C. J. Jagger

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.

Role for parathyroid hormone in mechanical responsiveness of rat bone

We investigated the relationship between parathyroid hormone (PTH) and mechanical stimulation in mechanically induced osteogenesis. In normal rats, mechanical stimulation of the eighth caudal vertebra induced an osteogenic response. This was augmented by a single injection of human PTH-(1-34) 30-45 min before loading. No osteogenic response was seen in thyroparathyroidectomized (TPTX) rats; the osteogenic response was restored by a single injection of PTH before stimulation, suggesting that physiological levels of PTH are necessary for the mechanical responsiveness of bone. c- fosexpression was detected only in the osteocytes of those rats that were both mechanically stimulated and given PTH. This suggests that PTH supports mechanically induced osteogenesis by sensitizing either the strain-sensing mechanism itself or early responses of bone to strain-generated signals. The osteogenic response was not augmented by two further daily injections of PTH and was not seen in TPTX rats in which PTH administration was started 3 days after loading. These results reveal a major role for PTH in the mechanical responsiveness of rat bone.

Estrogen suppresses activation but enhances formation phase of osteogenic response to mechanical stimulation in rat bone.

We used a model whereby mechanical stimulation induces bone formation in rat caudal vertebrae, to test the effect of estrogen on this osteogenic response. Unexpectedly, estrogen administered daily throughout the experiments (8-11 d) suppressed, and ovariectomy enhanced, mechanically induced osteogenesis. Osteogenesis was unaffected by the resorption-inhibitor pamidronate, suggesting that the suppression of bone formation caused by estrogen was not due to suppression of resorption. We found that estrogen did not significantly reduce the proportion of osteocytes that were induced by mechanical stimulation to express c-fos and IGF-I mRNA; and estrogen suppressed mechanically induced osteogenesis whether administration was started 24 h before or 24 h after loading. This suggests that estrogen acts primarily not on the strain-sensing mechanism itself, but on the osteogenic response to signals generated by strain-sensitive cells. We also found that when estrogen administration was started 3 d after mechanical stimulation, by which time osteogenesis is established, estrogen augmented the osteogenic response. This data is consistent with in vitro evidence for estrogen responsiveness in two phenotypically distinct bone cell types: stromal cells, whose functional activities are suppressed, and osteoblasts, which are stimulated, by estrogen.

Stimulation of bone formation by dynamic mechanical loading of rat caudal vertebrae is not suppressed by 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP).

We have recently developed an experimental system whereby pins inserted into the seventh and ninth caudal vertebrae of rat tails are used to load the eighth caudal vertebrae (C-8) in compression. In this model, a single 5-min period of dynamic loading, sufficient to induce strains within the range to which bones are exposed under physiological circumstances, stimulates lamellar bone formation in the cancellous bone of the vertebrae. The rapidity with which the increase in bone formation was induced raised the possibility that this bone formation might have occurred without prior resorption. To test the role of bone resorption in the response of the bone to mechanical stimulation, we compared the anabolic response to a single period of loading, of rats treated with 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP) or vehicle. We found that mechanical loading caused a significant increase in dynamic and static indices of bone formation. The same indices were unaffected by AHPrBP, while the bone formation rate in the tibiae was reduced by AHPrBP. These results suggest that the increased bone formation induced by mechanical stimulation in the cancellous bone of rat vertebrae is not dependent on bone resorption.

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.

REDUCTION IN DYNAMIC INDICES OF CANCELLOUS BONE FORMATION IN RAT TAIL VERTEBRAE AFTER CAUDAL NEURECTOMY

We have previously noted that a relatively large load (150 N) is required to induce a strain on the cortex of rat vertebrae similar to that induced on weight-bearing bones by normal mechanical usage. It seems unlikely that the musculature of the tail normally imposes loads of this magnitude, and this suggests that the quantity of bone in caudal vertebrae is maintained at a higher level than would be expected for the mechanical environment to which it is exposed. This high bone mass could represent a genetically determined minimum, or could be maintained through increased sensitivity to mechanical stimuli. To distinguish between these two possibilities, we denervated the tails of 13-week-old rats by neurectomy at L6, and assessed the response of the caudal vertebrae to mechanical disuse. We found that caudal neurectomy caused a reduction in the cancellous bone formation rate in the eighth caudal vertebrae to 12% of that seen in sham-operated animals. The cancellous bone formation rate in the thoracic vertebrae of neurectomized rats, which are not mechanically disused by caudal neurectomy, was not significantly reduced. This suggests that the cancellous bone formation rate in vertebrae is maintained by substantially less intense mechanical environments than those prevailing in weight-bearing bones, raising the possibility that bones may differ in their sensitivity to mechanical strain.