0021-972X/90/7004-1068$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 4 Printed in U.S.A.
25-Hydroxyvitamin D-24-Hydroxylase in Phytohemagglutinin-Stimulated Lymphocytes: Intermediate Bioresponse to 1,25-Dihydroxyvitamin D 3 of Cells from Parents of Patients with Vitamin DDependent Rickets type II* EIJI TAKEDA, ICHIRO YOKOTA, MICHINORI ITO, HIDEAKI KOBASHI, TAKAHIKO SAIJO, YASUHIRO KURODA Department of Pediatrics, University of Tokushima School of Medicine, Tokushima, Japan
ABSTRACT. A method for assay of 25-hydroxyvitamin D-24hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10~8 mol/L and (1.3 ± 0.4) x 10"9 mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10~8, 10~7, and 10~6 mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated
T
HE CHARACTERISTIC clinical features of vitamin D-dependent rickets type II (VDDR II) are early onset of rickets, alopecia, hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum levels of alkaline phosphatase activity and 1,25dihydroxyvitamin D [1,25-(OH)2D] (1). The existence of siblings with this disease (2-5) suggests that vitamin D resistance is an inherited disease, probably transmitted as an autosomal recessive trait. The presence of a heterozygous state in the parents' receptors has recently been demonstrated (6, 7). However, the method used for their detection is not suitable for detection of heterozygotes of this disease in the general population. Cultured skin fibroblasts have been used to analyze the receptor status of patients with this disease (1). Currently, available data indicate that various steps in the intracellular Received February 13, 1989. Address all correspondence and requests for reprints to: Eiji Takeda, M.D., Department of Pediatrics, University of Tokushima School of Medicine, Kuramoto-Cho 2, Tokushima City, Tokushima 770, Japan. * This work was supported by Grant 62570428 from the Ministry of Education, Science, and Culture of Japan and a grant from the Mother and Child Health Foundation.
the presence of an intracellular receptor-effector system for 1,25(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease. {J Clin Endocrinol Metab 70:10681074,1990)
mechanism of action of 1,25-(OH)2D are impaired in patients with this disease. Stimulation of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity by 1,25-(OH)2D3 has been investigated to evaluate the overall pathway of hormone action in cultured skin fibroblasts (2, 8, 9). 24-Hydroxylase activity appears to be a useful index of the 1,25(0H) 2 D effector pathway, because it changes in parallel with modulation of receptors during change in the rate of cell division (10). In addition, we demonstrated previously that the effect of 1,25-(OH)2D3 on DNA biosynthesis in phytohemagglutinin (PHA)-stimulated lymphocytes is another index of responsiveness to 1,25-(OH)2D3 and that the expression of the 1,25-(OH)2D3 receptor in PHA-stimulated lymphocytes, skin fibroblasts, and bone might be controlled by the same gene (11). These findings strongly suggested that PHA-stimulated lymphocytes have a receptor-effector system for 1,25-(OH)2D. In this study, a method for assay of 24-hydroxylase activity in PHA-stimulated lymphocytes is described, and its application for measurements of the induction of 24-hydroxylase and inhibition of DNA biosynthesis by 1068
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES
1,25-(OH)2D3 in cells from patients with VDDR II, their parents, and control subjects to determine the inheritance of VDDR II is reported.
Materials and Methods Materials 25-Hydroxy[26(27)-metM-3H]cholecalciferol([3H]25OHD3; 20.6 Ci/mmol), [14C]thymidine, and ACS were purchased from Amersham Corp. (Arlington Heights, IL). Preservative-free 25OHD3) 24,25-(OH)2D3, and 1,25-(OH)2D3 were generous gifts from Chugai Pharmaceutical Co. (Japan). These compounds were dissolved in 95% ethanol and stored in glass vials at —20 C. Lymphocyte culture PHA-stimulated lymphocytes of control subjects, patients with VDDR II, and their parents were prepared as described previously (11-13). Cultured skin fibroblasts of all patients with alopecia showed normal cytosol binding of [3H]1,25(OH)2D3. Of the patients, two were siblings, and three were isolated cases. The parents had normal phenotypes and normal serum levels of calcium, phosphorus, and alkaline phosphatase activity. Lymphocytes from peripheral blood were isolated by centrifugation on a Ficoll-Paque gradient. They were then washed twice and cultured at a density of 1 x 106 cells/ml in RPMI1640 medium with 10% fetal calf serum containing 1 ^g/mL PHA for 72 h at 37 C in a humidified atmosphere of 5% CO2 in air unless otherwise stated. 24-Hydroxylase induction and assay The procedure used was a modification of the technique of Chandler et al. (14). A solution of 1,25-(OH)2D3 was added to the culture medium (RPMI-1640 medium containing 10% fetal calf serum) in 5-mL culture tubes to give final concentrations of 10~n-10~6 mol/L. In the standard procedure, after incubation for 15 h, the cells were washed by suspension and centrifugation in medium A (Eagle's Minimum Essential Medium with glutamine at 0.15 g/L) and resuspended in medium A with 1% fetal calf serum. The 24-hydroxylase activity in suspensions of PHA-stimulated lymphocytes was quantitated by measuring the conversion of [3H]25OHD3 to [3H]24,25-(OH)2D3. Unless otherwise indicated, the assay conditions were as follows. One hundred picomoles of [3H]25OHD3 in ethanol were introduced into a borosilicated glass tube ( 1 x 7 cm) and allowed to dry. Then, 5 X 106 cells suspended in 0.2 mL medium A with 1% fetal calf serum were added, and the mixture was incubated in a water bath with shaking at 37 C. After 40 min, reference standards [25OHD3, 24,25-(OH)2D3, and 1,25-(OH)2D3; 400 pmol each] in 5 nL ethanol were added, and then 0.62 mL methanol-chloroform (2:1) was added. Calciferols were extracted by the method of Bligh and Dyer (15). The solvent-cell mixtures were transferred to 1.5-mL polypropylene tubes and incubated at 24 C for 1 h. Cell debris was precipitated by centrifugation for 1.5 min, and the supernatants were transferred to 1.5-mL polypropylene tubes con-
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taining 0.2 mL chloroform and 0.1 mL H2O. After vigorous mixing and centrifugation for 1 min, the lower organic phase was separated from the aqueous phase. The aqueous phase was reextracted as before with 0.4 ml chloroform, the two organic phases were combined and dried under N2) and the residue was dissolved in 100 y.h ethanol. Production of 24,25-(OH)2D3 was determined by high pressure liquid chromatography (HPLC; Waters Associates, Berkeley, CA) with a 3.9-mm X 30-cm ^Bondapak C18 column. The column was eluted isocratically with 80% methanol at a flow rate of 1 mL/min, and fractions of 0.5 mL were collected. Their absorbance was measured at 254 nm, with a sensitivity of 0.02 absorbance unit full scale. Figure 1 shows the resolutions and retention times of authentic radioinert reference standards. After the addition of 6 mL ACS II, the radioactivity of each fraction was measured in a liquid scintillation counting system. The synthesis of metabolites was calculated from the radioactivity eluted with the appropriate authentic compound. Identification of fH]24,25-(0H)2D3 by straight phase HPLC and periodate oxidation The fractions eluted in the region of authentic 24,25-(OH)2D3 on reverse phase HPLC were pooled and dried under a stream of N2. The dried residue was reconstituted in 100 /*L 8% isopropanol in hexane and subjected to HPLC on a /uPoracil column (3.9 mm x 30 cm; Waters Associates). The mobile phase was 8% isopropanol in hexane, and the flow rate was 1 mL/min. The radioactivity of an aliquot of the eluate that comigrated with the 24,25-(OH)2D3 reference standard was measured, and the remainder of the pooled sample from the peak was dried under N2 and dissolved in 0.1 mL methanol. Half the preparation was then treated with 0.1 mL H2O, and half with 0.1 mL 10% NaIO4, for 1 h at 24 C. Then, the preparations were reextracted and chromatographed, and their radioactivities were quantitated as before.
X.f\ 10 15 Elution Time (min)
20
25
FIG. 1. Resolutions and retention times of authentic radioinert reference standards on HPLC. Chromatography of a mixture of authentic 25OHD3) 1,25-(OH)2D3, and 24,25-(OH)2D3 (400 pmol each) was performed as described in Materials and Methods.
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Study of thymidine incorporation Viable lymphocytes that had been cultured for 72 h in the presence of 1 /xg/mL PHA and various concentrations of 1,25(OH)2D3 were pulsed with 0.05 MCi [14C]thymidine/106 cells as described previously (11). After 2-4 h, the cells were collected by centrifugation at 1500 rpm for 10 min, and the medium was discarded. The cells were washed three times with saline, suspended in 1 mL distilled water, and stored at —70 C. Radioactivity was determined in a liquid scintillation counter after the addition of 5 mL ACS.
Results Development of an assay for 24-hydroxylase in PHAstimulated lymphocytes. When the fractions in the region of the peak coeluted
with 24,25-(OH)2D3 were pooled and rechromatographed by straight phase HPLC as described in Materials and Methods, 85% of the radioactivity from the reverse phase HPLC peak was coeluted with authentic 24,25-(OH)2D3. By periodate oxidation, the putative [3H]24,25-(OH)2D3 peak eluted by the straight phase HPLC showed approximately 92% loss of radioactivity. Therefore, we concluded that the major radioactive metabolite was indeed 24,25-(OH)2D3 and that 1,25-(OH)2D3 treatment of PHA-stimulated lymphocytes caused a marked increase in 24-hydroxylase activity. With this protocol, other [3H] dihydroxyvitamin D3 metabolites, such as 1,25-(OH)2D3, were not produced. Production of more polar metabolites of [3H]24,25-(OH)2D3, including [3H]1,24,25-(OH)3D3 did not increase in l,25-(OH)2D3-treated PHA-stimulated lymphocytes. Suitable conditions for assay of 24-hydroxylase activity in PHA-stimulated lymphocytes from control subjects were determined. The formation of 24,25-(OH)2D3 was assessed as a function of the concentration of [3H] 25OHD3. The apparent Km value for the substrate in three experiments was 0.6 jumol/L (Fig. 2). The 24hydroxylase activity was linearly proportional to the number of cells with up to 7.5 X 106 cells and with time for at least 1 h after the addition of [3H]25OHD3 (Fig. 3). The effect of the time of culture with PHA on the induction of 24-hydroxylase by 10~8 mol/L 1,25-(OH)2D3 is shown in Fig. 4. Interestingly, 10"8 mol/L 1,25(OH)2D3 induced detectable activity in cells not cultured with PHA. During culture of lymphocytes with PHA, 10~8 mol/L 1,25-(OH)2D3 induced a 3-fold increase in 24hydroxylase on day 2, a maximum increase on day 3, and 20-30% of the maximum on day 4 of culture. In lymphocytes cultured with PHA for 72 h, induction of [3H] 24,25(OH)2D3 was detectable 5 h after the addition of 1,25(OH)2D3 and increased linearly for at least 25 h after its addition (Fig. 5).
FlG. 2. Lineweaver-Burk plot of the effect of 24-hydroxylase in PHAstimulated lymphocytes. Cells treated with 10~8 mol/L 1,25-(OH)2D3 for 15 h were incubated with the indicated concentrations of [3H] 25OHD3 for 30 min at 37 C. Synthesis of [3H]24,25-(OH)2D3 was quantitated as described in Materials and Methods. The Michaelis constant (Kro) for 25OHD3 was calculated from this plot.
Induction of 24-hydroxylase activity by various concentrations of 1,25-(OH)2D3 in PHA-stimulated lymphocytes of patients with VDDR II, their parents, and control subjects (Fig. 6) In PHA-stimulated lymphocytes of normal subjects, induction of 24-hydroxylase by exposure to 1,25-(OH)2D3 for 15 h was detectable at a concentration of 10~10 mol/ L; maximal induction occurred at a concentration of 10~8 mol/L 1,25-(OH)2D3. A concentration of 10"7 mol/L 1,25-(OH)2D3 also induced the maximal level, but with 10"6 mol/L 1,25-(OH)2D3, little or no [3H]24,25-(OH)2D3 was detectable. The mean activities ± SDs and the ranges (shown in parentheses) of 24-hydroxylase induced by 10"10,10"9,10"8,10"7, and 10~6 mol/L 1,25-(OH)2D3 were 63 ± 23 (range, 40-104; n = 6), 147 ± 48 (86-235; n = 7), 300 ± 51 (228-363; n = 12), 299 ± 73 (205-391; n = 10), and 45 ± 32 (n = 4) fmol/h-106 cells, respectively. The concentration of 1,25-(OH)2D3 for half-maximal induction of 24-hydroxylase was 1.3 ± 0.4 x 10~9 mol/L in control cells. In contrast, in PHA-stimulated lymphocytes from patients with VDDR II no enzyme activity was induced by 1,25-(OH)2D3 at 10"8, 10~7, or 10"6 mol/ L.
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES
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FiG. 3. Effect of cell concentration on [3H]24,25-(OH)2D3 synthesis (A) and the time course of [3H]24,25-(OH)2D3 synthesis (B) by PHA-stimulated lymphocytes. A, The indicated numbers of cells that had been cultured with 10~8 mol/L 1,25-(OH)2D3 for 15 h were incubated for 30 min at 37 C. B, Treated cells (5 x 10e) were incubated for the indicated times at 37 C. Then, formation of 24,25(OH)2D3 was measured as described in Materials and Methods.
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FIG. 4. Effect of time of culture with PHA on induction of 24-hydroxylase. Lymphocytes (5 x 10G) were cultured with PHA for the indicated times and then incubated with 10"8 mol/L 1,25-(OH)2D3 for 15 h. 24Hydroxylase activity was measured under standard conditions.
FlG. 5. Time course of l,25-(OH)2D3-mediated induction of 24-hydroxylase activity in PHA-stimulated lymphocytes. Lymphocytes (5 x 106) were cultured with PHA for 3 days and then incubated with 10"8 mol/ L 1,25-(OH)2D3 for the indicated times. Then, 24-hydroxylase activity was assayed as described in Materials and Methods.
A concentration of 10~9 mol/L 1,25-(OH)2D3 induced lower activity in cells from two parents of these patients than in control cells and did not induce activity in cells of four other parents of patients. In cells from the parents, the mean ± SDs and the ranges (shown in parentheses) of the activities induced by 10~8 and 10~7 mol/L
1,25-(OH)2D3 were 103 ± 32 (50-162; n = 12) and 143 ± 29 (80-179; n = 12) fmol/h-106 cells, respectively. Thus, the cells from all parents showed intermediate 24-hydroxylase bioresponses to 10~8 and 10~7 mol/L 1,25(OH)2D3 and no overlap values with those of the patients and controls, as shown in Fig. 6.
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TAKEDA ET AL.
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1,25-(OH)2D3 conc.(mol/L) FlG. 6. Dose-dependent induction of 24-hydroxylase activity by 1,25(OH)2D3 in PHA-stimulated lymphocytes. PHA-stimulated lymphocytes of control subjects (O) and patients with VDDR II (A, • , • , • , and • ) and their parents (A, A, U, U, 3 , and ) were incubated with the indicated concentrations of 1,25-(OH)2D3 for 15 h. The cells were then assayed for [3H]24,25-(OH)2D3 synthesis as described in Materials and Methods. Points and bars represent the mean ± SD for cells of control subjects. Points only represent means for duplicate determinations on cells from individual subjects.
Effect of 1,25-(OH)2D3 on the rate of thymidine incorporation into PHA-stimulated lymphocytes of the parents As shown in Fig. 7,1,25-(OH)2D3 caused dose-dependent inhibition of the incorporation of thymidine into PHA-stimulated lymphocytes of the parents. The lymphocytes of parents of patients with VDDR II were not distinguishable from normal lymphocytes by their levels of thymidine incorporation in the presence of 10~9,10~8, or 1(T7 mol/L 1,25-(OH)2D3.
Discussion There are reports that the kidney (10, 14, 16) and various other tissues and cells, including the intestine (17), bone cells (18), and skin fibroblasts (2, 8, 9) contain 24-hydroxylase that can be induced by 1,25-(OH)2D.
FlG. 7. Effect of dose of 1,25-(OH)2D3 on rate of thymidine incorporation into PHA-stimulated lymphocytes. The stippled area indicates mean ± SD for 11 control subjects, and the shaded area that for values in 5 patients with VDDR II. Values for parents (A—Zk, 01—U, 3—3, —O, and 0 —• ) are means of triplicate determinations on cells from each subject.
Cultured LLC-PKl kidney cells with reduced receptors show a reduced ability to respond to 1,25-(OH)2D3, as measured by induction of 24-hydroxylase by 1,25(OH)2D3 is also abnormal in cultured skin fibroblasts from patients with hereditary diseases causing resistance to 1,25-(OH)2D (1, 2, 8, 9). These findings indicate the essential role of the receptor in the action of 1,25(OH)2D3 and the close coupling of receptor content with functional responsiveness. In addition, induction of 24hydroxylase activity by 1,25-(OH)2D3 was reported to probably be receptor mediated (10, 16). However, the induction of 24-hydroxylase in skin fibroblasts of parents of patients with VDDR II was normal, except in one case that showed half the normal number of receptors and half the normal response to 1,25-(OH)2D3 (19). The demonstration of a heterozygous state by the elution pattern of [3H]1,25-(OH)2D3 from nuclei in parents has recently been reported (6, 7). In two families reported, a single nucleotide mutation was found in the DNA-binding domain of the receptor. However, this method cannot be used to identify heterozygotes of VDDR II in other types of receptor impairment. Thus, cultured skin fibroblasts are not appropriate for screening for heterozygotes in the general population. We and others have recently reported that in vitro
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES activation of human lymphocytes by mitogenic lectins or Ebstein-Barr virus induces expression of the 1,25(OH)2D receptor (11, 20, 21), and there is evidence that 1,25-(OH)2D3 has a positive regulatory effect on the level of vitamin D receptor in cultured mammalian cells (2224). Therefore, we measured the ability of 1,25-(OH)2D3 to induce 24-hydroxylase in PHA-stimulated lymphocytes as a marker of their functional response. The 1,25(OH) 2 D 3 concentrations necessary for maximal and halfmaximal 24-hydroxylase induction in lymphocytes from normal subjects were 10~8 and 1.3 ± 0.4 x 10~9 mol/L, respectively. Thus, there is an intracellular receptoreffector system for 1,25-(OH)2D3 in PHA-stimulated lymphocytes. These findings were similar to those in cultured skin fibroblasts. The maximal response of 24hydroxylase in dermal fibroblasts was observed after exposure to 10" 8 -10" 7 mol/L 1,25-(OH)2D3, the halfmaximal response was observed after exposure to 3 X 10~9 mol/L 1,25-(OH)2D3, and the minimal detectable response was observed after exposure to 3 x 10~10 mol/ L (2, 8). In this study, cells from patients with a defect in 1,25-(OH)2D3 uptake into the nucleus (11) did not respond to high concentrations of 1,25-(OH)2D3. Interestingly, the cells from their parents showed only half the normal response of 24-hydroxylase induction on treatment with 1,25-(OH)2D3. Thus, 24-hydroxylase induction in PHA-stimulated lymphocytes also appeared to be mediated through the receptor for 1,25-(OH)2D3, and with the present assay, heterozygotes of VDDR II could be detected. In contrast, obligate heterozygotes of this disease could not be identified by measuring thymidine incorporation as a marker of the action of 1,25-(OH)2D3 in PHAstimulated lymphocytes. 1,25-(OH)2D3 is a potent inhibitor of interleukin-2 and lymphocyte proliferation (25), and it also inhibits the generation of cytotoxic lymphocytes (26) and antibody production (26-28). The 1,25(OH) 2 D 3 concentrations for inhibitions of interleukin-2 and DNA biosynthesis in mitogen-stimulated lymphocytes were 2 x 10~12 and 10~8 mol/L, respectively. The difference in these concentrations of 1,25-(OH)2D3 indicates that there are several steps between the inhibition of the synthesis of interleukin-2 and that of DNA in lymphocytes. In addition, 10"8 mol/L 1,25-(OH)2D3 caused 24-hydroxylase amplification from an undetectable level to 300 fmol/h-10 6 cells, but caused 35-60% inhibition of DNA biosynthesis. Because mainly T cells are stimulated by PHA, the induction of 24-hydroxylase may reflect the receptor status of T cells. Provvedini and Manolagas (29) demonstrated that the time courses of expression of the receptor after activation in T helper and T suppressor subsets were very similar, but that 1,25-(OH)2D3 inhibited the rate of proliferation of the helper subset dose-dependently, without any effect on
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the proliferation of suppressor cells. Thus, peripheral T lymphocytes are homogeneous in receptor expression, but heterogeneous in their proliferative responses to 1,25-(OH)2D3. Intermediate activities of 24-hydroxylase were induced by 10"8 and 10"7 mol/L 1,25-(OH)2D3 in PHA-stimulated lymphocytes from all six parents of our patients with VDDR II. These findings may explain why assay of induction of this enzyme by 1,25-(OH)2D is better than assay of inhibition of DNA biosynthesis for identification of heterozygotes. It is concluded from the present study that the measurement of 24-hydroxylase activity in PHA-stimulated lymphocytes, which can easily be obtained, is useful in the diagnosis of cases of VDDR II and heterozygotes and that VDDR II shows autosomal recessive inheritance. References 1. Marx SJ, Liberman UA, Eil C, Degrange DE, Bliziotes MM. Resistance to 1,25-dihydroxycholecalciferol in man and in other species. In: Norman AW, Schaefer K, Grigoleit H-G, Herrath DV, eds. Vitamin D: chemical, biochemical and clinical update. New York: de Gruyter; 1985;107-16. 2. Feldman D, Chen T, Cone C, et al. Vitamin D resistant rickets with alopecia: cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25-(OH)2D3. J Clin Endocrinol Metab. 1982;55:1020-2. 3. Hochberg Z, Benderli A, Levy J, et al. 1,25-Dihydroxyvitamin D resistance, rickets, and alopecia. Am J Med. 1984;77:805-ll. 4. Rosen JF, Fleischman AR, Finberg L, Hamstra A, DeLuca HF. Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr. 1979;94:729-35. 5. Balsan S, Garabedian M, Liberherr M, Gueris J, Ulmann A. Serum 1,25-dihydroxyvitamin D concentrations in two different types of pseudo-deficiency rickets. In: Norman AW, Schaefer K, Grigoleit H-G, et al. eds. Vitamin D: basic research and its clinical application. New York: de Gruyter; 1979;1143-8. 6. Hughes MR, Malloy PJ, Kieback DG, et al. Point mutation in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 1988;242:1702-5. 7. Malloy PJ, Hochberg Z, Pike JW, Feldman D. Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II. J Clin Endocrinol Metab. 1989;68:263-9. 8. Gamblin GT, Liberman UA, Eil C, Downs Jr RW, DeGrange DA, Marx SJ. Vitamin D-dependent rickets type II: defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts. J Clin Invest. 1985;75:954-60. 9. Griffin JE, Zerwekh JE. Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. J Clin Invest. 1983;72:1190-9. 10. Hirst M, Feldman D. Regulation of 1,25-(OH)2D3 receptor content in cultured LLC-PKl kidney cells limits hormonal responsiveness. Biochem Biophys Res Commun. 1983;116:121-7. 11. Takeda E, Kuroda Y, Saijo T, et al. Rapid diagnosis of vitamin Ddependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes. Clin Chin Acta. 1986;155:245-50. 12. Takeda E, Kuroda Y, Saijo T, et al. la-Hydroxyvitamin D3 treatment of three patients with 1,25-dihydroxyvitamin D-receptordefect rickets and alopecia. Pediatrics. 1987;80:97-101. 13. Takeda E, Yokota I, Kawakami T, Hashimoto Y, Kuroda Y, Arase S. Two siblings with vitamin D-dependent rickets type II: no recurrence of rickets for 14 years after cessation of therapy. Eur J Pediatr. 1989;149:54-7. 14. Chandler JJ, Chandler SK, Pike JW, Haussler MR. 1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently defi-
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cient in the high affinity receptor for the hormone. J Biol Chem. 1984;259:2214-22. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911-7. Colston K, Feldman D. 1,25-Dihydroxyvitamin D3 receptors and function in cultured pig kidney cells (LLCPK): regulation of 24,25dihydroxyvitamin D3 production. J Biol Chem. 1981;257:2504-8. Kumar R, Schnoes HK, DeLuca HF. Rat intestinal 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3-24-hydroxylase. J Biol Chem. 1978;253:3804-9. Howard GA, Turner RT, Sherrard DJ, Baylink DJ. Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. J Biol Chem. 1981;256:7738-40. Chen TL, Hirst MA, Cone CM, Hochberg Z, Tietze H-U, Feldman D. 1,25-Dihydroxyvitamin D resistance, rickets, and alopecia: analysis of receptors and bioresponse in cultured fibroblasts from patients and parents. J Clin Endocrinol Metab. 1984;59:383-8. Provvedini DM, Tsoukas CD, Deftos LJ, Manolagas SC. 1,25Dihydroxyvitamin D3 receptors in human leukocytes. Science. 1983;221:1181-3. Bhalla AK, Amento EP, Clemens TL, Holik HF, Krane SM. Specific high affinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab. 1983;57:1308-10. Allegretto A, Pike JW. Trypsin cleavage of chick 1,25-dihydroxy-
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vitamin D3 receptors: generation of discrete polypeptides which retain hormone but are unreactive to DNA and monoclonal antibody. J Biol Chem. 1985;260:10139-45. Mangelsdorf DJ, Pike JW, Haussler MR. Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation. Proc Natl Acad Sci USA. 1987;84:354-8. Costa EM, Hirst MA, Feldman D. Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. Endocrinology. 1985;117:2203-10. Tsoukas CD, Provvedini DM, Manolagas SC. 1,25-Dihydroxyvitamin D3; a novel immunoregulatory hormone. Science. 1984;244:1438-40. Manolagas SC, Provvedini DM, Tsoukas CD. Interaction of 1,25dihydroxyvitamin D3 and the immune system. Mol Cell Endocrinol. 1985;43:113-22. Lemire JM, Adams JS, Sakai R, Jordan SC. 1,25-Dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells. J Clin Invest. 1984;74:657-61. Provvedini DM, Tsoukas CD, Deftos LJ, Manolagas SC. 1,25Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production. J Immunol. 1986;136:2734-40. Prowedini DM, Manolagas SC. la,25-Dihydroxyvitamin D3 receptor distribution and effects in subpopulations of normal human T lymphocytes. J Clin Endocrinol Metab. 1989;68:774-9.
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Vol. 70, No. 4 Printed in U.S.A.
25-Hydroxyvitamin D-24-Hydroxylase in Phytohemagglutinin-Stimulated Lymphocytes: Intermediate Bioresponse to 1,25-Dihydroxyvitamin D 3 of Cells from Parents of Patients with Vitamin DDependent Rickets type II* EIJI TAKEDA, ICHIRO YOKOTA, MICHINORI ITO, HIDEAKI KOBASHI, TAKAHIKO SAIJO, YASUHIRO KURODA Department of Pediatrics, University of Tokushima School of Medicine, Tokushima, Japan
ABSTRACT. A method for assay of 25-hydroxyvitamin D-24hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10~8 mol/L and (1.3 ± 0.4) x 10"9 mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10~8, 10~7, and 10~6 mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated
T
HE CHARACTERISTIC clinical features of vitamin D-dependent rickets type II (VDDR II) are early onset of rickets, alopecia, hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum levels of alkaline phosphatase activity and 1,25dihydroxyvitamin D [1,25-(OH)2D] (1). The existence of siblings with this disease (2-5) suggests that vitamin D resistance is an inherited disease, probably transmitted as an autosomal recessive trait. The presence of a heterozygous state in the parents' receptors has recently been demonstrated (6, 7). However, the method used for their detection is not suitable for detection of heterozygotes of this disease in the general population. Cultured skin fibroblasts have been used to analyze the receptor status of patients with this disease (1). Currently, available data indicate that various steps in the intracellular Received February 13, 1989. Address all correspondence and requests for reprints to: Eiji Takeda, M.D., Department of Pediatrics, University of Tokushima School of Medicine, Kuramoto-Cho 2, Tokushima City, Tokushima 770, Japan. * This work was supported by Grant 62570428 from the Ministry of Education, Science, and Culture of Japan and a grant from the Mother and Child Health Foundation.
the presence of an intracellular receptor-effector system for 1,25(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease. {J Clin Endocrinol Metab 70:10681074,1990)
mechanism of action of 1,25-(OH)2D are impaired in patients with this disease. Stimulation of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity by 1,25-(OH)2D3 has been investigated to evaluate the overall pathway of hormone action in cultured skin fibroblasts (2, 8, 9). 24-Hydroxylase activity appears to be a useful index of the 1,25(0H) 2 D effector pathway, because it changes in parallel with modulation of receptors during change in the rate of cell division (10). In addition, we demonstrated previously that the effect of 1,25-(OH)2D3 on DNA biosynthesis in phytohemagglutinin (PHA)-stimulated lymphocytes is another index of responsiveness to 1,25-(OH)2D3 and that the expression of the 1,25-(OH)2D3 receptor in PHA-stimulated lymphocytes, skin fibroblasts, and bone might be controlled by the same gene (11). These findings strongly suggested that PHA-stimulated lymphocytes have a receptor-effector system for 1,25-(OH)2D. In this study, a method for assay of 24-hydroxylase activity in PHA-stimulated lymphocytes is described, and its application for measurements of the induction of 24-hydroxylase and inhibition of DNA biosynthesis by 1068
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES
1,25-(OH)2D3 in cells from patients with VDDR II, their parents, and control subjects to determine the inheritance of VDDR II is reported.
Materials and Methods Materials 25-Hydroxy[26(27)-metM-3H]cholecalciferol([3H]25OHD3; 20.6 Ci/mmol), [14C]thymidine, and ACS were purchased from Amersham Corp. (Arlington Heights, IL). Preservative-free 25OHD3) 24,25-(OH)2D3, and 1,25-(OH)2D3 were generous gifts from Chugai Pharmaceutical Co. (Japan). These compounds were dissolved in 95% ethanol and stored in glass vials at —20 C. Lymphocyte culture PHA-stimulated lymphocytes of control subjects, patients with VDDR II, and their parents were prepared as described previously (11-13). Cultured skin fibroblasts of all patients with alopecia showed normal cytosol binding of [3H]1,25(OH)2D3. Of the patients, two were siblings, and three were isolated cases. The parents had normal phenotypes and normal serum levels of calcium, phosphorus, and alkaline phosphatase activity. Lymphocytes from peripheral blood were isolated by centrifugation on a Ficoll-Paque gradient. They were then washed twice and cultured at a density of 1 x 106 cells/ml in RPMI1640 medium with 10% fetal calf serum containing 1 ^g/mL PHA for 72 h at 37 C in a humidified atmosphere of 5% CO2 in air unless otherwise stated. 24-Hydroxylase induction and assay The procedure used was a modification of the technique of Chandler et al. (14). A solution of 1,25-(OH)2D3 was added to the culture medium (RPMI-1640 medium containing 10% fetal calf serum) in 5-mL culture tubes to give final concentrations of 10~n-10~6 mol/L. In the standard procedure, after incubation for 15 h, the cells were washed by suspension and centrifugation in medium A (Eagle's Minimum Essential Medium with glutamine at 0.15 g/L) and resuspended in medium A with 1% fetal calf serum. The 24-hydroxylase activity in suspensions of PHA-stimulated lymphocytes was quantitated by measuring the conversion of [3H]25OHD3 to [3H]24,25-(OH)2D3. Unless otherwise indicated, the assay conditions were as follows. One hundred picomoles of [3H]25OHD3 in ethanol were introduced into a borosilicated glass tube ( 1 x 7 cm) and allowed to dry. Then, 5 X 106 cells suspended in 0.2 mL medium A with 1% fetal calf serum were added, and the mixture was incubated in a water bath with shaking at 37 C. After 40 min, reference standards [25OHD3, 24,25-(OH)2D3, and 1,25-(OH)2D3; 400 pmol each] in 5 nL ethanol were added, and then 0.62 mL methanol-chloroform (2:1) was added. Calciferols were extracted by the method of Bligh and Dyer (15). The solvent-cell mixtures were transferred to 1.5-mL polypropylene tubes and incubated at 24 C for 1 h. Cell debris was precipitated by centrifugation for 1.5 min, and the supernatants were transferred to 1.5-mL polypropylene tubes con-
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taining 0.2 mL chloroform and 0.1 mL H2O. After vigorous mixing and centrifugation for 1 min, the lower organic phase was separated from the aqueous phase. The aqueous phase was reextracted as before with 0.4 ml chloroform, the two organic phases were combined and dried under N2) and the residue was dissolved in 100 y.h ethanol. Production of 24,25-(OH)2D3 was determined by high pressure liquid chromatography (HPLC; Waters Associates, Berkeley, CA) with a 3.9-mm X 30-cm ^Bondapak C18 column. The column was eluted isocratically with 80% methanol at a flow rate of 1 mL/min, and fractions of 0.5 mL were collected. Their absorbance was measured at 254 nm, with a sensitivity of 0.02 absorbance unit full scale. Figure 1 shows the resolutions and retention times of authentic radioinert reference standards. After the addition of 6 mL ACS II, the radioactivity of each fraction was measured in a liquid scintillation counting system. The synthesis of metabolites was calculated from the radioactivity eluted with the appropriate authentic compound. Identification of fH]24,25-(0H)2D3 by straight phase HPLC and periodate oxidation The fractions eluted in the region of authentic 24,25-(OH)2D3 on reverse phase HPLC were pooled and dried under a stream of N2. The dried residue was reconstituted in 100 /*L 8% isopropanol in hexane and subjected to HPLC on a /uPoracil column (3.9 mm x 30 cm; Waters Associates). The mobile phase was 8% isopropanol in hexane, and the flow rate was 1 mL/min. The radioactivity of an aliquot of the eluate that comigrated with the 24,25-(OH)2D3 reference standard was measured, and the remainder of the pooled sample from the peak was dried under N2 and dissolved in 0.1 mL methanol. Half the preparation was then treated with 0.1 mL H2O, and half with 0.1 mL 10% NaIO4, for 1 h at 24 C. Then, the preparations were reextracted and chromatographed, and their radioactivities were quantitated as before.
X.f\ 10 15 Elution Time (min)
20
25
FIG. 1. Resolutions and retention times of authentic radioinert reference standards on HPLC. Chromatography of a mixture of authentic 25OHD3) 1,25-(OH)2D3, and 24,25-(OH)2D3 (400 pmol each) was performed as described in Materials and Methods.
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Study of thymidine incorporation Viable lymphocytes that had been cultured for 72 h in the presence of 1 /xg/mL PHA and various concentrations of 1,25(OH)2D3 were pulsed with 0.05 MCi [14C]thymidine/106 cells as described previously (11). After 2-4 h, the cells were collected by centrifugation at 1500 rpm for 10 min, and the medium was discarded. The cells were washed three times with saline, suspended in 1 mL distilled water, and stored at —70 C. Radioactivity was determined in a liquid scintillation counter after the addition of 5 mL ACS.
Results Development of an assay for 24-hydroxylase in PHAstimulated lymphocytes. When the fractions in the region of the peak coeluted
with 24,25-(OH)2D3 were pooled and rechromatographed by straight phase HPLC as described in Materials and Methods, 85% of the radioactivity from the reverse phase HPLC peak was coeluted with authentic 24,25-(OH)2D3. By periodate oxidation, the putative [3H]24,25-(OH)2D3 peak eluted by the straight phase HPLC showed approximately 92% loss of radioactivity. Therefore, we concluded that the major radioactive metabolite was indeed 24,25-(OH)2D3 and that 1,25-(OH)2D3 treatment of PHA-stimulated lymphocytes caused a marked increase in 24-hydroxylase activity. With this protocol, other [3H] dihydroxyvitamin D3 metabolites, such as 1,25-(OH)2D3, were not produced. Production of more polar metabolites of [3H]24,25-(OH)2D3, including [3H]1,24,25-(OH)3D3 did not increase in l,25-(OH)2D3-treated PHA-stimulated lymphocytes. Suitable conditions for assay of 24-hydroxylase activity in PHA-stimulated lymphocytes from control subjects were determined. The formation of 24,25-(OH)2D3 was assessed as a function of the concentration of [3H] 25OHD3. The apparent Km value for the substrate in three experiments was 0.6 jumol/L (Fig. 2). The 24hydroxylase activity was linearly proportional to the number of cells with up to 7.5 X 106 cells and with time for at least 1 h after the addition of [3H]25OHD3 (Fig. 3). The effect of the time of culture with PHA on the induction of 24-hydroxylase by 10~8 mol/L 1,25-(OH)2D3 is shown in Fig. 4. Interestingly, 10"8 mol/L 1,25(OH)2D3 induced detectable activity in cells not cultured with PHA. During culture of lymphocytes with PHA, 10~8 mol/L 1,25-(OH)2D3 induced a 3-fold increase in 24hydroxylase on day 2, a maximum increase on day 3, and 20-30% of the maximum on day 4 of culture. In lymphocytes cultured with PHA for 72 h, induction of [3H] 24,25(OH)2D3 was detectable 5 h after the addition of 1,25(OH)2D3 and increased linearly for at least 25 h after its addition (Fig. 5).
FlG. 2. Lineweaver-Burk plot of the effect of 24-hydroxylase in PHAstimulated lymphocytes. Cells treated with 10~8 mol/L 1,25-(OH)2D3 for 15 h were incubated with the indicated concentrations of [3H] 25OHD3 for 30 min at 37 C. Synthesis of [3H]24,25-(OH)2D3 was quantitated as described in Materials and Methods. The Michaelis constant (Kro) for 25OHD3 was calculated from this plot.
Induction of 24-hydroxylase activity by various concentrations of 1,25-(OH)2D3 in PHA-stimulated lymphocytes of patients with VDDR II, their parents, and control subjects (Fig. 6) In PHA-stimulated lymphocytes of normal subjects, induction of 24-hydroxylase by exposure to 1,25-(OH)2D3 for 15 h was detectable at a concentration of 10~10 mol/ L; maximal induction occurred at a concentration of 10~8 mol/L 1,25-(OH)2D3. A concentration of 10"7 mol/L 1,25-(OH)2D3 also induced the maximal level, but with 10"6 mol/L 1,25-(OH)2D3, little or no [3H]24,25-(OH)2D3 was detectable. The mean activities ± SDs and the ranges (shown in parentheses) of 24-hydroxylase induced by 10"10,10"9,10"8,10"7, and 10~6 mol/L 1,25-(OH)2D3 were 63 ± 23 (range, 40-104; n = 6), 147 ± 48 (86-235; n = 7), 300 ± 51 (228-363; n = 12), 299 ± 73 (205-391; n = 10), and 45 ± 32 (n = 4) fmol/h-106 cells, respectively. The concentration of 1,25-(OH)2D3 for half-maximal induction of 24-hydroxylase was 1.3 ± 0.4 x 10~9 mol/L in control cells. In contrast, in PHA-stimulated lymphocytes from patients with VDDR II no enzyme activity was induced by 1,25-(OH)2D3 at 10"8, 10~7, or 10"6 mol/ L.
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES
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FiG. 3. Effect of cell concentration on [3H]24,25-(OH)2D3 synthesis (A) and the time course of [3H]24,25-(OH)2D3 synthesis (B) by PHA-stimulated lymphocytes. A, The indicated numbers of cells that had been cultured with 10~8 mol/L 1,25-(OH)2D3 for 15 h were incubated for 30 min at 37 C. B, Treated cells (5 x 10e) were incubated for the indicated times at 37 C. Then, formation of 24,25(OH)2D3 was measured as described in Materials and Methods.
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FIG. 4. Effect of time of culture with PHA on induction of 24-hydroxylase. Lymphocytes (5 x 10G) were cultured with PHA for the indicated times and then incubated with 10"8 mol/L 1,25-(OH)2D3 for 15 h. 24Hydroxylase activity was measured under standard conditions.
FlG. 5. Time course of l,25-(OH)2D3-mediated induction of 24-hydroxylase activity in PHA-stimulated lymphocytes. Lymphocytes (5 x 106) were cultured with PHA for 3 days and then incubated with 10"8 mol/ L 1,25-(OH)2D3 for the indicated times. Then, 24-hydroxylase activity was assayed as described in Materials and Methods.
A concentration of 10~9 mol/L 1,25-(OH)2D3 induced lower activity in cells from two parents of these patients than in control cells and did not induce activity in cells of four other parents of patients. In cells from the parents, the mean ± SDs and the ranges (shown in parentheses) of the activities induced by 10~8 and 10~7 mol/L
1,25-(OH)2D3 were 103 ± 32 (50-162; n = 12) and 143 ± 29 (80-179; n = 12) fmol/h-106 cells, respectively. Thus, the cells from all parents showed intermediate 24-hydroxylase bioresponses to 10~8 and 10~7 mol/L 1,25(OH)2D3 and no overlap values with those of the patients and controls, as shown in Fig. 6.
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TAKEDA ET AL.
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1,25-(OH)2D3 conc.(mol/L) FlG. 6. Dose-dependent induction of 24-hydroxylase activity by 1,25(OH)2D3 in PHA-stimulated lymphocytes. PHA-stimulated lymphocytes of control subjects (O) and patients with VDDR II (A, • , • , • , and • ) and their parents (A, A, U, U, 3 , and ) were incubated with the indicated concentrations of 1,25-(OH)2D3 for 15 h. The cells were then assayed for [3H]24,25-(OH)2D3 synthesis as described in Materials and Methods. Points and bars represent the mean ± SD for cells of control subjects. Points only represent means for duplicate determinations on cells from individual subjects.
Effect of 1,25-(OH)2D3 on the rate of thymidine incorporation into PHA-stimulated lymphocytes of the parents As shown in Fig. 7,1,25-(OH)2D3 caused dose-dependent inhibition of the incorporation of thymidine into PHA-stimulated lymphocytes of the parents. The lymphocytes of parents of patients with VDDR II were not distinguishable from normal lymphocytes by their levels of thymidine incorporation in the presence of 10~9,10~8, or 1(T7 mol/L 1,25-(OH)2D3.
Discussion There are reports that the kidney (10, 14, 16) and various other tissues and cells, including the intestine (17), bone cells (18), and skin fibroblasts (2, 8, 9) contain 24-hydroxylase that can be induced by 1,25-(OH)2D.
FlG. 7. Effect of dose of 1,25-(OH)2D3 on rate of thymidine incorporation into PHA-stimulated lymphocytes. The stippled area indicates mean ± SD for 11 control subjects, and the shaded area that for values in 5 patients with VDDR II. Values for parents (A—Zk, 01—U, 3—3, —O, and 0 —• ) are means of triplicate determinations on cells from each subject.
Cultured LLC-PKl kidney cells with reduced receptors show a reduced ability to respond to 1,25-(OH)2D3, as measured by induction of 24-hydroxylase by 1,25(OH)2D3 is also abnormal in cultured skin fibroblasts from patients with hereditary diseases causing resistance to 1,25-(OH)2D (1, 2, 8, 9). These findings indicate the essential role of the receptor in the action of 1,25(OH)2D3 and the close coupling of receptor content with functional responsiveness. In addition, induction of 24hydroxylase activity by 1,25-(OH)2D3 was reported to probably be receptor mediated (10, 16). However, the induction of 24-hydroxylase in skin fibroblasts of parents of patients with VDDR II was normal, except in one case that showed half the normal number of receptors and half the normal response to 1,25-(OH)2D3 (19). The demonstration of a heterozygous state by the elution pattern of [3H]1,25-(OH)2D3 from nuclei in parents has recently been reported (6, 7). In two families reported, a single nucleotide mutation was found in the DNA-binding domain of the receptor. However, this method cannot be used to identify heterozygotes of VDDR II in other types of receptor impairment. Thus, cultured skin fibroblasts are not appropriate for screening for heterozygotes in the general population. We and others have recently reported that in vitro
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25OHD-24-HYDROXYLASE IN LYMPHOCYTES activation of human lymphocytes by mitogenic lectins or Ebstein-Barr virus induces expression of the 1,25(OH)2D receptor (11, 20, 21), and there is evidence that 1,25-(OH)2D3 has a positive regulatory effect on the level of vitamin D receptor in cultured mammalian cells (2224). Therefore, we measured the ability of 1,25-(OH)2D3 to induce 24-hydroxylase in PHA-stimulated lymphocytes as a marker of their functional response. The 1,25(OH) 2 D 3 concentrations necessary for maximal and halfmaximal 24-hydroxylase induction in lymphocytes from normal subjects were 10~8 and 1.3 ± 0.4 x 10~9 mol/L, respectively. Thus, there is an intracellular receptoreffector system for 1,25-(OH)2D3 in PHA-stimulated lymphocytes. These findings were similar to those in cultured skin fibroblasts. The maximal response of 24hydroxylase in dermal fibroblasts was observed after exposure to 10" 8 -10" 7 mol/L 1,25-(OH)2D3, the halfmaximal response was observed after exposure to 3 X 10~9 mol/L 1,25-(OH)2D3, and the minimal detectable response was observed after exposure to 3 x 10~10 mol/ L (2, 8). In this study, cells from patients with a defect in 1,25-(OH)2D3 uptake into the nucleus (11) did not respond to high concentrations of 1,25-(OH)2D3. Interestingly, the cells from their parents showed only half the normal response of 24-hydroxylase induction on treatment with 1,25-(OH)2D3. Thus, 24-hydroxylase induction in PHA-stimulated lymphocytes also appeared to be mediated through the receptor for 1,25-(OH)2D3, and with the present assay, heterozygotes of VDDR II could be detected. In contrast, obligate heterozygotes of this disease could not be identified by measuring thymidine incorporation as a marker of the action of 1,25-(OH)2D3 in PHAstimulated lymphocytes. 1,25-(OH)2D3 is a potent inhibitor of interleukin-2 and lymphocyte proliferation (25), and it also inhibits the generation of cytotoxic lymphocytes (26) and antibody production (26-28). The 1,25(OH) 2 D 3 concentrations for inhibitions of interleukin-2 and DNA biosynthesis in mitogen-stimulated lymphocytes were 2 x 10~12 and 10~8 mol/L, respectively. The difference in these concentrations of 1,25-(OH)2D3 indicates that there are several steps between the inhibition of the synthesis of interleukin-2 and that of DNA in lymphocytes. In addition, 10"8 mol/L 1,25-(OH)2D3 caused 24-hydroxylase amplification from an undetectable level to 300 fmol/h-10 6 cells, but caused 35-60% inhibition of DNA biosynthesis. Because mainly T cells are stimulated by PHA, the induction of 24-hydroxylase may reflect the receptor status of T cells. Provvedini and Manolagas (29) demonstrated that the time courses of expression of the receptor after activation in T helper and T suppressor subsets were very similar, but that 1,25-(OH)2D3 inhibited the rate of proliferation of the helper subset dose-dependently, without any effect on
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the proliferation of suppressor cells. Thus, peripheral T lymphocytes are homogeneous in receptor expression, but heterogeneous in their proliferative responses to 1,25-(OH)2D3. Intermediate activities of 24-hydroxylase were induced by 10"8 and 10"7 mol/L 1,25-(OH)2D3 in PHA-stimulated lymphocytes from all six parents of our patients with VDDR II. These findings may explain why assay of induction of this enzyme by 1,25-(OH)2D is better than assay of inhibition of DNA biosynthesis for identification of heterozygotes. It is concluded from the present study that the measurement of 24-hydroxylase activity in PHA-stimulated lymphocytes, which can easily be obtained, is useful in the diagnosis of cases of VDDR II and heterozygotes and that VDDR II shows autosomal recessive inheritance. References 1. Marx SJ, Liberman UA, Eil C, Degrange DE, Bliziotes MM. Resistance to 1,25-dihydroxycholecalciferol in man and in other species. In: Norman AW, Schaefer K, Grigoleit H-G, Herrath DV, eds. Vitamin D: chemical, biochemical and clinical update. New York: de Gruyter; 1985;107-16. 2. Feldman D, Chen T, Cone C, et al. Vitamin D resistant rickets with alopecia: cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25-(OH)2D3. J Clin Endocrinol Metab. 1982;55:1020-2. 3. Hochberg Z, Benderli A, Levy J, et al. 1,25-Dihydroxyvitamin D resistance, rickets, and alopecia. Am J Med. 1984;77:805-ll. 4. Rosen JF, Fleischman AR, Finberg L, Hamstra A, DeLuca HF. Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr. 1979;94:729-35. 5. Balsan S, Garabedian M, Liberherr M, Gueris J, Ulmann A. Serum 1,25-dihydroxyvitamin D concentrations in two different types of pseudo-deficiency rickets. In: Norman AW, Schaefer K, Grigoleit H-G, et al. eds. Vitamin D: basic research and its clinical application. New York: de Gruyter; 1979;1143-8. 6. Hughes MR, Malloy PJ, Kieback DG, et al. Point mutation in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 1988;242:1702-5. 7. Malloy PJ, Hochberg Z, Pike JW, Feldman D. Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II. J Clin Endocrinol Metab. 1989;68:263-9. 8. Gamblin GT, Liberman UA, Eil C, Downs Jr RW, DeGrange DA, Marx SJ. Vitamin D-dependent rickets type II: defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts. J Clin Invest. 1985;75:954-60. 9. Griffin JE, Zerwekh JE. Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. J Clin Invest. 1983;72:1190-9. 10. Hirst M, Feldman D. Regulation of 1,25-(OH)2D3 receptor content in cultured LLC-PKl kidney cells limits hormonal responsiveness. Biochem Biophys Res Commun. 1983;116:121-7. 11. Takeda E, Kuroda Y, Saijo T, et al. Rapid diagnosis of vitamin Ddependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes. Clin Chin Acta. 1986;155:245-50. 12. Takeda E, Kuroda Y, Saijo T, et al. la-Hydroxyvitamin D3 treatment of three patients with 1,25-dihydroxyvitamin D-receptordefect rickets and alopecia. Pediatrics. 1987;80:97-101. 13. Takeda E, Yokota I, Kawakami T, Hashimoto Y, Kuroda Y, Arase S. Two siblings with vitamin D-dependent rickets type II: no recurrence of rickets for 14 years after cessation of therapy. Eur J Pediatr. 1989;149:54-7. 14. Chandler JJ, Chandler SK, Pike JW, Haussler MR. 1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently defi-
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15. 16. 17. 18.
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20. 21.
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