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Department of Biochemistry and Molecular Biology (K.R.T., T.E.H., K.M.W., J.A.R., M.S., T.C.S.), Department of Internal Medicine (B.L.R.), Division of Endocrinology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Thomas C. Spelsberg, Department of Biochemistry and Molecular Biology, Division of Endocrinology, Mayo Clinic and Mayo Foundation, Guggenheim Building, Room 1601A, Rochester, Minnesota 55905. E-mail: Spelsberg.Thomas{at}mayo.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many in vitro studies on the effects of E2 on osteoblast proliferation and differentiation have produced inconsistent results (for review, see Refs. 5 and 30). 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 (25, 31). To overcome these uncertainties, we have developed a human immortalized fetal cell line [human fetal osteoblastic/ER9 (hFOB/ER9)] that expresses the mature osteoblast phenotype and contains high levels of ER (32, 33). Recent studies in our laboratory have shown that during the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent inhibition of [3H]-thymidine incorporation (34). Further, E2 causes an increase in alkaline phosphatase activity and a decrease in osteocalcin protein levels. These results support the hypothesis that E2 does have an effect on osteoblastic growth and function by decreasing hFOB/ER9 cell proliferation and differentially regulating the production of extracellular matrix proteins, growth factors, and cytokines.
Our laboratory also has identified a novel TGF-ß inducible early gene (TIEG) in human osteoblasts (35). This gene encodes a 480-amino acid protein and contains three zinc finger motifs at the C-terminal end. The zinc finger region of this gene shows homology to a known transcription factor family of genes, e.g. SP-1, BTEB, and Wilm’s tumor gene. The messenger RNA (mRNA) steady-state levels for TIEG are induced as early as 30 min after TGF-ß treatment in human osteoblasts, with a maximal induction at 1–2 h (35). This induction displayed a growth factor specificity in osteoblasts, with TGF-ß, BMP-2 and EGF as the major inducers, whereas many other growth factors and cytokines showed only a minimal effect (35). In addition, the induction of TIEG mRNA levels occurs at the level of transcription and is independent of protein synthesis. The gene displays a tissue-specific expression, because the basal mRNA steady-state levels were shown to be high in several human organs (heart, liver, muscle, placenta, pancreas), but low to absent in others (kidney, brain, lung) (35).
Recent studies using immunoprecipitation methods with TIEG-specific polyclonal antibodies have demonstrated that, in the immortalized hFOB cells, TIEG encodes a 72-kDa protein, and TGF-ß treatment of hFOB cells increases the TIEG protein levels, by 2 h after the induction of TIEG mRNA levels (36). Further, Western blotting and immunohistochemical studies demonstrate that TIEG protein is expressed in many, but not all, tissues (following the pattern of mRNA levels) and, thus, is markedly cell-type-specific (35, 36). Interestingly, a stage-dependent expression of TIEG protein was found in a dozen breast cancer biopsies, which is inversely correlated with the stage of the disease, suggesting that TIEG might be a tumor-suppressor gene (36). Recent studies have demonstrated that overexpression of TIEG protein in pancreatic carcinoma cells inhibits cell proliferation and induced apoptosis (37).
Studies described in this report support the role of TIEG as a potential participant in the signaling pathway of the E2 regulation of DNA synthesis. This study demonstrates that E2 rapidly induces the expression of TIEG mRNA and protein in hFOB/ER9 cells. The TIEG protein is shown to be localized in and around the nucleus, with E2 treatment rapidly increasing its levels in the nucleus. The induction of TIEG mRNA by E2 is rapid and independent of new protein synthesis, and it acts via the ER. The induction of TIEG expression is followed by the E2-mediated decrease in DNA replication in these cells (34). To support the hypothesis that TIEG plays a primary role in the E2 inhibition of DNA synthesis, the overexpression of TIEG in the parent cell line (hFOB) was shown to result in a significant inhibition of DNA synthesis, mimicking the effects of the E2 treatment. These data support the fact that TIEG represents an early response gene in the E2 regulation of human osteoblast cells and, when overexpressed, can inhibit DNA synthesis.
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Materials and Methods |
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32P]deoxycytidine triphosphate, were purchased from
DuPont-New England Nuclear (Boston, MA). ICI 182,780 was a gift from
Zeneca Pharmaceuticals (Cheshire, UK). Tissue culture flasks and dishes
were obtained from Corning (Park Ridge, IL).
Cell culture and Northern analysis
The immortalized hFOB cells used in these studies were
engineered as a temperature-sensitive cell line which proliferates at
the permissive temperature (34 C), yet ceases proliferation when
incubated at the restrictive temperature (39.5 C) (24). The hFOB cells
were maintained in phenol-red free DMEM/F12 (1:1) supplemented with
10% (vol/vol) FBS and 300 µg/ml geneticin, and incubated at the
permissive temperature of 34 C. Two second-generation transfected hFOB
cell lines expressing wild-type human ER (hFOB/ER3 and hFOB/ER9) have
been developed recently and described previously (32). The hFOB/ER cell
lines were maintained in phenol red-free DMEM/F12 (1:1), supplemented
with 10% (vol/vol) charcoal-stripped FBS (FBS-cs) and continuous
selection, with alternating media changes containing either 300 µg/ml
geneticin or 150 µg/ml hygromycin B. The hFOB/ER cell lines were
cultured at the permissive temperature (34 C).
For Northern blot analysis, cells were first plated in 100-mm tissue culture dishes and grown to confluency using the media described above. The monolayers were washed twice with DMEM/F12 and once with DMEM/F12 containing 0.25% (vol/vol) BSA (BSA medium). Cells were incubated in 10 ml/plate BSA medium containing 10 nM ICI 182,780 for 48 h at 34 C. These serum-starved, ICI-pretreated cells were washed as described above, before steroid treatment. For steroid treatments, first E2 at concentrations of 0.1–100 nM, or IC1 182,780 at 100-fold excess concentrations of the E2 or an equivalent volume of ethanol (vehicle) were added to the cells. The cells were incubated at 34 C for the times indicated both in Results and in the figures. Total RNA (8–10 µg), as previously described (34), isolated from the hFOB/ER cells, was resolved on a 1% (wt/vol) agarose glyoxal gel (36, 37, 38, 39), transferred to nylon membrane by capillary diffusion in 20x saline-sodium citrate (3 M NaCl, 0.3 M sodium citrate, pH 7.0), and bound to the membrane by baking at 80 C under vacuum for 2 h. The blots were probed with [32P]-labeled TIEG complementary DNA (cDNA) (34). Densitometry was performed with a Shimadzu CS 9000 flying spot scanning laser densitometer (Kyoto, Japan).
To examine the effect of cycloheximide or actinomycin D on E2 regulation of TIEG mRNA, the hFOB/ER9 cells were plated in 100-mm dishes, followed by the steroid treatment, as previously described, using 10 nM E2 or vehicle alone with cycloheximide (10 µg/ml) or actinomycin D (1 µg/ml) added to the medium for 120 min. To examine the effect of antiestrogen upon E2 regulation of TIEG, hFOB/ER9 cells were pretreated, as described above, and either E2 (1 nM or 10 nM) alone or E2 with ICI 182,780 (100-fold molar excess of E2) was added to the cells and incubated at 34 C for 120 min. Total RNA was obtained, and Northern blot analysis was performed, as described above. To examine the antagonism between PTH and E2, hFOB/ER9 cells were treated as described above, except that human PTH (1–34) (0.001 nM-10 nM) was added directly to medium coincident with E2 or vehicle addition. The cells were then incubated at 34 C for 120 min and Northern analysis performed.
Localization of TIEG by immunofluorescence
The hFOB/ER9 cells were plated at a density of 25,000
cells/cm2 in 8-well permanox chamber slides (Nunc,
Naperville, IL) and grown for 48 h at 34 C in DMEM/F12 (1:1)
containing 10% FBS-cs. The cells were serum-starved and ICI 182,780
pretreated, as described above. The monolayers on chamber slides were
treated with E2 (10 nM) or vehicle alone,
incubated at 34 C for 120 min, and fixed with methanol (-20 C) for
1 h. Nonspecific protein binding was blocked with 10% normal goat
serum, and subsequently, slides were incubated with rabbit polyclonal
antihuman TIEG antibody (1 µg/ml) in a humid chamber for 2 h at
room temperature. Cells were extensively rinsed with PBS and were
incubated with the secondary goat antirabbit dichlorotriacinyl
aminofluorescein-conjugated antibody (Jackson ImmunoResearch, West
Grove, PA) at a dilution of 1:200 for 2 h at room temperature.
After extensive washing with PBS, the slides were mounted in glycerol
solution containing paraphenylenediamine, and they were analyzed with a
laser scanning confocal microscope (LSM 310, Carl Zeiss, Oberkochen,
Germany) equipped with an argon/krypton laser tuned to an excitation
wavelength of 488 nm. An emission filter of 530 nm was used in front of
the photomultiplier tube. The objective lens was a Zeiss plan-neofluar
(40x magnification, oil immersion).
Cell proliferation analyses
Cell proliferation was assessed using a
[3H]-thymidine incorporation assay. The hFOB/ER9 cells
were plated into 24-well culture dishes at 10,000 cells/cm2
in DMEM/F12 containing 10% (vol/vol) FBS-cs, and they were incubated
for 48 h. The cells were then washed twice with PBS and pretreated
with ICI 182,780 (10-7 M) in DMEM/F12
containing 1% (vol/vol) FBS-cs for 48 h, 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 (40).
Stable expression of TIEG in human osteoblasts
TIEG cDNA was cloned into an eukaryotic expression vector that
contains the hygromycin gene (32). The vector was linearized and
transfected into hFOB cells, using the electroporation method
previously described (32, 33). The transfected cells were selected for
hygromycin resistance. The selected clones were individually
ring-cloned and were expanded for further studies. To study
proliferation, hFOB and hFOB TIEG overexpression cells were plated at a
density of 30,000 cells/cm2 in 12-well plates and incubated
for 48 h at 34 C in DMEM/F12 containing 10%(vol/vol) FBS. The
[3H]thymidine was added 24 h before harvesting the
cells, and the levels of [3H]thymidine incorporation were
determined as previously described.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, E2 treatment of hFOB/ER9
cells resulted in a rapid increase in TIEG mRNA steady-state levels,
with a maximal induction at 120 min post treatment. Densitometric
analysis of multiple Northern blots indicated a 7- to 8-fold induction
of TIEG steady-state mRNA by E2 in hFOB/ER9 cells. The
levels of glyceraldehyde-3-phosphate-dehydrogenase (control) mRNA
demonstrate equivalent RNA loading. In half of the six independent
experiments, the levels of TIEG remained elevated at 4 h post
treatment, whereas in the remaining experiments, the E2
induction of TIEG was more transient, often declining to basal levels
shortly after 4 h post treatment (data not shown). The exact cause
of this variation is unknown.
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shows that, as with the hFOB/ER9
cells, E2 treatment of hFOB/ER3 cells results in a rapid
increase in the steady-state levels of TIEG mRNA. However,
densitometric analysis of TIEG mRNA levels demonstrated a maximal
stimulation between 2- to 5-fold in hFOB/ER3 cells, compared with 7- to
8-fold for hFOB/ER9 cells 120 min post treatment. Two approaches were
taken to support the premise that the E2 regulation of TIEG
requires an active ER for the TIEG induction. First, the ER-deficient
hFOB cells were treated with 10 nM E2 and
analyzed for TIEG expression. The hFOB cells were serum starved and ICI
pretreated for 48 h before treatment with either vehicle or 10
nM E2. As shown in Fig. 2B, E2 does
not induce TIEG in ER-deficient hFOB cells. Second, serum-starved,
subconfluent hFOB/ER9 cells were cotreated with E2 and a
100-fold excess of ICI 182,780 for 2 h, and Northern analyses were
performed. As shown in Fig. 3, the
addition of the ICI 182 780 completely blocks the E2
induction of TIEG mRNA levels, supporting the premise that the effect
is mediated through the ER.
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shows that there is a
steroid, dose-dependent induction of TIEG mRNA steady-state levels,
with a maximal stimulation between 1 and 10 nM
E2. To determine whether the E2 induction of
TIEG was dependent on new protein synthesis, the protein synthesis
inhibitor, cycloheximide, was added during E2 treatment at
a concentration which inhibits more than 95% of the protein synthesis.
The inhibition of protein synthesis was shown not to affect the
E2 induction of TIEG mRNA (Fig. 5). This suggests that the induction is
not dependent on protein synthesis. In contrast, the addition of RNA
synthesis inhibitor, actinomycin-D, during the 10 nM
E2 treatment of hFOB/ER9 cells, does abolish the
E2 induction of TIEG, suggesting that the E2
induction of TIEG is mediated, at least in part, at the level of
transcription (Fig. 5).
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). The 2-h E2 treatment
results in an enhanced nuclear localization of TIEG protein, as
demonstrated by the greatly increased punctate fluorescence observed in
the nuclei (Fig. 6C). There was no fluorescent staining observed with
the negative control antibody (Fig. 6A).
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, treatment of hFOB/ER9 cells for 120
min with PTH (1–34) alone moderately reduces the TIEG mRNA
steady-state levels in a dose-dependent manner. The concurrent
addition of PTH (1–34) to E2-treated cells blocked the
E2-induced increase in TIEG mRNA steady-state levels. The
lack of a dose response in these competition studies is probably caused
by the fact that the maximal inhibition was achieved at the lowest dose
of the PTH. These data suggest that PTH also may rapidly regulate TIEG,
and its antagonism of E2 action on osteoblast cells may
occur at the level of TIEG gene expression. Past studies have shown
that both E2 and TIEG inhibit DNA synthesis and cell
proliferation. The possibility arises that E2 inhibits DNA
synthesis via the TIEG gene. As shown in Fig. 8, the E2-induced TIEG mRNA
steady-state levels at 120 min is indeed followed by an
E2-induced inhibition of DNA replication after 5 days in
the hFOB/ER9 cells. Further, the antiestrogen is shown to block these
actions, suggesting that the TIEG may be directly involved in the
E2 inhibition of DNA replication. In support of this
possibility, the parent hFOB cell line was stably transfected with a
TIEG expression construct (as described in Materials and
Methods), and the rate of DNA synthesis was measured. The hFOB
cells were used in these studies because transfections of the already
stably transfected (with the SV-40 T-antigen and ER) hFOB/ER cells were
difficult to clone and displayed poor cell viability. As shown in Fig. 9, the nonconfluent cells, with stably
transfected TIEG, repressed DNA synthesis by 53%, compared with the
nontransfected (control) cells. Thus, TIEG overexpression seems to
inhibit DNA synthesis, and the E2-induced inhibition of
osteoblastic cell replication may be mediated by the E2
induction of the TIEG gene expression, which in turn, inhibits DNA
synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The E2 effects on osteoblast cell proliferation have had mixed reports. We have observed a faster cell doubling rate for the hFOB parental cell line, which expresses very low levels of ER, than the hFOB/ER9 subclone (32). A decrease in cell growth has been shown also in SAOS-2 and HTB 96 human osteosarcoma cells, transfected with the wild-type ER, compared with their parental cell counterpart (49, 50). The effect of E2 on cell proliferation in these bone cell models may be caused by many factors, which 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 caused by an increase in responsiveness of the cells to E2 and/or cross-talk between the ER and growth factor regulatory pathways (31, 51). This is possible, because recent work in our laboratory has shown that E2 stimulates both IGF-BP4 and TGF-ß production in hFOB/ER9 cells, and the addition of exogenous IGF-BP4 or TGF-ß to cultures inhibits cell proliferation in these cells (29, 30, and data not published). Alternately, the rate of proliferation also may 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 house-keeping genes (52). Lastly, in vivo studies involving E2-treated ovariectomized rats have demonstrated fewer [3H] thymidine-labeled preosteoblasts than those observed in control ovariectomized rats (12, 53). The authors suggested from these studies that the decrease in osteoblast number was probably caused by the marked inhibition of proliferation of the preosteoblast cells. This observation may be relevant to the results we observe with E2 regulation of cell proliferation in the hFOB/ER9 cells that were derived from fetal tissue and have properties of preosteoblasts (32, 33, 34). In any case, the biological function of the E2 inhibition of OB proliferation remains obscure. It is possible that, by this mechanism, the E2 might inhibit the loss of bone by reducing the level of bone turnover, which is enhanced in conditions of bone loss. In this case, the E2 inhibition of preosteoblasts would reduce the amount of functioning mature OB needed for bone turnover. The E2 induction of TIEG mRNA levels is shown here to be followed by an inhibition of cell proliferation/DNA synthesis. When the E2 induction of TIEG mRNA levels is blocked, using the antiestrogen ICI 182,780, the E2-induced inhibition of DNA synthesis also is blocked. A more direct connection between TIEG levels and DNA replication was achieved by studies wherein the stable overexpression of TIEG in hFOB cells caused an inhibition of DNA synthesis. Thus, there is a direct connection between E2 action and TIEG expression and, in turn, between TIEG expression and DNA synthesis. These correlations raise the possibility, but certainly do not prove, that TIEG may serve as a primary intracellular signal for E2 in the inhibition of DNA replication in the hFOB/ER9 cells.
The ability of a gene and its protein product to inhibit DNA replication is one property of a tumor suppressor gene. Interestingly, TIEG expression levels recently have been shown to have an inverse correlation with the stage of breast cancer disease (36) and to inhibit the DNA replication in pancreatic carcinoma cells (37). Some tumor suppressor genes recently have been reported to function as transcription factors (e.g. p53, WT-1; BRCA-1/2), whereas others seem to function as inhibitors of transcription factors (e.g. Rb), cell adhesion molecules (DCC), guanosine triphosphatase activators (NF-1), DNA repair enzymes, and/or intracellular kinase inhibitors (p16) (54). The TIEG protein is shown in this investigation to be a nuclear protein, and other studies have reported that its structure contains transcription factor motifs (35). The transcription factor class of tumor suppressor genes has been shown to be regulated by estrogens (48). As shown in this study, TIEG also is regulated by E2 in a fashion similar to other tumor suppressor genes.
Because TIEG seems to respond rapidly to E2 and TGF-ß in osteoblast cells, several questions have arisen that are currently under study. First, the antagonism between PTH and E2, known to occur in osteoblast cells and skeletal tissue, occurs at the rapidly regulated levels of TIEG mRNA. The question arises as to whether TIEG is a primary intracellular target for TGF-ß, E2, and PTH actions on osteoblast cell proliferation, and in a broader sense, whether it serves as the primary regulatory gene in various other TGF-ß/E2/PTH regulated functions. Second: is TIEG a tumor suppressor gene? The inverse expression levels of TIEG with the stage of breast cancer (36) and its repression of DNA synthesis/cell proliferation in pancreatic carcinoma cells (37) and bone cells, as demonstrated in this study, support this possibility. Third: would defects in the expression and functions of TIEG play a role in bone disease and cancer in general? This possibility is suspected, because TIEG has been localized to chromosome 8,q22.2, the site of a gene involved in osteopetrosis and a gene involved in acute myelocytic leukemia (36). The mechanisms by which E2 induces the TIEG transcription, using genomic clones of TIEG, and the target genes with which TIEG protein may act, are also currently under study.
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Acknowledgments |
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Footnotes |
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Received July 23, 1997.
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