A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

1 Alpha-25,26-trihydroxyvitamin D3: an in vivo and in vitro metabolite of vitamin D3

A new metabolite of vitamin D3 has been isolated from the plasma of vitamin D3 treated cows and has been generated from 25(S),26-dihydroxyvitamin D3 with homogenates of vitamin D deficient chick kidney. This metabolite has been identified as 1,25,26-trihydroxyvitamin D3 by comigration with synthetic 1,25(S),26-trihydroxyvitamin D3 in four chromatographic systems, ultraviolet spectroscopy, mass spectrometry, and high-pressure liquid chromatography and mass spectrometry of derivatives. 1,25(S),26-Trihydroxyvitamin D3 is one-tenth as effective as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol 1,25-dihydroxyvitamin D receptor. Either 25(S),26-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 can serve as precursor for in vitro production of 1,25,26-trihydroxyvitamin D3 by chick kidney tissue.     

Development of 1,25-dihydroxycholecalciferol receptor in the duodenal cytosol of chick embryo.

The development of 1,25-(OH)2D3 receptor in the duodenal cytosol of chick embryo was studied by the sucrose density gradient analysis. The binding profile for 1,25-(OH)2D3 in the cytosol of vitamin D-deficient chick duodenum on the sucrose density gradient revealed 3 binding components, and the sedimentation constant was estimated as 2.5, 3.5 and 5.5S respectively. The 3.5S binding component has high affinity and low capacity for 1,25-(OH)2D3 and is thought to be 1,25-(OH)2D3 receptor. During the development of chick embryo, the 3.5S binding component was not detected in 13-day embryonic duodenum, it appeared on 15th day of incubation and then gradually increased to the level of vitamin D-deficient chick on 19th day of incubation. The 5.5S binding component was specific for 25-OH-D3 and it was found even in 13-day embryo, but it did not show any significant change during development. On the other hand, the 2.5S component was not specific for either 1,25-(OH)2D3 or 25-OH-D3. However, it was main binding component in early stages of development and decreased during development. From these results, it is suggested that the receptor for 1,25-(OH)2D3 is available a few days before hatching and the inability to produce CaBP in the duodenum of chick embryo could not be ascribed to the absence of the receptor.     

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Terminal differentiation of chick embryo chondrocytes requires shedding of a cell surface protein that binds 1,25-dihydroxyvitamin D3

Endochondral ossification comprises a cascade of cell differentiation culminating in chondrocyte hypertrophy and is negatively controlled by soluble environmental mediators at several checkpoints. Proteinases modulate this control by processing protein signals and/or their receptors. Here, we show that insulin-like growth factor I can trigger hypertrophic development by stimulating production and/or activation of proteinases in some populations of chick embryo chondrocytes. Cell surface targets of the enzymes include 1,25-dihydroxyvitamin D3 membrane-associated rapid response steroid receptor (1,25 D3 MARRS receptor), also known as ERp57/GRp58/ERp60. This protein is anchored to the outer surface of plasma membranes and inhibits late chondrocyte differentiation after binding of 1,25-dihydroxyvitamin D3. Upon treatment with insulin-like growth factor I, 1,25 D3 MARRS receptor is cleaved into two fragments of approximately 30 and 22 kDa. This process is abrogated along with hypertrophic development by E-64 or cystatin C, inhibitors of cysteine proteinases. Cell differentiation is enhanced by treatment with antibodies to 1,25 D3 MARRS receptor that either block binding of the inhibitory ligand 1,25-dihydroxyvitamin D3 or inactivate 1,25 D3 MARRS receptor left intact after treatment with proteinase inhibitors. Therefore, proteolytic shedding of 1,25 D3 MARRS receptor constitutes a molecular mechanism eliminating the 1,25-dihydroxyvitamin D3-induced barrier against late cartilage differentiation and is a potentially important step during endochondral ossification or cartilage degeneration in osteoarthritis.     

Appearance of the intestinal cytosolic receptor for 1,25-dihydroxyvitamin D3 during neonatal development in the rat

During the early suckling period in the rat, active calcium uptake by the intestine is refractory to stimulation by 1,25-dihydroxyvitamin D3. To determine the role that the specific cytosolic receptor for 1,25-dihydroxyvitamin D3 plays in this refractory state, the concentration of the receptor and its binding characteristics were measured during the neonatal period. From Scatchard analysis, the concentration of receptor in the adult intestine was 563 +/- 64 fmol/mg of protein. At 28 and 21 days postpartum, receptor concentrations were 711 +/- 129 and 251 +/- 36 fmol/mg of protein, respectively. Estimates from sucrose density gradient profiles and Scatchard analyses indicated that, at 14 and 7 days postpartum, receptor concentrations were less than 50 fmol/mg of protein. Equilibrium dissociation constants were similar at all times measured and ranged from 0.38 to 0.52 nM. These results suggest that the lack of response observed in the early neonatal intestine to 1,25-dihydroxyvitamin D3 may stem, at least in part, from the relative absence of receptors for 1,25-dihydroxyvitamin D3. Furthermore, the appearance of the receptor may be the determining factor in the initiation of active calcium absorption in the intestine and its regulation.     

Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. Chick kidney and brain.

Chick kidney and brain were analyzed for the subcellular distribution (if any) of a putative plasma membrane receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Fractionation protocols were found to be based not only on differential centrifugation conditions, but also gentleness of resuspension procedures, and sufficiently dense Percoll gradients. The postnuclear pellets were resolved on 21.85% Percoll gradients overlayed on 2.4 M sucrose cushions. For both kidney and brain, fraction 1 (bottom of tube) was found to be enriched over whole homogenate 5.4- and 1.6-fold, respectively, in acid phosphatase activity, fractions 2 through 5 were enriched four- and eightfold, respectively, in succinate dehydrogenase activity, fraction 8 contained Golgi, as judged by a small peak of alpha-mannosidase activity, and fraction 9 was enriched sevenfold (for each tissue) in Na+,K+-ATPase activity. Western analyses, using a characterized antibody to the putative chick intestinal plasma membrane vitamin D receptor, revealed the highest levels of antigenicity in both chick kidney and brain in plasma membrane and Golgi fractions, followed by unidentified membranes in fractions 6 and 7 of Percoll gradients. Distribution of specific binding of [3H]1,25(OH)2D3 in Percoll gradient fractions paralleled that of antigenicity. Qualitatively, kidney plasma membrane contained more antigen than brain plasma membrane after Western blot analyses; these results were mirrored by differences in specific binding of the tritiated secosteroid (65 +/- 14.5 and 34 +/- 11.9 fmol/mg of protein, respectively).     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.     

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3 modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.     

Tissue distribution of the 1,25-dihydroxyvitamin D3 receptor in the male rat.

Tissue distribution of 1,25-dihydroxyvitamin D3 receptors was studied in male rats using a quantitative immunoradiometric assay. Extracts were prepared from 16 different rat tissues and assayed for 1,25-dihydroxyvitamin D3 receptor. Measurable levels of receptor were detected in intestine, stomach, kidney, bone thyroid/parathyroid, skin, liver, spleen, heart and lung. The highest levels were found in the proximal small intestine and colon, containing over 1000 fmol/mg total protein, while ileum and kidney contained one-half and one-fourth of this amount, respectively. Other parts of the vitamin D endocrine system, including bone, thyroid/parathyroid and skin, contained moderate levels of receptor of 40 to 80 fmol/mg, while lung, heart, stomach, spleen and liver had levels at or below 20 fmol/mg. No 1,25-dihydroxyvitamin D3 receptor was detected in cerebrum, cerebellum or skeletal muscle. The data support a wide-spread role for 1,25-dihydroxyvitamin D3 on cellular processes and suggest a more important role for vitamin D in colon.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.