Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

Oral 1,25(OH)2D3 pulse therapy

Conclusion In our experience, after a few months of therapy, every patient showed a marked improvement in both X-ray abnormalities derived from osteitis fibrosa and symptoms of renal osteodystrophy, especially bone pain, unless the serum phosphorus level was very high. The effectiveness of this therapy on the suppression of PTH secretion apparently depends on the initial PTH level, and also on the size of the gland itself. One of the major current difficulties in this therapy is the prevention of hypercalcemia when calcium carbonate is used. The calcium concentration of the dialysate must be reduced to 2.5 mEq/l not only for pulse therapy, but also for conventional therapy by vitamin D with calcium carbonate. Parathyroidectomy should be indicated only for the patient who does not respond to pulse therapy.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)2D3 and for glucocorticoids, and have examined the modulation of the 1,25(OH)2D3 receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)2D3 receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)2D3; this phenomenon occurred in a dose-dependent fashion for triamcinolone (10(-9) to 10(-7) M) with a maximum increase of 1,25(OH)2D3 receptor concentration of approximately equal to twofold. During the culture period, the 1,25(OH)2D3 receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)2D3 receptor concentration was higher in the triamcinolone-treated cells.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Biochemical characterization of 1,25(OH)2D3 receptors in chick embryonal duodenal cytosol

Summary This study presents measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)₂D₃ receptor in 15-, 18-, 19-, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks. The present results showed that: (a) serum 1,25(OH)₂D and 24,25(OH)₂D levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable; (b) serum calcium levels rise at hatching to adult levels; (c) the duodenal 1,25(OH)₂D₃ receptor is detectable in 15-day-old embryo and has a Kd similar to that of 118-day-old vitamin D-replete chicks; and (d) the activity of 1,25(OH)₂D₃ receptor in chick duodenal cytosol is maximal at hatching.     

1,25(OH)2D and 24,25(OH)2D production in the developing kidney

To clarify the state of vitamin D production by the developing kidney, firstly, we measured serum levels of 1,25(OH)₂D and 24,25(OH)₂D in humans of different ages (pregnant and nonpregnant women, adult males, children and newborn infants) and secondly, we measured 1- and 24-hydroxylase activity in the kidney mitochrondria of rats at different ages. The mean serum levels of 1,25(OH)₂D in pregnant women, cord blood and newborns were significantly higher than those in children and non-pregnant women and adult males. In newborns, the level increased with gestational age. Synthesis of 1,25(OH)₂D was, at least in part, under the control of the fetus and newborn, rather then being solely a reflection of the conditions prevailing in the mother. The 1-hydroxylase activity in mitochondria was highest in the 1- to 2-month-old rats, and it decreased gradually thereafter. The change in 1-hydroxylase activity with age was due to a change in the V_max of the system.     

Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2)

Summary Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)₂D₃ and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)₂D₃ and the initial⁴⁵Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)₂D₃ on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)₂D₃ for 48 hours and then incubated for 4 min in the culture medium containing 0.5 μCi/ml of⁴⁵CaCl₂. In high population density cultures, 0.25–1.0 pg/ml of 1,25(OH)₂D₃ stimulated the intracellular accumulation of⁴⁵Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of⁴⁵Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80–120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)₂D₃ are biphasic—low concentrations stimulating and high concentrations inhibiting⁴⁵Ca accumulation. The sensitivity of the cells to 1,25(OH)₂D₃ increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined inin vitro studies utilizing osteogenic cell culture systems.     

Early manifestations of vitamin D effects in rat osteogenic sarcoma cells

Summary We have recently demonstrated that 48 hour exposure of ROS 17/2 cells to low concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) (1.0 pg/ml) stimulated the cellular accumulation of⁴⁵Ca, and exposure to high concentrations (160 pg/ml) inhibited such accumulation. In the present study, short-term (15 min) effects of the sterol on⁴⁵Ca accumulation in ROS 17/2 cells were compared with the long-term (48 hours) effects in order to clarify mechanisms responsible for 1,25(OH)₂D₃ control of calcium metabolisms in ROS 17/2 cells. ROS 17/2 cells were grown for 48 hours in the presence and absence of 1,25(OH)₂D₃ and then incubated for an additional 15 min in the presence and absence of 1,25(OH)₂D₃ immediately before measuring⁴⁵Ca accumulation. Cellular⁴⁵Ca was measured after incubating the cells in the medium containing 0.5 μCi/ml of⁴⁵CaCl₂ for 4 min at 25°C. The effect of actinomycin D was determined by preincubating the cells in 0.1 μg/ml of actinomycin D for 45 min at 25°C. Exposure to low concentrations (1.0 pg/ml) of 1,25(OH)₂D₃ for either 48 hours or 15 min increased⁴⁵Ca in the cells by 10–20%. An additional 15 min exposure following 48 hour exposure yielded an increase in the cellular⁴⁵Ca similar to that after 48 hours or 15 min exposure. Exposure to high concentrations (160 pg/ml) for either 48 hour or 15 min decreased cell⁴⁵Ca by approximately 20%.An additional 15 min exposure to the high concentrations did not change the 48 hour effect. Actinomycin D reversed early inhibitory effects of high concentrations, but had no effect on the early stimulatory effects of low concentrations. These results suggest that the mechanisms underlying the 15 min and 48 hour effects of 1,25(OH)₂D₃ are the same. Moreover, the mechanism responsible for the inhibitory effect of high concentrations is dependent onde novo protein synthesis whereas that for the stimulatory effects of low concentrations is not.     

Histological observations on the failure of rachitic rat bones to respond to 1,25/OH)2D3

Summary Rachitic rats, maintained on diets with low or normal P contents, were given daily intraperitoneal doses of 1,25(OH)₂D₃ or 25OHD₃ at levels of 100 or 200 ng. Plasma chemistry was measured and the ash content and histological appearance of the bones investigated. Using labeled material it was shown that the dosing levels of 1,25(OH)₂D₃ employed ensured a higher than normal plasma concentration of that metabolite over the period between doses. 1,25(OH)₂D₃ was not as effective as 25OHD₃ in raising bone ash or reducing the amount of osteoid. The difference between the effects of the metabolites was evident at both dietary P levels, but more marked at the higher P level. In contrast, the metabolites reduced the width of the epiphyseal plate to an approximately similar degree, and this is possibly the reason why there are discrepancies between previous reports of the effectiveness of 1,25(OH)₂D₃ compared with 25OHD₃ or vitamin D₃. Dosing with 1,25(OH)₂D₃ failed to maintain a constant plasma P_i value over the period between doses in animals fed the low P diet.     

Comparison of 1,25-, 25-, and 24,25-hydroxylated vitamin D3 binding in fetal rat calvariae and osteogenic sarcoma cells

Summary The hormonal metabolite of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], exerts its biological effects by binding to a cytosolic receptor protein. Such a protein has been demonstrated in vitamin D₃ target organs including fetal rat calvariae and more recently in rat osteogenic sarcoma cells. In this study we have compared the binding of 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] to that of 1,25-(OH)₂D₃ in fetal rat calvariae and osteogenic sarcoma (OS) cells. Sucrose density sedimentation, DNA-cellulose chromatography, and intracellular uptake studies have been employed to evaluate these interactions. In cytosol preparations from calvariae, [³H]-1,25(OH)₂D₃ bound to a 3.3S macromolecule and to a much greater extent to a 5.8S macromolecule while both [³H]25(OH)D₃ and [³H]24,25(OH)₂D₃ bound to the 5.8S macromolecule. By incubating intact calvariae and OS cells with labeled metabolites and thus establishing binding intracellularly prior to cell disruption, we have found that the 3.3S protein which has high specificity for 1,25(OH)₂D₃ occurs inside the cells; the 5.8S protein, however, does not occur inside the cells but is generated after cell disruption. The [³H]-1,25(OH)₂D₃-receptor complex adsorbed to DNA-cellulose and was eluted from this affinity resin at 0.28M KCl. In contrast, [³H]25(OH)D₃ and [³H]-24,25(OH)₂D₃ binding activity did not adsorb to DNA-cellulose. We conclude that, in contrast to the 3.3S protein, the 5.8S macromolecule does not fulfill receptor criteria but is rather generated by the experimental manipulation of the bone cells. Our data suggest that the vitamin D₃ actions on bone are mediated only via the 3.3S receptor, and hence quantitative but not qualitative differences of the effects of the various metabolites are feasible. With technical assistance by M. Larsen, D. Meler, and M. LaFrance.     

Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

Effects of 1,25(OH)2D3 on bone tissue in the rabbit: Studies on fracture healing,disuse osteoporosis,and prednisone osteoporosis

Summary A closed tibial fracture, which was controlled by an intramedullary stainless steel pin, was created in 16 rabbits. Eight rabbits were treated with 75 ng of 1,25(OH)₂D₃ daily as subcutaneous (s.c.) injections. After three weeks, the fractured tibia resisted a force of 101,7±21.0 Newtons in the control group and 57.3±8.0 Newtons in animals given 1,25(OH)₂D₃ (m±SE,P<0.05). In another group of eight rabbits, the left hindleg was immobilized in a plastic splint. Four rabbits were given 75 ng of 1,25(OH)₂D₃/day s.c. and the effect of immobilization was studied on the calcaneus. Bone ash/cm³ of the calcaneus on the immobilized side was decreased by 11±2% in control rabbits and by 20±2% in the group treated with 1,25(OH)₂D₃ indicating a more advanced immobilization osteoporosis (m±SE,P<0.05), which was also demonstrated by studies of bone density. Eighteen rabbits were used in a study of the effects of 1,25(OH)₂D₃ on the development of prednisolone osteoporosis. The dose of prednisolone was 2.5 mg per day, given by the oral route. After four months, the density of the femur was 1.53±0.02 g/cm² in control rabbits and 1.42±0.01 in prednisolonetreated animals (P<0.01). In rabbits additionally given 1,25(OH)₂D₃, the mean value for bone density was further lowered (n.s.). It appears that 1,25(OH)₂D₃ exaggerates disuse osteoporosis and prednisolone osteoporosis and impairs fracture healing in rabbits. These results differ from what has been shown earlier with 1,25(OH)₂D₃ treatment in the rat.     

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

Summary Parathyroid 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.     

Enhanced suppression of 1,25(OH)2D3 and intact parathyroid hormone in Graves' disease as compared to toxic nodular goiter

Summary 1,25(OH)₂D₃, 25OHD₃, and intact parathyroid hormone, as well as various parameters of calcium-phosphorus metabolism were measured in 38 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Plasma 1,25(OH)₂D₃ levels were lower in GD patients (82 ±29 pmol/liter) than in those with TNG (155±32 pmol/liter) (P<0.0005). The mean value of 1,25(OH)₂D₃ in 45 controls was intermediate between the two groups of patients (140±41) and the difference was statistically significant. GD patients before and after treatment had higher alkaline phosphatase (P<0.05), lower intact parathyroid hormone (PTH) (P<0.05), and lower 1,25(OH)₂D₃ levels (P<0.0005 in the hyperthyroid andP<0.01 in the euthyroid state) than TNG patients. We conclude that increased skeletal calcium resorption is due to elevated levels of T₃ causing suppression of 1,25(OH)₂D₃ production and of PTH levels in both groups of patients albeit of different degrees. Furthermore, we postulate that the profound suppression of 1,25(OH)₂D₃ in GD is secondary to an immune-mediated phenomenon.     

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by ⁴⁵Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.     

Growth hormone and triiodothyronine permit an increase in plasma 1,25(OH)2D concentrations in response to dietary phosphate deprivation in hypophysectomized rats

Summary Hypophysectomy abolishes the four- to fivefold increase in plasma 1,25(OH)₂D levels that normally accompanies dietary phosphate deprivation in rats despite a smaller but significant decrease in plasma phosphate in these animals. This effect appears within 1 week of hypophysectomy and may be the result of a lack of GH, T₃, or some other pituitary hormone. In hypothyroid rats (2 weeks after TPTX) not given replacement T₃, plasma 1,25(OH)₂D levels rose threefold from 148±57 pmol/l to 402±96 pmol/l (mean±SD) after 4 days of dietary phosphate deprivation. However, in hypophysectomized animals given replacement T₃ alone, plasma 1,25(OH)₂D levels rose fourfold from 82±13 to 333±230 pmol/l after 4 days of phosphate deprivation. In addition, in hypophysectomized animals replaced with GH alone, plasma 1,25(OH)₂D levels rose from 243±86 to 525±85 pmol/l during phosphate deprivation. These results would suggest that both GH and T₃ must be absent to prevent enhanced renal 1,25(OH)₂D synthesis during phosphate deprivation. GH and T₃ appear to play a permissive role since plasma levels of these hormones do not increase when intact rats are deprived of phosphate. Furthermore, bioassayable somatomedin levels are also not increased in intact rats during phosphate deprivation as well as plasma levels of prolactin. As observed previously, plasma 1,25(OH)₂D levels were inversely correlated to plasma phosphate concentrations (r=0.46,P<0.025), despite the inclusion of data points for unreplaced hypophysectomized animals who were hypophosphatemic but showed no increase in plasma 1,25(OH)₂D. Thus the possibility remains that GH and T₃ may exert their effect by permitting the renal 25OHD-1α-hydroxylase to respond to a change in phosphate concentrations during dietary phosphate deprivation, that, in turn, may ultimately increase renal 1,25(OH)₂D synthesis and plasma levels of this hormone.     

Control of plasma 1,25-(OH)2-vitamin D concentrations by calcium and phosphorus in the rat: Effects of hypophysectomy

Summary The mechanism by which dietary phosphate deprivation elevates plasma 1,25-(OH)₂-D levels is not known. To evaluate the role of the pituitary in regulating plasma 1,25-(OH)₂-D concentrations, the responses of plasma 1,25-(OH)₂-D to dietary phosphate deprivation and, separately, to dietary calcium deprivation were evaluated in intact and hypophysectomized male rats. Among intact and hypophysectomized rats eating normal diets, plasma 1,25-(OH)₂-D levels averaged 228±76 and 148±62 pmol/1, respectively (P<0.01). During dietary phosphate deprivation, plasma 1,25-(OH)₂-D levels rose to 1160±260 in intact rats and fell to 90±26 pmol/l in hypophysectomized rats (P<0.001). By contrast, during dietary calcium deprivation, plasma 1,25-(OH)₂-D levels rose in both intact and hypophysectomized animals to 856±107 and 742±279 pmol/l, respectively (NS). In response to dietary phosphate deprivation, serum calcium concentrations rose as 1,25-(OH)₂-D concentrations rose in intact rats but remained at control levels in hypophysectomized rats. These results support the hypothesis that a pituitary hormone acting either directly or indirectly on the kidney mediates the increase in plasma 1,25-(OH)₂-D during dietary phosphate deprivation. The hypercalcemia that occurs in rats during dietary phosphate deprivation appears to depend on the elevation of plasma 1,25-(OH)₂-D.