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Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine.

ABSTRACT: 1. The intranuclear distribution of cholecalciferol and its metabolites was studied in the intestine of rachitic chicks. 2. At high doses of cholecalciferol the nuclei contain the vitamin and its 25-hydroxy metabolite, but over 80% of this is localized on the nuclear membranes. The hormone, 1,25-dihydroxycholecalciferol, is found within the cell nuclei irrespective of the intake of cholecalciferol, but significant amounts could not be found with chromatin isolated free of nuclear membranes. 3. 1,25-Dihydroxycholecalciferol is associated in the nucleus with an acidic protein. Since one of the actions of 1,25-dihydroxycholecalciferol is to control the synthesis of mRNA for calcium-binding protein it was to be expected that the hormone would be bound to chromatin, as with the other steroid hormones. It is suggested that the hormone-receptor complex exists as part of an equilibrium mixture of the complex bound to the DNA and in a free form. 4. A protein extract of nuclei was obtained, which when incubated at 4 degrees C for 1h took up the 1,25-dihydroxycholecalciferol. The nature of this binding was studied. 5. There appear to be two nuclear proteins able to bind the hormone one of which is the intestinal nuclear receptor. The binding sites on this protein are saturable with the hormone, have an association constant of 2x10(9)m(-1) and show a high chemical specificity for the 1,25-dihydroxycholecalciferol. The number of nuclear binding sites for the hormone provided by this receptor is similar to the maximum intestinal hormone concentration so far observed. Its sedimentation coefficient is 3.5S, and is very close to that observed for the nuclear protein to which is attached the 1,25-dihydroxycholecalciferol formed in vivo from vitamin D. 6. The cytoplasmic protein has an association constant of 1x10(9)m(-1)and a sedimentation coefficient of 3.0S, but its relation with the nuclear receptor is not yet clear.

SUBMITTER: Lawson DE

PROVIDER: S-EPMC1168536 | biostudies-other | 1974 Dec

REPOSITORIES: biostudies-other

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Project description:1. A simple technique has been developed to obtain subcellular fractions of chick bone. The method yielded 60-70% of total DNA in the nuclear debris fraction and 80-90% of total (14)C recovered in bone after a dose of radioactive vitamin D. 2. After a dose of [4-(14)C,1,2-(3)H(2)]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the time-course of total (14)C radioactivity in the epiphysis, metaphysis and diaphysis of proximal tibiae was measured. The maximum concentrations were reached at 6h, corresponding to a similar peak of radioactivity in blood, decreasing until 24h and indicating the dependence on the circulating (14)C and on the blood supply of the three bone components. 3. The (14)C radioactivity of cholecalciferol and 25-hydroxycholecalciferol (expressed per mg of DNA) followed the pattern of incorporation of total (14)C radioactivity in all three bone components. The more polar metabolite fraction reached a peak of radioactivity at 6-9h and maintained its concentration over the 24h period studied in all anatomical bone components. 4. After a dose of [4-(14)C,1-(3)H]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the subcellular distribution was studied. At 24h after dosing, the nuclear fraction contained 27% and the supernatant fraction had 67% of total (14)C recovered in the bone filtrate. When the (14)C in the residual bone fragments was included, the nuclear fraction contained up to 35% of the total radioactivity in the bone. 5. The subcellular distribution pattern of individual vitamin D metabolites indicated that the purified nuclear fraction concentrated the polar metabolite, which lost (3)H at C-1, so that 77% of the radioactivity could be accounted for by 1,25-dihydroxycholecalciferol. The supernatant fraction contained smaller amounts of 1,25-dihydroxycholecalciferol (9%), with 66% of 25-hydroxycholecalciferol forming the major metabolite, corresponding to its concentration found in blood at 24h. 6. The preferential accumulation of 1,25-dihydroxycholecalciferol in the nuclear fraction and the overall pattern of other metabolites, found previously in intestinal tissue, suggests a similar mechanism of action in bone to that postulated for the intestinal cell in calcium translocation.

Project description:Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH)(2)D(3)] have been described recently in broken-cell preparations of several cultured human breast cancer cell lines including the T47 D line. It was necessary to determine whether intact breast cancer cells in culture would bind 1,25-(OH)(2)D(3) specifically and whether the next step in the proposed scheme of action, i.e. nuclear translocation, occurred. The following results were obtained. (1) Specific uptake of 1,25-(OH)(2)D(3) by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH)(2)D(3) concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5nm-1,25-(OH)(2)D(3) in the medium, at least 4h are required to reach maximum compared with less than 1h for oestradiol binding. (3) Estimation of binding characteristics by Scatchard analysis indicates a single class of binding sites with K(d) of 68pm and 11800 binding sites/cell, which are similar results to those obtained with broken-cell preparations. (4) Inclusion of various vitamin D metabolites in the incubation medium decreased specific binding of 1,25-(OH)(2)D(3) by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca(2+) transport and bone resorption in vivo. Order of potency was 1,25-(OH)(2)D(3)>(24R)-1,24,25-trihydroxycholecalciferol >>25-hydroxycholecalciferol>(25R)-24,25-dihydroxycholecalciferol >>(25R)-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH)(2)D(3)-depleted state, 80% of the 1,25(OH)(2)D(3) receptor is found in the cytosol fraction of the cells even when the subcellular fractionation is performed under low-salt conditions. By contrast after incubation with [(3)H]1,25-(OH)(2)D(3), 59% of the specific 1,25-(OH)(2)D(3) binding is found in the partially purified nuclei fraction. These data indicate that nuclear translocation of the receptor-hormone complex takes place in the intact T47 D cell. The results also support the hypothesis that the 1,25-(OH)(2)D(3) receptor is functional in this cultured breast cancer cell line, which may provide a useful model for further study of the early biochemical events in 1,25-(OH)(2)D(3) action.

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Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine.

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