oestrogen stimulates bone formation under physiological circumstances, and that the osteopaenia that follows oestrogen deficiency may be attributable not only to an increase in bone resorption, but also to a relative deficiency in bone formation.

Journal of Endocrinology (1994) 142, 119–125

ABSTRACT

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

Abstract

Although short-term administration of oestradiol-17β (OE₂) stimulates cancellous bone formation in the rat, this is replaced by a tendency to suppression after prolonged treatment. Hence, in rats rendered osteopaenic by ovariectomy, OE₂ administration fails either to induce a sustained increase in bone formation or to restore bone volume. A possible explanation for this failure is that OE₂ also inhibits bone resorption, secondarily suppressing bone formation through coupling mechanisms. We therefore investigated whether the effects of OE₂ treatment might be modified by intermittently stimulating bone resorption with retinoic acid (120mg/kg daily) for 4 out of every 20 days. We found, in a preliminary experiment using intact animals, that intermittent retinoic acid reduced cancellous bone volume, consistent with previously documented stimulation of bone resorption by retinoic acid. Rats were then rendered osteopaenic by ovariectomy, and given vehicle, retinoic acid and/or OE₂. We found that animals treated with intermittent retinoic acid and OE₂ showed a substantial increase in cancellous bone volume compared with ovariectomized animals treated with vehicle, retinoic acid alone or OE₂ alone. Therefore, intermittent retinoic acid appears to cause a net increase in bone formation over resorption when given to ovariectomized animals in conjunction with OE₂. We conclude that the effects of OE₂ on cancellous bone are modified by intermittent treatment with retinoic acid, resulting in a substantial increase in bone volume.

Journal of Endocrinology (1994) 142, 61–67

Abstract

Although hormone replacement therapy can prevent postmenopausal bone loss, it does not restore bone mass to normal in patients with established osteoporosis. This might reflect a failure to reproduce certain aspects of gonadal function. One method of investigating this possibility would be to examine the effect of ovarian transplantation on the skeleton of osteopaenic ovariectomized rats. However, ovarian transplantation may not fully restore ovarian function to normal, and it is not known whether transplanted ovaries reproduce the action of native ovaries on the skeleton. Therefore, we investigated whether renal capsular or subcutaneous ovarian transplants prevent the effects of ovariectomy on histomorphometric indices of rat tibiae over 44 days. Daily vaginal smears showed that oestrous cycles returned in all but two of 25 animals receiving ovarian transplants. We found that ovarian transplantation prevented the reduction in cancellous bone volume following ovariectomy. While trabecular number was reduced in ovariectomized animals receiving renal capsular ovarian transplants compared to intact animals, trabecular thickness was increased in both transplant groups. Ovarian transplantation also prevented the increase in cancellous and cortical bone formation, cancellous bone resorption and longitudinal growth rate caused by ovariectomy. We conclude that restoration of ovarian function by ovarian transplantation largely prevents the effects of ovariectomy on histomorphometric indices of rat tibiae, suggesting that transplanted ovaries can substitute for the action of native ovaries on the skeleton.

Journal of Endocrinology (1994) 142, 187–192

ABSTRACT

We have previously demonstrated that administration of oestrogen, at doses sufficient to raise serum concentrations to those seen in late pregnancy, increases trabecular bone formation in the metaphysis of adult rats. To determine whether prostaglandins (PGs), which have been shown to induce osteogenesis in vivo, play a role in the induction of bone formation by oestrogen, 13-week-old female rats were given daily doses of 4 mg 17β-oestradiol (OE₂)/kg for 17 days, alone or with indomethacin (1 mg/kg). The rats were also given double fluorochrome labels and at the end of the experiment tibias were subjected to histomorphometric assessment. Treatment with OE₂ suppressed longitudinal bone growth and increased uterine wet weight, as expected, and neither response was affected by indomethacin. Oestrogen also induced a threefold increase in trabecular bone formation in the proximal tibial metaphysis, which resulted in a substantial increase in trabecular bone volume. As previously observed, the increase in bone formation was predominantly due to an increase in osteoblast recruitment (as judged by an increase in the percentage of bone surface showing double fluorochrome labels), with only a minor increase in the activity of mature osteoblasts (as judged by the mineral apposition rate). Indomethacin abolished the increase in osteoblastic recruitment, but the activity of mature osteoblastic cells remained high. The bone formation rate and bone volume remained similar to controls. The results suggest that PG production may be necessary for the increased osteoblastic recruitment induced by oestrogen, but not to mediate the effects of oestrogen on the activity of mature osteoblasts.

Journal of Endocrinology (1992) 133, 189–195

ABSTRACT

Osteoclasts, the major agents of bone resorption, were isolated from neonatal rat bone, and the cytoplasmic spreading of these cells was measured after incubation in the presence or absence of hormones or other cell types. Salmon calcitonin, which inhibits osteoclastic bone resorption, reduced spreading in a dose-dependent manner and caused significant inhibition at concentrations as low as 6·7 pg/ml. Parathyroid hormone (PTH) had no effect on the spreading of isolated osteoclasts but if osteoblasts and osteoclasts were co-cultured the addition of PTH caused a marked increase in spreading at concentrations of 0·025 i.u./ml and above. The results suggest that while calcitonin is a direct inhibitor of osteoclastic activity, PTH may stimulate osteoclasts through a primary action on osteoblasts.

J. Endocr. (1984) 102, 281–286

ABSTRACT

Tri-iodothyronine (T₃) increases bone resorption in vivo and in vitro. In order to understand further the mechanisms by which this occurs we studied the effects of T₃ at concentrations in the range of 1 pmol/l–1 μmol/l on bone resorption by osteoclasts isolated from neonatal rat long bones. Osteoclasts were disaggregated and incubated either with or without UMR 106 cells or with mixed bone cells. We found that there was no effect of T₃ on bone resorption by osteoclasts incubated alone or co-cultured with UMR 106 cells. However, in culture with mixed bone cells there was a significant relationship between the concentration of T₃ and bone resorption (r = 0·54, P= 0·01) The greatest effect was observed at a T₃ concentration of 1 μmol/l at which a 1·8-fold increase in resorption was seen compared with control (P <0·005; paired t-test). We conclude that the ability of T₃ to increase osteoclastic bone resorption is not due to a direct action of T₃ on osteoclasts but is mediated by another cell present in bone. The observation that UMR 106 cells are unable to mediate this effect suggests that either the mediating cell is not osteoblastic or the phenotype of UMR 106 does not conform to the phenotype of osteoblastic cells that mediate the T₃ responsiveness of bone.

Journal of Endocrinology (1992) 133, 327–331

ABSTRACT

The calcitonin gene encodes a small family of peptides: calcitonin, calcitonin gene-related peptide (CGRP) and katacalcin. Whereas calcitonin is concerned with skeletal maintenance, the function, if any, of katacalcin is still unknown. In the present study we have assessed resorption of human cortical bone substrate by isolated rat osteoclasts and have shown that CGRP acts directly on the osteoclast to inhibit bone resorption. The three CGRP peptides (rat, human(a) and human(β)) caused an almost equivalent decrease in osteoclastic bone resorption and were approximately 1000-fold less potent than human calcitonin in this respect. The responses of human calcitonin and human CGRP(α) were additive. Furthermore, prior treatment with trypsin to destroy receptors abolished the responsiveness of osteoclasts to CGRP and calcitonin. The carboxyl- and amino-terminal fragments of CGRP were found not to inhibit bone resorption, suggesting that the whole molecule of CGRP is necessary for biological activity. We have therefore suggested that the calcitonin-like effects of CGRP, seen both in vivo in the rat bioassay and in vitro in organ cultures, are due to the direct action of CGRP on the osteoclast, probably mediated through the calcitonin receptor. Though it is unlikely that CGRP is involved in the regulation of plasma calcium, the peptide may be an important local regulator of bone cell function.

J. Endocr. (1987) 115,511–518