An estrogen-stimulated 1,25-dihydroxyvitamin D3 receptor in rat uterus

Sucrose density gradient analysis was utilized to determine whether 1,25-dihydroxyvitamin D₃ receptors are present in the rat uterus. A distinct 3.6S [³H]1,25-dihydroxyvitamin D₃ binding component was observed in chromatin extracts of estrogen-primed, ovariectomized rat uteri. Binding to this putative 1,25-dihydroxyvitamin D₃ receptor was inhibited by excess 1,25-dihydroxyvitamin D₃, but not by 25-hydroxyvitamin D₃, estradiol-17β, promegestone, or cortisol. Low levels of the receptor seemed to be present in the unprimed uterus. Estrogen injection significantly increased the number of 1,25-dihydroxyvitamin D₃ receptors and progesterone co-administration reduced, but did not abolish, this effect.     

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.     

Vitamin K-dependent gamma-carboxylation of the 1,25-dihydroxyvitamin D3 receptor.

It has been reported that vitamin K deficiency in the rat markedly increases the 1,25-dihydroxyvitamin D3 receptor (VDR) binding to DNA and that vitamin K-dependent gamma-carboxylation of endogenous substrates of the intestinal and renal cytosol, also containing VDR, sharply reduced that binding (Sergeev, I.N., and Spirichev, V.B. (1989) Nutr. Res. 9, 725-733). In the present study we have evaluated vitamin K-dependent 14CO2 incorporation to VDR quantitated by immunoprecipitation with anti-VDR monoclonal antibodies. The results obtained strongly suggest that VDR in vitro can undergo gamma-carboxylation in the presence of vitamin K1 and that 15-25% of Glu residues in the VDR are carboxylated in vivo. Taking into account our earlier findings, it is likely that the VDR gamma-carboxylation modulates its binding to DNA.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.     

Modulation of 1,25-dihydroxyvitamin D3 receptor by phospholipids and fatty acids

The structural relationship between several lipids and their respective capacities to inhibit the specific binding of [3H]-1,25 (OH)2 vitamin D3 to chick intestinal cytosol preparations was investigated. The lipids investigated were: synthetic 3-sn-phosphatidylcholine and 3-sn-phosphatidic acid, egg yolk 3-sn-phosphatidylcholine and its corresponding phosphatidic acid, and free unsaturated fatty acids and their esters. The results indicate that at least three structural elements in the phospholipid molecule appear to be important; these are: 1) the structure of the fatty acid, 2) the anionic properties of the phospholipid phosphate group, and 3) the glycerol phosphate portion of the molecule. Our data also demonstrate that the position (1 or 2) and the amount (single vs. double) of unsaturated fatty acids in the phospholipid molecule do not play a major role in the receptor-1,25 (OH)2 vitamin D3 interaction. Furthermore, under equilibrium conditions, kinetic and Scatchard analysis suggest that phospholipids or free fatty acids may bind at a site different from the 1,25 (OH)2 vitamin D3 binding site, and therefore inhibit the hormone binding via a noncompetitive conformational change in the receptor molecule. A model for this phospholipid/free fatty acid binding site is proposed.     

Developmental changes in rat kidney 1,25-dihydroxyvitamin D receptor

Kidney 1,25-dihydroxyvitamin D receptor (VDR) was examined in both young and aged male Fischer 344 rats. Cytosols prepared by direct homogenization of the kidney indicated no significant difference in the amount of unoccupied VDR in young (149 +/- 8 fmol/mg) and aged (155 +/- 8 fmol/mg) rats. Binding of kidney VDR to DNA-cellulose, however, was significantly different for the two groups. The assay indicated that about 44% and 24% of the VDR prepared from young and aged rats, respectively, were bound to calf thymus DNA. Elution profiles from DNA-cellulose chromatography displayed the presence of two peaks from young kidneys, while a single broad peak was evident from aged rats. Immunoblot analysis confirmed the existence of two receptor bands at 52K and 50K. The presence of the 50K band was greatly diminished or absent in aged samples. The 50K receptor form was observed to elute from DNA-cellulose at a higher salt concentration than the 52K-form. Similarly, prepared receptor extracts from intestinal tissue produced only a single band at 52K. These results demonstrate for the first time that the rat kidney possesses two forms of the receptor which have different affinities for DNA.     

Receptor for 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

Cytosol prepared in 0.3 M KCl from pituitary GH3 cells, but not from AtT-20 cells contains a receptor-like macromolecule that binds 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with specificity and high affinity (Kd = 2.9 x 10(-10) M). The GH3 cytosolic binding component sediments at 3.3 S in high-salt sucrose gradients and adsorbs to DNA-cellulose; its elution profile from DNA-cellulose and other biochemical properties are indistinguishable from those of classical 1,25(OH)2D3 hormone receptors. The presence of the 1,25(OH)2D3 receptor in pituitary cells which secrete primarily growth hormone and prolactin (GH3), but not in a line which secretes the 31,000-dalton ACTH precursor and its derived peptides (AtT-20), suggests that 1,25(OH)2D3 may play a regulatory role in specific pituitary cells.     

Immunomodulatory role of 1,25-dihydroxyvitamin D3.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25-D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. At a molecular level, 1,25-D3 inhibits the accumulation of mRNA for IL-2, IFN-gamma, and GM-CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th-clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone. When given in vivo, 1,25-D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25-D3-receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.     

Ligand-dependent regulation of the 1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells

The specific binding of radiolabeled 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to intact rat osteosarcoma (ROS 17/2) cells was followed for 24 h. In the presence of 0.5-1.5 nM 1,25(OH)2D3, hormone binding increased over a period of 12 h, from 1.1 X 10(4) to 1.3 X 10(5) receptors/cell. The elevated level of hormone binding persisted through 24 h provided that the initial concentration of hormone was maintained. The concentration dependence of this increase in receptor level was centered between 10 and 30 pM 1,25(OH)2D3, and the binding at 12 h exhibited the metabolite specificity expected for a 1,25(OH)2D3 receptor. The t 1/2 values for the disappearance of unoccupied and occupied receptors were roughly the same, approximately 2.7 h; therefore, the increase in hormone binding was not due to receptor stabilization. In comparison, hormone-receptor complexes appeared to dissociate with a t 1/2 of 1 h. alpha-Amanitin treatment reduced the magnitude of receptor accumulation by 50-60%, indicating that mRNA synthesis was required to achieve the maximal response. Ligand-dependent regulation of cellular receptor levels provides a mechanism for amplifying the primary hormonal signal and is predicted to influence the kinetics, magnitude, and dose dependence of cellular responses.     

Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system

In addition to its role in calcium and skeletal homeostasis, there is increasing evidence that the hormonal form of vitamin D, 1, 25-dihydroxyvitamin D(3), appears to serve as a modulator of the immune system. We have determined the level of the 1, 25-dihydroxyvitamin D(3) receptor (VDR) in resting and activated lymphocytes by immuno- and ligand-binding assays. As expected from previous work, the total T lymphocyte population contains VDR whose levels are increased when activated and treated with 1, 25-dihydroxyvitamin D(3). Surprisingly, the highest concentrations of VDR are found in CD8 lymphocytes, although significant amounts are also present in CD4 lymphocytes. Furthermore, B lymphocytes do not contain detectable amounts of VDR. Cells of the monocyte/macrophage lineage possess small amounts of VDR that are not affected by activation but are increased by treatment with 1, 25-dihydroxyvitamin D(3). These results suggest that CD8 lymphocytes may be a major site of 1,25-dihydroxyvitamin D(3) action, while B lymphocytes are likely not directly regulated by 1, 25-dihydroxyvitamin D(3).     

Evidence for multiple molecular weight forms of the chick intestinal 1,25-dihydroxyvitamin D3 receptor

Studies from many laboratories have reported apparent molecular weights for the chick intestinal 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] receptor varying from 47,000 to 67,000 daltons. We report here that in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.3 mM) and in the presence or absence of ligand, the apparent molecular weight of the receptor is 99,700 ± 9,400 (SD) daltons (as determined by gel filtration). In the absence of PMSF, however, the unoccupied receptor migrates with an apparent molecular weight of 51,400 ± 5,700 (SD) daltons. This smaller form of the 1,25(OH)₂D₃ receptor, upon incubation with [³H]-1,25(OH)₂D₃ in the presence of PMSF, then migrates with an apparent molecular weight of 95,900 ± 7,300 (SD) daltons. These results suggest the presence of heretofore unappreciated multiple molecular forms of the chick intestinal 1,25(OH)₂D₃ receptor.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains

Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.