Parathyroid Hormone Down-Regulates. 1,25-Dihydroxyvitamin D Receptors (VDR) and. VDR Messenger Ribonucleic Acid in Vitro and. Blocks Homologous ...
0013-7227/90/1272-0942$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 2 Printed in U.S.A.
Parathyroid Hormone Down-Regulates 1,25-Dihydroxyvitamin D Receptors (VDR) and VDR Messenger Ribonucleic Acid in Vitro and Blocks Homologous Up-Regulation of VDR in Vivo TIMOTHY A. REINHARDT AND RONALD L. HORST National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, Iowa 50010
by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the l,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the l,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the l,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent downregulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes. (Endocrinology 127: 942-948, 1990)
ABSTRACT. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 ± 9 to 35 ± 5 fmol/mg protein at 18 h of exposure. The ED60 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTHmediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected
R
which has been shown to limit VDR occupancy, shorten the cellular half-life of occupied VDR, and limit the maximum response to 1,25(OH)2D3 stimuli (14-17). Furthermore, these data showed that entry of intact 1,25(OH)2D3 is restricted to a much greater extent during a secondary introduction of hormone (17). 1,25(OH)2D3 is degraded so rapidly that, when intact cell VDR assays are employed, one concludes that VDR has been downregulated when, in fact, VDR levels have increased dramatically as measured in a cytosol assay (17). Homologous up-regulation of VDR by 1,25(OH)2D3 has also been shown to occur in vivo (12,13,18, 19), and self-induced metabolism of 1,25(OH)2D3 has been shown to limit this cellular response to 1,25(OH)2D3 in vivo (19). The physiological significance of these observations remains unclear, as pharmacological doses of 1,25(OH)2D3 were used in these studies. In contrast to the VDR up-regulation observed after exogenous administration of 1,25(OH)2D3, Goff et al. (12) have recently shown that increased 1,25(OH)2D3 resulting from in-
EGULATION of cell l,25-(OH)2D3-receptor (VDR) numbers is considered an important mechanism for modulating cellular responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], as the biological activity of 1,25(OH)2D3 in cells has been shown to be proportional to cell VDR number (1-3). Several hormones [1,25(OH)2D3, retinoic acid, glucocorticoids, and estrogen] and different physiological states (aging, pregnancy, lactation, and dietary calcium restriction) have been shown to alter target tissue VDR number (4-13) and presumably tissue responsiveness to 1,25(OH)2D3. Recent reports have shown that not all regulated changes in tissue VDR numbers result in proportional changes in cellular responses to 1,25(OH)2D3. Homologous up-regulation of VDR by 1,25(OH)2D3 is paralleled by increased 1,25(OH)2D3 catabolic enzyme activity, Received March 2, 1990. Address all correspondence and requests for reprints to: Dr. T. A. Reinhardt, National Animal Disease Center, P.O. Box 70, Ames, Iowa 50010.
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PTH REGULATES 1,25(OH)2D3 RECEPTORS creased endogenous 1,25(OH)2D3 production, stimulated by dietary calcium restriction, fails to up-regulate intestinal VDR while VDR in renal tissue is significantly down-regulated. The present study shows that PTH is the likely mediator of these responses, as PTH was shown to down-regulate VDR in ROS 17/2.8 cells and partially block l,25(OH)2D3-mediated up-regulation of VDR in ROS 17/2.8 cells. In addition, in vivo PTH administration was shown to block l,25(OH)2D3-mediated up-regulation of both intestinal and kidney VDR.
Materials and Methods General
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stripped FCS. Cells were washed two times with Hank's balanced salt solution (without Ca++ and Mg++) at 4 C. Infusion experiment In order to study the effects of PTH on in vivo VDR regulation, 36 male Holtzman rats (150 g) were divided into four treatment groups and implanted with Alzet miniosmotic pumps. Group 1 received vehicle. Group 2 was infused with 1.8 IU/h PTH in 1 mM HC1, 0.15 M NaCl, and 2% cysteine. Group 3 was infused with 1.5 ng/h 1,25(OH)2D3 in propylene glycol with 2% ethanol. Group 4 was implanted with one pump delivering 1.8 IU/h PTH and one pump delivering 1.5 ng/h 1,25(OH)2D3. At the end of the 5-day infusion, blood was collected for the determination of plasma calcium and 1,25(OH)2D3 (21, 22). Total intestinal and renal VDR were determined as described below. Renal 24-hydroxylase (24OHase) activities were determined as described previously (23).
Pure synthetic bovine PTH (1-34) and human PTH-relatedprotein (PTH-RP) for cell experiments was obtained from Peninsula Laboratories (San Carlos, CA). Crude synthetic bovine PTH for in vivo rat experiments was obtained from Peninsula Labs and determined to be 700 IU/mg peptide. The vehicle for both forms of PTH was a solution of 1 mM HC1, 0.15 M NaCl, and 2% cysteine (20). Synthetic 1,25(OH)2D3 was a gift from Dr. Milan P. Uskokovic, Hoffmann-La Roche (Nutley, NJ). l,25-Dihydroxy[26,273H]vitamin D3 (85 Ci/ mmol) (1,25(OH)2[3H]D3) was prepared as previously described (17). Retinoic acid and triamcinolone were obtained from Sigma Chemical Co. (St. Louis, MO). Triamcinolone acetonide, [6,73 H] (42 Ci/mmol) was obtained from NEN Research Products (Wilmington, DE). 1,25(OH)2D3, retinoic acid, and triamcinolone were prepared in ethanol and standardized using molar extinction coefficients of 18,200 M^cnT1 at 265 nm, 45,000
Washed cells were counted using a hemocytometer and adjusted to a final concentration of 2 x 107 cells/ml in a hypertonic buffer containing 500 mM KC1, 10 mM Tris, 1.0 mM EDTA, and 5 mM dithiothreitol (DTT) at pH 7.5 (KTED). VDR was extracted by sonication of the cells with a Branson Sonifier. Cell cytosol was obtained after centrifugation of cell homogenates at 220,000 X g for 20 min using a Beckman Ti 70.1 rotor and an L8-80 M ultracentrifuge. Triplicate aliquots of cytosol (250 /LII) were treated immediately with hydroxyapatite in order to estimate occupied VDR. Additional aliquots (250 fj\) of
M^cm"1 at 351 nm, and 14,600 M^cm"1 at 238 nM, respectively.
cytosol were incubated in triplicate with a saturating dose of
Radioactivity was measured in Beckman Ready Solv HP using a Beckman LS-8000 liquid Radioactivity scintillation counter. Counting efficiencies averaged 40%. Fetal calf serum (FCS), tissue culture medium (RPMI-1640), and antibiotics were purchased from Grand Island Biological Co. (Grand Island, NY).
1,25(OH)2[3H]D3 (8 nM in the presence or absence of a 100fold molar excess of cold 1,25(OH)2D3 for 16 h at 4 C in order to estimate total VDR binding sites. Bound hormone was separated from free hormone using hydroxyapatite. Rat duodenal mucosa and kidneys were minced and washed with Tris-buffered saline (10 mM Tris, 150 M NaCl, pH 7.2) containing 500 IU/ml Trasylol (Mobay Chemical Corp., New York, NY). Twenty percent homogenates of the two tissues were prepared in KTED containing 200 y.g/m\. soybean trypsin inhibitor (24). Each sample was homogenized with three 10-sec bursts at setting 7 with cooling between each step. From this point on, tissue cytosols were prepared and assayed as described for cells. Cell or tissue protein was determined by the method of Bradford (25) using a BSA standard. All VDR assays were corrected for nonspecific binding and presented as femtomoles per mg protein.
Cell culture experiments Rat osteosarcoma cells (ROS 17/2.8) were grown in RPMI1640 supplemented with 10% FCS and antibiotics. Incubation conditions were 37 C, 5% CO2 in a humidified atmosphere. Cell medium was changed every 2 days, and experiments were performed with confluent cultures at 7 to 8 days of growth. Twenty-four hours before the start of an experiment, cell media was changed to RPMI-1640 containing 25 mM HEPES and 1% FCS. Cells were treated with vehicle (1 mM HC1, 0.15 M NaCl, and 2% cysteine), or PTH at concentrations and times indicated. For the induction of homologous VDR up-regulation, only 1,25(OH)2[3H]D3 was used in order to obtain a direct measure of occupied VDR. Cells were treated with ethanol vehicle, 1,25(OH)2[3H]D3, or a combination of 1,25(OH)2[3H] D3 and 100-fold molar excess of nonradioactive 1,25(OH)2D3. The final concentration of ethanol did not exceed 0.1% in any flasks. Cells were harvested for VDR assay at the indicated times by a 10-min incubation in trypsin-EDTA at 37 C followed by trypsin inactivation with RPMI-1640 plus 5% charcoal-
VDR assay in cells and tissue
Glucocorticoid receptor assay Cell cytosols were prepared as described for the VDR assay with the exception of the homogenization buffer which contained 10 mM Tris, 1 mM EDTA, and 5 mM DTT, at pH 7.5 (TED). Cell cytosols were incubated with 20 nM triamcinolone acetonide [6,7-3H] ± 200-fold molar excess of cold hormone for 16 h at 4 C. Separation of bound hormone from free hormone was performed using dextran-coated charcoal (22).
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PTH REGULATES 1,25(OH)2D3 RECEPTORS
Northern blot analysis Total RNA was extracted from cells as described (26). RNA (20 Mg) was fractionated under denaturing conditions on a 1.2% formaldehyde-agarose gel and transferred to Optibind nitrocellulose (Schleicher and Schuell, Keene, NH). Membranes with bound RNA were irradiated for 5 min by UV light to cross-link RNA to the membranes. Complementary DNA probe for rat VDR was kindly provided by Dr. Wesley Pike (Baylor College of Medicine, Houston, TX). A 1.7 kilobase rat VDR complementary DNA (cDNA) insert from the EcoRl site pIBI76 (27) and a 2.1 kilobase chick /3-actin cDNA insert from the Hind III site of pBR 322 (28) were obtained by restriction-enzyme digestion of the respective plasmids. The cDNA was labeled to a specific activity of 108-109 cpm/fig of DNA using Pharmacia oligolabeling (Pharmacia, Piscataway, NJ). The filters were prehybridized for 2-4 h at 42 C in 50% foramide, 5x Denhardts solution, 0.5% sodium dodecyl sulfate (SDS), 5x (SSPE = 0.18 M NaCl, 10 DIM NaPO4, lmM EDTA pH 7.7), and 100 ng/ ml sonicated herring sperm DNA. Hybridization was carried out for 18 h in fresh prehybridization solution to which 1 x 5 x 106 cpm/ml 32P-labeled probe was added along with carrier transfer RNA (tRNA). Filters were washed two times in 2XSSPE, 0.1% SDS at room temperature for 10 min each, and two times in O.lxSSPE, 0.1% SDS at room temperature for 10 min each. The filters were then counted and imaged and bands quantitated using an Ambis Radioanalytic Imaging System (Ambis Systems, San Diego, CA). For auto radiographs, the films were exposed to XOMAT X-ray films (Eastman Kodak Co., Rochester, NY) with intensifying screens for 1-5 days.
Results The effect of PTH treatment on VDR concentration was examined in confluent ROS 17/2.8 cells. Figure 1 shows that treatment of cells with 4 X 10~9 M bovine (b)PTH (1-34) resulted in a time-dependent down-regulation of cell VDR from 95 ± 9 at time zero to 35 ± 5
Endo• 1990 Vol 127-No 2
fmol/mg protein after 18 h of treatment. Cell VDR remained significantly reduced for up to 36 h after PTH treatment. Vehicle-treated cell VDR remained constant during this 36-h experiment. Treatment of cells with increasing concentrations (0-5 nM) of PTH or PTH-RP resulted in a dose-dependent down-regulation of cell VDR. The ED50 for both peptides was approximately 1 to 2 nM (Fig. 2). Figure 3 shows that 18 h of PTH treatment down-regulates VDR 50% or more without affecting the affinity of VDR for 1,25(OH)2D3 as measured by Scatchard analysis. We next determined whether PTH's effects were specific for VDR or if this effect was of a general nature for steroid receptors. Eighteen hours of PTH (10~8 M) treat120 -I
%
80
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• PTH O hPTH-RP
40
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20
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1 10 Peptide (nM) FIG. 2. Effect of increasing doses of PTH or PTH-RP on VDR expression by ROS 17/2.8 cells. Confluent cultures of cells were treated with either increasing doses of PTH or PTH-RP for 24 h. Cells were then harvested and assayed for VDR. Values presented are the mean ± SEM for three experiments. O Control •
PTH
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0.80
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13 O CQ
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Time (h) FIG. 1. Time course of PTH effect on VDR expression by ROS 17/2.8 cells. At time zero, cells were treated with either vehicle or 4 x 10~9 M PTH. Cells were harvested and assayed for VDR at the times indicated. Values presented are the mean ± SEM for three experiments. *, Means significantly different from time zero. P < 0.01.
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fmol/Tube FIG. 3. Effect of PTH on the affinity of VDR for 1,25(OH)2D3. Cells were treated for 18 h with vehicle or 10"8 M PTH and then assayed in triplicate.
PTH REGULATES 1,25(OH)2D3 RECEPTORS ment resulted in a significant 60% down-regulation of VDR (Fig. 4, left panel), while cell glucocorticoid receptor concentrations were not affected by PTH treatment (Fig. 4, right panel). The effect of PTH treatment on l,25(OH)2D3-mediated up-regulation of VDR in vitro was also examined (Table 1). Treatment for 18 h with 4 nM PTH downregulated cell VDR from control concentrations of 95 fmol/mg protein to 27 fmol/mg protein. Treatment for 18 h with 0.5 nM 1,25(OH)2[3H]D3 homologously upregulated VDR from 95 fmol/mg to 322 fmol/mg. Treatment of cells with 4 nM PTH 4 h before 0.5 nM 1,25(OH)2[3H]D3 treatment (data not shown) or simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of VDR up-regulation. In addition, the combined PTH and 1,25(OH)2[3H]D3 treatment reduced by 50% the amount of occupied VDR as compared to cells treated with 1,25(OH)2[3H]D3 alone (Table 1). In a similar manner, PTH treatment blocked retinoic acidmediated VDR up-regulation in ROS cells. In this experiment, we also examined the effect of PTH treatment on VDR mRNA expression. Figure 5 shows that PTH treatment of ROS cells led to a 50% decline in VDR mRNA as compared to control cells. In contrast, retinoic acid or 1,25(OH)2D3 treatment resulted in a 3- to 5-fold increase in VDR mRNA (Fig. 5). However, both 1,25(OH)2D3 and retinoic acid-mediated in800 _
110 ,
I2
Treatments Vehicle PTH
1,25(OH)2[3H]D3 PTH + 1,25(OH)2[3H]D3 Retinoic acid PTH + retinoic acid
Total 1,25(OH)2D3 receptor
Occupied 1,25(OH)2D3 receptor
95 ± 9" 27 + lfc 322 ± 22° 121 ± 19° 192 ± 4d 118 ± 10c
ND ND 157 ± 13° 70 ± 11" ND ND
Means with different superscripts (a-e) within the same column differ, P < 0.05. Values are femtomoles per mg of protein (mean ± SEM for three experiments); ND, Not determined.
creases in VDR mRNA were attenuated significantly by PTH addition. We next designed an experiment to determine to what extent PTH regulates VDR in vivo. The effect of 5-day infusions of vehicle, PTH (1.8 IU/h), 1,25(OH)2D3 (1.5 ng/h), or simultaneous infusion of PTH and 1,25(OH)2D3 on intestinal and kidney VDR and kidney 24-OHase are shown in Table 2. Infusion of PTH alone was without effect on intestinal VDR concentrations. PTH infusions did, however, significantly elevate kidney VDR and kidney 24-OHase to 93 fmol/mg and 73 pmol/min-g tissue, respectively, as compared to vehicle-treated kidney VDR and 24 OHase concentrations of 52 fmol/mg and 19 increased plasma 1,25(OH)2D3 and plasma calcium (Table 3). Infusion with 1,25(OH)2D3 alone significantly increased intestinal VDR (586 fmol/mg) kidney VDR (175 fmol/mg), kidney 24-OHase (743 pmol/min-g), and plasma 1,25(OH)2D3 (296 pg/ml) over that observed for either PTH or vehicle-infused rats (Tables 1 and 2). Infusion of PTH along with 1,25(OH)2D3 completely blocked l,25(OH)2D3-mediated VDR up-regulation in intestine and l,25(OH)2D3-mediated induction of kidney 24-OHase. PTH treatment reduced kidney VDR concentrations to concentrations observed in rats receiving PTH alone (Table 2). This inhibition of 1,25(OH)2D3mediated effects by PTH occurred in the face of the highest 1,25(OH)2D3 concentrations observed in the experiment (Table 3).
600
a. on
TABLE 1. 1,25(OH)2D3 receptor concentrations in ROS 17/2.8 cells as effected by 18 h of treatment with PTH (4 nM), 1,25(OH)2[3H]D3 (0.5 nM), retinoic acid (7 MM), or combined treatment with PTH and 1,25(OH)2[3H]D3 or PTH and retinoic acid
pmol/min-g tissues. PTH treatment also significantly
700 .
I
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S00 .
400 . 300 200
100
Discussion PTH
PTH
FIG. 4. Effect of PTH on both VDR and glucocorticoid receptor expression in ROS 17/2.8 cells. Confluent cultures were treated for 18 h with vehicle (c) or 10~8 M PTH. Cells were then harvested and assayed for both VDR and glucocorticoid receptor content. Values presented are the mean ± SEM for three expeirments. *, Means significantly different from controls. P < 0.01.
This study demonstrates that PTH is a potent and specific regulator of VDR both in vitro and in vivo. We also demonstrate that PTH-RP acts in a similar manner to PTH on VDR regulation. In ROS 17/2.8 cells, PTH and/or PTH-RP down-regulates VDR in a dose- and time-dependent manner. Also, in ROS cells, PTH blocks
PTH REGULATES 1,25(OH)2D3 RECEPTORS
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Endo • 1990 Vol 127-No 2
TABLE 2. The effect of PTH, 1,25(OH)2D3, and combined PTH and 1,25(OH)2D3 administration on in vivo regulation of VDR and the vitamin D-24-OHase
Treatments
Vehicle PTH
1,25(OH)2D3 PTH + 1,25(OH)2D3
P
2
3
Kidney 24-OHase (pmol/min/ g tissue)
446 ± 25° 481 ± 29° 586 ± 42" 469 ± 77°
52 ± 7° 93 ± 15" 175 ± 19C 101 ± 12"
19 ± 5° 73 ± 19" 743 ± 132C 21 ± 7°
TABLE 3. The effect of PTH, 1,25(OH)2D3, and combined PTH and 1,25(OH)2D3 administration on in vivo regulation of VDR and the vitamin D-24-OHase
! i I
Kidney receptor (fmol/mg protein)
Using miniosmotic pump, rats were infused for 5 days with vehicle, PTH (1.8 IU/h), 1,25(OH)2D3 (1.5 ng/h), or a combination of PTH and 1,25(OH)2D3. Assays were performed as described in Materials and Methods. Means with different superscripts within the same column differ, P < 0.05. Mean ± SEM, n = 6-9.
xooo.,
2000 .
Intestinal receptors (fmol/mg protein)
4
Treatments
5
6
Treatment FIG. 5. Effect of PTH treatment of ROS 17/2.8 cells on VDR mRNA expression. Cells were treated for 18 h with; lane 1, vehicle; lane 2, 0.5 nM 1,25(OH)2D3; lane 3, 4 nM PTH; lane 4, 4 nM PTH and 0.5 nM 1,25(OH)2D3; lane 5, 7 ixM retinoic acid; and lane 6, 4 nM PTH and 7 ^M retinoid acid. The Northern blot of VDR mRNA (top panel) is representative of three experiments. The Northern blot of /3-actin (middle panel) was obtained by stripping and rep robing the VDR Northern blot with a /3-actin probe in order to normalize the data for differences in RNA loading. The quantification of treatment effects (bottom panel) was achieved by direct proportional counting of the two blots using an Ambis Radioanalytic Imaging System. The data are presented as counts/24 h ± SEM, corrected for background and normalized to actin.
both homologous up-regulation of VDR by 1,25(OH)2D3 and heterologous up-regulation by retinoic acid (Table 1). In rats, PTH inhibits l,25(OH)2D3-mediated up-regulation of intestinal and kidney VDR and abolishes l,25(OH)2D3-mediated induction of renal 24-OHase. The data in our experiments are in marked contrast to the results of Pols et al. (29) who showed that in vitro PTH up-regulates VDR in UMR-106 cells. Their use of UMR106 cells vs. our use of ROS 17/2.8 cells could account, in part, for the contrasting results. The confirmation of our in vitro ROS cell data by an in vivo rat study, however, supports our contention that PTH inhibits l,25(OH)2D3-mediated up-regulation of VDR. The likelihood that PTH functions as a down-regulator of VDR both in vitro and in vivo is also substantiated by the
Plasma 1,25(OH)2D
Plasma calcium
Vehicle 55 ± 10° 10.2 ± 0.2° 71 ± 21fc PTH 13.7 ± 0.7" 296 ± 40c 1,25(OH)2D3 12.8 ± 0.2° 428 ± 45d PTH + 1,25(OH)2D3 15.2 ± 0.4c Using miniosmotic pump, rats were infused for 5 days with vehicle, PTH (1.8 IU/h), 1,25(OH)2D3 (1.5 ng/h), or a combination of PTH and 1,25(OH)2D3. Assays were performed as described in Materials and Methods. Means with different superscripts within the same column differ, P < 0.05. Mean ± SEM, n = 6-9.
recent data of Goff et al. (12). They showed that nutritional hyperparathyroidism, secondary to a calcium deficiency, resulted in a significant down-regulation of kidney VDR despite plasma 1,25(OH)2D3 levels of 829 pg/ ml. Furthermore, intestinal VDR did not up-regulate in response to this large increase in plasma 1,25(OH)2D3. These results led them to suggest that PTH may block l,25(OH)2D3-mediated up-regulation of VDR, since exogenous administration of 1,25(OH)2D3 with its associated hypercalcemia and hypoparathyroidism did result in up-regulation of intestinal and kidney VDR. We confirmed their hypothesis by demonstrating that coinfusion of both PTH and 1,25(OH)2D3 does block the up-regulation observed in these tissues when 1,25(OH)2D3 is infused alone. In our in vivo experiments, infusion of PTH alone resulted in mild hypercalcemia which limited the PTHmediated rise in plasma 1,25(OH)2D3. It has been shown by Hove et al. (30) that hypercalcemia antagonized the effects of high PTH on plasma 1,25(OH)2D3. Therefore, iatrogenic primary hyperparathyroidism with its associated hypercalcemia may explain the inability of PTH infusion to down-regulate kidney VDR in vivo. This suggests that serum calcium may also have a role in VDR
PTH REGULATES 1,25(OH)2D3 RECEPTORS
regulation. The present data also showed that PTH treatment of ROS cells led to a 50% decline in VDR mRNA. The suppression of VDR mRNA levels by PTH was also seen when basal VDR mRNA levels were increased by 1,25(OH)2D3 or retinoic acid. Therefore, PTH downregulates VDR, at least in part, through reducing VDR mRNA levels. Whether PTH reduces VDR mRNA through transcriptional control and/or by altering mRNA stability remains to be determined. However, PTH has been shown to regulate gene expression by both mechanisms (31, 32). For example, PTH increases the expression of osteocalcin by increasing the stability of osteocalcin mRNA (31), while it suppresses the production of osteopontin by reducing transcription of osteopontin mRNA (32). The observed effect that PTH infusions blocked l,25(OH)2D3-mediated up-regulation of VDR in rat intestine was somewhat surprising. PTH receptors have not been demonstrated in intestinal tissue, and workers in the field of calcium homeostasis generally consider any described effects of PTH on the intestine to its wellknown effect on 1,25(OH)2D3 production. There are data, however, providing support for receptor-mediated action of PTH in intestine (33, 34). Mok et al. (33) have shown that PTH is a potent relaxant of rat and pig small intestinal smooth muscle, and that the PTH antagonist (PTH-3-34) blocks PTH effects. Also, Nemere and Norman (34) have shown that PTH stimulates calcium transport in isolated perfused duodena from chicks, and the PTH antagonist (PTH 3-34) could block PTH effects on calcium transport. These data taken together with our data and those of Goff et al. (12) suggest a need to reexamine the intestine for the presence of PTH receptors. Another widely accepted and experimentally useful effect of 1,25(OH)2D3 in target tissues is 1,25(OH)2D3 induction of 24-OHase (35, 36). In vivo PTH treatment completely inhibited l,25(OH)2D3-mediated induction of kidney 24-OHase. These observations are consistent with results observed during nutritional hyperparathyroidism due to calcium restriction. Under these conditions, kidney 24-OHase is depressed despite dramatic elevations in kidney lcv-OHase activity (37). PTH appears to be the mediator of this reciprocal control and therefore likely blocks the self-induction of 1,25(OH)2D3 metabolism observed in several experimental models of VDR regulation (14-17, 19). Therefore, we propose that when 1,25(OH)2D3 rises, due to its normal physiological regulation by PTH, target tissue VDR is not up-regulated by 1,25(OH)2D3, and selfinduced 1,25(OH)2D3 metabolism does not initially occur. As plasma calcium rises and PTH secretion slows, the 1,25(OH)2D3 starts to induce its own metabolism via
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induction of 24-OHase degradative pathway (17, 38). This regulated increase in target tissue metabolism of 1,25(OH)2D3, in conjunction with reduced 1,25(OH)2D3 production, leads to termination of the biological response to 1,25(OH)2D3. Finally, the data presented clearly show that PTH is a potent down-regulator of VDR in vitro and that PTH and 1,25(OH)2D, both in vitro and in vivo, have opposing effects on the expression of certain genes. References 1. Haussler MR 1986 Vitamin D receptors: nature and function. Annu Rev Nutr 6:527 2. Dokah S, Donaldson CA, Haussler MR 1984 Influence of 1,25dihydroxyvitamin D3 on cultured osteogenic sarcoma cells: correlation with the 1,25-dihydroxyvitamin D3 receptor. Cancer Res 44:2103 3. Chen TL, Li JM, Ye TV, Cone CM, Feldman D 1986 Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells. J Cell Physiol 126:21 4. Petkovich PM, Heersche JN, Tinker DO, Joens C 1984 Retinoic acid stimulates 1,25-(OH)2D3 binding in rat osteosarcoma cells. J Biol Chem 259 5. Hirst MA, Feldman D 1982 Glucocorticoid regulation of 1,25-(OH)2 vitamin D3 receptors: divergent effects on mouse and rat intestine. Endocrinology 111:12400 6. Walters MR 1981 An estrogen-stimulated 1,25-dihydroxyvitamin D3 receptor in rat uterus. Biochem Biophys Res Commun 103:721 7. Costa EM, Hirst MA, Feldman D 1985 Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. Endocrinology 117:2203 8. Horst RL, Reinhardt TA 1988 Changes in intestinal 1,25-dihydroxyvitamin D receptor during aging, gestation and pregnancy in rats. In: Norman AW, Schaefer K, Grigoleit H-G, Herrath, Dv (eds) Vitamin D: Molecular, Cellular and Clinical Endocrinology. Seventh Workshop on Vitamin D, Rancho Mirage, CA, de Gruyter, Berlin, p 229 9. Goff JP, Horst RL, Reinhardt TA 1988 Duodenum and colon 1,25dihydroxyvitamin D receptor concentration is increased during lactation in the rat. In: Norman AW, Schaefer K, Grigoleit HG, Herrath, DV (eds) Vitamin D: Molecular, Cellular and Clinical Endocrinology. Seventh Workshop on Vitamin D, Rancho Mirage, CA, de Gruyter, Berlin, p 246 10. Favus MJ, Mangelsdorf DJ, Tembe F, Coe BJ, Haussler MR 1988 Evidence for in vivo upregulation of the intestinal vitamin D receptor during dietary calcium restriction in the rat. J Clin Invest 82:218 11. McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, O'Malley BW 1987 Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science 235:1214 12. Goff JP, Reinhardt TA, Beckman MJ, Horst RL 1990 Contrasting effects of exogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D] versus endogenous 1,25-(OH)2D, induced by dietary calcium restriction on vitamin D receptors. Endocrinology 126:1031 13. Horst RL, Goff JP, Reinhardt TA 1990 Advancing age results in reduction of intestinal and bone 1,25-dihydroxyvitamin D receptor. Endocrinology 126:1053 14. Pols HAP, Burkenhager JC, Schilte JP, Visser TJ 1988 Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells. Biochim Biophys Acta 970:122 15. Pols HAP, Schilte HP, Visser TJ, Birkenhager JC 1987 Effect of ketoconazole on metabolism and binding of 1,25-dihydroxyvitamin D3 by intact rat osteogenic sarcoma cells. Biochim Biophys Acta 931:115 16. Reinhardt TA, Horst RL 1989 Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells.
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Arch Biochem Biophys 272:459 17. Reinhardt TA, Horst RL 1989 Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness. J Biol Chem 264:15917 18. Costa EM, Feldman D 1986 Homologous up-regulation of the 1,25(OH)2 vitamin D3 receptor in rats. Biochem Biophys Res Commun 137:742 19. Reinhardt TA, Horst RL, Engstrom GW, Atkins KS 1988 Ketoconazole potentiates l,25-(OH)2D3-directed upregulation of 1,25(OH)2D3-receptors in rat intestine and bone. In: Norman AW, Schaefer K, Grigoleit HG, Herrath DV (eds) Vitamin D: Molecular, Cellular and Clinical Endocrinology. Seventh Workshop on Vitamin D, Rancho Mirage, CA, de Gruyter, Berlin, p 233 20. Ibrahim MM, Forte LR, Thomas ML 1982 Maintenance of normocalcemia by continuous infusion of the synthetic bovine parathyroid hormone (1-34) in parathyroidectomized rats. Calcif Tissue Int 3:553 21. Perkin-Elmer Corp 1965 Analytical Methods for Atomic Absorption Spectrophotometry. Perkin-Elmer, Norwalk, CT 22. Reinhardt TA, Horst RL, Orf JW, Hollis BW 1984 A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J Clin Endocrinol Metab 58:91 23. Engstrom GW, Horst RL, Reinhardt TA, Littledike ET 1984 25hydroxyvitamin D la- and 24-hydroxylase activities in pig kidney homogenates: effect of vitamin D deficiency. J Nutr 114:119 24. Hirst MA, Feldman D 1986 Cleavage of the rat intestinal 1,25dihydroxyvitamin D3 receptor by an endogenous protease to a form with defective DNA binding. Arch Biochem Biophys 250:153 25. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid gwanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 27. Pike JW, Kesterson RA, Scott RA, Kerner SA, McDonnell DP, O'Malley BW 1988 Vitamin D3 receptors: molecular structure of the protein and its chromosomal gene. In: Norman AW, Schaefer K, Grigoleit HG, Herrath, DV (eds) Vitamin D: Molecular, Cellular and Clinical Endocrinology. Seventh Workshop on Vitamin D, Rancho Mirage, CA, de Gruyter, Berlin, p 215 28. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter
29.
30. 31. 32. 33.
34.
35. 36.
37.
38.
Endo • 1990 Voll27«No2
WJ, Kirschmer MW 1980 Number and evolutionary conservation of a- and /3-tubulin and cytoplasmic and 5-actin genes using specific cloned cDNA probes. Cell 20:95 Pols HAP, van Leeuwen JPTM, Schilte JP, Visser TJ, Birkenhager JC 1988 Heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor by parathyroid hormone (PTH) and PTH-like peptide in osteoblast-like cells. Biochem Biophys Res Commun 156:588 Hove K, Horst RL, Little ET, Beitz DC 1984 Infusions of parathyroid hormone in ruminants: hypercalcemia reduced plasma 1,25dihydroxyvitamin D concentrations. Endocrinology 114:897 Noda M, Yoon K, Rodan GA 1988 Cyclic AMP-mediated stabilization of osteocalcin in RNA in rat osteoblast-like cells treated with parathyroid hormone. J Biol Chem 263:18574 Noda M, Rodan GA 1989 Transcriptional regulation of osteopontin production in rat osteoblast-like cells by parathyroid hormone. J Cell Biol 108:713 Mok LL, Cooper CW, Thompson JC 1989 Parathyroid hormone and parathyroid hormone-related protein inhibit phasic contraction of pig duodenal smooth muscle. Proc Soc Exp Biol Med 191:337 Nemere I, Norman AW 1986 Parathyroid hormone stimulates calcium transport in perfused duodena from normal chicks: comparison with the rapid (transcaltachic) effect of 1,25-dihydroxyvitamin D3. Endocrinology 119:1406 Hirst M, Feldman D 1983 Regulation of 1,25(OH)2 vitamin D3 receptor content in cultured LLC-PKl kidney cells limits hormonal responsiveness. Biochem Biophys Res Commun 116:121 Chandler JS, Chandler SK, Pike JW, Haussler MR 1984 1,25Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone. J Biol Chem 259:2214 Engstrom GW, Horst RL, Reinhardt TA, Littledike ET 1982 Renal 25-hydroxyvitamin D la- and 24-hydroxylase activity in pigs fed diets varying in vitamin D, calcium, and phosphorus. In: Norman AW, Schaefer K, Herrath Dv, Grigoleit H-G, (eds) Vitamin D: Chemical Biochemical and Clinical Endocrinology of Calcium Metabolism, Fifth Workshop on Vitamin D. Williamsburg, VA. Walter de Gruyter, Berlin-New York, p 499 Reddy GS, Tserng KY 1989 Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry 28:1763