JOURNAL OF BONE A N D MINERAL RESEARCH Volume 7, Number 1 , 1992 Mary Ann Liebert, Inc., Publishers

Glucocorticoids Decrease Vitamin D Receptor Number and Gene Expression in Human Osteosarcoma Cells MICHAEL GODSCHALK,1,2J A M E S R. LEVY,',* and R O B E R T W. DOWNS, JR.'

ABSTRACT The mechanisms by which glucocorticoids (GC) inhibit some actions of vitamin D [ 1,25-(OH),D31 are not well understood, but there is growing evidence that G C alter vitamin D receptor (VDR) number. We studied the effects of dexamethasone (DEX) on VDR number and mRNA in the human osteosarcoma cell line, MG-63. The effects of DEX on 1,25-(OH),D3 binding were examined by incubating confluent cells overnight M DEX. DEX decreased VDR number (Bmax)by 70% (1 10 versus 32 fmol/ in media without or with mg cellular protein, p < 0.001) without significantly changing the apparent affinity ( K b ) of 1,25-(OH),D3 for its receptor (3.8 versus 2.2 x M, p > 0.05). Overnight incubation of MG-63 cells with DEX produced a time- and dose-responsive decrease in VDR mRNA compared to untreated controls (p < 0.01). T o determine the mechanism of the DEX-mediated decrease in VDR mRNA, the effect of DEX on VDR mRNA stability was studied. We found that the half-life for the VDR mRNA was -5.7 h and was not significantly changed when the cells were incubated with DEX (-6.3 h). We conclude that DEX decreases both VDR number and mRNA in MG-63 osteosarcoma cells. Since the half-life of VDR mRNA was not significantly modified by dexamethasone, glucocorticoids appear to decrease VDR mRNA by inhibiting VDR gene transcription or by affecting the processing of VDR mRNA.

-

INTRODUCTION

glucocorticoid-mediated increases in VDR number in rat(4) and dog") intestine and in rat bone.(*) T o better understand the effects of glucocorticoids on the VDR in human HROUGH REGULATION of intestinal calcium absorption, inhibition of parathyroid hormone (PTH) secretion, bone cells, we studied the effect of dexamethasone (DEX) and direct effects on bone, 1,25-dihydroxycholecalciferol on both VDR number and gene expression in the human [ 1,25-(OH),D,] plays an important role in mineral metabo- MG-63 osteosarcoma cell line. MG-63 cells have proven to lism.(') Like other steroid hormones, it exerts its biologic be a good model for the human osteoblast phenotype since effects by binding to an intracellular receptor. The hor- 1,25-(OH),D3 stimulates alkaline phosphatase producmone-receptor complex binds to DNA to increase or de- tionl9) and osteocalcin synthesis,('o) both markers of crease transcription of target genes.(2) osteoblastic function. Our data demonstrate that dexaGlucocorticodis inhibit various actions of 1,25-(OH),D,; methasone decreases VDR number and that at least part of for example, they decrease vitamin D-mediated intestinal this decline in VDR number is due to dexamethasone-mecalcium a b ~ o r p t i o n . 'The ~ ) mechanisms by which glucocor- diated inhibition of VDR mRNA abundance. ticoids inhibit vitamin D actions are not well understood, however. A growing body of evidence suggests that glucoMATERIALS AND METHODS corticoids alter cellular vitamin D receptor number (VDR). Cell culture Previous studies have shown that glucocorticoids decrease VDR number in mouse intestine(4)and bone(5)and in huHuman osteogenic sarcoma cells (MG-63) were obtained man monocytes. 1 6 ) In contrast, other investigators report from American Type Culture Collection (Rockville, MD)

T

'Divisions of Geriatrics and Endocrinology, Department of Internal Medicine, McGuire VA Medical Center, Richmond, Virginia. 'Medical College of Virginia, Richmond.

21

22 and cultured in 10 x 200 mm Petri dishes under 5% CO, and 95% air at 37°C in 45% Dulbecco’s modified Eagle’s medium (DMEM) and 45% Ham’s F-12 nutrient mixture supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 pg/ml), and 20 nM glutamine (Sigma, St. Louis, MO). The culture medium was changed daily. Cells were grown to confluence (6.5-10 million/dish) for the experiments described. Cell viability measured by trypan blue exclusion was not affected by incubation with either 0.1 Yo ethanol or dexamethasone in the concentrations used for the experiments described.

Assay of 1,25-(OH),D, receptor The day before the binding experiments, the culture medium was changed to serum-free medium without or with various concentrations of dexamethasone (Sigma) in ethanol (the highest concentration of ethanol used was 0.1070). Preliminary experiments showed that an ethanol concentration of 0.1070 had no significant effect on VDR binding. VDR number was measured by a modification of a method described by Eil and Marx.(ll) Cells were incubated with 18,000 cpm of [’H]1,25-(OH),D3 (Amersham, Arlington Heights, IL), and varying concentrations (zero to 2 x M) of unlabeled 1,25-(OH),D, (kindly provided by Dr. Milan Uskokovic, Hoffmann-LaRoche, Inc., Nutley, NJ) at 37°C for 2 h in DMEM with 0.25% bovine serum albumin. At the end of the incubation, the medium was removed and the cells were then lysed with a buffer containing 0.25 M sucrose, 0.5% Triton X-100, 20 mM Tris (pH 7.85), and 1.1 mM MgCI, at 4°C. The cell suspension was scraped from the culture dishes and centrifuged (1000 x g at 4°C) for 10 minutes. The supernatant was aspirated, and 0.5 ml ethanol was added to the nuclear pellet to extract [’HI1 ,25-(OH),D3. After 30 minutes, the ethanol extract was transferred to scintillation vials, to which 10 ml scintillation cocktail aliquots were added. The vials were counted in a liquid scintillation counter. Data from three separate experiments were analyzed together and curves fitted by weighted nonlinear leastsquares iterative regression analysis using the LIGAND and ALLFIT computer programs.(12.13)Weighting was chosen to assume a constant proportional error in bound ligand concentration. LIGAND models “nonspecific” binding directly from saturation and displacement binding results. The “goodness of fit” was determined using a number of runs test on residuals and by evaluation of the rootmean-square residual error. LIGAND can easily compare the difference between two fits for the same set of data but does not compare two separate curves for different data sets. To d o this, we transferred the binding data as corrected for protein by the LIGAND analysis to tables for analysis by ALLFIT, a program specifically designed for the comparison of curves from different data Results were normalized for protein and quantitated using the Bio-Rad protein assay (Bio-Rad, Richmond, CA).

-

GODSCHALK ET AL.

R N A preparation, northern transfer, and slot-blot analysis Confluent cells were incubated for 0, 6 , or 24 h in the absence or presence of various concentrations of dexamethasone. The cells were then washed with phosphatebuffered saline (0.15 M) and lysed with guanidine isothiocyanate buffer. Total RNA was extracted by spinning overnight at 32,000 rpm against a cesium chloride gradient. The RNA pellet was suspended in 0.3 M sodium acetate (pH 6) and purified by precipitating with ethanol. For northern transfers and slot-blots, 5 , 10, and 20 pg total RNA was denatured in formaldehyde and either electrophoretically separated in a 1070 agarose gel and transferred to nitrocellulose by northern transfer or directly applied to nitrocellulose by slot-blot using a vacuum manifold (Minifold 11, Schleicher and Schuell).(14)

cDNA labeling and hybridizations The cDNA for the full-length VDR (kindly provided by Dr. Wesley Pike)(15)p-actin,(’6) and histone H2B(I7)were labeled by nick translation with 32P-dCTP (New England Nuclear, Wilmington, DE) by standard methods.(18)The nitrocellulose filters were hybridized overnight with 2 x lo7 cpm of the labeled probe, washed, and exposed to Kodak XAR film with Quanta I11 screens at -70”C.(19’ Band density was quantitated with a computer-assisted Hoefer scanning densitometer, and the area under the optical density curve was calculated using GS 350 densitometry software (Hoefer Scientific Instruments).

Measurement of the effect of dexamethasone on VDR mRNA half-life After incubation of cells for 8 h at 37°C in serum-free media without or with dexamethasone, 12.5 pg actinomycin D (Sigma) was added to each dish to block RNA synthesis. RNA was collected as described earlier at 0, 2, 4, and 6 h following the addition of actinomycin D. Total RNA (10 pg) was applied to nitrocellulose with a slot-blot apparatus as described and was hybridized to nick-translated 32P-labeled VDR, histone h2B, and p-actin cDNAs. Autoradiography was performed and band density quantitated.

RESULTS

Dexamethasone decreases VDR binding to vitamin D Under the conditions of these experiments, our binding data were fit best to a single-site binding model; addition of a second component of binding into the model did not result in any better fit to the data by an extra sum of squares F test.‘’,) In these experiments, the number of

23

GLUCOCORTICOIDS DECREASE VITAMIN D RECEPTOR NUMBER VDR (B,,,) was 110 femtomol/mg cellular protein (17% coefficient of variation, CV), with an estimated K b of 3.8 x M (23% CV). This average value for the Kb for the VDR in MG-63 osteosarcoma cells incubated without glucocorticoids was similar to what has been reported in the mouse and rat (2.0 x M)IZ1and in human monocytes (1.2-4.5 x M).(6.20) After incubation of MG-63 cells with medium containM dexamethasone, specific nuclear binding of ing [3H]1,25-(OH),D, (Bmax)decreased by about 70% (Table 1 and Fig. 1). Under these conditions, the B,,, was 32 femtomoVmg cellular protein (21% CV), but the Kb was not significantly changed at 2.2 x M (38% CV). Binding of [3H]1,25-(OH)2D3was clearly different for cells incubated with dexamethasone by analysis using ALLFIT (p < 0.001). B,,, for cells incubated with dexamethasone was significantly lower than B,,, for control cells.

0.5

I

0.4

; o.3

0

LL

2 5 2

0.2

0.1

Dexamethasone decreases VDR mRNA abundance To examine the effect of dexamethasone on the level of VDR mRNA, MG-63 cells were incubated for 0, 6, or 24 h in the absence or presence of various concentrations of dexamethasone and total RNA was extracted. Initially, RNA was separated on an agarose gel for northern transfer to nitrocellulose and hybridized to the cDNA for the VDR and p-actin. As shown in Fig. 2, the autoradiogram of the northern transfer reveals one band at approximately 4.4 kb, consistent with the size of the VDR mRNA found in other human t i s s ~ e s . " ~ ~ To facilitate quantification of VDR mRNA, 5 , 10, and 20 pg total RNA was also directly applied to nitrocellulose via a slot-blot apparatus and hybridized t o either the cDNA for the VDR or P-actin. A representative autoradiogram of a slot-blot analysis from one experiment is shown in Fig. 3. Quantitation by densitometry of the 10 pg mRNA slot-blot data from several experiments is shown in Table 2. In the absence of dexamethasone, VDR mRNA levels rise to 141% of unincubated controls after incu-

1

kWo\ OO

0

,

00

0

0.0

, ~

Bound (frnoles/rng

ptn)

FIG. 1. Scatchard plot showing the effect of DEX on binding of [3H]1 ,25-(OH),D3 to MG-63 human osteosarcoma nuclei. Intact cells were grown to confluence and incubated overnight in serum-free DMEM containing 0.25% bovine serum albumin (BSA) without or with M DEX. Cells were then incubated for 2 h with [3H]1,25-(OH)zD3 and with increasing concentrations of unlabeled 1,25(OH),D, from 0 to 2 x M (as described in Materials and Methods). Nuclear uptake of 1,25-(OH),D3 was determined, and the data were fit by LIGAND to a single-site binding model after correction for nonspecific tracer binding. Data shown here are from three separate experiments, which were combined for analysis (two of which contained data points for cells incubated with and without DEX, the other with control cell data only). Open symbols, controls; closed symbols, DEX.

TABLE1. ANALYSIS OF BINDING OF ['HI 1 ,25-(OH),D, TO NUCLEAR SITES IN MG-63 HUMAN OSTEOSARCOMA CELLSa

Control

DEX

M)

Concentration range 1,25-(OH),D3, M

10-11-10-7

10-1 1- 10-7

Number of concentration points B,,, fmol/mg cellular protein (Vo) KD, M VoRMS, degrees of freedom

20

18 32 (21) 2.2 x 10-'O (38) 30%, 24

110 (17) 3.8 x 10''o (23)

27%, 33

aCells were grown to confluence, treated overnight in serum-free medium without or with M dexamethasone, and then incubated with ['H]1,25-(OH),D3 for 2 h as described in Materials and Methods. Data were pooled from three experiments and analyzed with LIGAND for calculation of maximum binding capacity and K b . The percentage coefficient of variation (Vo CV) is shown in parentheses, and the root-mean-square error (RMS error), the average deviation of data points from the fitted curve, is expressed as a percentage.

24

GODSCHALK ET AL. from three separate experiments were analyzed together and are shown in Fig. 4. At baseline, VDR mRNA was 30% less in the cells incubated with M dexamethasone compared to untreated controls. After the addition of actinomycin D, the levels of VDR mRNA decreased exponentially in both control and dexamethasone-treated cells, and the slopes of the decay curves were similar. The halflife for the VDR mRNA was -5.7 h and was not significantly changed by treatment with dexamethasone ( 6.3 h). In comparison, the half-lives of the p-actin and histone H2B mRNA were approximately 40 and 2.3 h, respectively, in both control and dexamethasone-treated cells.

DEX

- +

-

DISCUSSION We demonstrated that incubation with glucocorticoids decreases the binding of 1,25-(OH),D, to its receptor in the MG-63 osteosarcoma, a human osteoblast-like cell. The glucocorticoid-mediated inhibition of binding is due to a decline in VDR number with little change in binding affinity. At least some of the reduced VDR number can be explained by a glucocorticoid-mediated decline in VDR FIG. 2. Autoradiogram demonstrating the specificity of mRNA abundance. Since glucocorticoids did not affect the VDR probe. MG-63 were incubated Over- VDR mRNA in our experiments, we propose that night without or with M DEX. Total RNA (10 pg) was separated on a formal- glucocorticoids most likely inhibit the production of VDR denatured and e~ectrophoretically dehyde-agarose gel. The RNA was transferred to nitrocel- mRNA by inhibiting VDR gene transcription or by perM lulose and hybridized with '*P-labeled cDNA for the VDR turbing VDR mRNA processing. In our studies, (14 x 10' corn) and for actin (3.5 x 107 cDm). After dry- dexamethasone decreased VDR number analyzed by ligand in,, the nit;ocellulose was exposed to Kodak XAR film binding by approximately 70% but decreased VDR mRNA with Quanta I11 intensifying screens for 2 days. The band abundance by only 40-50%. Since we did not assess the at -4.4 kb is VDR mRNA and the band at -2.2 kb is rate of synthesis of new VDR protein from mRNA or the actin mRNA. rate of degradation of VDR during incubation with glucocorticoids, the data here do not allow us to determine whether the dexamethasone-induced decline in mRNA bating 24 h in serum-free medium. Incubation with dexa- abundance can fully account for the decrease in VDR methasone suppresses the rise of VDR mRNA, particularly number. at the 6 h point. When compared to time-matched controls T o demonstrate the effects of excess glucocorticoids, we (incubated without DEX for 6 h), dexamethasone de- used high doses of dexamethasone in this study. Our data creases VDR mRNA in a dose-responsive manner; lo-'" indicate that glucocorticoid concentrations within the lo-', and M dexamethasone suppress VDR mRNA to physiologic range are not likely to cause significant 88, 56 (p = 0.04), and 52% (p = 0.01) of control mRNA changes in VDR mRNA abundance. Pharmacologic levels M dexamethasone of dexamethasone, however, which may be achieved durlevels, respectively. At 24 h, only significantly inhibits the rise of VDR mRNA (p = 0.04). ing drug therapy in humans, induced significant effects on Actin mRNA does not increase during incubation in VDR mRNA levels. The high doses of dexamethasone serum-free medium. Rather, actin mRNA levels fall to ap- used in these experients were not directly toxic to the proximately 80% of control mRNA levels at 6 h and 50% osteosarcoma cells, but there is a possibility that some of at 24 h when cells are incubated in serum-free medium. the effects we observed could be mediated through nongluThis has been observed in other cell lines.'21)Actin mRNA cocorticoid receptor mechanisms. Some recent studies have is not affected by treatment with dexamethasone (Table 2). suggested that there may be a distinct low-affinity receptor for a glucocorticoid metabolite that could mediate actions that occur at high steroid concentrations.(22JThus far, Dexamethasone has no effecr on VDR however, there is no evidence for such a receptor in bone mRNA stability cells. T o determine the mechanism of the dexamethasone-meA glucocorticoid-mediated decrease in VDR number was diated decrease in VDR mRNA levels, the effect of dexa- also observed in an in vivo study by Nielsen et al.(6JThey methasone on VDR mRNA stability was assessed using ac- found that 40 mg prednisone given daily for 5 days to nine tinomycin D to inhibit gene transcription. Messenger RNA healthy subjects decreased ['HI 1,25-(OH),D, binding by levels for p-actin and histone H2B were used as internal 40% in peripheral monocytes. Other nonhuman in vivo long- and short-half-life controls. Densitometric analysis studies have shown species-specific results, with glucocorti-

2.0 Kb

25

GLUCOCORTICOIDS DECREASE VITAMIN D RECEPTOR NUMBER

lncubation Time

DEX Concentration

(hours)

(MI

1

0

Actin 20

-

-

10 l o

10-6

0

6

1 1

24

FIG. 3. Effect of DEX on VDR and 0-actin mRNA abundance. MG-63 cells were grown to confluence and incubated for 0, 6, and 24 h in medium without or with M DEX. Total RNA ( 5 , 10, and 20 pg) was applied to nitrocellulose filters using a slot-blot manifold and hybridized with a nick-translated 3'P-labeled cDNA for VDR (7 x lo7cpm) and actin (7 x lo7 cpm) as described in Materials and Methods. To optimize quantification by densitometry, the nitrocellulose containing slots hybridized to the 32P-VDRcDNA required 10 days of exposure to the XAR film versus 24 h for the actin probe. The autoradiographs shown are representative samples.

TABLF 2. EFFECT OF DEX ~

~~~

ON

VDR

~

AND

A C T I NM R N A ~

~~

24 H

6 H

DEX (M) 0 (n

=

Actin

VDR

128

*

16

78 f 14

141 f 20

54*

4

113

*

14

75*

115

19

63f

9

Actin

7)

IO-'O

( n = 6) lo-* ( n = 5)

o-6

VDR

1 ( n = 8)

* 66 * 72

8b

68

4'

72

*

7 14 7

f

125 f 15 81 f 12b

41 f 7 75

17

"Cell5 were incubated for 0, 6 , or 24 h without or with various concentrations of DEX. Total RNA was extracted and 10 pg applied to nitrocellulose by a slotblot method. After hybridization with 3zP-cDNA, autoradiography was performed and band density was measured with a Hoefer computer-assisted scanning densitonieter. Band density is expressed in arbitrary density units. with a value of 100 assigned to the bands that represent mRNA from control cells at time 0, before incubation (not shown). The table shows the mean standard error of the mean (SEM) for five to eight separate experiments as indicated. hSignificantly different from untreated samples, p = 0.04. LSignificantly different from untreated samples, p = 0.02.

GODSCHALK ET AL.

26

C .-

100 90 80 70 60

0) V)

50

Q)

0

.c

40

0

bp

30

;

& W

1

Y

0

0 J

0

-

A I

I

I

1

0

2

4

6

TIME (hours)

50 40

0

M

30

Y

0

2

20

10

V)

20

1

10'

'

0

I

I

2

4

I

1

6

TIME ( h o u r s )

FIG. 4. Effect of actinomycin D on VDR, actin, and histone gene mRNA synthesis. MG-63 cells were grown to confluence and then incubated 8 hr in medium without (circles) or with M DEX (squares). Actinomycin D was added to each dish and RNA collected and probed using 32Plabeled cDNAs as described in Materials and Methods. Data from three separate experiments were analyzed together. (A) The effects of actinomycin D on VDR mRNA. (B) Histone (dashed line) and actin (solid line) mRNA were used as short- and long-half-life controls.

coids increasing intestinal VDR number (and mRNA) in the rat and decreasing VDR number in mouse intestinal cells.(') However, it has not been clear whether changes in VDR number are due to direct effects of prednisone on VDR synthesis or degradation or to indirect effects, such as changes in serum 1 ,25-(OH),D, or P T H levels. Previous in vitro studies in which conditions can be better controlled have shown a doubling in VDR number in rat calvarial osteoblast-like culturess incubated with glucocorticoids. I * ) In mouse bone cell cultures, glucocorticoids decreased VDR number by 60-70%,15) similar to our findings in human bone cells. Vitamin D plays an important role in bone mineralization. 1,25-(OH),D, increases intestinal calcium absorption and renal calcium resorption and decreases P T H synthesis.") Osteoblasts contain VDR, and 1,25-(OH),D, has direct effects on bone metabolism, including increased synthesis of the bone matrix protein, o s t e o ~ a l c i n . Phar(~~~ macologic doses of glucocorticoids have been shown to have an "antivitamin D" effect, decreasing collagen synthesis,'24) the incorporation of radiolabeled nucleotides into DNA(2S) and RNA,(26) and alkaline phosphatase activity. ( 2 5 ) In addition, glucocorticoids have been shown to inhibit 1,25-(OH),D,-stimulated osteocalcin secretion in cultures of human osteoblasts. (23) The mechanisms for these glucocorticoid effects on VDR action are likely to be diverse and multifactorial. The present study demonstrates that one mechanism that contributes to these effects is that glucocorticoids decrease VDR mRNA levels and VDR protein.

ACKNOWLEDGMENTS This study was presented at the annual meeting of the American Society for Bone and Mineral Research (1990) and published in abstract form (Journal of Bone and Mineral Research 5:S267, 1990). We thank Frank Dastvan for his excellent technical assistance. We also thank Drs. Peter J . Munson and David Rodbard for kindly providing the software for LIGAND and ALLFIT. Finally, we thank Dr. Geoffrey Krystal for his expert advice and Dr. Robert Adler for reviewing the manuscript. This study was supported by a Geriatric Leadership Academic Award 1K07AGO0404 from the National Institute on Aging (to Godschalk) and funds provided by the Veterans Administration Merit Review (to Levy) and the American Diabetes Association, Virginia Affiliate (to Levy). The order of the second and third coauthors was arbitrary.

REFERENCES 1. Reichel H, Koeffler HP, Norman AW 1989 The role of the vitamin D endocrine system in health and disease. N Engl J Med 320:980-991. 2. Pike JW 1985 Intracellular receptors mediate the biologic action of 1,25-dihydroxyvitamin D,. Nutr Rev 43:161-167. 3. Kimberg DV, Baerg RD, Gershon E, Graudusius RT 1971 Effect of cortisone treatment on the active transport of calcium by the small intestine. J Clin Invest 50:1309-1321. 4. Hirst M, Feldman D 1982 Glucocorticoid regulation of 1,25(OH),vitamin D, receptors: Divergent effects on mouse and

GLUCOCORTICOIDS DECREASE VITAMIN D RECEPTOR NUMBER

21

17. Thompson CB, Challoner PB, Neiman PE, Groudine M rat intestine. Endocrinology 111:1400-1402. 1985 Levels of c-tnyc oncogene mRNA are invariant 5 . Chen TL, Cone CM, Morey-Holton E, Feldman D 1982 Gluthroughout the cell cycle. Nature 314:363-366. cocorticoid regulation of 1,25(OH),-vitamin D, receptors in 18. Davis LG, Dibner MD, Battey J F 1986 Nick translation. In: cultured mouse bone cells. J Biol Chem 257:13564-13569. Basic methods in molecular biology. Elsevier, New York, pp. 6. Nielsen HK, Eriksen EF, Storm T , Mosekilde L 1988 The effects of short-term, high-dose treatment with prednisone on 80-83. 19. Davis LG, Dibner MD, Battey J F 1986 Probing nucleic acids the nuclear uptake of I ,25-dihydroxyvitamin D, in monowith plasmid-derived probes. In: Basic methods in molecular cytes from normal human subjects. Metabolism 37:109-1 14. biology. Elsevier, New York, pp. 79-87. 7. Korkor AB, Kuchibotla J, Arrieh M, Gray RW, Gleason WA 1985 The effects of chronic prednisone administration 20. Eriksen EF, Nielsen HK, Mosekilde L, Nielsen HB, Melsen F 1986 Nuclear uptake of 1,25-dihydro~y[~H]-cholecalciferol in on intestinal receptors for I ,25-dihydroxyvitamin D, in the peripheral blood monocyres. Scand J Immunol 24:171-177. dog. Endocrinology 117:2267-2273. 8. Chen TL, Hauschka PV, Feldman D 1986 Dexamethasone 21. Levy JR, Krystal G , Glickman P, Dastvan F 1991 Effects of media conditions, inwlin. and dexamethasone on insulin-reincreases 1,25-dihydroxyvitamin D, receptor levels and augceptor mRNA and promoter activity in HepG2 cells. Diaments bioresponses in rat osteoblast-like cells. Endocrinology betes 4058-65. 118: 1 1 19-1 126. 9. Franceschi RT, James WM, Zerlauth G 1985 lalpha, 25-di- 22. Duncan RL, Grogan WM, Kramer LB, Watlington CO 1988 Corticosterone’s metabolite is an agonist for NA+ transport hydroxyvitamin D, specific regulation of growth, morpholstimulation in A6 cells. Am J Physiol 255:F736-F748. ogy, and fibronectin in a human osteosarcoma cell line. J 23. Beresford JN, Gallagher JA, Poser JW, Russell RGG 1984 Cell Physiol 123:401-409. Production of osteocalcin by human bone cells in vitro. Ef10. Franceschi RT, Romano PR, Park K 1988 Regulation of type fects of I ,25(OH),D,, 24,25(OH),D,, parathyroid hormone, 1 collagen synthesis by 1,25-dihydroxyvitamin D, in human and glucocorticoids. Metab Bone Dis 5:229-234. osteosarcoma cells. J Biol Chem 263: 18938-18945. 11. Eil C , Marx SJ 1981 Nuclear uptake of 1,25-dihydro~y[~H]- 24. Dietrich JW, Canalis EM, Maina DM, Raisz LG I979 Effects of glucocorticoids on fetal rat bone collagen Fynthesis in cholecalciferol in dispersed fibroblasts cultured from normal vitro. Endocrinology 104:715-721. human skin. Proc Natl Acad Sci USA 78:2562-2566. 12. Munson P J , Rodbard D 1980 LIGAND: A versatile com- 25. Canalis E 1983 Effect of glucocorticoids on type I collagen synthesis, alkaline phosphatase activity, and deoxyribonuputerized approach for characterization of ligand-binding cleic acid content in cultured rat calvariae. Endocrinology systems. Anal Biochem 107:200-239. 112:931-939. 13. De Lean A, Munson P J , Rodbard D 1978 Simultaneous 26. Peck WA, Messinger K, Brandt J , Carpenter J H 1969 Imanalysis of families of sigmoidal curves: Application to biopaired accumulation of ribonucleic acid precursors and deassay, radioligand assay, and physiological dose-response pletion of ribonucleic acid in glucocorticoid-treated bone curves. Am J Physiol 235:E97-E102. cells. J Biol Chem 244:4174-4184. 14. Davis LG, Dibner MD, Battey J F 1986 Preparation and analysis of RNA from eukaryotic cells. In: Basic methods in molecular biology. Elsevier, New York, pp. 129-156. Address reprint requests to: IS. Baker AR, McDonnell DP. Hughes M, Crisp TM, MangelsMichael F. Godschalk, M. D. dorf DJ, Haussler MR, Pike JW, Shine J , O’Malley BW Division of Gerialrics 1988 Cloning and expression of full-length cDNA encoding McGuire VA Medical Center ( I l l s ) human vitamin D receptor. Proc Natl Acad Sci USA 85: 3294-3298. 1201 Broad Rock Blvd. 16. Gunning P , Ponte P, Okayama H , Engel J , Blau H, Kedes L Richmond, V A 23249 1983 Isolation and characterization of full-length cDNA clones for human a-,p-. and y-actin mRNAs: Skeletal but not cytoplasmic actins have an amino-terminal cysteine that Received for publication August 27, 1990; in revi\ed form July is subsequently removed. Mol Cell Biol 3:787-795. 16, 1991; accepted August 2, 1991.