(25(OH)D3), 61%; 24,25-dihydroxycholecalciferol, 29%; cholecalciferol,

Transcription

1 HIGHLY SPECIFIC BINDING OF 1,25-DIHYDROXYCHOLECALCIFEROL IN BONE CYTOSOL S. C. MANOLAGAS, C. M. TAYLOR AND D. C. ANDERSON Department of Medicine, University of Manchester School of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL and * Department of Medicine, University of Manchester School of Medicine, Hope Hospital, Eccles Old Road, Salford, M68HD (Received 1 March 1978) SUMMARY A method developed initially for the detection of high-affinity binding of glucocorticoids in the cytosol from foetal rat calvaria has been adapted for metabolites of vitamin D. Consistent displacement of [3H]1,25-dihydroxycholecalciferol ([3H]1,25(OH)2D3) by unlabelled 1,25(OH)2D3 was obtained with an apparent dissociation constant (Kd) 2\m=.\3\m=x\10\m=-\9mol/l. of The specificity of this binding was examined by competition experiments. Displacement of labelled 1,25(OH)2D3 by a 100-fold excess of unlabelled metabolites, expressing the fall with unlabelled 1,25(OH)2D3 as 100%, was as follows: 25-hydroxycholecalciferol (25(OH)D3), 61%; 24,25-dihydroxycholecalciferol, 29%; cholecalciferol, 3%. These are similar to results for the chick mucosa nuclear 1,25(OH)2D3 receptor. No displacement was obtained with corticosterone, testosterone, oestradiol or progesterone. When [3H]25(OH)D3 was used as ligand, a displacement curve with unlabelled 25(OH)D3 indicated only binding with a greater Kd (approximately 10\m=-\7mol/l). These data suggest a direct action of 1,25(OH)2D3 on bone which is similar to that of steroid hormones on their target tissues. INTRODUCTION It has recently been established that the principal biologically active metabolite of vitamin D3 is 1,25-dihydroxycholecalciferol (l,25(oh)2d3) (Fraser & Kodicek, 1970; Kodicek, Lawson & Wilson, 1970; Holick, Schnoes & DeLuca, 1971 ; Lawson, Fraser, Kodicek, Morris & Williams, 1971) and that it has the greatest stimulatory effect on the absorption of calcium in the upper gastrointestinal tract (Boyle, Miravet, Gray, Holick & DeLuca, 1972). Furthermore, it has been shown that l,25(oh)2d3 is the most potent agent in inducing bone résorption in vitro, a process probably directly involved in bone remodelling (Reynolds, 1974). Specific cytosol receptors for l,25(oh)2d3 have been reported in the gut mucosa of the chick (Brumbaugh & Haussler, 1974, 1975) and the rat (Shimura, Moriuchi & Hosoya, 1977), as well as in chick parathyroid tissue (Brumbaugh, Hughes & Haussler, 1975) and bovine and human adenomas (Hughes & Haussler, 1977). It appears that l,25(oh)2d3 acts in these systems in a manner similar to other steroids on their target organs. In a recent autoradiographic study, the uptake of [3H]l,25(OH)2D3 was demonstrated in the long bones of rachitic rats, but was not seen in rats replete with this metabolite of vitamin D (Favus & Wezeman, 1977). A simple method for the detection of the high-affinity binding of glucocorticoids in foetal rat calvaría has recently been developed (Manolagas & Anderson, 1978). Such binding had already been described for glucocorticoids by Feldman, Dziak, Koehler & Stern (1975) in cytosol obtained from a mixed population of collagenase-digested bone cells, but a maximum t Please address requests for reprints to this author.

2 value of only 1% for the ratio bound : free dexamethasone was obtained. By setting up all incubations in intact calvaría, the bound : free ratio in the cytosol was increased to 30 ± 5 (s.e.m.) % with displacement down to 3 + 2% by a 100-fold excess of unlabelled glucocorticoids. The characteristics of this binding were otherwise identical with those found by Feldman et al. (1975). In this paper the method described by Manolagas & Anderson (1978) has been adapted to examine the high-affinity binding of l,25(oh)2d3 in foetal rat calvaría. The specificity of this binding is compared with that of the high-affinity nuclear receptor protein for l,25(oh)2d3 from chick intestinal mucosa. MATERIALS AND METHODS Preparation of l,25-[26,27-3h]dihydroxycholecalciferol Tritiated l,25(oh)2d3 was prepared from 25-[26,27-3H]hydroxycholecalciferol ([3H]25(OH)D3; 12-2 Ci/mmol, The Radiochemical Centre, Amersham, Bucks) in kidney homogenates from chicks deficient in vitamin D (Fraser & Kodicek, 1970) and purified by chromatography on Sephadex LH-20 (Taylor, Mawer & Reeve, 1975). Incubation of calvaría and study of 1,25(0H) D3 binding Pregnant rats (Sprague-Dawley strain, days of gestation) were provided by the animal unit of Manchester University. The animals were killed by concussion and the foetal calvaría were dissected, rinsed and incubated as described for glucocorticoid receptors (Manolagas & Anderson, 1978). As in those studies, calvaría were incubated intact rather than cells either isolated by collagenase, since this treatment might destroy receptors, or after the preparation free ratio will be reduced of cytosol. From the law of mass action, it follows that the bound : as a function of protein dilution when cytosol is prepared before incubation with ligand. The present conditions were therefore designed to maximize the chance of detecting the high-affinity binding of l,25(oh)2d3. The calvaría were incubated overnight with tritiumlabelled ligand (concentration 1 IO-9 mol/1) with or without unlabelled steroid. Afterwards the calvaría were rinsed, sonicated and the sonicate was centrifuged at # for 1 prepare a soluble supernatant (cytosol) fraction. Bound and free steroid were separated with charcoal and the radioactivity was counted (see Manolagas & Anderson, 1978, for detailed method). Demonstration of the effect ofprotein-active agents on l,25(oh)2d3 binding Cytosol, prepared after incubation of calvaría with labelled l,25(oh)2d3 as described above, was exposed to Pronase (protease VI, Sigma P5130; 5-7 mg/ml) for 20 min at 37 C. Specificity of binding To test the specificity of the binding of l,25(oh)2d3 (1 IO-9 mol/1), 10, 100 and 1000-fold excesses of cholecalciferol (D3), 24,25-dihydroxycholecalciferol (24R,25(OH)2D3) and 25(OH)D3 were used as unlabelled competitors for the binding sites. In a further experiment, [3H]l,25(OH)2D3 was incubated with calvaría in the presence of a 100-fold excess of corticosterone, oestradiol, testosterone or progesterone. Experiment with the nuclear receptors preparedfrom the intestinal mucosa of the chick The nuclear receptors were obtained from the intestinal mucosa of chicks made deficient in vitamin D by the method of Lawson & Wilson (1974). After solubilization, 0-5 ml portions of diluted solution were equilibrated in plastic tubes at 4 C for 1 h with [3H] 1,25(OH)2D3 (2-5 x mol/1) either alone or with 10, 100 and 1000-fold excess unlabelled metabolites of vitamin D. Bound and free steroid were separated with dextran-coated charcoal and portions of the supernatant fraction counted for tritium. h to

3 RESULTS Consistent displacement of labelled l,25(oh)2d3 (1 IO-9 mol/1) in the presence of low concentrations of unlabelled l,25(oh)2d3 was obtained in five experiments with foetal rat calvaría. An initial bound : free ratio of 34-3 ± 6-3 (s.e.m.)% was obtained. In the presence of a 100-fold excess of unlabelled steroid the value of this ratio fell to %. In two experiments more detailed data were obtained (Fig. la), which indicated high-affinity binding of l,25(oh)2d3, comparable to that observed previously for glucocorticoids (a) (b) 30 3 O pa J Bound 109 (mol/1) Fig. 1. The graphical analysis (Rosenthal, 1967) of Scatchard (1949) plots obtained for the binding of (a) 1,25-dihydroxycholecalciferol ( ^=2 3 10"9mol/I) and ( >) dexamethasone (ATd===5x 10-9 mol/l) in intact calvaría of the rat. Each point represents the mean of duplicate incubations and different symbols in (a) refer to separate experiments. See text for assumptions made in calculating apparent K. 30 ^ 20 o 10-0J D3 24,25(OH)2D3 25(OH)D3 l,25(oh)3d Ds 24,25(OH)2D3 25(OH)D3 l,25(oh)2d Excess unlabelled sterol Fig. 2. Comparison of the displacement of [3H]l,25-dihydroxycholecalciferol (l,25(oh)2d3) by unlabelled cholecalciferol (D ) and its metabolites [24,25-dihydroxycholecalciferol (24,25(OH)2D3) and 25-hydroxycholecalciferoi (25(OH)D3)] from (a) cytosol from foetal rat bone and (b) nuclear receptor from chick intestinal mucosa at 10-, 100- and 1000-fold excess. Arrows indicate addition of [ah]l,25(oh)2d3 alone (IO-9 mol/1). Assuming that the bound : free ratio did not change after disruption of the cells and that the intracellular concentration of steroids is equal to that in the incubation medium, an apparent dissociation constant (Kd) at 4 C of approximately 2-3 IO-9 mol/1 was calculated, compared with 5 IO-9 mol/1 for dexamethasone in the same system (Fig. 16). The apparent intracellular concentration of binding sites for l,25(oh)2d3 was 0-85 IO-9 mol/1, com parable again with the value of 1-4 IO-9 mol/1 calculated for dexamethasone. In three separate experiments, the total protein concentration in the cytosol prepared from four

4 calvaría before separation with charcoal varied from 0-42 to 0-58, 0-63 to 0-67 and 0-60 to 0-78 mg/ml. Values for the displacement of labelled l,25(oh)2d3 by a 100-fold excess of related metabolites (Fig. 2a), expressing the fall in the presence of unlabelled l,25(oh)2d3 as 100% were: 25(OH)D3, 61%, 24R,25(OH)2D3,29%, and D3, 3%. These results and the shape of the displacement curves are similar to those observed using nuclear receptors prepared from the intestinal mucosa of the chick (Fig. 2b). In this system the Kà was 2-2 IO-9 mol/1 (C. M. Taylor, F. S. Jones & E. B. Mawer, unpublished observations). When corticosterone, oestradiol, testosterone and progesterone were allowed to compete with labelled 1,25(OH)2D3, no significant displacement of binding was seen. The binding was completely inhibited by incubation with Pronase at 37 C for 20 min. Finally, when [3H]25(OH)D3 was incubated in this system with increasing amounts of unlabelled 25(OH)D3, a much shallower displacement curve resulted which, making the same assumptions as for l,25(oh)2d3, gave an approximate Kà of 10~7 mol/1. DISCUSSION The binding data presented here demonstrate the presence of high-affinity proteinaceous binding sites for l,25(oh)2d3 in the cytosol of foetal rat bone. The characteristics of this binding are very similar to those described for the binding of glucocorticoids, which suggests a mode of action of l,25(oh)2d3 on bone similar to that of hormonal steroids on their target tissues (O'Malley & Schrader, 1976). The results obtained with 25(OH)D3 may be due to a specific binding protein for 25(OH)D3 in bone or more probably to contamination with binding proteins for 25(OH)D3 normally found in the plasma, as has been suggested by Van Baelen & Bouillon (1977). The high specificity of the receptor in bone for l,25(oh)2d3 is demonstrated in the competition experiments and it appears to be very similar to the nuclear receptor prepared from the intestinal mucosa of the chick. Moreover, when hormonal steroids were tested none of them exhibited significant affinity for the l,25(oh)2d3 binding sites and it is thought that this at the receptor level excludes any direct functional interrelationship between l,25(oh)2d3 and either the glucocorticoids or sex steroids. The present work suggests a mode of action for vitamin D in bone, but the direct role of l,25(oh)2d3 in inducing the mobilization of minerals either in vivo in the presence of parathyroid hormone (PTH) (Garabedian, Tanaka, Holick & DeLuca, 1974) or / vitro in the absence of PTH (Reynolds, 1974) is not yet understood. Furthermore, the process whereby calcitonin inhibits the résorption of bone induced by l,25(oh)2d3 in vitro (Reynolds, 1974) is also unclear. This study does not provide information on whether receptors for l,25(oh)2d3 are only present in certain specific types of bone cell (osteoclasts or osteoblasts) or whether all bone cells contain them. Nevertheless, we believe that this simple method will allow further studies on the function of these receptors, although direct hormonal manipulation will be necessary to reveal the possible interactions between polypeptide and steroid hormones in intact bone. During the course of our experiments, results were obtained by Kream, José, Yamada & DeLuca (1977) concerning the occurrence of high-affinity receptors for l,25(oh)2d3 in foetal rat and chick calvaría. These receptors were detected by sucrose-density centrifugation of cytosol prepared from calvaría and incubated with [3H]l,25(OH)2D3. In their study, labelled l,25(oh)2d3 could be displaced from a 3-5S macromolecule by unlabelled l,25(oh)2d3 but not by unlabelled 25(OH)D3; no estimate of the Kd for the receptorl,25(oh)2d3 complex, nor of the concentration of binding sites was made. We thank the Medical Research Council, the Wellcome Trust and the United Manchester Hospital Research Fund for their support, Mrs C. M. Case for technical assistance and Professor S. W. Stanbury for his advice. This work was presented to the Endocrine Section

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