ER{alpha} Gene Expression in Human Primary Osteoblasts: Evidence for the Expression of Two Receptor Proteins

Stefanie Denger, George Reid, Martin Kos, Gilles Flouriot, Dominik Parsch, Heike Brand, Kenneth S. Korach, Vera Sonntag-Buck and Frank Gannon

European Molecular Biology Laboratory (S.D., G.R., M.K., H.B., V.S.-B., F.G.), 69117 Heidelberg, Germany; Endocrinologie Moléculaire de la Reproduction (G.F.), UPRES-A CNRS 6026, Campus de Beaulieu, 35042 Rennes cedex, France; Department of Orthopedics (D.P.), University of Heidelberg, 69118 Heidelberg, Germany; and National Institute of Environmental Health Sciences (K.S.K.), Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Stefanie Denger, EMBL, Meyerhofstrasse 1, 69012 Heidelberg, Germany. E-mail: denger{at}embl-heidelberg.de


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ER{alpha} in osteoblasts clearly demonstrated that the well characterized 66-kDa ER{alpha} was only one of the ER{alpha} isoforms present. Here we describe a 46-kDa isoform of ER{alpha}, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ER{alpha} gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ER{alpha} isoform is absent. Functional analysis revealed that human (h)ER{alpha}46 is able to heterodimerize with the full-length ER{alpha} and also with ERß. Further, a DNA-binding complex that corresponds to hER{alpha}46 is detectable in human osteoblasts. We have shown that hER{alpha}46 is a strong inhibitor of hER{alpha}66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hER{alpha}46 are cotransfected with hER{alpha}66. In addition to human bone, the expression of the alternatively spliced ER{alpha} mRNA variant is also detectable in bone of ER{alpha} knockout mice.

These data suggest that, in osteoblasts, E2 can act in part through an ER{alpha} isoform that is markedly different from the 66-kDa receptor. The expression of two ER{alpha} protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ER{alpha} protein isoforms in bone and perhaps in other tissues.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OSTEOPOROSIS AFFECTS ONE third of the postmenopausal female population and arises as a consequence of accelerated bone loss that follows menopause or ovariectomy. The development of osteoporosis can be prevented by early hormone replacement therapy (1, 2). However, the detailed cellular and molecular mechanisms underlying the positive effect that estrogens have in maintaining bone density are poorly defined. Estrogens act to decrease bone resorption and by doing so, contribute to the prevention of osteoporosis. E2 binds to specific intracellular receptors, the ERs. To date, two ERs (ER{alpha} and ERß), encoded by different genes, have been characterized (3, 4, 5). Both belong to the nuclear receptor superfamily of ligand-inducible transcription factors that include the steroid, thyroid hormone, and retinoic acid receptors. Nuclear receptors regulate gene expression by interacting directly with cognate DNA sequences [responsive elements (6, 7)] or through protein/protein interactions with other transcriptional factors (8, 9). ERs are modular proteins and can be subdivided into six distinct regions (A–F) (10) based on homology with other members of the nuclear hormone superfamily. Regions C and E are responsible for DNA and hormone binding, respectively. The A/B region contains a ligand-independent transactivation domain [activating function 1 (AF-1)] whereas a hormone-inducible transcription activating function (AF-2) is present in the hormone-binding domain.

Antiestrogens, such as tamoxifen and raloxifene [also known as selective ER modulators (SERMs)] are clinically used to block the actions of estrogen in ER-positive breast tumors while conferring agonist-like effects in bone tissue (11, 12).

It was recently shown that the ER{alpha} gene in human (h), chicken (c), and mouse (m) is a complex genomic unit showing differential promoter usage and alternative splicing (13, 14, 15). This results in a cell-specific variation in the expression level of the ER{alpha} gene in different tissues. Generally, the consequence of alternative splicing has been the expression of the classical and well studied 66-kDa ER{alpha} (hER{alpha}66). In addition, we have recently described a 61-kDa ER{alpha} (cER{alpha}61) that is present in chicken liver and which is transcribed from a specific promoter (16). However, in contrast to the isoforms described in this study, the responsible promoter is located in a region downstream from the translation start site in exon 1 of the 66-kDa chicken ER{alpha}. The resulting cER{alpha}61 protein is characterized by an increased basal level of ligand-independent activity (16). However, the internal ATG that is responsible for the expression of cER{alpha}61 is exclusively conserved in oviparous species and is not found in mammals.

In this study we evaluated the expression profile of ER{alpha} present in human osteoblasts and show that an alternative hER{alpha} isoform, 46 kDa in size (hER{alpha}46), is expressed in osteoblasts at a significant level and constitutes a significant proportion of the total ER{alpha}. Analogous studies showed that MCF-7 cells also have the potential to express hER{alpha}46 (17), albeit to a lower level than in osteoblasts. The hER{alpha}46 arises from an alternative splicing event that excludes the first exon of hER{alpha}. The differential expression of this isoform in primary osteoblasts suggests alternative explanations for the differential action of SERMs in a variety of tissues.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Selective Amplification and Quantitative Analysis of ER{alpha} cDNA Demonstrates the Expression of Alternatively Spliced Receptor Variants
To determine the promoter(s) used in the expression of hER{alpha} in osteoblasts and in bone tissue, RT-PCR analysis was performed using specific upstream primers from the different 5'-exons of ER{alpha} (A–F) (13). The only mRNA isoforms detected using RNA directly isolated from bone tissue or from cultivated primary osteoblast cells were derived from the F-hER{alpha} promoter (Fig. 1BGo). It should be noted that exon 1F splices to exon 1E before splicing to a common splice site within exon 1 at position +163. Therefore, all F-hER{alpha} variants include a partial sequence of exon 1E. All exon-specific 5'-untranslated regions (UTRs) (A–F) were detected in the MCF-7 mRNA. Primary smooth muscle cells, isolated from the medial layer of human aorta and used as an alternative primary culture, showed a PCR band with A- and C-specific primers, whereas no signal was obtained with the other variants tested (Fig. 1BGo). In the osteoblast-like cell line SaOs, both E-hER{alpha} and F-hER{alpha} variants were detectable. S1 nuclease protection results confirmed that the F-hER{alpha} mRNA variant was the only ER{alpha} mRNA transcript observed in osteoblasts (Fig. 2Go) and indicates that the F-hER{alpha} mRNA plays the major role in the generation of ER{alpha} in bone.



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Figure 1. RT-PCR Analysis of hER{alpha} Variants

A, Schematic representation of the experimental design for RT-PCR. The location of the primers are indicated by short arrows. Open boxes represent the unique (1A–1F) and common exons (1 2 3 4 5 6 7 8 ) encoding each isoform. Primer (PRT), located in the 3'-untranslated region of hER{alpha} (exon 8) was used for reverse transcription. Primers A1–F1 were used in the first round of PCR in parallel with a common 3'-primer (P1). A second round of nested PCR amplification was then performed using primers A2–F2 as well as primer P2. Southern blotting, using a specific oligonucleotide probe from exon 1 (probe), showed the specificity of the PCR products. B, Using total RNA from different tissues and cells, the hER{alpha} isoforms were amplified as described above. Yeast tRNA was used as a negative control. The amplification products were separated on a 1% agarose gel and transferred to a membrane, which was hybridized using the oligonucleotide probe ex. 1. The size of the expected PCR-products is indicated in the figure.

 


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Figure 2. S1 Analysis of hER{alpha} Variant Expression

A, Experimental design for the detection of hER{alpha} mRNA isoforms, showing the location and the size of each variant-specific probe (A–F) as well as the corresponding protected fragments after S1 digestion of the probe/hER{alpha} mRNA hybrids. Each variant-specific probe also allowed the protection of the other variants, due to their common sequence up to the splice site position. Each probe was designed to contain vector sequences in their 5'-end to discriminate between undigested probe and protected fragments. ts, Transcription start site. B, S1 protection analysis was performed as described in Materials and Methods on 20 µg of MCF-7 total RNA and 50 µg of osteoblast total RNA. Yeast tRNA (50 µg) was used as a negative control.

 
As is clear from RT-PCR analysis, shorter products of F-hER{alpha} cDNAs were amplified from primary osteoblasts, SaOs and bone tissue. To characterize these products, an S1 nuclease protection analysis, designed to detect potential alternative internal splicing where the F-transcript spliced directly to exon 2, was performed. It was found that this alternative mRNA splice variant was present in osteoblast cells and also in MCF-7 cells (Fig. 3Go). The resulting F-hER{alpha} mRNA transcripts from exon 2 to exon 8 are therefore identical to the previously described full-length transcript. In contrast to MCF-7, where splicing of exon 1F to exon 2 represents only a minority of the transcripts (as shown by the ratio of the specific splice fragment to that of all other forms of ER{alpha}), this splice form corresponds to approximately 50% of total transcripts in osteoblasts. These data were also confirmed by a quantitative real-time RT-PCR approach (Fig. 3BGo), where it is demonstrated that hER{alpha}66 mRNA levels in MCF-7 cells are 200-fold more abundant as compared with primary osteoblasts. The alternative splicing event of F splicing to exon 2 results in the formation of an mRNA transcript that encodes hER{alpha}46 and represents a minor component in MCF-7 cells, whereas in primary osteoblasts this splicing event generates 50% of total transcripts coding for ER{alpha}. In light of this observation, it was then investigated whether this alternative splicing event resulted in the expression of an alternative ER{alpha} protein at significant levels.



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Figure 3. Quantitative Analysis of hER{alpha}F-mRNA Variants

A, S1 analysis using a probe specific for the splicing of exon 1F/E hER{alpha} mRNA directly to exon 2 was performed. Note that this probe results in an additional protected fragment as E and F also share one part of exon 1E. The probe was designed to contain vector sequence in their extremity (denoted by the thinner gray line) to allow discrimination between undigested probe and specific protected fragments. B, A quantitative RT-PCR approach was used for the amplification of hER{alpha} F-mRNA variants, where exon 1F splices either to the common splice site at position +163 in exon 1 or directly to position +685 in exon 2. A fluorescence labeled primer was used as a probe. The amplification of glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard for normalization.

 
Alternative Splicing Events Result in the Expression of ER{alpha} Isoforms in Human Primary Osteoblasts
Two internal ATG codons, in frame with the remainder of the hER{alpha} open reading frame, occur in exon 2 at positions +752 and +758. Examination of the sequence surrounding these ATGs showed that both have a favorable Kozak sequence for translation initiation (18). Use of either of these ATGs would result in a hER{alpha} protein with a predicted size of approximately 46 kDa lacking the first 173 amino acids of the hER{alpha}66. Human ER{alpha} cDNA starting from exon 2 was cloned into an eukaryotic expression vector (pSG5) to test whether this protein could be expressed. As a positive control, the hER{alpha} cDNA expression vector (HEO), which generates a hER{alpha} protein of 66 kDa, was used. Analysis of the in vitro translation products that resulted from HEO (IVT66) showed a protein of 66 kDa (hER{alpha}66) and a shorter hER{alpha} protein of 46 kDa (hER{alpha}46) at a significantly lower level. This shorter hER{alpha} isoform was the only product expressed by pSGhER{alpha}46, confirming that translation could begin from the downstream initiation codon. To investigate whether the high levels of F/2 transcript correspond to the expression of a hER{alpha}46 protein isoform in vivo, immunoprecipitation of radiolabeled total protein from osteoblasts using the ER{alpha}-specific H222 monoclonal antibody [which is directed against the hormone-binding domain of the hER{alpha} protein] was performed. As shown in Fig. 4Go, the alternative isoform hER{alpha}46 is expressed to a very significant level in human osteoblasts. Densitometric evaluation of the hER{alpha}66 and hER{alpha}46 protein levels showed similar amounts of each in osteoblasts. Surprisingly, another protein of approximately 39 kDa was also immunoprecipitated. The origin of this band is currently under investigation. In summary, the hER{alpha} F promoter is the predominant promoter used by osteoblasts, and this results in the expression of either the 66-kDa hER{alpha} protein or a 46-kDa hER{alpha} isoform that lacks the A/B domain of the full-length receptor.



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Figure 4. Two Protein Isoforms Are Expressed in Vitro and in Vivo

A, Schematic representation of the cDNAs inserted within the expression vector pSGhER{alpha}66, and pSGhER{alpha}46, encoding the hER{alpha}66 and hER{alpha}46 proteins. The position of the two initiator methionines are indicated. Both plasmids were in vitro transcribed and translated by rabbit reticulocyte lysates in the presence of 35S-methionine. B, 35S-labeled osteoblast whole cell protein and in vitro translated constructs were immunoprecipitated using H222 monoclonal antibody and separated on a 10% polyacrylamide gel. Immunoreactive bands of 66 kDa, 46 kDa, and 39 kDa were visualized by autoradiography.

 
Heterodimerization of hER{alpha}46 with hER{alpha}66 and ERß
ER acts as a dimer when it activates transcription via DNA binding. To ascertain whether hER{alpha}46 is a functionally active ER{alpha} protein isoform, the ability of hER{alpha}46 to form homodimers or heterodimers with hER{alpha}66 and with hERß that are able, in turn, to bind to a responsive element [estrogen response element (ERE)] was evaluated in vitro by EMSAs. Heterodimers were synthesized in vitro by rabbit reticulocyte lysates using extracts containing equimolar concentrations of constructs encoding hER{alpha}66/hER{alpha}46, hER{alpha}66/hERß, or hERß/hER{alpha}46. Homodimers were generated by including the appropriate single constructs. As shown in Fig. 5Go, these in vitro translated extracts were able to form stable DNA/protein complexes with a radiolabeled consensus ERE. The specificity of these complexes was confirmed by supershift experiments using antibodies directed against the N terminus (ER21) and C terminus (H222) of ER{alpha}. The potential to form hER{alpha}66/hERß heterodimers (19) was confirmed. Interestingly, hER{alpha}46 was able to form heterodimers with both hER{alpha}66 and hERß in addition to forming homodimers. A shift of the heterodimer complexes hER{alpha}46/hER{alpha}66 and hER{alpha}46/hERß was observed using the H222 monoclonal antibody. As H222 does not recognize ERß, the supershift of hER{alpha}46/hERß complexes is due to H222 binding to the hER{alpha}46 component of the heterodimer. A complex of intermediate mobility was generated when hER{alpha}46 and hER{alpha}66 were cotranslated, resulting from the formation of hER{alpha}46/hER{alpha}66 heterodimers. When the complexes were incubated with the polyclonal antibody ER21, which is directed against the N terminus of ER-{alpha}, a supershift was detectable only in complexes where hER{alpha}66 was present (hER{alpha}66 homodimers and hER{alpha}66/46 heterodimers, respectively). Due to the lack of the A/B domain, hER{alpha}46 homodimers were not supershifted in the presence of ER21. Although the hERß protein is not expressed at detectable levels in osteoblasts (Denger, S., manuscript in preparation), the formation of hER{alpha}46/hERß complexes that are detectable in vitro could be involved in other cell- and tissue-specific ER signaling processes in which hER{alpha} and hERß are synthesized concomitantly (21).



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Figure 5. In Vitro Heterodimerization of hER{alpha}46 with hER{alpha}66 and hERß

Plasmids (0.75 µg) containing either vector alone (pSG5), pSGhER{alpha}46, pSGhER{alpha}66, or hERß were in vitro transcribed and translated using the reticulocyte lysate system. Four microliters of each product were incubated with 60,000 cpm of labeled ERE. Specificity was determined by supershift using monoclonal H222 antibody, directed against the C terminus of ER{alpha} (A) and the polyclonal antibody ER21, which is directed against the N terminus (B). Both antibodies show no cross-reactivity to ERß. The unspecific band corresponds to the vector (pSG5) alone. The positions of the specific hER{alpha}46, hER{alpha}66, and ERß homodimers complexed with ERE, the heterodimers consisting of hER{alpha}46/66, hER{alpha}66/ß, or hER{alpha}46/ß bound to ERE as well as the supershifts are indicated by arrows.

 
hER{alpha}46 Is Expressed in Primary Human Osteoblasts ex Vivo and Binds to an ERE
We evaluated, using a gel shift assay, which ER{alpha} protein isoforms that are expressed in human osteoblasts ex vivo bind to an ERE. In vitro translated constructs coding for hER{alpha}66 and hER{alpha}46 served as controls. Interestingly, a homodimer complex in the same region of hER{alpha}46 was detectable after incubation of whole cell extracts of osteoblasts with radiolabeled ERE in addition to higher and lower complexes (Fig. 6Go), demonstrating that the shorter ER{alpha} isoform is functionally active and can bind to an ERE. In contrast, no hER{alpha}66/ERE complex was detectable in extracts prepared from osteoblasts. This is in agreement with other results, which show that the hER{alpha}46 has a higher affinity for DNA compared with the full-length receptor and, consequently, hER{alpha}46 homodimers are preferentially formed as shown in Fig. 5Go (Denger, S., and G. Flouriot, unpublished results). The formation of the additional smaller complex could be due to homodimerization of the 39-kDa isoform that was seen in immunoprecipitation of osteoblast ER{alpha} protein, although this complex needs to be further characterized (Fig. 6Go).



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Figure 6. Complex Formation Of Osteoblast Extracts with ERE

Lysates of human primary osteoblasts and in vitro translated hER{alpha}66 and hER{alpha}46 proteins were incubated with 60,000 cpm of labeled ERE. Specificity was determined by supershift using monoclonal H222 antibody, directed against the C terminus of ER{alpha}.

 
Inhibition of hER{alpha} 66 Transactivation by hER{alpha}46 in SaOs
Both hER{alpha}66 and hER{alpha}46 receptor isoforms were evaluated in transient transfection assays using a luciferase reporter gene construct in which two EREs are placed upstream of the thymidine kinase minimal promoter (ERE-tk-Luc). The osteosarcoma cell line SaOs was used in these experiments as primary osteoblasts proved refractory to transfection. As hER{alpha}46 is devoid of the A/B domain, it lacks one of the major transactivation functions (AF-1) of hER{alpha}66. The results show that hER{alpha}46 can transactivate ERE-mediated reporter gene expression in an estrogen-dependent manner in this cell context, although to a lower extent than to hER{alpha}66 (Fig. 7Go). To assess the ability of hER{alpha}46 to influence hER{alpha}66 transactivation of reporter genes, both hER{alpha} receptors were cotransfected into SaOs cells in various ratios. The results obtained demonstrated that hER{alpha}46 acts as a potent inhibitor of hER{alpha}66 transactivation with a dose- dependent mode of action. In addition, the effects of the SERM 4-hydroxytamoxifen (4-OHT) on the transactivation capability of both isoforms were evaluated. While 4-OHT had no effect on reporter gene activation of hER{alpha}46, a moderate transactivation of hER{alpha}66 was detectable in the presence of 4-OHT. In contrast to the result in the presence of E2, increasing concentrations of hER{alpha}46 had only a minor inhibitory effect on luciferase expression modulated by hER{alpha}66 in the presence of 4-OHT, which indicates a distinct, differential action of SERMs on these two ER{alpha} isoforms. In SaOs, hER{alpha}46 was unable to transactivate luciferase reporter gene expression from an ERE in response to 4-OHT, in keeping with the described agonistic effects of 4-OHT that are mediated through AF-1 (22). In contrast, hER{alpha}46 was able to transactivate reporter gene expression in response to E2 through AF-2. These results show that transactivation of reporter genes in SaOs is mediated by both transactivation domains, AF-1 and AF-2.



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Figure 7. SaOs Cells Were Transiently Transfected Using 5 µg of Reporter Plasmid EREtkLuc, Together with 0.5 µg of the Expression Vector pSG5, pSGhER{alpha}46, or pSG5hER{alpha}66

Cotransfection experiments were performed using hER{alpha}66 (0.5 µg) with increasing amounts of hER{alpha}46 (0.06, 0.5, 1.0, and 2.0 µg plasmid DNA). Cells were cultivated in DMEM + 2.5% charcoal-stripped, E2-free serum for 48 h and subsequently treated either with (+) or without (-) E2 (10-8 M), or 4-OHT (OHT 10-6 M) before being assayed for luciferase and CAT activities. Luciferase activities were normalized using EF-1{alpha}-CAT plasmid construct as an internal reference control. Results are expressed as means of six separate transfection experiments ± SD.

 
Cotransfection of hER{alpha}66 and hER{alpha}46 Results in Decreased Proliferation of SaOs Cells in a Dose-Dependent Manner
We next addressed whether the coexpression of both hER{alpha} isoforms would have a functional impact on SaOs cells. Proliferation of the cells was evaluated after immunostaining using an antibody directed against the proliferation marker Ki67 (MIB). As shown in Fig. 8Go, proliferation of SaOs cells was slightly increased when low concentrations of hER{alpha}46 were cotransfected with a constant amount of hER{alpha}66. When higher concentrations of hER{alpha}46 were cotransfected, proliferation decreased in a dose-dependent manner. This suggests that hER{alpha}46 acts as a regulatory element that can control the proliferative activity of hER{alpha}66.



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Figure 8. SaOs Cells Were Transiently Transfected Using 5 µg of Reporter Plasmid EREtkLuc, Together with 0.5 µg of the Expression Vector pSG5, pSGhER{alpha}46, or pSG5hER{alpha}66

Cotransfection experiments were performed using hER{alpha}66 (0.5 µg) with increasing amounts of hER{alpha}46 (0.5, 1.0, and 2.0 µg plasmid DNA). Cells were cultivated in DMEM +2.5% charcoal-stripped, E2-free serum for 48 h and subsequently treated either with (+) or without (-) E2 (10-8 M), before being assayed for luciferase and CAT activities. Luciferase activities were normalized using EF-1{alpha}-CAT plasmid construct as an internal reference control. Results are expressed as means of six separate transfection experiments ± SD. Proliferation of cells was analyzed by quantification of MIB-positive cells (line graph, right y-axis).

 
To rule out that the inhibitory action of hER{alpha}46 was due to a squelching effect, we performed transfection experiments with a total of 300 ng of DNA, a concentration that had been previously reported to be an optimal concentration for maximal transactivation without an inhibitory effect in SaOs cells (23). Similar to the previous results shown in Figs. 7Go and 8Go, we observed increased cellular proliferation at low ratios of hER{alpha}46 to hER{alpha}66, followed by a decrease when the ratios transfected increased such that hER{alpha}46 was the predominant isoform present (data not shown). Therefore, we conclude that a squelching effect does not occur under these experimental conditions and that the observed effects are due to a synergistic effect of hER{alpha}46 when present at low concentrations and an inhibitory activity when this isoform is the predominant ER{alpha} present.

hER{alpha}46 Is Expressed in ERKO Bone
The generation of a mouse knockout model for ER{alpha} ({alpha}ERKO) has been a useful tool to study the impact of ER on different tissues including bone. It was assumed that the {alpha}ERKO mouse would be completely devoid of ER{alpha} expression due to the insertion of a neomycin cassette in exon 1 (24). As it has been demonstrated that alternative splicing events in human bone result in the formation of hER{alpha}46 (Fig. 4Go), the question of whether a corresponding mER{alpha} variant could be expressed in {alpha}ERKO mice was addressed. Therefore, an RT-PCR analysis of primary {alpha}ERKO mouse bone cells using primers from the 5'-untranslated exon 1F and 3' primers from exon 2 of the mER{alpha} coding region was performed using sequences obtained for the mER{alpha} (15). PCR amplification resulted in a fragment of 250 bp. Using Southern blotting, the amplification products were screened with oligonucleotides corresponding to exon 1 and to exon 2 of mER{alpha}, respectively. As shown in Fig. 9Go, Southern blotting resulted in a positive signal when the PCR products were probed with an oligonucleotide primer from exon 2. Alternatively, a primer sequence from exon 1 was used as a probe in keeping with the construct used for the generation of the {alpha}ERKO mouse. No hybridization of the smaller product was detectable when a primer from exon 1 was used as a probe in ERKO tissue, whereas the wild-type control resulted in a positive signal. Thus an alternatively spliced mRNA, which potentially encodes a protein equivalent to the hER{alpha}46, also exists in wild-type and in {alpha}ERKO mice.



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Figure 9. Analysis of mER{alpha} Using Total RNA from {alpha}ERKO Bone Cells

RT-PCR from mER{alpha} 5'-upstream exon 1F to exon 2 was performed as described in Materials and Methods. The amplification products were separated on a 1% agarose gel and transferred to a membrane, which was hybridized using oligonucleotide primers from mER{alpha} exon 2 and exon 1, respectively. Kidney RNA from wild-type mice (Co) was used as a positive control. m, Male; f, female.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The importance of ER{alpha} in human bone metabolism is supported by the report of a man unresponsive to estrogen as a consequence of a homozygous mutation in both ER{alpha} alleles that results phenotypically in a decrease in bone mineral density and in an increase in bone turnover (25). Sequence analysis revealed an identical mutation of a single base pair within the second exon of both copies of his hER{alpha} gene, causing a premature stop codon upstream of the start codon for the hER{alpha}46. Despite the importance of ER in a wide range of tissues, this mutation did not result in lethality to the individual. We show evidence that the mild effects observed may be explained by the potential expression of hER{alpha}46 in this individual due to the alternative splicing event to exon 2. It is possible that this surprising phenotype is due to a compensating role played by hER{alpha}46, which can be generated within this individual.

A detailed analysis of the ER status in bone has not been reported to date but is required particularly as there is a growing trend to use hormone replacement therapy or SERMs for the treatment and prevention of osteoporosis. Such studies are hampered by the fact that bone tissue consists of a mixture of cell types and by the low expression of ER. In this study, we have focused predominantly on osteoblasts obtained from primary culture of human bone. Using methods of greater sensitivity, the relative roles of different ER{alpha} isoforms have been clarified. Previous work described the existence of a new 46-kDa isoform of ER{alpha} (17). This hER{alpha}46 protein isoform is identical to the well characterized hER{alpha}66 (3) except that it lacks the A/B domain and therefore is devoid of AF-1 activity. However, as the DNA-binding and ligand-binding domains are not affected by deletion of this region, hER{alpha}46 may act as a ligand-inducible transcription factor (17). S1 nuclease protection assays and immunoprecipitation indicate that hER{alpha}46 mRNA and protein are expressed at a level similar to hER{alpha}66 in osteoblasts. This contrasts with the previous study in MCF-7 in which hER{alpha}46 was a minor component and shows that hER{alpha}46 has the potential to be a major element in some tissues (perhaps not only in osteoblasts) as the mediator of SERM or hormone action. These findings provide, at least in part, a potential explanation for the selective action of E2 and SERMs in a variety of physiological and pathological processes. In the light of these results, the role of ER in bone must be re-evaluated to take into account the presence of all ER{alpha} protein isoforms that can influence bone metabolism.

In this paper we show that both hER{alpha} isoforms are functionally active. In addition to having the capacity to transactivate a luciferase reporter through an ERE, hER{alpha}46 was also able to influence the proliferation of SaOs cells in a dose-dependent manner (Fig. 8Go).

Dimerization of full-length ER{alpha} and ERß has been previously demonstrated (19). Our results further show that in vitro heterodimerization of hER{alpha}46 and hER{alpha}66 complexes can occur. The results shown in Fig. 6Go demonstrate that the hER{alpha}46/46 homodimer and hER{alpha}46/66 heterodimer preferentially bind to the ERE. This is also reflected in the extract prepared from osteoblasts, in which shorter isoforms of ER{alpha} appear to preferentially bind to the ERE.

The demonstration of the significant level of expression of hER{alpha}46 in osteoblasts may shed a light on open questions regarding how estrogens act in a cell-specific manner in bone and in other tissues. Obviously, the 66-kDa ER{alpha} receptor is only one component of the pool of ER{alpha} proteins in osteoblasts. Recently, we have demonstrated that hER{alpha}46 expression is not restricted to osteoblasts but is also present in other cells. For example, hER{alpha}46 is expressed in MCF-7 cells, although a different expression profile of both hER{alpha} isoforms occurs compared with bone, as a consequence of other promoters being used preferentially in these cells (17). Internal ribosome entry in exon 2 of the full-length mRNA that encodes hER{alpha}66 can also result in the generation of hER{alpha}46. Such a mechanism has recently been described (26) and is likely to be responsible for the presence of low amounts of hER{alpha}46 that are seen when plasmid DNA coding for hER{alpha}66 is translated in vitro (Fig. 5Go). This indicates that alternative splicing of exon 1F to exon 2 is the major mechanism by which hER{alpha}46 is generated in osteoblasts, although both mechanisms result in the formation of hER{alpha}46. In osteoblasts, however, hER{alpha}66 and hER{alpha}46 are present at similar levels, as are the respective mRNA species that directly encode these proteins. Other tissues need to be reexamined to determine the levels of hER{alpha}46 as this can have functional consequences.

Recent studies indicate that ERß does not make a significant contribution to the actions of estrogens in bone (20). The current data suggest that at least two isoforms of ER{alpha} must be considered in the response of osteoblasts to estrogens. However, hER{alpha}46 can also form heterodimers with ERß, suggesting that a broad functional activity of this shorter ER{alpha} isoform within ER-mediated signaling in a tissue-specific manner is possible. Therefore, although the ER{alpha}/ERß function plays a minor role in osteoblasts, heterodimer formation of ER{alpha} and ERß may reflect the in vivo situation in other tissues such as testis (20) or during embryogenesis (27), where both receptors are expressed.

The N-terminal domain of hER{alpha} is important for ligand-independent activation through the MAPK pathway (28). The hER{alpha}46 isoform lacks the N-terminal 173 amino acids. However, ligand-inducible transactivation occurs despite the deletion of the A/B domain in SaOs cells. Therefore, hER{alpha}46 can act as a transcription factor that can bypass this cytokine- induced signaling while maintaining intact the ligand-dependent activation property of hER{alpha} (Fig. 7Go). Other experiments show that, in some cellular contexts, such as HepG2, where transactivation domain 1 (AF-1) is predominantly used in ER signaling, hER{alpha}46 does not have a transactivation effect (17). In contrast, analysis of hER{alpha}46-mediated transactivation demonstrated that a cell context such as HeLa, which is mainly sensitive to AF-2, showed that hER{alpha}46 induced transcriptional activity in a ligand-dependent manner to a level similar to hER{alpha}66 (17). The relative contributions of both AF-1 and AF-2 on transcriptional control is known to vary in a cell- and promoter-specific manner (29, 30). One consequence of these data and of the present study is that the presence of an AF-2 domain in hER{alpha} appears to be sufficient to activate transcription after binding of ligand. The presence of AF-2 can therefore provide a sufficient response to E2 to allow, albeit limited, function and development. In transient transfection experiments performed in this study, coexpression of hER{alpha}46 and hER{alpha}66 revealed that hER{alpha}46 can act as a powerful competitor that efficiently suppresses the transactivation capacity of hER{alpha}66 in SaOs cells in a dose-dependent manner. These results show that, in SaOs cells, transfected ER{alpha} isoforms mediate their action via both AF-1 and AF-2 and consequently hER{alpha}46 is able to transactivate ERE-mediated reporter gene expression after binding of estrogen. This indicates that the influence of the cell context on the spectrum of action of ERs is more complex than previously anticipated with three scenarios now described: HeLa as an AF-2 context, HepG2 as an AF-1 context, and SaOs acting in a mixed AF-1/AF-2 context. As a functional consequence of hER{alpha} isoform expression, the proliferation of SaOs cells is altered in a dose-dependent manner.

The generation of transgenic mice devoid of ER{alpha} was an important experiment by which to define the role of ER{alpha} (24). One possible outcome could have been that the {alpha}ERKO would be lethal given the broad range of tissues influenced by E2 action through ER. However, both sexes of the homozygous {alpha}ERKO mice show no grossly altered phenotype and develop normally, with the exception of being infertile. After the identification of ERß, this result and that of the human individual with a disrupted ER{alpha} gene have been ascribed to the rescue of the lack of functional ER{alpha} by ERß (31, 32). From our data and reports of other groups (33, 34), it is now evident that ERß plays only a minor role in bone. In the construction used for the knockout mouse, the disruption of the ER{alpha} gene was performed within mouse exon 2, which corresponds to hER{alpha} exon 1. Our data indicate that the modest effects on bone in the {alpha}ERKO mouse may be due, in part, to the occurrence of shorter isoforms whose translation is initiated downstream from the insertional disruption. In this regard, uterine tissue from {alpha}ERKO mice was able to specifically bind 3H-E2 (up to 10% of levels detected in the wild-type mice) that could be immunoprecipitated by H222 antibody (35). Recently, we performed a detailed analysis of the 5'-upstream region of the mER{alpha} gene, which showed that it is a complex genomic unit exhibiting multiple alternative splicing and promoter usage (15). The results obtained from primary {alpha}ERKO bone cells demonstrate that the mER{alpha} gene also generates alternatively spliced transcripts that are characterized by a deletion of the A/B domain and therefore correspond to the shorter hER{alpha} isoform in bone that is described in this study. Other transcripts have also been described and could yield open reading frames that contribute to an overall minor impact of the {alpha}ERKO (35, 36). Recent data demonstrate that another ER{alpha} isoform is expressed in the uterus of ERKO mice (Kos, M., unpublished data); therefore, it can be speculated that other mER{alpha} isoforms can mediate the effects of estrogens in other tissues such as bone.

Further work addressing ER isoform expression in osteoblasts and in osteoclasts is required to fully elucidate the physiological role of these two receptor proteins in the cell lineages that regulate the balance of bone metabolism. The benefit of studying ER in tissues, as opposed to the standard cell lines in culture, is also highlighted. We present evidence that 1) hER{alpha}46 is expressed at a significant level and 2) is able to form heterodimers with hER{alpha}66 and hERß and 3) has the capacity to modulate ER{alpha}- and ERß-mediated transactivation and increases the complexity of ER signaling. The results of this study contribute to the knowledge and understanding of how E2 and E2 analogs mediate their pleiotropic effects in a variety of physiological and pathological processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Tissues
All cell lines were maintained in DMEM (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% FCS (Life Technologies, Inc.), penicillin (100 U/ml, Life Technologies, Inc.), and streptomycin (100 µg/ml, Life Technologies, Inc.) at 37 C and 5% CO2. Human primary osteoblasts were isolated from trabecular bone samples from patients who underwent hip or knee operations under sterile conditions. Outgrowing osteoblasts were cultivated in DMEM + 20% FCS and characterized by staining for alkaline phosphatase activity (Sigma, Deisenhofen, Germany). Primary osteoblasts were trypsinized and propagated when local confluency was obtained and were used in experiments until passage 3.

ER{alpha} Expression Vector Preparation
The expression vector plasmid HEO (kindly provided by P. Chambon) was used to obtain the full-length hER{alpha} receptor protein with a molecular mass of 66 kDa. To create the expression vector coding for hER{alpha} of 46 kDa (hER{alpha}46), the hER{alpha} coding region from position +519 up to +1,788 was amplified by PCR, using HEO as template DNA. Primers were designed to introduce BamHI restriction sites at the ends of the resulting PCR product. The nested primers contained EcoRI and BamHI restriction sites, which were used to subclone the amplified fragment into the polylinker of pSG5 expression plasmid, downstream of SV40 promoter to obtain the expression vectors pSGhER{alpha}66 and pSGhER{alpha}46. All cloned PCR products were validated by dideoxy sequencing.

S1 Nuclease Assay
A modified S1 nuclease mapping procedure was followed as described by Flouriot et al. (37). This method allows the generation of highly labeled single-stranded DNA probes by extension from a specific primer by T7 DNA polymerase in the presence of [{alpha}32-P]deoxy-CTP (3,000 Ci/mmol). The probes are then hybridized with the appropriate RNA sample and subjected to S1 nuclease digestion. To prepare templates for different probes, RT-PCR reactions were performed. The PCR products were subcloned into the TA cloning vector pCR 2.1 (Invitrogen, San Diego, CA). A PCR reaction was then performed using a biotinylated forward primer together with a reverse primer either from vector (M13) or hER{alpha} coding region. To characterize the abundance of the different 5'-variants, the probes were designed to contain specific 5'-sequences of exon 1A-1F up to exon 2. All probes were constructed by PCR with a 5'-biotinylated primer located within the vector sequence to allow discrimination of free probe from protected fragments.

RNA Isolation and RT-PCR of hER{alpha} Isoforms
Expression of different hER{alpha} isoforms was determined using reverse transcription of RNA followed by PCR and Southern blotting. Reverse transcription of total RNA was performed using 1 µg of total RNA, an oligonucleotide primer (PRT) from exon 8, located within the 3'-UTR of hER{alpha} (5'-TTGGCTAAAGTGGTGCATGATGAGG) with 50 U of Expand reverse transcriptase (Roche Diagnostics, Mannheim, Germany) following the protocol of the supplier. Two microliters of this reaction were then used for two rounds of 35 cycles of PCR amplification. The 5'-primers and nested primers used for the amplification of hER{alpha} isoforms A, B, C, E, and F were A1 (5'-CTCGCGTGTCGGCGGGACAT and A2 (5'-GCTGCG TCGCCTCTAACCTC), B1 (5'-CTGGCCGTGAAACTCA GCCT) and B2 (5'-ATCCAGCAGCGACGACAAGT), C1 (5'-TCTCTCGGCCCTTGACTTCT and C2 (5'-CAAGCCCATGGAACATTTCTG), E1 (5'-AGCCTCAAATATCTCCAAAATCT) and E2 (5'-AATTATATTCTGTAGCTACCAAAGAAG) and F1 (5'-TTCTAT- AGCAT AAG AAGACAG) and F2 (5'-GAGTGATAATCTTC), respectively. The 3'-primer P1 (5'-ATTATCTGAACCGTGTGGGAG) was chosen within exon 8 of the hER{alpha} gene. A nested primer P2 (5'-CGTGAAGTACGACATGTCTAC) was selected upstream of primer P1. The Expand Long Template PCR system (Roche Diagnostics) was used for amplification as recommended by the manufacturer. Five microliters of each reaction were analyzed on a 1% agarose gel.

Southern Blotting
After separation on agarose gels, PCR products were transferred to nylon membranes (Hybond N+, Amersham Pharmacia Biotech, Arlington Heights, IL) with 6x saline sodium citrate (SSC) as transfer solution. The membranes were incubated in a prehybridization buffer containing 6x SSC, 5x Denhardt’s solution, 0.05% sodium pyrophosphate, 100 µg/ml salmon sperm DNA, and 0.5% SDS at 37 C for 1 h. Then, the membranes were hybridized in 6x SSC, 1x Denhardt’s solution, 0.05% sodium pyrophosphate, 100 µg/ml yeast tRNA with an oligonucleotide probe selected in exon 1, which had been end-labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech). The most stringent wash was carried out for 1 min at 55 C in 6x SSC, 0.05% sodium pyrophosphate. Specific PCR products were visualized by exposing the membranes to x-ray films.

For Southern blotting of {alpha}ERKO bone, primers from mER{alpha} exon 1 and exon 2 were used to probe the PCR products that were amplified from mouse 5'-upstream exon 1F to exon 2.

Quantitative RT-PCR
To quantitate hER{alpha} mRNA levels, we used a quantitative RT-PCR approach, based on the incorporation of a labeled fluorogenic probe. After RNA isolation, reverse transcription was performed with Expand reverse transcriptase (Roche Diagnostics) using a primer located within the 3'-UTR of ER{alpha} as described above. For the amplification of hER{alpha} F-mRNA variants, where exon 1F splices either to the common splice site at position +163 in exon 1 or directly to position +685 in exon 2, PCR was performed in a SMARTCycler (Eurogentec, Seraing, Belgium) using the downstream primers 5'-GCAGGGCAGAAGGCTCAGAA-3' (exon 1) and 5'-CCACCCTGGCGTCGATTATCT-3' (exon 2) and an upstream primer from exon 1F 5'-TGCAGGCTCCATGCTCAGAA-3'. The sequence of the probe (5'-TET-CCATGCTCCTTTCTCCTGCCCATTC-DABSYL-3') was chosen as a nested primer to the exon 1F-primer. The amplification of glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal standard for normalization.

In Vitro Transcription and Translation
In vitro transcription and translation were accomplished with the TNT coupled Reticulocyte Lysate system from Promega Corp. (Madison, WI) following the manufacturer’s protocol. The expression vectors pSGhER66 and pSGhER46 were used as templates for transcription with T7 RNA polymerase followed by translation to generate human ER{alpha} proteins (hER{alpha}66 and hER{alpha}46).

Immunoprecipitation
Human primary osteoblasts were cultivated in 15-cm TC-plates in DMEM+ 10% FCS until subconfluency. After washing twice with PBS, the cells were incubated in methionine-free DMEM + 10% methionine-free FCS for 10 h. Then, 1,000 µCi radiolabeled Pro-mix (35S-methionine/ 35S-cysteine-mix, Amersham Pharmacia Biotech) were added to each plate, and the cells were incubated overnight at 37 C and 5% CO2. After washing with cold PBS, the cells were harvested and lysed in 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for 30 min at 4 C. The in vitro transcibed and translated hER{alpha}46 and hER{alpha}66, which were translated in the presence of 35S-labeled methionine and served as positive controls, as well as the osteoblast whole cell proteins, were incubated with an unspecific antibody for 1 h (anti-{alpha}-Actin, 1 µg/ml, Roche Diagnostics), followed by binding to 5% protein-A-sepharose (Amersham Pharmacia Biotech) and centrifuged for 5 min at 10,000 rpm. The supernatants were incubated with hER{alpha}-specific antibody (H222, 1 µg/ml), followed by binding to protein A-sepharose for 1 h at 4 C. After extensive washes, Laemmli buffer was added to the precipitates, and the samples were boiled for 5 min. After centrifugation, the supernatants were separated on a 10% SDS-PAGE gel. The gel was then dried and subjected to autoradiography.

Transient Transfections
For transfection experiments, SaOs cells were plated into 6-cm dishes (Nunc, Wiesbaden, Germany) at a density of 1 x 105 cells per plate and grown in DMEM media supplemented with 10% FCS. After 3 d, the cells were washed with 1x PBS, and the medium was replaced by phenol-red free DMEM + 2.5% charcoal stripped, E2-free FCS. After an additional 24 h, transient transfections were carried out using the calcium phosphate precipitate method (38). Transfections were performed using 5 µg of luciferase reporter plasmid containing two EREs (EREtkLuc) together with 0.25 µg of a CAT plasmid to correct for transfection efficiency along with 0.5 µg of pSG5 expression plasmid encoding hER{alpha}66 and hER{alpha}46. After overnight incubation, the transfection media was removed, the cells were washed twice with PBS, and 3 ml of phenol-red free DMEM supplemented with 2.5% charcoal stripped FCS and 10-8 M E2 were added. After 48 h cells were harvested and luciferase assays and CAT ELISAs were performed using commercial kits (Roche Diagnostics). Quantification of cellular proliferation was performed by counting immunostained SaOs using the KI67 proliferation antigen that is localized in the nucleus. The number of Ki67-positive cells per 150 cells per visual field was evaluated using an Axiophot fluorescence microscope (Carl Zeiss, Jena, Germany).

EMSA
In vitro translated products or extracts of human primary osteoblasts were preincubated in GSA buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA, 3 mM MgCl2, 12% glycerol, 100 µg/ml BSA, 1 mM DTT, 107 M E2, 100 mM KCl] with 1 µg of poly (dI/dC) for 5 min at room temperature. The samples were then incubated for 20 min at room temperature with 1 ng of radioactive oligonucleotide probe (6 x 104 cpm) end labeled with [{gamma}-32P]ATP (3,000 Ci/mM) using T4 polynucleotide kinase (Roche). For supershift experiments, ER{alpha}-specific antibodies (1 mg/ml) were added 10 min after initial start of incubation. Protein-DNA complexes were then separated from free probe by nondenaturing electrophoresis on 4% polyacrylamide gels in 1x TBE. The gels were prerun at 4 C for 30 min followed by electrophoresis for 2 h at 200 V, dried subsequently, and exposed to Kodak Biomax film (Eastman Kodak Co., Rochester, NY). The sequence of the consensus ERE 30-bp oligonucleotide was derived from the 5'-flanking region of chicken apo-VLDL II gene (-186 to -156) (39). The nucleotide sequence was 5'-ctgtgctcaGGTCAgacTGACCttccatta-3' with the wild-type consensus ERE palindrome shown in capital letters.


    ACKNOWLEDGMENTS
 
The helpful discussions of Dr. Gaetano Clavenna, Dr. Francesco Colotta and Dr. Gianfranco Caselli contributed to the development of the work presented in this report. We also thank Dr. Geoffrey Greene for the generous gift of hER{alpha} antibodies.


    FOOTNOTES
 
This work, which was supported by the European Molecular Biology Organization, forms part of the GENOSPORA network program funded by the EC 5th framework program. S.D. received a fellowship from Dompé SPA (L’Aquila, Italy).

Abbreviations: AF-1, AF-2, Activating function-1 and -2; CAT, chloramphenicol acetyltransferase; {alpha}ERKO, mouse knockout model for ER{alpha}; 4-OHT, 4-hydroxytamoxifen; SERM, selective ER modulator; SSC, saline sodium citrate; UTR, untranslated region.

Received for publication February 23, 2001. Accepted for publication August 22, 2001.


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 INTRODUCTION
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 MATERIALS AND METHODS
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