Homologous and heterologous regulation of 1,25-dihydroxyvitamin D-3 receptor mRNA levels in human osteosarcoma cells

Biochimica et Biophysica A cta, 1088 (1991 ) ! ! 1-118 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4781/91/$03.50 ADONIS 016747819100064N

111

BBAEXP 92196

Homologous and heterologous regulation of 1,25-dihydroxyvitamin D-3 receptor mRNA levels in human osteosarcoma cells A n i t t a M a h o n e n , A s t a P i r s k a n e n a n d P e k k a H. M~enp~i~i Department of Biochemistry and Biotechnology, University of Kuopio, Kuopio (Finland) (Received 15 May 1990)

Key words: 1,25-dihydroxyvitamin D-3 receptor; Steroid hormone; Retinoic acid: Triiodothyronine: Oncogene; (Human osteosarcomacell}

The heterologous regulation of hormone receptors is well described in the hormone receptor literature. We were interested in determining whether human 1,25-dihydroxyvitamin D-3 receptor (hVDR) and glneocorticoid receptor (GR), members of the steroid/thyroid hormone receptor family, are heterologously regulated by other steroids and related hormones. We used human osteosareoma cells (MG-63) and measured hVDR and GR mRNA levels after androgen, estrogen, glucorficoid, progesterone, thyroid hormone, vitamin A and vitamin D treatments. Each honmme, except androgen and progesterone, was capable of increasing hVDR mRNA levels like the natural ligand in human osteosarcoma cells. On the other hand, GR gene expression was not affected by these hormones. To study whether the cells responded to the 1,25(OH)2D3-treatment with changes in differentiation and proliferation,we also studied c-myc and c-~fos gene expression, Both genes were only regulated by 1,25(OH)2D3. 1,25(OH)2D3 slightly increased the accumulation of c-]os mRNA within 4-12 h from the hormone addition, while the increase in c-myc mRNA appeared at 24h.

lntreduetinn Steroid hormones exert complex effects on transcription during development, growth and differentiation in higher organisms, anct modulate cellular activities in response to changing environmental conditions. The mediators for the hormones are the intracellular receptors, which are activated when the steroid hormone is bound, and which are then capable of modifying specific target gene expression. The molecular cloning of several steroid hormone [1-3], thyroid hormone [4], and vitamin A and D [5-7] receptors indica,e that they are

Abbreviations: Dex, dexamethasone; DHT, 5a-dihydrotestosterone: 1,25(OH)2D3; 1,25-dihydroxyvitamin D-3; DME, Dulbecco's modified Eagle's medium; E2, lT~estradiol; FCS, fetal calf serum; GR, glucocorticoid receptor; hVDR. human 1,25-dihydroxyvitamin 1%3 receptor; Mops, 3-(morpholino)propanesuifonic acid; PBS, phosphate-baffered saline; Pro8. progesterone; RA, retinoic acid; SDS, sodium dodecyl sulfate: SSC, sodium chloride-sodium citrate; T3,

3.3',5'-triiodothyronine. Correspondence:A. Mahonen, Department of Biochemistryand Biotechnology, University of Kuopio, P.O. Box 6, SF-702ll Kuopio, Finland.

members of a common gene family regulating transcription by an analogous way. An important mechanism, which modulates target cell responsiveness to hormones, is the ligand-dependent autoregulation. This phenomenon has been described both for peptide and steroid receptors [8,9]. Previous studies have suggested that 1,25-dihydroxyvitamin D-3 (1,2~(OH)2D3) regulates its own receptors in vitro and in vivo. 1,25(OH)2D3 is capable of inducing its receptors in a number of cultured mammalian cells including mouse fibroblasts [5], human and rat osteosarcoma cells [10,11], pig kidney cells [12! and human promyelocytic leukemia cells [13]. Induction of VDR levels has also been demonstrated in vivo in rat intestine [14] and kidneys [15]. Further, it has been recently reported that treatment with estrogens and glucocorticoids may suppress their respective receptors at transcriptional and post-translatioval levels in a number of tissues [16-20]. Examples of one hormone regulating the receptors of another hormone (heterologous regulation) are well described in the hormone receptor literature. The heteroIogous regulation occurs regardless of the nature (peptide or steroid) of the receptor [8,21-23]. We were interested in detcnu~ning whether hVDR and GR,

112 members of the steroid/thyroid hormone family, are heterologously regulated by other steroids and related hormones. According to previous studies using radiolabelled hormone binding assays, glucocorticoids regulate VDR levels in primary cultures of mouse and rat osteoblast-like bone cells [24,25]. Retinoic acid, a naturally occurring vitamin A metabolite, has also been found to elicit a dose-dependent increase in 1,25(OH)2D3-binding in rat osteesarcoma cells (ROS 17/2) [26]. We investigated the heterologous regulation of hVDR mRNA levels in human osteoblastic-like osteosarcoma cells. Glucocorticoids, estrogen, vitamin A and thyroid hormone were capable of stimulating hVDR levels, while progesterone and dihydrotestosterone were without effect on hVDR mRNA levels. At the same time, the expression of the glucocorticoid receptor gene was not affected by these hormones. The cellular oncogenes (proto-oncogenes) seem to play a role in cellular proliferation and differentiation [27]. To examine the possibility that the cells responded to the hormone treatment with changes in differentiation and proliferation, we also determined c-fos and c-myc mRNA levels. 1,25(OH)2D 3 increased c-fos mRNA accumulation within 4-12 h from the hormone addition, while the increase in c-myc mRNA levels appeared at 24 h. The other hormones did not influence c-fos or c-myc mRNA levels. Materials and Methods

Chemicals and cells The cell culture medium, fetal calf serum (FCS), L-glutamine, nonessential amino acids, penicillin and streptomycin were from Gibco (Paisley, U.K.). MG-63 human osteosarcoma cells were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). 1,25-Dihydroxyvitamin D-3 was from Hoffmann La Roche (Nufley, N J, U.S.A.). 17fl-estradiol, dexamethasone, 5a-dihydrotestosterone, progesterone and 3,3',5'-triiodothyronine were obtained from Sigma Chemical (St. Louis, MO, U.S.A). [a-32p]dCTP ( > 3000 Ci/mmol) was purchased from Amersham International (Amersham, U.K.) and the nick-translation kits were from Boehringer-Mannheim (F.R.G.). Cell culture MG-63 cells were cultured in Dulbecco's modified Eagle's medium (DME) supplemented with 10~ irradiated FCS, 2 mM L-glutamine, 100 U / n i l penicillin, 0.1 m g / m l streptomycin and nonessential amino acids in a humidified 95~ a i r / 5 ~ CO 2 incubator. 3.105 cells were seeded onto 100-mm culture plates and cultured to about 80~ confluency. The culture medium was replaced for 24 h before each experiment with a medium containing 2~ charcoal-treated FCS to minimize the

effects of endogenous hormones. The hormones were added into the media in ethanol and the control cells were treated with 0.1% ethanol.

RNA preparation and hybridization Total RNA was prepared from the cultured cells according to the method of Anderson et al. [28] and denatured in 3 vol. of 66~ formamide, 8~ formaldehyde, and 1.3 × Mops ( 1 0 × Mops: 0.2 M morpholinopropanesulfonic acid (pH 7.0), 50 mM sodium acetate, 10 mM EDTA) at 65°C for 5 rain, chilled on ice and made 10 x for SSC (1 x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate). The amount and purity of total RNA was measured spectrophotometrically. Slot-blot hybridization analysis was performed as described previously [10]. For Northern-blot analysis, RNA was fractionated by electrophoresis in the presence of formaldehyde in 1.4~-agarose gels, transferred onto nylon membranes (Hybond N, Amersham International) and hybridized with a nick-translated hVDR eDNA probe [29]. The probe for VDR was a eDNA clone (hVDR 1/'3) containing the entire coding sequence of the hVDR, 115 bp of the 5'-nontranslated region and 800 bp of the 3'-region [6]. The eDNA probe for the glucocorticoid receptor was a 2.8 kb BamHI fragment of the complete rat glucocorticoid receptor region (pBal 117) [30]. The eDNA probe for c-los proto-oncogene was a 3.1 kb DNA fragment of the human c-fos gene, and for c-myc, a 1.5 kb DNA fragment of the human c-myc gene (Amersham International). The specific radioactivity of the nick-translated probes ([a-32p]dCTP > 3000 Ci/mmol) was ( 2 - 6 ) - l 0 s cpm//~g DNA. The filters were washed twice with 5 x SSC at 42°C, once with 2 x SSC + 0.1~ SDS at 42°C, and once with 0.1 x SSC + 0.1t~ SDS at room temperature. After the washes, the filters were exposed to X-ray films at - 8 0 ° C using intensifying screens. The relative amounts of mRNAs were determined by densitometric scanning at 550 nm (Shimadzu DuaI-Wavelenght TLC Scanner CS-930) of the autoradiogram films and by integrating the areas of the peaks. R ~ The aim of this study was to determine the ability of steroids and related hormones to regulate hVDR gene expression. We used human osteosarcoma cells (MG-63) and determined hVDR m R N A levels after the hormone treatments, hVDR m R N A levels were analyzed by slotblot hybridization using a radiolabelled eDNA probe for hVDR. The specificity of the probe was tested by Northern-blot analysis shown in Fig. 1. Hybridization with the hVDR 1 / 3 cDNA revealed a single species of mRNA, approx. 4.5 kb, which agrees with the Northern-blot results of Baker et al. [6]. Lanes 1-3 represent

113 Control ceils

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Fig. l. Northern-blot analysis of hVDR mRNA from MG-63 cells. Lanes 1-3, control MG-63 cells grown in the absence of 1,25(OH)2D3; lanes 4-6 MG-63 cells treated with l0 nM 1,25(OH)2D3 for 48 b. The transcript size for hVDR mRNA is about 4.5 kb. The results are representative of three separate RNA isolations. The amount of total RNA loaded on each lane ~as evaluated relative to ,8-actin mRNA content. control cells grown in the absence of 1,25(OH)2D 3. In lanes 4 - 6 , where M G - 6 3 cells were treated with 10 n M 1,25(OH)2D 3 (48 h), a typical ligand-dependent accu-

mulation of h V D R m R N A is seen. h V D R c D N A alst, binds to 18S r R N A - f r a g m e n t , which can be seen as a 2.1 k b b a n d in the Northern-blots. More efficient washes with 0.1 × SSC + 0.1~ SDS at 65°C did not significantly reduce the nonspecific binding of the h V D R c D N A probe. The level of ~-actin m R N A , which is not affected by the 1,25(OH)2D 3 treatment, was used to determine the a m o u n t of total R N A in the N o r t h e r n - b l o t analysis. T h e effects of dexamethasone, dihydrotestosterone, estradiol, progesterone, retinoic acid a n d triiodothyronine on cellular h V D R m R N A levels were investigated by treating the M G - 6 3 cells with increasing concentrations (from 1 p M to f / ~ M ) of the h o r m o n e s for 48 h, a n d 10 n M h o r m o n e concentration was used in the time-course experiments. Both experiments were performed with daily changes of m e d i u m containing fresh hormones. 1,25(OH)2D 3 t r e a t m e n t was used as a positive control for the ligand-dependent accumulation of h V D R m R N A . In Fig. 2, autoradiograms from typical slot blot-hybridization results are shown. After dexamethasone, estradiol, retinoic acid or triiodothyronine treatment, we found that h V D R m R N A was induced in MG-63 cells in a time- a n d dose-dependent m a n n e r (Fig. 3A and B). However, progesterone and dihydrotestosterone did not have any effect on h V D R m R N A levels. T h e m a x i m u m increase was seen in 48 h with 10 n M dexamethasone (about 375~), a n d with 1 / t M estradiol a n d retinoic acid (about 350~ a n d 275~, respectively). Tri-

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Fig. 2. Effects of steroids and related hormones on hVDR mRNA levels in MG-63 cells assayed by slot-blot hybridization. The cells were grown in the presence of the hormones for the indicated times and at the indicated concentrations. RNA was prepared and assayed for hVDR mRNA as described in Materials and Methods. Autoradiograms represent individual determinations from three separate experiments. 1,25. 1,25(OH)2D3; Dex, dexamethasone; DHT, dihydrotestosterone; E2, estradiol; Prof, progesterone; RA, retinoic acid: and T 3, triiodothyronine.

114 1,25(OH)zD3-dependent continuous stimulation of hVDR mRNA is shown. If 1,25(OH)2D 3 was added to the culture medium as one dose, the maximal upregulation of hVDR mRNA level occurred at 48 h, whereafter the level decreased, being at the control level by 96 h (Fig. 4A, one dose). According to these results, the half-life of hVDR mRNA after the 1,25(OH)2D 3treatment ~: about 72 h (Fig. 4B). The same effect was seen with the other hormones. If the hormones were added as one dose, the elevations in the hVDR mRNA levels occurred at about 48 h, whereafter the levels decreased, being at the control level by 96 h. With daily

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iodothyronine had an effect with as low concentration as 1 pM (about 200%), whereafter the cells did not respond with further elevations in the hVDR mRNA levels. It was somewhat surprising that the cells responded with more pronounced elevations in hVDR mRNA levels at 1 pM concentration of dexamethasone, estradiol or triiodothyronine than with 1 pM 1,25(OH)2D3. The time-course of hVDR mRNA upregulation by the hormones is shown in Fig. 3B. The elevation in hVDR mRNA with each hormone was first detected at 6 h. The maximal response was reached with triiodothyronine at 24 h, and thereafter the level of hVDR mRNA slightly decreased. With dexamethasone and retinoic acid, the response was increased during the treatment by 480~ and 300~, respectively, at 96 h. After estradiol treatment, the mRNA levels were maximally elevated at 72 h (425~). Combination of 1,25(OH)2D3 (10 nM) with any other hormone (10 riM) used in this study did not result in an additive increase in hVDR mRNA levels (not shown). MG-63 cells were grown up to 144 h in the absence or presence of 10 nM 1,25(OH)2D3 to determine the half-life of hVDR mRNA after the treatment. The hVDR mRNA levels were analyzed at various times from the hormone addition, in Fig. 4A (daily doses),

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Fig. 4. Regulation of h V D R m R N A levels by the 1,25(OH)2D3-treatment, (A) Autoradiograms of slot-blot hybridization analysis. Daily doses: MG-63 cells were cultured up to 144 h with daily medium changes with addition of fresh 10 nM 1,25(OH)2D3. One dose: MG-63 cells were cultured without medium changes in a medium where l 0 nM 1,25(OH)2D 3 was added as one dose. After the treatments total R N A was isolated a n d analyzed as described in Materials and Methods. (B) Determination of h V D R m R N A half-life after the 1,25(OH)2D3-treatment. Each d a t a point represents the mean "4-S.E of three separate experiments.

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Fig. 5. Effects of steroids and related hormones on GR mRNA levels in MG-63 cells assayed by slot-blot hybridization. The autoradiogram represents one (6 h) of four measured time points (6 h, 24 h, 72 h or 96 h). The MG-63 cells were treated with l 0 nM hormone up to 96 h, whereafter the GR mRNA levels were analyzed.

doses, the hVDR mRNA levels were continuously elevated throughout the experiment up to 144 h. There were no significant differences in the half-life of the hVDR mRNA level after dexamethasone, estradiol, retinoic acid or tciiodothyronine treatment compared with the 1,25(OH)2D3 treatment (not shown). To characterize corresponding effects on glucocorticoid receptors, we analyzed GR mRNA levels as described for hVDR. The MG-63 cells were treated with 10 nM hormone for various times (from 6 h to 96 h) before the analysis of RNA. The results in Fig. 5 represent one (6 h) of four measured time points (6 h, 24 h, 72 h or 96 h). The G R mRNA levels did not respond to any of the hormones at any time point studied. Osteosarcoma cells may respond to treatments with the hormones with changes in cellular differentiation or proliferation. We therefore determined mRNA levels of the cellular oncogenes c-myc and c.fos. In these experiments, FCS in the culture media was replaced with bovine serum albumin to eliminate effects of endogenous hormones and growth factors. The MG-63 cells were treated with each hormone (10 riM) for 1 h, 4 h, 12 h, 24 h and 72 h with daily medium changes. We found that both c-los and c-myc genes are expressed in these cells and are regulated only by 1,25(OH)2D3 (Fig. 6).

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~tme (tt) Fig. 6. Regulation of c.fosand c-myc m R N A levelsby 1,25(OH)2D 3 in MG-63 cells.The cells were treated with 10 n M hormone for the

times indicated.OncogenemRNA levelswere analyzedby slot-blot hybridization.Each bar represents the mean± S.E of three separate e.xpcrirt~ents.

1,25(OH)2D3 caused a 60~ increase in c-fos mRNA accumulation within 4-12 h from the hormone addition, while the increase (about 50~) in c-myc mRNA levels appeared at 24 h. Increased c-los and c-myc mRNA levels were no longer seen 72 h after the hormone addition. Discussion

These data describe heterologous regulation of hVDR mRNA levels in human osteoblast-like osteosarcoma cells. Steroids and other related hormones were capable of stimulating hVDR mRNA levels in a dose- and time.dependent manner. The regulation of VDR numbers is an important mechanism in modulating cellular responsiveness to 1,25(OH)2D3, as the biological activity of 1,25(OH)2D3 in cells has been shown to be proportional to the amount of cellular VDR [31-33]. Several hormones, e.g., retinoic acid, glucocorticoids and estrogen, have previously been shown to increase the synthesis of VDR [24-26,34-36], thus increasing cell responsiveness to 1,25(OH)2D3 in the specific tissues affected. Chen et al. [37] have reported that osteoblast-like cells have receptors for glucocorticoids. The same group has also demonstrated that there is a difference in VDR regulation by glucocorticoids in rat and mouse ostec~ blast-like bone cells [24,25]. After glucocorticoid treatment, the VDR concentration was elevated in rat osteoblast-like cells, in contrast to the decline they found in mouse osteoblast-like cells. Also, in vivo experiments have indicated a species difference; in intestinal mucosa cells, glucocorticoids reduce the numbers of VDR in the mouse [38], but increase the receptor levels in the rat [39]. Our results indicate that dexamethasone has a stimulating effect on VDR mRNA levels in human osteosarcoma cells. Maximal upregulation of hVDR mRNA levels occurred at 10 nM dexamethasone concentration and, in the time-course experiment, a slight elevation of mRNA was first detected at 6 h. Thereafter VDR mRNA levels increased, being maximal at 96 h. We did not see any effect on VDR mRNA concentrations after progesterone treatment, which agrees with the results of Chen et al. [24]. Also dihydrotestosterone was without effect, despite the presence of androgen receptors in the osteoblast-like cells [40].

116 Furthermore, we studied homologous and t,,eterologous regulation of the G R gene expression after treatments with the same hormones. GR mRNA levels did not respond to any of the hormones at any time point studied. The ligand-induced homologous regulation of the glueocortico~d receptor has been detected using classical ligand binding techniques and later by Northern-blot and nuclear run-on transcription assays. Those results have suggested that down-regulation of glucocorticoid receptors by its cognate ligand is complex and occurs at both transcriptional and pest-translational levels in hepatoma cells and in rat liver in vivo [19,41431. The apparent absence of estrogen receptors in bone has been previously reported [44]. in 1988, however, Erikson et al. 1451 and Komm et at. [461 showed, using a sensitive receptor assay and RNA blot analysis, that cultured human bone cells and osteosarcoma cells contain estrogen receptors, which did not depend on estrogen treatment. Further, testosterone was the only steroid hormone, which competed significantly with labelled estrogen for the binding to the receptor-like sites in osteosarcoma cells. This could be the result of either the presence of a second sex-steroid binding protein or the existence of a unique estrogen receptor in bone that also binds certain androgens. On the other hand, Komm et at. [46] suggested that the presence of an aromatase in the extracts could account for the testosterone competition by conversion of testosterone to estradiol. Ernst et al. [47] found in long-term bone cell cultures that estradiol stimulates cell proliferation in a dose-dependent fashion and increases steady-state levels of mRNA encoding the al-~hain of type I procollagen. In the clonal osteoblastic cell line, UMR-106, estradiol reduces growth rates and increases alkaline phosphatase activity [481. After the treatment up to 96 h with vmi~,as concentrations of estradiol, the MG-63 cells responded with increased hVDR levels. The increa.~e was dose-dependent and was first detected at l pM cone :ntration. Similar estrogen regulation of VDR levels has been demonstrated in rat uterus [36]. Phenol red, a prl indicator in culture media, is a weak estrogenic compound [49] and it may cause an elevation of hVDR mRNA levels. On the other hand, estrogen stimulation of progesterone receptors was not found to be affected by phenol red, but the basal progesterone receptor level was 3-times higher in cells grown in the presence of phenol red [49]. According to the results presented and cited above, estrogen seems to stimulate bone formation by a direct action on osteoblasts. The action of retinoic acid on the stimulation of bone resorption and inhibition of collagen synthesis is similar to the effects of 1,25(OH)2D 3 [50,51]. A possible relationship between the two bone-acting agents was uncovered by Petkovich et al. [26], who demonstrated

that retinoic acid increases the binding capacity of VDR in rat osteogenic sarcoma cells (ROS 17/2) and decreases it in nontransformed cells derived from normal rat calvariae, in normal rat and mouse bone cells, opposite effects of retinoic acid on VDR binding and biological function has been found [341. Retinoic acid downregulated VDR in rat bone cells and upregulated the receptor in mouse bone cells. Protein and RNA synthesis is required for the augmentation of the VDR levels by retinoic acid in mouse bone cells [34]. Further, the regulation of VDR based on a species difference in these rat and mouse cells was opposite to the action of glucocorticoids demonstrated by Chen et al. [24,251, and the response to retinoic acid m normal rat cells was opposite to that reported for malignant rat cells by Petkovich et al. [261. Our results indicate that hVDR gene expression is regulated time- and dose-dependently by retinoic acid in human osteogenic sarcoma cells. The elevated hVDR mRNA levels in human osteosarcoma cells support the finding of Petkovich et al. [26] that the increased 1,25(OH)2D3-binding after retinoic acid treatment is dependent on continuous production of VDR mRNA in rat osteosarcoma cells. Rizzoli and Burki [52] showed in 1986 that the maximum triiodothyronine nuclear binding capacity was 0.13 + 0.02 n g / m g DNA in rat osteosarcoma cells. They also found that the production of collagen and the n.'~st abundant noncollagen protein, osteocaicin, was enhanced by triiodothyronine in these cells. Our findings suggest that triiodothyronine is capable of regulating VDR at the transcriptional level in human osteosarcoma cells. The cellular oncogene products play an important role in the regulation of growth, differentiation and functioning of normal cells. The amino acid sequences of steroid receptors are similar to the erb-A oncogene product, a thyroid hormone receptor I53]. These results have suggested that steroid receptors and the c-erb A product are members of a superfamily of transcription factors, which regulate normal and malignant growth during differentiation [541. The c-los gene product seems to play a significant role in the process of differentiation. The expression of the c-los gene has been shown to be induced during monocytic differentiation {55]. in contrast to the induction of the c-los gene expression, c-myc mRNA levels decrease during terminal differentiation [56]. However, unlike the rapid c-los expression, the c-myc transcription decreases relatively slowly (over an 8 h period). The decreased c-myc expression may not be obligatory to the differentiated state, but may reflect proliferation of the differentiating cells. We found that VDR is homologously and heterologously regulated by steroids and related hormones in human osteosarcoma cells. The continuous treatment of the cells with these hormones led to permanent eleva-

117

tions of VDR mRNA, which may indicate changes in the cellular differentiation stage. To examine the possibility that the hormones influence the cells to differentiate toward more mature osteoblasts in which the numbers of VDR remain permanently increased, we measured the proto-oncogene c-los and c-myc mRNA levels. Both oncogenes are expressed in this human osteosarcoma cell line. Only 1,25(OH)2D 3 influences both proto-oncogene mRNA levels. The slight accumulation of c-los mRNA appeared within 4-12 h from the hormone addition, while the increase in the c-myc m R N A levels was seen at 24 h. The more rapid effects in the c-los gene expression suggest a direct regulatory action of the VDR-complex on the c-fos gene. The c-myc gene expression was possibly stimulated by a different mechanism. The other hormones, despite their effects on hVDR mRNA levels, did not affect the c-los or the c-myc expression. Studies using rat osteogenic sarcoma cell lines have shown that cell lines, which express high levels of VDR, also express c-myc mRNA, and cell lines, in which VDR is not detectable, do not express the c.myc gene [57]. Since the c-myc gene is thought to regulate transcription of genes involved in cell growth and differentiation [27] and since c-myc m R N A and VDR are expressed together, Manolagas et al. [57] have suggested that, by acting through its receptor, 1,25(OH)2D 3 may be a natural counterregulatory signal for cell proliferation and differentiation controlled by the c-myc oncogene. In this study, we show that VDR gene expression is regulated by steroids and related hormones. However, the mechanism by which these hormones exert their effects on the osteoblastic-like bone cells is at the moment not known. The elevated hVDR mRNA levels indicate the regulation by these hormones. It does not, however, allow a determination of whether these changes are caused by a direct action on the VDR gene, prem R N A processing, or mRNA stabilization. Further studies are, therefore, necessary to determine steadystate levels of hVDR m R N A and rates of hVDR gene transcription during the hormonal induction. The increased c-los gene expression after the 1,25(OH)2D 3treatment may indicate cellular differentiation during the treatment, although the results on the stimulation of c-myc mRNA levels are somewhat conflicting with the literature [56]. However, the elevated c-myc and hVDR m R N A levels during the 1,25(OH)2D3-treatment support the possibility that these genes are regulated coordinately. Acknowledgements This work was supported in part by grants from the Academy of Finland. We thank Dr. Sam Okret (Karoliniska Institutet) for the glucocorticoid receptor cDNA, and Dr. Bert O'Malley and Dr. Donald Mc-

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