Theresa L. Chen

1,25-dihydroxyvitamin D resistance, rickets, and alopecia

Two unrelated kindreds with four affected children having 1,25-dihydroxyvitamin D resistance, rickets, and alopecia are described. The children exhibited early onset of severe rickets with hypocalcemia, hypophosphatemia, elevated serum alkaline phosphatase levels, and secondary hyperparathyroidism. Radiography showed diffuse demineralization and classic changes of rickets. All affected children had total-body alopecia. Serum levels of 1,25-dihydroxyvitamin D3 were elevated and rose to extremely high values during treatment, with no apparent change in the mineral disorder. However, secondary hyperparathyroidism and hypophosphatemia did remit during treatment despite persistently low calcium levels. Skin biopsy was performed in the parents and affected children in one kindred. Analysis of 1,25-dihydroxyvitamin D3 receptors in cultured fibroblasts indicated apparent normal receptors in the parents and undetectable receptors in both affected children. After long periods of treatment with vitamin D metabolites and mineral replacement, healing took place in the older child in each kindred. These data suggest that the healing occurred spontaneously as the children reached seven to nine years of age rather than as a result of the treatment. The biochemical lesion in these children appeared to be a genetically transmitted defect in the 1,25-dihydroxyvitamin D3 receptor. The mechanisms by which healing was initiated and maintained remain to be elucidated.

Dexamethasone and 1,25(OH)2 vitamin D3 modulate the synthesis of insulin-like growth factor-I in osteoblast-like cells

SummaryIn the present study, we have shown that insulin-like growth factor-I (IGF-I) was released by primary cultures of rat osteoblast-like (ROB) cells into the conditioned medium (CM). Dexamethasone (DEX) caused a dose-dependent inhibition of the IGF-I. At 10−8 M, DEX reduced IGF-I level to 70% of the control value (P<0.05); at 10−7 M DEX, the IGF-I level was further reduced to 60% of the control (P<0.01). The active vitamin D metabolite 1,25-dihydroxycholecalceferol [1,25(OH)2D2] slightly increased the IGF-I level, but the increase was not statistically significant. However, in combined treatments of 10−7 M DEX and 10−8 M of 1,25(OH)2D3, the inhibition of DEX was partially antagonized by the presence of 1,25(OH)2D3. Studies with metabolically radiolabeled IGF-I by immunoprecipitation indicated the changes of IGF-I in the CM reflected synthesis of the protein by the cells. The alteration of IGF-I level may mediate some of the actions of these steroid hormones on bone cells.

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

SummaryThe effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its interaction with glucocorticoids to regulate bone cell growth were studied in osteoblast-like (OB) 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 >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 thein 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 treatmentin vitro.

Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: Species differences between rat and mouse

Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells.

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: the effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids.

SummaryParathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)2D3 are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED50 ∼ 1.0 nm). On the other hand, 1,25(OH)2D3 decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)2D3 treatment. Introduction of physiological concentrations of 1,25(OH)2D3 (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)2D3 and a complex interplay among PTH, dexamethasone, and 1,25(OH)2D3.

Fetal rat bone in organ culture: Effect of bone growth and bone atrophy on the comparative losses of45Ca and3H-tetracycline

SummaryFetal rat bones were cultured in either growth-inducing or resorption-inducing media to study mineral losses during bone growth and atrophy in vitro. Whole radii and ulnae from 19-day-old fetal rats, prelabeled with45Ca and/or3H-tetracycline, were cultured intact or cut, and then digested by collagenase to obtain the calcified portion of the bones. Three-to five-fold more3H-tetracycline than45Ca was lost from the calcified portion when the bones were cultured for 4 days in growth-inducing media. Similar small amounts of45Ca were lost from live and killed bones, but more3H-tetracycline was lost from live bones than from killed bones. More3H-tetracycline was released into the growth medium with a low concentration of calcium (0.5 mM) than when the calcium concentration was high (1.0 mM); no significant difference was seen in the release of45Ca into the medium at different calcium concentrations. Larger amounts of both isotopes were lost when the prelabeled bones were cultured in resorption-inducing media than in growth-inducing media. When parathyroid hormone stimulated bone resorption in a resorption-inducing medium, equal proportions of both isotopes and bone collagen were lost. Greater losses of3H-tetracycline than of45Ca suggest that45Ca was conserved locally during the resorption that accompanies bone growth, but not during resorption that accompanies bone atrophy.

Lawrence G. Raisz November 13, 1925-August 25, 2010.

No one person in the world of bone has had a greater impact than Larry on us all. To honor his memory, we asked some of Larry’s closest friends and colleagues to reflect upon his extraordinary life. What follows is an expression of that meaning sprinkled with Larry’s inimitable wit and reflections on life, science, and the future.