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Endocrinology Vol. 138, No. 7 2919-2927
Copyright © 1997 by The Endocrine Society

ARTICLES

Estrogen Regulation of Human Osteoblastic Cell Proliferation and Differentiation1

John A. Robinson, Steven A. Harris, B. Lawrence Riggs and Thomas C. Spelsberg

Departments of Biochemistry and Molecular Biology (J.A.R., T.C.S.) and Orthopedic Research (S.A.H.), and Division of Endocrinology (B.L.R.), Mayo Foundation, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: John A. Robinson, Ph.D., Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: robinson.john{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen (E2) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E2 receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E2. Previous studies on the effects of E2 on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E2 on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E2 on osteoblast proliferation and differentiation. The basal levels and E2 effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10–14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [3H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10-9 M E2 for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from ~200–500%. In contrast, E2 decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E2 treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E2 on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E2 responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E2 (ICI 182,780) abrogated these effects. These results support the hypothesis that E2 does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AFTER THE MENOPAUSE, reduction in circulating levels of estrogen (E2) results in bone loss (1, 2, 3, 4, 5). This loss of bone has been attributed to uncoupling between the functions of osteoblasts and osteoclasts and is characterized by an increase in bone formation and a further increase in bone resorption (1, 2). It has been well established by both in vivo and in vitro studies that E2 decreases bone resorption (1, 2, 6, 7, 8, 9, 10). However, the effects of E2 on bone formation are less clear. Whereas most in vivo studies in rats and humans have shown that E2 inhibits (1, 11, 12, 13, 14) bone formation, other studies have reported a stimulation (15, 16, 17) of bone formation by E2. The discovery of E2 receptors (ER) in osteoblasts (18, 19) has implicated the osteoblast as a direct target for E2. Many in vitro studies, investigating the effect of E2 on osteoblast proliferation and differentiation, have produced inconsistent results. E2 has been reported to stimulate, inhibit, and have no effect on cell proliferation (reviewed in Ref. 20). Osteoblast differentiation markers have also varied in their responses to E2. Alkaline phosphatase (AP), type I collagen (Col I), and osteocalcin (OC) gene expressions have been shown to be stimulated by, inhibited by, or unresponsive to E2 (reviewed in Ref. 20). Other studies have reported that E2 has various effects on the production of cytokines and growth factors as well as their binding proteins by osteoblasts. These include interleukin-6 (IL-6) (21, 22), insulin-like growth factor I (IGF-I) (23, 24, 25, 26), IGF-binding proteins (IGFBPs) (27, 28), and transforming growth factor-ß (26, 29).

The conflicting responses by osteoblasts to E2 may in part be attributed to species differences, cellular heterogeneity, incomplete differentiation, and/or low or variable ER content among the various cell lines and primary cultures (30). To overcome these uncertainties we have developed a human immortalized fetal cell line (hFOB/ER9) that expresses the mature osteoblast phenotype and contains high levels of ER (31, 32). Here we report further characterization of extracellular matrix (ECM) expression during hFOB/ER9 cell differentiation as well as the E2 regulation of that process and cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DMEM-Ham’s F-12 (DMEM/F12; 1:1, wt/wt) mix, FBS, menadione (vitamin K3), ascorbic acid (vitamin C), trypsin-EDTA reagent, 17ß-estradiol (E2), and AP enzyme assay kit were purchased from Sigma Chemical Co. (St. Louis, MO). Neomycin G418 (geneticin) and T3 and T7 RNA polymerase were purchased from Life Technologies (Gaithersburg, MD), and hygromycin B was purchased from Boehringer Mannheim (Indianapolis, IN). The Bradford protein reagent was purchased from Bio-Rad (Hercules, CA), and 1,25-dihydroxyvitamin D3 was obtained from Biomol (Plymouth Meeting, PA). The phenol-guanidine isothiocyanate (Tri-reagent) solution for RNA isolation was purchased from Molecular Research Center (Cincinnati, OH). Radiolabeled reagents, including [3H]thymidine and [{alpha}-32P]deoxy-CTP were purchased from DuPont-New England Nuclear (Boston, MA). The [{alpha}-32P]UTP was obtained from Amersham Life Science (Arlington Heights, IL). The human OC RIA kits were purchased from Nichols Institute Diagnostics (San Juan Capistrano, CA), and the human type I procollagen RIA kits were obtained from Incstar (Stillwater, MN). ICI 182,780 was a gift from Zeneca Pharmaceuticals (Cheshire, UK). The human AP complementary DNA (cDNA) probe was a gift from Dr. Gideon Rodan (Merck, Sharp, and Dohme, West Point, PA), and the osteonectin cDNA probe was a gift from Dr. George Long (University of Vermont, Burlington, VT). The ribonuclease (RNase) protection assay kit and 18S ribosomal RNA (rRNA) probe were purchased from Ambion (Austin, TX). Deoxyribonuclease I (DNase I) was purchased from Promega (Madison, WI).

Cell culture
The hFOB/ER9 cell line contains the T antigen expression vector with the neomycin resistance gene (32) and the wild-type ER expression vector with the hygromycin B resistance gene (31). To maintain selection, cells were cultured in a 1:1 mixture of phenol red-free DMEM/F12 containing 10% (vol/vol) charcoal-stripped FBS (FBS-cs) and supplemented with either geneticin (300 µg/ml) or hygromycin (100 µg/ml). The medium was changed every 2 days, with these two antibiotics used alternately. The cells were maintained at 34 C, the permissive temperature for the T antigen protein in this cell line (32). The hFOB/ER9 cells express 3931 binding sites/nucleus, as demonstrated by nuclear binding assay (31).

Cell proliferation
Cell proliferation was assessed using a [3H]thymidine incorporation assay. The hFOB/ER9 cells were plated into 24-well culture dishes at 2 x 104 cells/well in phenol red-free DMEM/F12 containing 10% (vol/vol) FBS-cs and incubated for 48 h. The cells were then washed twice with PBS and pretreated with the type II anti-E2 ICI 182,780 (10-7 M) in DMEM/F12 containing 1% (vol/vol) FBS-cs for 2 days to minimize the effects of residual E2 remaining after charcoal stripping. The cells were washed twice with PBS and treated with either hormone or vehicle for 5 days in DMEM/F12 containing 1% (vol/vol) FBS-cs at 34 C. Then, [3H]thymidine (0.5 µCi) was added to each well for 24 h before completion of the assay. The cells were rinsed four times with 10% (wt/vol) trichloroacetic acid, solubilized in 0.2% (wt/vol) NaOH, and mixed with scintillation cocktail for 3H quantitation. The incorporation of [3H]thymidine into the trichloroacetic acid-precipitable material was used as an indicator of cellular DNA synthesis (33).

AP, Col I, and OC assays
The hFOB/ER9 cells were plated at either 25,000 or 86,000 cells/well (as indicated) in 12-well dishes with phenol red-free DMEM/F12 containing 10% (vol/vol) FBS-cs and cultured at 34 C. Before harvesting the cells at the indicated times, the cells were pretreated for 2 days with 10-8 ICI 182,780 in DMEM/F12 containing 10% FBS-cs (vol/vol) or with DMEM/F12 containing 0.25% (wt/vol) BSA (as indicated). The cells were subsequently treated with steroid or vehicle for 2 days in DMEM/F12 containing 0.25% (wt/vol) BSA. The medium was collected, centrifuged to remove cell debris, and used for type I procollagen assays. The type I procollagen assay, which measures the propeptide portion of the molecule, reflects the synthesis of the mature form of the protein. Type I procollagen assays were carried out as described by the manufacturer. The AP activity was measured after rinsing the cells twice with PBS, then adding 0.3 ml alkaline lysis buffer (0.75 M 2-amino-2-methyl-1-propanol, pH 10.3) containing p-nitrophenol phosphate substrate (2 mg/ml) and incubating for 30 min at 37 C. To stop the reaction, 0.3 ml 50 mM NaOH was added to each well. The samples and standards were diluted in 20 mM NaOH, and the absorbance was measured at 410 nM. To normalize matrix protein expression to total cellular protein, a fraction of the reaction solution was used in a Bradford protein assay.

To determine OC levels, hFOB/ER9 cells were cultured and steroid treated as described above with some modifications. These modifications include adding 10-7 M vitamin D3, 100 µg/ml ascorbic acid, and 10-8 M vitamin K when steroid was added. After 2 days of steroid treatment, the medium was collected, centrifuged to remove cell debris, and dialyzed extensively in 50 mM ammonium bicarbonate. The samples were dried using a Savant Speed-Vac concentrator (Savant Instruments, Farmingdale, NY). The concentrated samples were reconstituted in a minimum volume of PBS and assayed for OC as described by the manufacturer, except that samples and standards were incubated for 18 h at 4 C.

Mineralized matrix staining
After the indicated time intervals, hFOB/ER9 cell cultures were fixed with 1% (wt/vol) paraformaldehyde in 20 mM Tris, pH 7.4, and 0.15 M NaCl (TBS) for 10 min. The cells were rinsed with TBS and stained by the von Kossa procedure as modified by Schenk et al. (34). Briefly, the cells were treated with 5% (wt/vol) silver nitrate in the dark for 15 min. The cells were washed with distilled water, exposed to UV light for 5 min, and treated with a solution containing sodium carbonate and formaldehyde for 2 min. Finally, the cells were treated with Farmer’s reducer for 30 sec, allowed to dry, and then photographed using a Nikon Diaphot 300 inverted microscope (Nikon Corp., Melville, NY).

RNA isolation and Northern blot analysis
The hFOB/ER9 cells were plated at 1 x 106 cells/100-mm diameter culture dish and were grown for 8 days (2 days postconfluence) in DMEM/F12 containing 10% (vol/vol) FBS-cs. The medium was changed to DMEM/F12 containing 0.25% (wt/vol) BSA for 2 days, and then the cells were treated for various time intervals with steroid or vehicle in the same fresh medium. Total cellular RNA was isolated as previously described (35) using a phenol-guanidine isothiocyanate extraction method. Briefly, after the first extraction, there was an additional extraction with an equal volume of chloroform, followed by lithium chloride precipitation and ethanol precipitation. Ten micrograms of total RNA was denatured in glyoxal-dimethylsulfoxide buffer and separated with a 1% (wt/vol) agarose-glyoxal gel (36). The RNA was then transferred by capillary diffusion in 20 x SSC (3 M NaCl and 0.3 M sodium citrate, pH 7.0) and subsequently vacuum baked at 80 C for 2 h. Hybridization was performed in 15 ml hybridization buffer [50% (vol/vol) formamide, 5 x Denhardt’s, 3 x SSC, 0.1% (wt/vol) SDS, 0.01 mg/ml polyadenylic acid, and 0.05 mg/ml salmon sperm DNA] for a minimum of 4 h at 42 C in a hybridization incubator. Labeling of cDNAs was performed with a random labeling kit (DuPont-New England Nuclear, Boston, MA) as described by the manufacturer using [{alpha}-32P]deoxy-CTP (3000 Ci/mmol). Labeled cDNAs were purified using a nick gel filtration column (Pharmacia, Piscataway, NJ) as described by the manufacturer. Each hybridization contained approximately 5 x 107 cpm (100 ng) labeled cDNA. The Northern blots were washed with 0.5 x SSC-0.1% (wt/vol) SDS at 42 C, exposed to Kodak X-Omat ARS film (Eastman Kodak, Rochester, NY) with intensifying screens at -70 C, and then developed in a Kodak X-Omat M2O film processor. Loading and integrity of RNA were assessed by hybridization with 18S rRNA cDNA.

RNase protection assay
The hFOB/ER9 cells were plated at 1 x 106 cells/100-mm diameter culture dish and were grown for 8 days (2 days postconfluence) in DMEM/F12 containing 10% (vol/vol) FBS-cs. The cells were pretreated for 2 days with 10-8 M ICI 182,780 in DMEM/F12 containing 10% (vol/vol) FBS-cs and then treated with steroid, 10-7 M vitamin D3, 100 µg/ml ascorbic acid, and 10-8 M vitamin K for the times indicated. Sample RNA was isolated as previously described. The human OC probe comprised nucleotides 6–336 of the coding region and 73 bp of vector sequence. OC riboprobe labeling was initiated with the addition of 15 U T3 RNA polymerase to 100 ng linearized OC DNA template, 100 µCi [{alpha}-32P]UTP (800 Ci/mmol), and 26 U RNAsin, all in buffer containing 50 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 25 mM NaCl, 2 mM spermidine, 5 mM dithiothreitol, and 1 mM NTPs (ATP, CTP, and GTP). The reaction mixture was incubated for 20 min at 37 C and subsequently stopped with 0.04 ml TE buffer, 10 µg transfer RNA, 100 U RNAsin, and 1 mM MgCl2. All DNA templates were digested with 10 U RNase-free DNase I for 20 min at 37 C. The unincorporated [{alpha}-32P]UTP was removed from the radiolabeled OC antisense RNA probe using a nick gel filtration column. The 18S RNA control probe was made using the same procedure, except with T7 RNA polymerase.

Probe hybridization and RNase digestion of sample RNAs were performed as described by the manufacturer. Briefly, 5 x 104 cpm radiolabeled OC RNA probe were added to 20 µg sample RNA in 0.02 ml hybridization buffer. The reaction mixture was heated to 95 C for 3 min and then incubated for 18 h at 43 C. All samples except the full-length probe sample were digested with 0.17 U RNase A/6.7 U RNase T1 mixture in RNase digestion buffer for 30 min at 37 C. The reactions were stopped, and RNA was precipitated with RNase inactivation/precipitation solution. The hybridization and RNase digestion procedures were also performed for the 18S RNA probe, except 10 ng sample RNA were used in those reactions.

The OC messenger RNA (mRNA) and 18S rRNA samples were denatured in gel loading buffer and resolved by PAGE using 6% or 8% (wt/vol) acrylamide-50% (wt/vol) urea gels, respectively. After electrophoresis, the gels were soaked in 10% (vol/vol) acetic acid for 20 min to remove the urea. The gels were subsequently dried using a Bio-Rad gel dryer (Bio-Rad, Hercules, CA) and exposed to both Kodak X-Omat ARS film and storage phosphor screens. Densitometry was performed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Statistical analysis
The results represent the mean ± SD of three separate experiments (n = 3). The mean and SD corresponding to the vehicle control values were derived from the mean of three replicates of three experiments. The effect of treatment was compared to control values by unpaired Student’s t test. Statistics for multiple comparisons were performed using one-way ANOVA followed by Fisher’s least significant difference (PLSD) analysis. Statistical analysis of AP data were performed on the log transformation values to stabilize the variance. Tests were performed using StatView II software (Abacus Concepts, Cupertino, CA). Linear regression was performed using SAS software (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fetal osteoblast cell differentiation
The mineralization of ECM and the formation of bone nodules are osteoblastic phenotypic markers and represent the final stages of osteoblast differentiation. Therefore, bone nodule formation was assessed in hFOB/ER9 cell cultures to establish a differentiation end point for these cells. As shown in Fig. 1Go, von Kossa staining performed at 10, 14, and 18 days of culture displayed nodule formation only after 14 days of culture and became more prominent by day 18. Ascorbic acid and ß-glycerol phosphate were not required for mineralization to occur. To further characterize hFOB/ER9 cell differentiation, bone matrix protein expression, including AP, OC, and Col I, was measured during the stages of rapid cell proliferation (day 6) through mineralization (day 18). To maintain similar cell culture conditions for subsequent differentiation experiments requiring E2, cells used in these experiments were pretreated with ICI 182,780. As shown in Fig. 2AGo, AP activity decreased by day 10 and subsequently increased to a maximum level by day 18, when the cells were actively mineralizing. In contrast to AP activity, the secreted levels of OC (Fig. 2BGo) increased from days 6–10 and then steadily decreased by day 18. Col I expression (Fig. 2CGo) was nearly maximal and showed little change between days 6–14. Furthermore, there was no clear pattern of expression on day 18, as both increases and decreases in Col I levels were observed.



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  		Figure 1. Histochemical changes during hFOB/ER9 cell differentiation. Phase contrast micrographs (x10) were taken of von Kossa-stained samples after 10, 14, and 18 days of culture. Cells were plated at 25,000 cells/well and grown in DMEM/F12 containing 10% FBS-cs. Two days before staining, the cells were placed in DMEM/F12 containing 0.25% BSA. Arrows indicate regions of mineralized matrix. Bar = 300 µm.

 


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  		Figure 2. Expression of ECM proteins during hFOB/ER9 cell differentiation. Levels of expression of AP activity (A), OC (B), and Col I (C) were presented as a percentage of maximum expression after normalizing values to total cellular protein. The mean values of maximum expression were 1.08 µmol AP/mg total cellular protein, 4.07 ng OC/mg total cellular protein, and 4.05 µg Col I/mg total cellular protein. Cells were plated at 25,000 cells/well and grown at 34 C in DMEM/F12 containing 10% FBS-cs. Before cell harvest at the indicated times, the cells were pretreated with ICI 182,780 (10-8 M) for 2 days in DMEM/F12 containing 10% FBS-cs and then placed for 2 days in DMEM/F12 containing 0.25% BSA. To determine OC levels, the cultures were treated for the final 2 days with 10-7 M vitamin D3, 100 µg/ml ascorbic acid, and 10-8 M vitamin K. Each symbol type in each panel represents the mean value of three replicates of an individual experiment of three performed.

 
Effects of E2 on cell proliferation
Having characterized the gene expression of various osteoblastic markers throughout each stage of hFOB/ER9 cell differentiation, experiments were performed to investigate whether E2 could modulate the proliferation of hFOB/ER9 cells during the initial stages of cell growth (days 0–6). During the stage of rapid cell proliferation when the cells were not contact inhibited, 5 days of E2 treatment caused a dose-dependent decrease (r = -0.82; P < 0.001) in hFOB/ER9 cell proliferation by as much as 72 ± 3.9% with 10-8 M E2 (Fig. 3Go). A significant decrease in proliferation was observed with E2 treatment as low as 10-11 M. Cell proliferation was not affected when cells were treated for less than 5 days with E2 (data not shown). In addition, E2 had no effect on hFOB/ER9 cell proliferation if the cultures were not pretreated for 2 days with ICI 182,780 (data not shown). To demonstrate whether the effects of E2 on proliferation were mediated specifically through the ER, hFOB/ER9 cells were cotreated with E2 (10-8 M) and a 10-fold molar excess of the anti-E2 ICI 182,780. Cotreatment of these cultures with ICI 182,780 completely abrogated the effect of E2 on proliferation, whereas ICI 182,780 (10-7 M) alone increased cell proliferation slightly compared to the effect of the vehicle control.



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  		Figure 3. Regulation of hFOB/ER9 cell proliferation by E2. Cells were plated at 2 x 104 cells/well, cultured for 2 days at 34 C in DMEM/F12 containing 10% FBS-cs, and then pretreated for 2 days with 10-7 M ICI 182,780 in DMEM/F12 containing 1% FBS-cs. The cells were subsequently treated with ethanol (control; {square}), various concentrations of E2 (E; {blacksquare}), 10-8 M E2 plus 10-7 M ICI 182,780 (E+I; ), or 10-7 M ICI 182,780 alone (I; ) for 5 days in DMEM/F12 containing 1% FBS-cs. [3H]Thymidine was added 24 h before cell harvest. Each treatment was performed in triplicate, and these data represent the mean ± SD of three experiments, expressed as a percentage of the vehicle control value. The mean value for 100% control was 6,247 cpm. *, P < 0.05; ***, P < 0.001 (compared to vehicle control, by one-way ANOVA and Fisher’s PLSD analysis).

 
Effects of E2 on expression of matrix proteins
To identify whether bone matrix protein expression in hFOB/ER9 cells was regulated by E2, postconfluent cultures were treated with steroid and assayed for cell-associated AP activity and secreted levels of OC and Col I. As shown in Fig. 4Go, E2 concentrations as low as 10-10 M began to show significant increases in AP activity (P < 0.001) and elicited a maximal response (416 ± 173% compared to vehicle control) with 10-9–10-8 M E2. However, the latter was not statistically different from the response observed with 10-10 M E2. Cultures cotreated with a 100-fold molar excess of ICI 182,780 (10-7 M) completely abrogated the E2 increase in AP activity, whereas ICI 182,780 alone had no effect on AP activity.



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  		Figure 4. Regulation of AP activity by E2 in hFOB/ER9 cells. Cells were plated at 86,000 cells/well and cultured at 34 C in DMEM/F12 containing 10% FBS-cs for 7 days (2 days postconfluence) and then pretreated for 2 days in DMEM/F12 containing 0.25% BSA. The cells were subsequently treated with ethanol (control; {square}), various concentrations of E2 (E; {blacksquare}), 10-9 M E2 plus 10-7 M ICI 182,780 (E+I; ), or 10-7 M ICI 182,780 alone (I; ) for 2 days in DMEM/F12 containing 0.25% BSA. Each treatment was performed in triplicate, and these data represent the mean ± SD of three experiments, expressed as a percentage of the vehicle control value. The mean value for 100% control was 0.42 µmol AP/mg total cellular protein. ***, P < 0.001 compared to vehicle control, by one-way ANOVA and Fisher’s PLSD analysis.

 
As the maximal effect of E2 on AP activity was observed with 10-9–10-8 M E2, all subsequent experiments were performed with these E2 concentrations. As shown in Fig. 5Go, 10-9 M E2 decreased OC secretion by hFOB/ER9 cells by 45 ± 10.7% compared to the vehicle control value. Cotreating cells with E2 (10-9 M) and a 100-fold molar excess of ICI 182,780 nearly blocked all of the E2-dependent decrease in secreted OC. ICI 182,780 (10-7 M) alone did not effect OC levels compared to those in the vehicle control. Col I was also measured in the medium of hFOB/ER9 cultures treated with E2 (data not shown). E2 (10-8 M) had a minimal effect on Col I secretion (116 ± 6.6%) compared to that in vehicle-treated control cultures. The small increase in Col I secretion, however, was statistically significant (P < 0.01). The mean value of Col I at 100% control was 2.53 µg/mg total cellular protein.



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  		Figure 5. Regulation of OC by E2 in hFOB/ER9 cells. Cells were plated at 25,000 cells/well, grown at 34 C in DMEM/F12 containing 10% FBS-cs for 10 days (2 days postconfluence), and then pretreated with ICI 182,780 (10-8 M) for 2 days in DMEM/F12 containing 10% FBS-cs. Cells were then treated for 2 days with ethanol (control; {square}), 10-9 M E2 (E; {blacksquare}), 10-9 M E2 plus 10-7 M ICI 182,780 (E+I; ), or 10-7 M ICI 182,780 alone (I; ) in DMEM/F12 containing 10-7 M vitamin D3, 100 µg/ml ascorbic acid, 10-8 M vitamin K, and 0.25% BSA. Each treatment was performed in triplicate, and these data represent the mean ± SD of three experiments, expressed as a percentage of the vehicle control value. The mean value for 100% control was 2.24 ng OC/mg total cellular protein. *, P < 0.05; ***, P < 0.001 (compared to vehicle control, by one-way ANOVA and Fisher’s PLSD analysis).

 
Effects of E2 on matrix protein mRNA expression
To determine whether the effect of E2 on AP activity and OC levels reflects parallel changes in gene expression, their respective steady state mRNAs were measured after various periods of steroid treatment. As demonstrated by Northern analysis in Fig. 6Go, AP mRNA expression was increased by E2 as early as 15 h after treatment compared to the vehicle control value. The increase in mRNA was observed throughout each period studied. Furthermore, cotreatment of culture with E2 (10-9 M) and a 100-fold molar excess of ICI 182,780 blocked the increase in AP mRNA expression by E2. In contrast, steady state mRNA levels for osteonectin were not regulated by E2 at the time intervals studied. As the levels of OC mRNA levels are significantly lower than those of other ECM proteins expressed in these cells, RNase protection assays were performed to measure OC mRNA levels. As demonstrated in Fig. 7AGo, E2 decreased OC mRNA levels throughout each period examined. The levels of 18S rRNA in those samples showed equivalent loading efficiency. Densitometric analyses of these data, when normalized to their respective 18S rRNA levels, demonstrated a 43% decrease in OC mRNA levels after 12 h of E2 treatment followed by a maximal decrease of 77% by 24 h (Fig. 7BGo). After 48 h, the E2-mediated decrease in OC mRNA returned to the levels observed after 12 h of treatment.



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  		Figure 6. E2 effects on AP (Alk. Phos.) and osteonectin mRNA levels in hFOB/ER9 cells. Cells plated at 1 x 106 cells/100-mm diameter culture dish were grown at 34 C in DMEM/F12 containing 10% FBS-cs for 8 days (2 days postconfluence) and then pretreated with ICI 182,780 (10-8 M) for 2 days in DMEM/F12 containing 10% FBS-cs. Total RNA (10 µg) from cultures treated for the indicated times with ethanol (control; V), 10-9 M E2 (E), 10-9 M E2 plus 10-7 M ICI 182,780 (E+I), or 10-7 M ICI 182,780 alone (I) in DMEM/F12 containing 0.25% BSA was analyzed by Northern blotting using AP, osteonectin, and 18S rRNA cDNA probes. This experiment was performed twice, and these data are representative of the results.

 


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  		Figure 7. Effects of E2 on OC mRNA levels in hFOB/ER9 cells. Cells were plated at 1 x 106 cells/100-mm diameter culture dish, grown at 34 C in DMEM/F12 containing 10% FBS-cs for 8 days (2 days postconfluence), and then pretreated with ICI 182,780 (10-8 M) for 2 days in DMEM/F12 containing 10% FBS-cs. Cells were treated for the indicated times with ethanol (control; V) or 10-9 M E2 (E) in DMEM/F12 containing 10-7 M vitamin D3, 100 µg/ml ascorbic acid, 10-8 M vitamin K, and 0.25% BSA. A, OC mRNA and 18S rRNA levels were analyzed by RNase protection assay using 20 µg and 10 ng total RNA, respectively. The OC mRNA and 18S rRNA images were taken from X-Omat film and PhosphorImager scanning, respectively. This experiment was performed twice, and these data are representative of the results. B, Scanning densitometry was performed to quantitate changes in mRNA levels. Results are expressed as a percentage of the vehicle control value and were obtained by calculating the ratio of E2 treatment to control treatment after standardization to 18S band intensity. This experiment was performed twice; each symbol type represents a separate experiment.

 
Effects of E2 on overall cell differentiation
To determine the effects of E2 on each stage of hFOB/ER9 cell differentiation, cultures at the various stages were treated with vehicle or E2 for 2 days before the day of harvest (as indicated) and then assessed for matrix protein expression. The values were calculated and normalized to their respective untreated cell values at each stage. At each time interval examined, except on day 14, E2 significantly increased AP activity (Fig. 8AGo). The magnitude of AP induction by E2 varied from ~200–500% of the control value depending on the stage of differentiation. Day 10, during the early stage of matrix maturation, showed the highest induction of AP activity by E2. In contrast to AP activity, E2 decreased OC levels throughout each stage of differentiation (Fig. 8BGo). Furthermore, the magnitudes of these responses were very similar (ranging from 46–68% of control) over the whole time course. There was little or no effect of E2 on Col I levels, as the only statistical increase was 118 ± 3% (compared to control), which was observed in day 18 cultures.



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  		Figure 8. Regulation of ECM protein expression by E2 during hFOB/ER9 cell differentiation. A, Changes in expression of AP activity. B, Col I ({square}) and OC ({diamondsuit}). Results were presented as a percentage of the vehicle control value (for the corresponding time point) after normalizing values to total cellular protein. Cells were plated at 25,000 cells/well and grown at 34 C in DMEM/F12 containing 10% FBS-cs. Before cell harvest at the indicated times, the cells were pretreated with ICI 182,780 (10-8 M) for 2 days in DMEM/F12 containing 10% FBS-cs and subsequently treated with either ethanol (control) or 10-9 M E2 for 2 days in DMEM/F12 containing 0.25% BSA. To assess changes in OC levels, the cell were treated with steroid in the presence of 10-7 M vitamin D3, 100 µg/ml ascorbic acid, and 10-8 M vitamin K for the final 2 days of culture. Each treatment was performed in triplicate, and these data represent the mean ± SD of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to vehicle control, by unpaired Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goal of this study was 2-fold: 1) to determine the basal levels of ECM protein expression during various stages of hFOB/ER9 cell differentiation from rapidly proliferating, relatively undifferentiated cells to cells that were mineralizing the matrix; and 2) to investigate the effects of E2 on cell proliferation and ECM protein expression during these stages of differentiation. The chronology for hFOB/ER9 cell differentiation is divided into three stages of osteoblast differentiation. These stages, which have been previously described for fetal rat calvaria cultures (37), include proliferative/relatively undifferentiated (day 6), matrix maturation (days 10 to 14), and mineralization (day 18).

The temporal pattern of matrix protein expressed by hFOB/ER9 cells differed from the pattern of gene expression observed in fetal rat calvaria cells (37). In fetal rat calvaria cultures, AP and Col I were expressed maximally during early stages of differentiation and then declined during mineralization. In contrast, AP activity increased during the mineralization of hFOB/ER9 cell cultures, whereas Col I was expressed in relatively high levels throughout all stages of hFOB/ER9 cell differentiation. Differences were also observed for OC expression. OC expression was low during early differentiation stages in fetal rat calvaria cultures and subsequently increased to a maximal level during mineralization. In contrast, OC was expressed maximally early and then steadily declined during mineralization of hFOB/ER9 cultures.

The differences in matrix protein gene expression observed between the hFOB/ER9 and fetal rat calvaria culture systems are probably due to a variety of factors. Differences in protein expression may be attributed to differences in the time required for mineralization to occur. Nodule formation in hFOB/ER9 cell cultures was prominent 10 days after cell confluence. However, mineralization in fetal rat calvaria cultures was not observed until ~28 days after cell confluence. These differences may be reflective of cell proliferative capacity or cellular homogeneity. However, it is not surprising to find differences in the pattern of ECM expression between the two cell models. These differences are probably due to differences in species, the culture conditions used, and the homogeneity of the cell population. It is possible that the simian virus 40 large T antigen influences ECM gene expression and, therefore, differentiation (38). However, this is not likely, as hFOB/ER9 cell proliferation decreases significantly with cell contact, and matrix mineralization occurs without inactivating the large T antigen at the restrictive temperature (31). Furthermore, the growth factors that have been examined in hFOB/ER9 cells have a pattern of expression similar to that of normal human osteoblasts in culture (22, 28, 39).

Having established the pattern of ECM protein expression, we took advantage of the high levels of ER in hFOB/ER9 cells to examine the effects of E2 on cell proliferation and ECM protein expression during cell differentiation. We have shown that E2 decreased hFOB/ER9 cell proliferation in a dose-dependent manner. However, E2 had no effect on cell proliferation if the cultures were not pretreated with the type II anti-E2 ICI 182,780 before E2 treatment. This observation may be due to the low levels of E2 (10-11 M) sufficient to regulate cell proliferation. We have previously reported (31) that residual amounts of E2 remain in FBS after charcoal stripping. The specificity of the E2 effects on cell proliferation, through the ER, was tested using ICI 182,780. Cotreatment of cultures with E2 and ICI 182,780 blocked the E2 response on proliferation. In fact, ICI 182,780 alone significantly increased hFOB/ER9 cell proliferation, although moderately compared to that in control cultures. This response may be attributed to antagonism of residual levels of E2 by ICI 182,780. In addition, prolonged exposure of the cells to ICI 182,780 occupied all of the available ER, thereby mimicking a cell with no ER. This possibility is also likely, as we observed a faster cell doubling rate for the hFOB parental cell line, which expresses lower levels of ER than the hFOB/ER9 subclone (31). A decrease in cell growth has also been shown in SAOS-2 and HTB 96 human osteosarcoma cells transfected with the wild-type ER compared to their parental cell counterpart (40, 41).

The effect of E2 on cell proliferation in these cell models may be due to many factors that could act alone or in concert with one another. For example, the E2-mediated decrease in proliferation that was observed in the hFOB/ER9 cells may have been due to an increase in responsiveness of the cells to E2 and/or cross-talk between the ER and growth factor regulatory pathways (30, 42). This is possible, as recent work in our laboratory has shown that E2 stimulates IGFBP-4 production in hFOB/ER9 cells and the addition of exogenous IGFBP-4 to cultures inhibited cell proliferation in these cells (28). Transforming growth factor-ß expression is also regulated by E2 in osteoblasts (29) and may be another factor involved in mediating the effects of E2 on hFOB/ER9 cell proliferation. However, at this time, this possibility has not been investigated using hFOB/ER9 cells. The rate of proliferation may also be influenced by the presence of E2 and high levels of ER, which could sequester important transcription factors required for expression of genes controlling proliferation or other housekeeping genes (43). This explanation would probably not account for all the effects of E2 on cell proliferation. In vivo studies involving E2-treated ovariectomized rats have demonstrated fewer [3H]thymidine-labeled preosteoblasts than those observed in control ovariectomized rats (11, 44). The researchers suggested from these studies that the decrease in osteoblast number was probably due in part to a decrease in preosteoblast proliferation. This observation may be relevant to the results we observed with E2 regulation of cell proliferation. The hFOB/ER9 cells were derived from fetal tissue and, therefore, may have a higher proliferative capacity than mature osteoblasts.

In addition to proliferation, E2 also regulated ECM protein gene expression. Unlike other studies, we investigated E2 regulation of human osteoblast ECM protein expression throughout differentiation. The effects of E2 observed were gene specific, as the steroid altered AP and OC expression, but did not significantly alter Col I synthesis and osteonectin gene expression. Furthermore, AP activity was induced by E2, whereas OC expression was suppressed throughout differentiation. Although AP activity was increased at each stage of differentiation by E2, the day 14 cultures did not demonstrate significance compared to control cultures. Contrary to the proliferation studies, there was no difference in E2 response on matrix gene expression if the hFOB/ER9 cell cultures were pretreated with BSA-containing medium or with FBS-containing medium and ICI 182,780. This observation may be due to the concentration of E2 (10-10–10-9 M) required to affect matrix gene expression.

The regulation of AP and OC by E2 was shown to be at the mRNA level and, furthermore, mediated through the ER. The ICI 182,780 compound completely abrogated the induction of AP activity by E2, but only partially blocked the E2 regulation of OC expression. The reason for the latter is not clear, but may be due to several causes. First, it has been demonstrated that the actions of anti-E2, like that of ICI 182,780, are not only cell specific, but are also gene promoter specific (45). These compounds act at multiple steps in the ER signal transduction pathway, which collectively account for its different effects on the transcription of different genes (45). Second, the OC experiments were performed in the presence of vitamin D3, which has been shown to regulate a wide range of genes, including protooncogenes (46). These genes may be down-regulated or up-regulated and sequestered by other vitamin D3-dependent transcription machinery and, therefore, influence ER-E2 and ER-ICI 182,780 interactions.

The hFOB/ER9 cells are shown here to be a target cell for E2 and demonstrate the capacity of human osteoblasts to respond to E2 treatment if sufficient receptors are present. Some in vivo studies have reported E2-mediated increases in bone formation (15, 16, 17); however, the majority of the in vivo studies in both rats and humans have demonstrated E2-mediated decreases in bone formation (1, 11, 12, 13, 14). In terms of osteoblast number and function, the effects of E2 in these studies were associated with decreases in osteoblast number and in serum levels of AP, OC, and Col I and/or gene expression. Our results in part resemble these in vivo observations, as E2 decreased hFOB/ER9 cell proliferation and OC expression. Interestingly, we observed an increase in AP activity and little or no change in Col I levels with E2 treatment. The significance of the E2 induction of AP activity is unclear even in light of recent data indicating a lower correlation of AP levels than OC or Col I levels to bone formation (13, 47). In the case of the Col I results, E2-treated ovariectomized rats exhibited decreased Col I mRNA levels after only a week of E2 treatment (11). As E2 treatment of hFOB/ER9 cells was carried out for 2 days, longer exposure to E2 may be required to see significant effects on Col I expression. It is also possible that the assay used to measure changes in Col I levels was not sensitive enough to detect changes after 2 days of hormone treatment. Further studies need to be performed to address these issues.

Although E2 inhibited hFOB/ER9 cell proliferation but had a mixed effect on differentiation markers, collectively, the data would be consistent with previous reports of E2-mediated decreases in bone formation. Furthermore, the findings of this study indicate that the hFOB/ER9 cell line is a good model for analysis of the effects of E2 and E2 analogs on human osteoblastic cells.


   Acknowledgments
 
We thank Sue Bonde and Larry Pederson for their excellent technical assistance, Ms. Jackie House for manuscript preparation, and Dr. Peter Wollan for statistical analysis assistance. We also thank Dr. Gideon Rodan for kindly providing the human alkaline phosphatase cDNA probe, Dr. George Long for kindly providing the human osteonectin cDNA probe, and Zeneca Pharmaceuticals (Cheshire, UK) for generously providing the anti-E2 ICI 182,780.


   Footnotes
 
1 This work was supported in part by NIH Grants AG-04875 and HD-07108 and by an institutional grant from the Mayo Foundation. Back

Received January 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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