Estradiol Regulation of Insulin-Like Growth Factor-I Expression in Osteoblastic Cells: Evidence for Transcriptional Control

Matthias Ernst* and Gideon A. Rodan Department of Bone Biology and Osteoporosis Research Merck, Sharp, and Dohme Research Laboratories West Point, Pennsylvania 19486

Insulin-like growth factor-l (IGF-I) has anabolic effects on skeletal tissues, acting as both a systemic hormone and an autocrine/paracrine regulator of cellular function. We have previously reported that estradiol (E2) stimulation of rat osteoblast proliferation in vitro was inhibited by IGF-I antibodies. We show here that E2, similar to IGF-I, also increases a^l) procollagen mRNA levels in primary cultures of rat calvarial osteoblasts. The E2 effect on collagen mRNA lags behind that produced by recombinant IGF-I by about 12 h and was also abolished in the presence of cycloheximide or by the addition of antibodies against IGF-I. Furthermore, 17/?-E2 induced a 2- to 2.5-fold elevation of the level of IGF-l mRNA within 2-4 h, which persisted thereafter. The E2 stimulation of IGF-I mRNA was not blocked by cycloheximide, suggesting that de novo protein synthesis of an intermediate protein was not required. The IGF-I mRNA half-life, estimated by treating the cells with the RNA polymerase inhibitor 5,6-dichloro1/tf-D-ribofuranosylbenzimidazole, was about 7 h and was not altered by E2 treatment. On the other hand, nuclear run-on assays indicated that E2 increased the transcriptional activity of the IGF-I gene, and this effect was further enhanced in cells overexpressing E2 receptors after transient transfection. These findings suggest that IGF-I may serve as a mediator for the anabolic effects of E2 on bone, and that E2 stimulates IGF-I gene expression at least in part through transcriptional control. (Molecular Endocrinology 5: 1081-1089, 1991)

growth factor for cartilage. The regulation of serum IGF-I levels by its production in the liver under the control of GH and its role as a systemic mediator of growth hormone action are well recognized (2). Thus, in GH-deficient rats, growth, including that of long bones, can be restored by the administration of either GH or IGF-I (3). In addition, transgenic mice, which overexpress genes encoding either GH-releasing factor or GH, have elevated levels of GH as well as IGF-I and can grow to twice the normal size (4). After GH administration, tissue-extractable IGF-I increases earlier than serum IGF-I, although serum levels are normally much higher than tissue levels (5). These findings together with the distribution patterns of IGF-I mRNA in multiple organs (6) suggest that IGF-I may also have an important paracrine/autocrine function. Skeletal tissues synthesize and respond to IGF-I both in vivo and in vitro (3, 7-10). IGF-I is a mitogen for osteoblasts (11, 12) and stimulates the synthesis of type I collagen (13,14), the major organic constituent of bone matrix. In addition, the synthesis of IGF-I by osteoblasts appears to be regulated by systemic hormones (15-17). Estradiol (E2) is the biologically active form of estrogen, and its deficiency causes the bone loss observed after menopause. Estrogen replacement therapy efficiently delays or prevents postmenopausal bone loss, but its mode of action is unknown. The effects of estrogens on the skeleton were believed to be indirect; however, recent evidence suggests that E2 may act directly on bone cells (18, 19). Osteoblasts posses a low number of binding sites for E2 (20-22), and E2 treatment increases the proliferation (17, 23) and elevates the level of ai(l) procollagen mRNA in these cells in vitro (17). Similar to its effects on classical estrogen target tissues, such as the uterus (24) and mammary glands (25), E2 also increased IGF-I mRNA levels in cultures of calvarial osteoblasts, and the E2-induced cell proliferation was abolished by the presence of IGF-I antibodies (17). In this study the regulation of IGF-I gene expression by E2 in osteoblasts in vitro is further characterized. We present additional evidence that locally produced IGF-I may serve as a mediator for the anabolic effects of E2

INTRODUCTION

Insulin-like growth factor-l (IGF-I), a 7649-dalton mitogenic polypeptide, circulates in the serum and is widely distributed in many tissues of all mammals examined so far (1). IGF-I was discovered as a putative serum 0888-8809/91/1081-1089$03.00/0 Molecular Endocrinology Copyright ffi 1991 by The Endocrine Society

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Vol 5 No. 8

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on bone and show that E2 increases the abundance of IGF-I mRNA at least in part through increased gene transcription.

RESULTS

Estrogen-induced stimulation of cell proliferation in primary cultures enriched for calvarial osteoblasts is dependent on IGF-I production (17). If the E2-induced elevation of collagen mRNA also depends on de novo IGF-I production, it should lag behind that induced by exogenously added IGF-I. A comparison of the time course of the effects of IGF-I and E2 on collagen mRNA in cultured osteoblasts showed indeed that 20 nM IGFI increased the abundance of collagen mRNA after about 9 h, whereas after the addition of 10 nM E2 an increase was apparent only at 24 h (Fig. 1). Treatment for 36 h with 10 nM E2 increased «i(l) procollagen mRNA by about 3-fold (Fig. 2, left panels). The presence of 2 Mg/ml cycloheximide, which inhibits about 90% of new protein synthesis (data not shown), abolished the effect of E2 on collagen mRNA. Cycloheximide also consistently decreased the steady state level of /3-tubulin mRNA (Fig. 2, left panels; compare also Fig. 5), as previously reported (26). The E2-induced elevation of collagen mRNA was abolished by addition of the immunoglobulin G (IgG) fraction (100 Mg/ml) of an antiserum against IGF-I to the culture medium (Fig. 2, right panels). These findings suggest that the effect of E2 on collagen mRNA is mediated by locally produced IGF-I. Hybridization of rat prepro-IGF-l cDNA to total cytoplasmic RNA prepared from confluent cultures of calvarial osteoblasts showed IGF-I mRNA species of predominantly 7.5 and 1 kilobases (kb), with less abundant species of about 2.5 kb, corresponding to the previously demonstrated IGF-I transcripts in RNA prepared

IGF-I 0

3

6 9

ESTRADIOL 12 24 h

coll

3-tub

from rat liver (27). 17/3-E2 treatment elevated the steady state level of IGF-I mRNA in a dose-dependent manner, reaching a half-maximal stimulation at 0.1 nM and maximal stimulation at concentrations of 10-100 nM E2 (Fig. 3). In contrast, the biologically less active stereoisomer 17a-E2 had no effect on IGF-I mRNA (data not shown). A single treatment of confluent cultures of osteoblastic cells with 10 nM 17/?-E2 caused a rapid increase in IGFI mRNA levels of about 2- to 2.5-fold after 2-4 h, which persisted for at least 48 h (Fig. 4). Under the same experimental conditions, there was no change in the abundance of /3-actin or /3-tubulin mRNA (with the exception of cycloheximide treatment), which were, therefore, used as references for estimating the changes in IGF-I mRNA. These filters were also reprobed with a 32 P-labeled cDNA for transforming growth factor-/31, which was not altered by E2 treatment of these cells (data not shown). To test whether E2 stimulation of IGF-I mRNA required new protein synthesis, the cells were treated with 10 nM E2 for 6 h in the presence or absence of 2 Mg/ml cycloheximide. As shown in Fig. 5, E2 stimulation of IGF-I mRNA was not blocked by cycloheximide, indicating that the E2-induced increase in IGF-I mRNA was not dependent on the synthesis of an intermediate protein. To further examine the mode of IGF-I mRNA regulation by E2 in osteoblasts, the mRNA synthesis inhibitor 5,6-dichloro-1i8-D-ribofuranosylbenzimidazole (DRB; 25 Mg/ml) was added to cultures, and the decay of IGF-I mRNA was estimated by densitometric scanning of Northern blot autoradiograms. As shown in Fig. 6, the decay pattern of IGF-I mRNA in cells treated with 10 nM E2 was similar to that in untreated control cells, indicating that the stability of IGF-I mRNA was not affected by E2. The estimated half-life for IGF-I transcripts in calvarial osteoblasts was about 7 h. In contrast, the rate of transcription of the IGF-I gene, esti-

0 3 6 12 24 48 h

^28 S

*§«§*•

-18S

28 S

18S

Fig. 1. Comparison of the Time Course for the E2- and IGF-l-lnduced Increases in Collagen mRNA Abundance Confluent primary cultures of rat osteoblasts were treated with 20 nM recombinant human IGF-I or 10 nM E2 in medium containing 0.5 mg/ml CS-BSA. At the indicated time, cytoplasmic RNA was prepared, and Northern analysis of 10 ^g RNA was carried out, as described in Materials and Methods. The upper panels represent the hybridization patterns for«,(!) procollagen mRNA (coll); the lower panels represent those for /3-tubulin (/3-tub). The positions of the 18S and 28S ribosomal RNA are indicated. These findings were reproduced in another similar experiment.

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Transcriptional Regulation of IGF-I by E2

c E

-

1083

Ab

E 2 [M]

E 0

coll

10"l110~1310~910"810'710'6

'28S

*

28S IGF-I ft-tub

-18S

'18S

Fig. 2. The E2-lnduced Increase in Collagen mRNA Depends on IGF-I Synthesis Left panel, Confluent primary cultures of calvarial osteoblasts were treated with vehicle (-) or 10 nM E2 (E) for 36 h in the absence or presence (C) of 2 /ig/ml cycloheximide. Northern analysis of 10 ng total cytoplasmic RNA was carried out as described in Materials and Methods. Right panel, Confluent cultures of osteoblasts were maintained for 72 h in medium containing 0.5 mg/ml CS-BSA in the absence (-) or presence of 10 nM E2 (E) together with 100 Mg/ml of the IgG fraction of an antiserum raised against IGF-I (Ab; described in Ref. 15). Northern analysis of 10 ng cytoplasmic RNA was carried out as described above. In both figures, the upper panels represent the hybridization patterns for ^(l) procollagen (coll); the lower panels represent the hybridization patterns for /3-tubulin (/3tub) of the same filters. The positions of the 18S and 28S ribosomal RNA are indicated. These findings were reproduced in another similar experiment.

18S

JH|^^^ ^^^^l^^^^^tfMttgk 18S

actin

B 2.5

1-5

0 1 0 " 1 l 1 0 " 1 ° 1 0 ' 9 10

mated by in vitro nuclear transcription (run-on) assays, was increased 2- to 2.5-fold (Fig. 7) after treatment with E2. This effect was observed 4 and 22 h after the addition of E2 and was specific for IGF-I gene transcription, since the rate of /3-tubulin gene transcription was not altered. It is well established that E2 regulation of gene expression is mediated by ligand-induced binding of the E2 receptor to a specific DNA sequence, termed the estrogen response element (ERE), in the 5'-flanking region of target genes (28, 29). Since it has also been shown that the rate of transcription from steroid receptor-responsive promoters depends on the cellular concentration of the respective receptor (30), we overexpressed E2 receptors in our primary cultures of osteoblastic cells and estimated E2-dependent IGF-I regulation. After transient cotransfection of cells with an E2 receptor expression plasmid (pJ3MOR) and an ERE-containing reporter plasmid (pERE-BLCAT), we first estimated the time needed for maximal expression of E2 receptors. As judged by induction of the chloramphenicol acetyl transferase (CAT) reporter gene, E2induced CAT activity was maximal 48-96 h after transfection (Fig. 8). Therefore, 48 h after cotransfection of primary cultures of calvarial osteoblasts with pJ3MOR and pERE-BLCAT, cultures were stimulated with 100 nM E2, and nuclear run-on experiments were performed. E2-induced IGF-I transcription in nuclei from cells overexpressing E2 receptors about twice as much as in nuclei of mock-transfected cells (Fig. 9). Since enhanced E2-dependent IGF-I transcription coincided with enhanced E2-dependent CAT induction in receptor-ov-

10"7 1 0 6

E 2 [M] Fig. 3. Dose-Dependent Increase in IGF-I mRNA by E2 Confluent cultures of calvarial osteoblasts were precultured as outlined in Materials and Methods. Twelve hours before E2 treatment, the cultures were switched to medium containing 0.5 mg/ml CS-BSA. At time zero, the medium was changed, and new medium containing CS-BSA and 17/3-E2 at the indicated concentrations was added. Twenty-four hours later, total cytoplasmic RNA was prepared, 20 ^g/'ane were used for Northern blot hybridization to 32P-labeled probes. A, The upper panel shows the hybridization pattern for IGF-I; the lower panel shows that for /^-actin. The positions of the 28S and 18S ribosomal RNA are indicated by arrowheads. B, Quantitation of IGF-I mRNA bands in A by densitometric scanning relative to the actin bands. These findings were reproduced in another similar experiment.

erexpressing cells, these results suggest that the abundance of receptors derived from the endogenous gene may limit E2-dependent IGF-I transcription in osteoblasts.

DISCUSSION

The results of this study show for the first time that E2 enhances the level of IGF-I gene transcription and suggest that IGF-I mediates the increase in collagen gene expression observed in primary cultures of osteoblastlike calvaria cells after E2 treatment. This report, thus, also suggests that estrogens may act directly on bone

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MOL ENDO-1991 1084

Vol 5 No. 8

0

1 2 4 6 8 24 48 96 h

IGFX}

18S

3-tub

it

-«18S 4

8

I I 20 40

J I 60 80 100 h

Time of Estradiol Treatment Fig. 4. Time Course of the Effects of E;2 on IGF-I mRNA Levels Confluent cultures of calvarial osteoblasts were precultured as outlined in Fig. 3, and the medium was changed 2 h before experiments. E2 (10 nwi) was then added, and cytoplasmic RNA was prepared from two identically treated culture dishes at the indicated time. Total RNA (20 ng) was used for Northern analysis, as described in Materials and Methods. A, The filter was hybridized with a 32P-labeled cDNA for rat prepro-IGF-l (upper panel) and later rehybridized with a 32P-labeled cDNA for /3-tubulin (/3-tub; lower panel). The positions of 18S and 28S ribosomal RNA are indicated. B, The autoradiograms were densitometrically scanned, and the intensity of the band of the 7.5-kb IGF-I transcripts was normalized for the intensity of the /3-tubulin signal. The points represent the means of two readings of filters prepared from RNA from two identically treated dishes, and the bars indicate the SD. These findings were reproduced in another similar experiment.

C E -E

IGF-I

-* 28S - 18S

B-lub

18S

and supports recent findings by several investigators that estrogen induces biological effects in these as well as in transformed osteoblastic cells in vitro (17-19, 23, 31,32). Osteoblasts posess receptors for IGFs (33) and have been shown to synthezise IGF-I and specific IGF-binding proteins in response to systemic hormones (17,32). Thus, IGF-I is likely to function as a local autocrine/ paracrine mediator, since certain anabolic effects of GH (15) and PTH (16) on osteoblasts in vitro are abolished by the presence of IGF-I antibodies. We have previously shown that the onset of E2-stimulated osteoblast proliferation lagged behind that of IGF-I (34), and that IGF-I antibodies abolished the effect of E2 (17). Here, we show similar findings for the E2-induced stimulation of

1085

0.75"

0.5

-

0.250 -

i

0

7

l

14h

time after ORB (25 (.tg/ml) addition Fig. 6. Effects of E2 on IGF-I mRNA Stability Confluent cultures of calvarial osteoblasts were treated with 10 nM E2 for 12 h in medium containing 0.5 mg/ml CS-BSA. DRB (25 fig/m\) was then added to culture medium, and cytoplasmic RNA was isolated 0, 7, and 14 h later. The cells maintained unchanged morphology and attachment throughout the drug treatment period. Cytoplasmic RNA was prepared as described in Materials and Methods, and 20 fig total RNA were separated through a 1 % agarose gel under denaturing conditions. The levels of IGF-I mRNA in control (•) and E2treated (•) cultures were quantitated by densitometric scanning of Northern blots. Each point represents the mean value from two filters of RNA prepared from two cultures, and the vertical bars indicate the SD. Similar results were obtained in a second experiment.

ulated IGF-I gene expression acting at least in part via transcriptional control. These results would be consistent with the presence of an ERE in the regulatory region of the IGF-I gene. The presence of such an element(s) has not yet been reported in the large and complex IGF-I gene, which may allow several levels of regulation (40). Differences in the hormone-dependent regulation of IGF-I mRNA in various tissues are consistent with the existence of IGF-I mRNAs with alternative 5' untranslated regions and with the alternative usage of exons at the 3' end (41). The time course for IGF-I mRNA regulation by E2 is consistent with a delay of 1 4 h observed between steroid uptake by cells and changes in the rate of transcription of structural genes (42). An increase in the abundance of mRNAs of early genes, such as c-fos in the rat uterus, is seen within 1 h after E2 administration in vitro (43). Binding studies of radiolabeled ligand have suggested that osteoblastic cells in vitro contain only a few hundred E2-binding sites (20, 21) which correspond to functional E2 receptors, as indicated by the E2-depend-

C

I

IGF tub »•

GH and E2 were reported to increase IGF-I mRNA in the rat uterus in vivo; however, in the presence of the protein synthesis inhibitor cycloheximide, only E2 injection caused an elevation of IGF-I mRNA (35). Similarly, the results of this study suggest that E2 stimulation of IGF-I mRNA in osteoblasts does not require de novo protein synthesis of a putative mediator of E2 action. As previously reported by Murphy and Luo (35), cycloheximide did not cause an appreciable superinduction of IGF-I mRNA, contrary to its effects on other mRNAs (36). In contrast, cycloheximide consistently reduced the steady state level of /3-tubulin mRNA, as previously reported by Pachter et al. (26). These investigators showed that tubulin autoregulates the stability of ribosome-bound tubulin mRNA, and that cycloheximide traps mRNAs onto stalled polyribosomes, thereby making tubulin mRNAs more susceptible to the effects of ribonucleases (26, 37). The estrogen receptor is a member of the steroid/ thyroid hormone receptor superfamily (28). Ligand-occupied estrogen receptors are, thus, expected to act as frans-activating factors which bind to specific regulatory DNA sequences and regulate gene transcription by modulating the activity of transcription factors and/ or altering chromatin structure (29). However, ovarian steroids are also known to regulate gene expression by posttranscriptional mechanisms, including the control of both mRNA translocation from the nucleus (38) and mRNA stability (39). Our data show no effects of E2 on IGF-I mRNA stability in osteoblasts. In contrast, the in vitro transcription assays indicated that E2 stim-

E

pGEM

W

I

IGF*-, tub

4h

22h

I

3h

o

I

2

CO

T

CD DC

0

T

L

Fig. 7. E2 Increases the Rate of Transcription of the IGF-I Gene Confluent cultures of calvarial osteoblasts were cultured in the absence (C) or presence (E) of 10 nM E2 for 4 and 22 h. Nuclei from 2-3 x 107 cells were isolated, and an in vitro nuclear run-on assay was carried out, as described in Materials and Methods. IGF-I, Linearized pGEM vector containing the cDNA insert for the rat preprolGF-l; pGEM, plasmid vector; tub, /3-tubulin. The figure represents one of two sets of filters from each transcription reaction run in duplicates. Two similar experiments were carried out from independent cell preparations, in which the stimulation by E2 was 2.8- and 2.3-fold, respectively. B, The autoradiogram in A was densitometrically scanned, and the intensity of the IGF-I signal was normalized for the /3-tubulin signal. The bars represent the mean and SD of four filters, each derived from two identically treated cultures.

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Vol 5 No. 8

MOL ENDO-1991 1086

Time (h)

24 -

48

72

-

-

96 -

+

m m Induction (fold)

5.2

9.1

11.8

12.1

Fig. 8. Time Course of E2-Dependent CAT Activation in Calvarial Osteoblasts Cells were precultured in 35-mm diameter dishes, as described in Materials and Methods. The cells were then transiently transfected with 0.7 ^g/dish pJ3MOR and 2.4 ^g/dish pERE-BLCAT and kept in phenol red-free medium containing 3% CS-FBS and 1 HM tamoxifen. Twenty-four hours before scraping the cells into CAT buffer at the indicated times, cultures were treated with 100 nM E2 or vehicle. The percent conversion reflects the counts of converted product divided by the total number of counts times 100. Similar findings were obtained in a second similar experiment.

ent activation of an ERE-containing reporter gene (Fig. 9C). Based on the CAT assay, the number of functional receptors resulting from the endogenous E2 receptor gene appeared to be rate limiting, since enhanced E2dependent CAT activation was observed in osteoblasts overexpressing E2 receptors from the transfected pJ3MOR plasmid. In addition, our results suggest that the abundance of E2 receptors expressed from the endogenous gene is not sufficient to stimulate IGF-I transcription maximally in these cells, supporting earlier findings that the rate of transcription of a glucocorticoidresponsive promoter correlates with the abundance of glucocorticoid receptors (30). Thus, the level of E2dependent gene transcription may relate to the number of available receptors, since within the same gene, several EREs may act in a cooperative manner (44). Overexpression of E2 receptors may, therefore, increase the probability that ligand-occupied E2 receptors dimerize and bind to enhancer elements in the IGF-I gene. In the classical estrogen target tissues, uterus and breast, estrogens regulate several factors that modulate cellular proliferation. In human breast cancer cells E2 regulates at least the production of IGF-I, plateletderived growth factor, and transforming growth factor/3 (45). Increased accumulation of radioimmunologically detectable IGF-I in response to E2 was observed in several breast cancer cell lines and in the rat osteosarcoma cell line UMR-106 (46). TGF/3 has been suggested as an E2-dependent negative regulator in MCF-7 cells (45) and as an E2-induced inhibitor of proliferation in rat osteosarcoma cell lines (47). In the uterus of ovariectomized rats in vivo, E2 elevates the mRNA level for

epidermal growth factor (48) and its respective receptor (49). In the same tissue, E2 induces the expression of IGF-I (24) and regulates the binding of IGF-I to its receptor (50). Finally, IGF-I is regulated in an estrous cycle-dependent manner (being maximal on estrus) in rat ovaries (51). Thus, the stimulation of IGF-I expression in osteoblastic cells is consistent with IGF-I mediation of E2 effects and with the mode of action of E2 in other E2 target organs, such as the uterus. The observations reported here indicate that cells from the uterus, mammary tissue, and bone may share at least one common locally acting estrogen mediator, namely IGF-I. It remains to be established whether regulation of IGF-I gene expression by E2 is restricted to these tissues and, if so, to elucidate the molecular nature of this tissue specificity.

MATERIALS AND METHODS 17j3-E2, 17a-E2, DRB, BSA, and cycloheximide were purchased from Sigma (St. Louis, MO); phenol red-free a-Modified Minimum Essential Medium, phenol red-free F-12 medium, and Kanamycin were obained from Gibco (Grand Island, NY); fetal bovine serum (FBS) was purchased from Hazelton, Lenexa, KS; DNase was obtained from Worthington (Freehold, NJ); and proteinase-K was purchased from Bethesda Research Laboratories (Gaithersburg, MD). Recombinant human IGF-I was a generous gift from Dr. Margaret Cascieri (52). The antiserum against IGF-I was supplied by Dr. Jurgen Zapf, and the IgG fraction was prepared as previously described (15). The cDNAs for rat prepro-IGF-l, rat a{\)^ procollagen, and rat i8-tubulin were generous gifts from Drs. Liam Murphy (27), David Rowe and Barbara Kream (53), and Stephen Farmer (54), respectively. FBS and BSA (10 g/liter) were charcoal

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Transcriptional Regulation of IGF-I by E2

stripped (CS-) as follows. Charcoal was activated by incubating 1 % Norit-A and 0.1% Dextran T-70 in 10 mM Tris, pH 7.6, overnight at 4 C. One gram of pelleted activated charcoal was added to 100 ml FBS or BSA, incubated for 30 min at 45 C, and pelleted by centrifugation before sterile filtration (0.22 MM) to obtain CS-FBS or CS-BSA.

1087

A

r

Plasmid

pGEM

pERE-BLCAT

;pERE-BLCAT + pJ3MOR

•

IGF-I •

Cell Culture Osteoblastic cells were obtained by sequential digestion of calvariae of 1-day-old rats, as previously described (10, 17). Cells were seeded at 25,000 cells/cm2 in 150-cm2 dishes (Nunc, Naperville, IL) in phenol red-free (31) a-Minimum Essential Medium-F-12 (1:1) medium containing 5% CS-FBS and 1 % Kanamycin. After 1 and 3 days, the media were changed, and the concentration of CS-FBS was reduced to 2.5% or replaced by 0.5 mg/ml CS-BSA, respectively. After an additional 2 days, 1 nwi E2 (as a 10-nriM stock solution in ethanol) or 20 nwi IGF-I (as a 100-MM stock solution in 0.1 M acetic acid, pH 4) was added to fresh medium containing 0.5 mg/ml CSBSA, and RNA was prepared at the indicated time. Northern Analysis Cytoplasmic RNA was extracted as described previously (17). Total RNA (10 or 20 Mg) was fractionated in formaldehyde (0.44 M) containing 1 % agarose gels and transferred to nylon filters (Hybond N, Amersham, Arlington Heights, IL) by electroblotting or to nitrocellulose (Schleicher and Schuell, Keene, NH) by capillary blotting. Filters were prehybridized overnight at 42 C in a buffer containing 50% formamide, 5 x SSC (1 x SSC = 0.15 M NaCI and 0.015 M sodium citrate, pH 7.0), 200 ^ g / ml sonicated salmon sperm DNA, and 5 x Denhardt's solution. Hybridization was carried out for 24 h in a buffer containing the above ingredients in addition to 106 cpm/ml 32P-labeled cDNA for IGF-I or a(\), procollagen. Filters were washed at 65 C in 0.1 x SSC-0.1% sodium dodecyl sulfate and were exposed to x-ray films (Kodak AR, Eastman Kodak, Rochester, NY) at - 7 0 C using intensifying screens. The amounts of RNA loaded and transferred were assessed by ethidium bromide staining of gels and rehybridization of the filters with a 32 P-labeled cDNA for /3-tubulin. Bands on the Northern autoradiograms were quantitated by densitometric scanning. Determination of mRNA Stability Five days after seeding, cell cultures (80-90% confluence) were incubated in fresh medium containing 0.5 mg/ml CSBSA in the presence or absence of 10 nM E2 for 12 h, followed by the addition of 25 Mg/ml DRB, and cytoplasmic RNA was prepared 0, 7, and 14 h after addition of the drug. In Vitro Transcription Assay The isolation of nuclei, purification of radiolabeled transcripts, and hybridization were carried out according to previously described protocols (55, 56). Briefly, confluent cells (7 days after seeding) were rinsed three times with cold PBS, scraped, and centrifuged at 1000 rpm. The nuclei were isolated by gentle homogenization of the cells on ice in a Dowex homogenizer in a Nonidet P-40 buffer (0.25%) containing 10 mM KCI, 10 mM Tris (pH 7.5), 1.5 mM MgCI2, and 0.3 M sucrose. The isolated nuclei were incubated at 25 C for 30 min in a transcription buffer containing 50 mM Tris (pH 7.4); 100 mM ammonium sulfate; 1.8 mM dithiothreitol; 1.8 mM MnCI2; 80 U RNasin (Amersham); 0.3 mM each of ATP, GTP, and CTP; and 100 nC\ [32P]UTP (800 Ci/mmol; Amersham). The reaction mixtures were treated with DNase-l and proteinase-K, extracted with phenol-chloroform, and ethanol precipitated. The pellets were dissolved in 6 M guanidinium hydrochloride, followed by the addition of 0.5 vol ethanol, and reprecipitated at - 2 0 C overnight. Two micrograms of linearized plasmids

Conversion °o

18.6

Fig. 9. E2-Dependent Transcriptional Activation of IGF-I in Osteoblasts Overexpressing E2 Receptors Primary cultures of calvarial osteoblasts were established and precultured in 150-mm diameter dishes, as described in Materials and Methods. A, Cultures were transiently transfected with 36 Mg/dish pERE-BLCAT and 11.7 Mg/dish pJ3MOR (right) or mock transfected with 36 Mg/dish pEREBLCAT and 11.7 Mg/dish pJ3 (left). After a recovery period of 48 h, the cultures were stimulated with 100 nM E2 or vehicle for 4 h. The cell nuclei were then prepared, and the in vitro transcription assay was carried out, as described in Materials and Methods. Equal amounts of radiolabeled RNA (two aliquots from each dish) were hybridized for 72 h at 42 C to linearized plasmid DNA (pGEM; 10 Mg), containing a rat preproIGF-I cDNA insert (10 Mg) or a rat /Mubulin cDNA insert (2 Mg), previously immobilized onto nitrocellulose. B, Quantitation of the hybridization signals obtained in A. The areas corresponding to the signals of IGF-I and /3-tubulin (/3-tub) were cut out, solubilized in sctintillation liquid (Atomlight, DuPont, Wilmington, DE), and counted in a ^-counter. Counts for /3-tubulin were used as a reference to estimate the effect of E2 on IGFI mRNA. In a repeat experiment, IFG-I mRNA levels from mock-transfected cells treated with E2 were 2.0-fold higher than control values, whereas in estrogen receptor-tranfected cells (PJ3MOR) they were 3.6-fold higher. C, Parallel cultures to those used for transcription analysis were established in 35-mm diameter dishes for monitoring the E2-dependent induction of CAT activity. Cells were transfected as described in Fig. 8, and CAT activity was determined 48 h later, as described in Materials and Methods.

containing cDNAs were spotted onto nitrocellulose (Dotblotter, Schleicher and Schuell) and were bound to the filters by baking at 80 C for 2 h. The filters were prehybridized overnight in 5 x SSC, 5 x Denhardt's solution, 50% formamide, 10 mM EDTA, 100 mM Tris (pH 7.4), 20 Mg/ml tRNA, 10 Mg/ml poliadenylic acid, 10 Mg/ml sonicated salmon sperm DNA, and

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Vol 5 No. 8

MOL ENDO-1991 1088

0.1% sodium dodecyl sulfate at 42 C. Hybridization was carried out for 72 h in fresh prehybridization buffer with isolated labeled transcripts (4-6 x 106 cpm/ml). Filters were washed, as described above, and exposed to x-ray films at - 7 0 C using intensifying screens, and quantification of signals was performed by densitometric analysis. Transient Transfection Assays

6.

7. 2

Primary calvarial osteoblasts were seeded at 25,000 cells/cm and kept for the first 72 h in phenol red-free medium in the presence of 2.5% CS-FBS. Twenty-four hours before transfection, the cultures were switched to fresh medium containing 5% CS-FBS and then washed three times with serum-free medium. Subsequently, the cells were transiently transfected by the calcium phosphate coprecipitation method and glycerol (15%) shocking with the indicated amounts of pJ3MOR, an expression plasmid containing the entire mouse E2-receptor open reading frame (57) cloned into the eukaryotic expression vector pJ3 and pERE-BLCAT, and a reporter plasmid containing the ERE derived from the vitellogenin A2 promoter (57). The pJ3 plasmid was used for mock transfection of control cultures. After transfection, the cells were allowed to recover for 48 h in phenol red-free medium containing 3% CS-FBS before the cultures were stimulated with 100 nM E2 or vehicle in fresh medium supplemented with 3% CS-FBS. The cells were harvested in CAT buffer (100 mw Tris, pH 7.4) at the indicated time, and aliquots of the cell extracts were assayed for CAT activity after incubation for 2 h at 37 C according to previously described procedures (58). The products of the CAT reaction were separated by TLC and visualized by autoradiography. The radioactivity associated with the spots was determined by liquid sctintillation counting, and the counts were normalized for the protein content of each sample.

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Acknowledgments We thank Ms. Dianne McDonald for the preparation of this manuscript.

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Received August 17, 1990. Revision received May 24, 1991. Accepted May 24,1991. Address requests for reprints to: Dr. Gideon A. Rodan, Department of Bone Biology and Osteoporosis Research, Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486. *Present address: Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, Royal Melbourne Hospital, Victoria 3050, Australia.

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