Bone response to normal weight bearing after a period of skeletal unloading.

Skeletal unloading in the growing rat induces a temporary inhibition of bone formation and thereby a deficit in bone calcium compared with age-matched, normally loaded animals. To determine whether this deficit can be restored by skeletal reloading we measured bone formation rate at the tibiofibular junction and total bone calcium in the tibia and lumbar vertebra in rats whose hindlimbs were unloaded for 2 wk and then reloaded by return to normal weight bearing. Continuously loaded or unloaded animals were also studied. Skeletal unloading reduced bone formation by 34% and tibial and vertebral calcium by 12 and 22%, respectively. Reloading significantly increased the rates of bone formation and calcium accretion 30-34% above normally loaded animals, and by 2 wk had decreased the deficit in tibial and vertebral calcium by 36 and 23%, respectively. These data indicate that the deficit in bone calcium induced by skeletal unloading in the growing rat can be restored in part by return to normal weight bearing. However, the time required to restore bone calcium exceeds the time required to produce the original calcium deficit.

Glucocorticoids and inhibition of bone formation induced by skeletal unloading.

Skeletal unloading or loss of normal weight bearing in the growing animal inhibits bone formation and reduces bone calcium. To determine whether the inhibition of bone formation induced by skeletal unloading is a consequence of an increase in plasma glucocorticoids and/or an increase in bone sensitivity to glucocorticoids, we measured plasma corticosterone throughout the day in unloaded and normally loaded rats (hindlimb elevation model) and examined the effect of adrenalectomy on the response of bone to skeletal unloading. Plasma corticosterone levels were similar in normally loaded and unloaded rats at all times. Skeletal unloading in sham-adrenalectomized animals reduced tibial and vertebral calcium by 11.5 and 11.1%, respectively, and in adrenalectomized animals by 15.3 and 20.3%, respectively. Uptake of 45Ca and [3H]proline in the tibia was reduced by 8 and 14%, respectively, in the sham-adrenalectomized animals and by 13 and 19% in the adrenalectomized animals. Bone formation and apposition rates were reduced to the same level in sham- and adrenalectomized animals. These results suggest that the inhibition of bone formation induced by skeletal unloading is not a consequence of increased plasma glucocorticoids or an increase in bone sensitivity to the glucocorticoids but, rather, point to a local mediator in bone that senses mechanical load and transmits that information to the bone-forming cells directly.

The effects of simulated weightlessness on bone maturation.

In earlier studies we showed that elevating the hind limbs of growing rats for up to 2 weeks results in a temporary cessation of bone growth in the hind limbs and a transient fall in the serum levels of 1,25-dihydroxyvitamin D. To determine whether such skeletal unloading also retards the maturation of bone, as seen in vitamin D-deprived animals, we fractionated by density the tibiae from rats whose hind limbs had been elevated for up to 15 days. These fractions were analyzed for dry weight, calcium content, and calcium and proline uptake. The most dense fraction (fraction 4) had the highest degree of mineralization (ratio of calcium to dry weight) and comprised 82% of the total dry weight of the control tibiae. The total incorporation of [3H]proline administered in vivo 24 h before removing the tibiae was evenly distributed among all of the fractions, although it was highest in the least dense fraction (fraction 1) when normalized to dry weight. Total incorporation of 45Ca was highest in fraction 4, although when normalized to dry weight it was highest in fraction 3. With skeletal unloading, the proportions of bone and 45Ca incorporation in fraction 4 decreased, while the proportions in less dense fractions increased. [3H]Proline incorporation fell in all fractions. These effects were maximal after 10 days of unloading and returned toward the control values after that time. We conclude that skeletal unloading transiently reduced bone formation and retarded mineralization in the growing rat, which resulted in a decrease in mature bone.

Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level.

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3-induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.

1,25-dihydroxyvitamin D resistance, rickets, and alopecia: analysis of receptors and bioresponse in cultured fibroblasts from patients and parents.

To investigate further the cellular defects of vitamin D-dependent rickets type II with alopecia, we studied 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and the response to 1,25-(OH)2D3 in cultured skin fibroblasts from rachitic patients. Our studies included cells from four affected patients from three kindreds and their parents and cells from five normal subjects. We measured total 1,25-(OH)2D3 receptor binding in cell extracts and the capacity of 1,25-(OH)2D3 to induce the enzyme 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) as a marker of functional response. In normal fibroblasts, the 1,25-(OH)2D3 maximal binding capacity was 52 +/- 5 fmol/100 micrograms DNA (mean +/- SE), and the apparent dissociation constant (Kd) was 0.05 +/- 0.01 nM. The maximal induced 24-hydroxylase activity after 1,25-(OH)2D3 treatment was 11.5 +/- 1 fmol/10(6) cells X 30 min, and the dose of 1,25-(OH)2D3 that achieved half-maximal induction was 2.3 +/- 0.3 nM. Fibroblasts from all four rachitic patients had the same defect: no measurable 1,25-(OH)2D3 receptor binding and no detectable response above basal activity even after high doses of 1,25-(OH)2D3. Cells from all parents except one had normal 1,25-(OH)2D3 binding characteristics and normal 24-hydroxylase bioresponse to 1,25-(OH)2D3. One parent despite a normal phenotype had only half the normal level of binding sites and only half the normal bioresponse. In summary, the cultured fibroblasts from four affected children representing three different kindreds with 1,25-(OH)2D3 resistance failed to exhibit detectable 1,25-(OH)2D3 receptors. We postulate that this biochemical defect produced both the inability to respond to 1,25-(OH)2D3 in vitro and the 1,25-(OH)2D3 resistance in vivo. The obligate heterozygotic parents were normal, except for one who had both half the normal number of receptors and half the normal response to 1,25-(OH)2D3. The data confirm the critical role of the receptor in 1,25-(OH)2D3 action and the close coupling of receptor content and functional responsiveness.

Effects of 1 alpha,25-dihydroxyvitamin D3 and glucocorticoids on the growth of rat and mouse osteoblast-like bone cells.

The effects of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its interaction with glucocorticoids to regulate bone cell growth were studied in osteoblast-like (OH) cell cultures. Owing to our earlier findings that species difference and cell density at the time of treatment modified hormonal responses, comparisons were made between rat and mouse cells and sparse and dense cultures. 1,25(OH)2D3 inhibited cell proliferation in both species regardless of cell density. The magnitude of inhibition was larger in mouse cells, but the sensitivity to 1,25(OH)2D3 was the same for both species. Other metabolites, 25(OH)D3 and 24R,25(OH)2D3, were greater than 100-fold less potent than 1,25(OH)2D3 even in serum-free medium, which is similar to their ratio of affinity for the 1,25(OH)2D3 receptor. Dexamethasone, as previously shown, inhibited sparse and dense mouse cell cultures and sparse rat cell cultures while stimulating dense rat cell cultures to grow. The inhibitory actions of 1,25(OH)2D3 were not additive to the inhibitory dexamethasone effects. However, 1,25(OH)2D3 addition resulted in attenuation of the stimulatory effect of dexamethasone. These responses to 1,25(OH)2D3 and dexamethasone were dependent on cell density and not selective attachment of certain cell types at either plating density. In conclusion, the findings demonstrated that 1,25(OH)2D3 exerts an inhibiting action on both mouse and rat bone cell proliferation. This effect must be reconciled with the in vivo beneficial actions of 1,25(OH)2D3 on bone metabolism. Also, the likelihood of decreased cell number must be considered when biochemical activities are assessed after vitamin D treatment in vitro.

Glucocorticoid modulation of cell proliferation in cultured osteoblast-like bone cells: differences between rat and mouse.

Glucocorticoids are important regulators of cell proliferation, but divergent effects have been noted in various systems. In this study, the actions of glucocorticoids on cell proliferation were examined in primary cultures of osteoblast-like cells from perinatal rodents. The aim of the study was to determine whether rat and mouse bone cells exhibit species differences in their responses to glucocorticoids and whether the response of either species was influenced by the cell density and time of treatment. Cultured osteoblast-like cells were treated with dexamethasone or ethanol vehicle (less than 0.05%). Changes in the proliferation rate were assessed by measurement of cell number, DNA content, and thymidine incorporation rate. We found that the response to dexamethasone was dependent on the cell density at the time of treatment in rat cultures: inhibitory in sparse and stimulatory in dense cultures. In contrast, dexamethasone inhibited mouse cell proliferation at all cell densities. The stimulatory effects in rat cells were seen after 4 days of treatment but not after 2 days. Both the stimulatory and inhibitory effects in rat and mouse cells occurred between dexamethasone concentrations of 1.3-13 nM, with half-maximal effects seen at 6 nM. The divergent responses in sparse and dense rat cultures were found not to result from the selective attachment of cell subpopulations at different plating densities. Epidermal growth factor was mitogenic in both species. The effects of dexamethasone were not modified by the presence of epidermal growth factor in either low (1%) or high (10%) serum concentrations. In conclusion, these findings reemphasize the importance of species differences and the difficulty in comparing one system to another, even with closely related species such as mouse and rat. Furthermore, the data show that the culture conditions at the time of treatment dramatically influence the responses of these cells to glucocorticoids. These factors must be considered in studies of the hormonal regulation of bone cell proliferation.

1 alpha,25-dihydroxyvitamin D3 receptors in cultured rat osteoblast-like cells. Glucocorticoid treatment increases receptor content.

The direct actions of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells are initiated by the binding of hormone to specific receptor sites. The aims of this study were to characterize the receptor for 1,25(OH)2D3 in cultured rat osteoblast-like (OB) cells and to determine whether these receptors are regulated by either the culture cycle itself or glucocorticoid treatment. The 1,25(OH)2D3 receptor in rat OB cells exhibited the same apparent binding affinity (Kd = 0.1 nM) and sedimentation coefficient (3.2 S) as mouse OB cells and receptors in other target organs. However, the receptor concentration in rat OB cells was substantially lower than mouse OB cells (approximately 20%). The concentration of receptors in rat OB cells did not show a correlation with the rate of DNA synthesis and therefore did not exhibit an endogenous rhythm in receptor concentration as was previously seen in mouse OB cells. Also in contrast to mouse cells, where glucocorticoids caused a decrease in receptor level, dexamethasone induced a marked increase in receptor binding throughout the culture cycle. This increase was due to an increase in receptor number with no change in receptor affinity. The change was glucocorticoid-specific, dose-dependent with half-maximal stimulation occurring between 1.3 and 13 nM and exhibited a latent period of at least 4 h. The independence of receptor concentration from DNA synthesis rate was established by assessing receptors after stimulating cell proliferation with epidermal growth factor and inhibiting it with hydroxyurea. Neither treatment altered basal 1,25(OH)2D3 receptor concentration or prevented the marked increase in receptor levels elicited by dexamethasone. We conclude that although the biochemical characteristics of 1,25(OH)2D3 receptors are indistinguishable in rat and mouse OB cells, there are genuine species differences in the regulation of the receptor number as it relates to DNA synthesis rate and response to glucocorticoids.

Blockage of depolarization-induced mitogenesis in CNS neurons by 5-fluoro-2'-deoxyuridine.

Experiments designed to provide further evidence, at the basic metabolic level, that true mitogenesis and mitotic activity are being induced in CNS neurons in response to sustained ionic depolarization were conducted. The ability of 5-fluoro-2'-deoxyuridine (FUdR), a well-studied inhibitor of normal mitogenesis in naturally proliferating cells, to block induction of DNA synthesis (and subsequent nuclear division) in culture-matured neurons depolarized with ouabain was ascertained, as well as the ability of exogenously supplied thymidine to permit effective bypass of such blockage. Observations of the sequence of intracellular morphological changes induced by ouabain were also made, along with a determination of the alterations in this sequence introduced by FUdR. The results indicate that ouabain mediated depolarization rapidly induces and/or activates the key mitogenic enzyme thymidylate synthetase in the mitotically quiescent neurons, along with all other mitogenesis-specific enzymes required for DNA synthesis and nuclear division. A probable mechanism by which such mitogenic induction may proceed is elaborated. The early morphological changes observed correlate well with the early time-sequence of mitogenic metabolic events, while development of the later changes appears to be dependent upon the progress of mitogenesis activity. The results support the possibility that CNS neurons of adult origin may also be induced to initiate normal mitogenesis by appropriately imposed depolarization treatments.

Induction of mitosis in mature neurons in central nervous system by sustained depolarization.

DNA synthesis and mitosis have been induced in vitro in fully differentiated neurons from the central nervous system by depolarization with a variety of agents that produce a sustained rise in the intracellular sodium ion concentration and a decrease in the potassium ion concentration. Depolarization was followed in less than 1 hour by an increase in RNA synthesis and in 3 hours by initiation of DNA synthesis. Apparently normal nuclear mitosis ensued, but cytokinesis was not completed in most cells; this resulted in the formation of binucleate neurons. The daughter nuclei each contained the same amount of DNA as the diploid preinduction parental neurons; this implies that true mitogenic replication was induced.