Characterization of Transiently and Constitutively Expressed Progesterone Receptors: Evidence for Two Functional States

Catharine L. Smith, Ronald G. Wolford, Tara B. O’Neill and Gordon L. Hager

Laboratory of Receptor Biology and Gene Expression National Cancer Institute National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
Activated steroid receptors induce chromatin remodeling events in the promoters of some target genes. We previously reported that transiently expressed progesterone receptor (PR) cannot activate mouse mammary tumor virus (MMTV) promoter when it adopts the form of ordered chromatin. However, when expressed continuously, the PR acquires this ability. In this study we explored whether this gain of function occurs through alterations in nucleoprotein structure at the MMTV promoter or through changes in receptor status. We observed no major structural differences at the MMTV promoter in the presence of constitutively expressed PR and found its mechanism of activation to be very similar to that of the glucocorticoid receptor (GR). However, a systematic comparison of the functional behavior of the transiently and constitutively expressed PR elucidated significant differences. The transiently expressed PR is activated in the absence of ligand by cAMP and by components in FBS and has significantly increased sensitivity to progestins. In contrast, the constitutively expressed PR is refractory to activation by cAMP and serum and has normal sensitivity to its ligand. In addition, while the PR is localized to the nucleus in both cases, a significant fraction of the transiently expressed PR is tightly bound to the nucleus even in the absence of ligand, while the majority of constitutively expressed PR is not. These results strongly suggest that the PR undergoes processing in the cell subsequent to its initial expression and that this processing is important for various aspects of its function, including its ability to productively interact with target genes that require chromatin remodeling for activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
Steroid receptors have provided a very fruitful model for the action of ligand-inducible transcription factors. The classic model for steroid receptor action (reviewed in Ref. 1 ) is that these proteins exist in complexes with various heat shock proteins in the uninduced state. In this form they are inactive, unable to bind DNA and modulate transcription. Binding of their specific ligands causes a conformational change that leads to dissociation from the repressive complex. Although the exact order has not been firmly established, ligand-bound steroid receptors undergo the following events: dimerization, increased phosphorylation, sequence-specific binding to DNA, and interactions with other factors, the end result being the modulation of transcription.

This classic model became considerably more complex with the discovery that some steroid receptors can be activated by other hormones and signal transduction pathways in the absence of their specific ligands (reviewed in Ref. 2 ). While the receptors for glucocorticoids (GR) and mineralocorticoids (MR) are generally refractory to activation by agents other than their specific ligands, receptors for progesterone (PR), estrogen (ER), and androgens (AR) can be activated through several other signaling pathways. These receptors have been shown to be activated by some inducers of cAMP signaling such as 8-Br-cAMP (3 4 5 6 ) and dopamine (7 ), and also by peptide growth factors such as epidermal growth factor (4 8 ), insulin growth factor (6 9 ), and keratinocyte growth factor (10 ). The mechanism of ligand-independent activation may not be uniform. While ligand-independent activation of the ER is correlated with changes in receptor phosphorylation (6 11 ), such activation of the chicken PR is independent of receptor phosphorylation (12 ).

Activation of steroid receptors can lead to changes in the chromatin structure of some target genes in vivo, as manifested by the appearance of hormone-inducible nuclease hypersensitive sites (13 14 15 16 17 ). Recently, a number of chromatin modifying factors have been identified that functionally interact with steroid receptors. Various members of large multi-subunit complexes known as SWI/SNF have been shown to be necessary for GR activation of transcription in both yeast and mammalian cells (18 19 20 21 ). These complexes have been shown to disrupt reconstituted nucleosomes and thereby facilitate the interaction of various transcription factors with their DNA binding sites (22 23 24 ).

Steroid receptors also interact with histone acetyltransferases (HATs). Acetylation of lysine residues in the tails of core histones is thought to reduce the electrostatic interactions between DNA and the very basic histone tails, thereby loosening contacts between the DNA and the core histones and increasing access of transcription factors (25 26 ). Several HATs, including CREB-binding protein (CBP)/p300, p300/CBP associated factor (PCAF), activator of thyroid and retinoic acid receptors (ACTR), and steroid receptor coactivator 1 (SRC-1) (27 28 29 30 31 ) can interact with each other as well as with the carboxy-terminal activation domains [activation function 2 (AF-2)] of various steroid receptors. In addition, the amino-terminal transcriptional activation domain of the GR (AF1 or {tau}1), can interact with the factor Ada2 both physically and functionally (32 ). Ada2 forms a complex with at least two other proteins, Ada3 and Gcn5 (33 34 ), which can acetylate histones in nucleosome cores (35 ).

The mouse mammary tumor virus (MMTV) promoter has been used as a model for GR activation mechanisms in chromatin. When this promoter exists in cells in a stably replicating form, it has a highly ordered nucleoprotein structure consisting of six nonrandomly positioned nucleosome families (36 37 ). Upon activation of the GR a region of the promoter containing its binding sites undergoes a chromatin remodeling event characterized by the formation of a nuclease-hypersensitive site (36 ). As a result, other transcription factors previously excluded from their sites are able to gain access to the promoter (38 39 40 ). The GR, PR, AR, and MR are members of a nuclear receptor subgroup that bind to the same DNA sequences. However, the mechanisms by which target gene specificity is achieved in vivo are not clearly understood. We have previously shown that the ability of the PR to activate the MMTV promoter in replicating chromatin is conditional (41 ). When it is transiently expressed (less than 48 h) in transfected cells, it is a very poor activator of the MMTV template in chromatin because it fails to induce the remodeling event in the promoter (42 ). Transiently transfected GR activates this template efficiently, indicating that the GR and PR have different requirements for productive interactions with chromatin. In contrast, both receptors efficiently activate transiently transfected MMTV reporter constructs that do not have a repressed nucleoprotein conformation and thus do not require remodeling (43 ). However, the PR gains the ability to activate the replicating MMTV template upon constitutive expression in stably transfected cells (41 ).

Since all endogenous genes exist in the context of complex nucleoprotein structure, it is important to understand how steroid receptors function in this environment. We are interested in defining the requirements for PR interaction with chromatin. One possible mechanism by which the PR becomes competent to activate the MMTV promoter in ordered chromatin is that it alters the nucleoprotein structure of the template. In the course of multiple rounds of DNA replication, when nucleosomal restraints are temporarily removed, the PR might access its binding sites and influence the reassembly of the chromatin. In fact, the PR has been shown to maintain an integrated MMTV promoter in a constitutively remodeled state in a human mammary adenocarcinoma cell line (44 45 ). An alternative mechanism is that some feature of intracellular PR status may change over time. In this model the PR would be processed by the cell to make it a fully functional transcription factor, able to activate target genes that require the type of chromatin remodeling associated with the formation of nuclease-hypersensitive sites, as well as those, such as the transient MMTV template, that do not require this type of remodeling.

To distinguish between these possibilities, we have characterized various aspects of PR function when it is transiently or constitutively expressed. We find no major structural alterations in the replicating MMTV template in the presence of constitutively expressed PR. In fact, this receptor appears to activate the template by a basic mechanism highly similar to that used by the GR. In contrast, we observed major differences in the functional activity of the PR, indicating that some type of time-dependent PR processing takes place, which changes its response to extracellular signals and its ability to interact productively with chromatin. This work provides new insights into the regulation of PR function and has important implications for the way in which we study steroid receptor function in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
Generation of Cell Lines Constitutively Expressing PR
In our previous report we examined PR activity in fibroblast (NIH3T3)-derived cell lines (41 ). However, more biologically relevant insight into PR function might be gained by using cell lines derived from tissues in which PR would normally be expressed. Therefore, we generated clonal cell lines by stable transfection of 1470.2 cells with a chicken PR expression vector (Fig. 1AGo). This cell line is derived from a murine mammary adenocarcinoma line (C127i), expresses moderate levels of GR, and contains multiple copies of stably replicating MMTV-chloramphenicol acetyltransferase (CAT) transcription units in the context of bovine papilloma virus (BPV) sequences (46 ). Drug- resistant cell pools were expanded and assayed for activation of transiently transfected and replicating MMTV templates by progestin. In individual cell pools we observed that both templates were activated by progestins or that neither was activated (data not shown). One of the cell pools that showed activation was then subjected to single cell cloning to isolate clonal cell lines, of which cell line 3017.1 is representative in terms of progestin-induced activation of the MMTV promoter. Table 1Go shows the results of hormone binding analysis. These cells express slightly more PR (133.1 fmol/mg) than GR (93.3 fmol/mg) as determined by Scatchard analysis. The calculated dissociation constants for each receptor are in the expected range (47 48 49 50 ).



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Figure 1. Comparison of PR Expression and Activity at the Replicating MMTV Template in Highly Related Cell Lines

A, Cell line 3017.1 was derived from cell line 1470.2 after stable transfection with a chicken PR expression vector, pcPRO, and subsequent drug selection and clonal expansion. Both cell lines contain stably replicating MMTV-CAT transcription cassettes and express GR, but only 3017.1 cells constitutively express PR. B, RNA was isolated from either 3017.1 cells or 1470.2 cells that had been transfected with pcPRO and pCMVIL2R, grown in the presence of charcoal/dextran-treated serum, and sorted by the magnetic affinity method. Cells had been treated as shown for 3 h at the following concentrations: 100 nM Dex, 30 nM R5020. ß-Actin and MMTV-CAT mRNA levels were determined by S1 nuclease analysis. C, Fold inductions of MMTV-CAT mRNA generated by Dex or R5020 treatment of 3017.1 cells in three independent experiments were subjected to statistical analysis to calculate averages and SEs. The average induction from R5020 treatment was divided by the average induction from Dex treatment to obtain the R5020/Dex ratio. D, Whole-cell extracts isolated from 3017.1 cells or sorted 1470.2 cells that had been transfected with pcPRO were subjected to SDS-PAGE, Western transfer, and immunoblotting with an antibody directed against both A and B forms of the chicken PR.

 

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Table 1. Hormone Binding Analysis-3017.1 Cells

 
Ability of Transiently and Constitutively Expressed PR to Activate a Replicating MMTV Template
With our two highly related cell lines, we sought to compare the relative abilities of transiently expressed and constitutively expressed PR to activate the replicating MMTV template when expressed at the same level. In addition, we wanted to determine how the constitutively expressed PR activated the replicating template relative to the mechanism determined for GR. Therefore, we titrated the amount of PR expression vector transfected into 1470.2 cells so that expression of the PR in the transfected population of cells would be roughly equal to that of the PR in 3017.1 cells. All further transfections were carried out using these conditions. Immunoblot analysis demonstrates that transfected 1470.2 cells, which were sorted by magnetic affinity (see Materials and Methods), express the same amount of PR as the 3017.1 cells (Fig. 1DGo). Under the same transfection conditions, 1470.2 cells were treated as shown in Fig. 1BGo and sorted to isolate the transfected population. RNA from the various treatment conditions was subjected to S1 nuclease analysis using MMTV- and actin-specific probes. At this level of PR expression there was no significant induction of MMTV mRNA in transfected 1470.2 cells treated with the synthetic progestin, R5020. In sharp contrast, the constitutively expressed PR in 3017.1 cells was able to induce significant amounts of mRNA from the replicating MMTV template when activated by R5020. The differences in activation of the MMTV promoter were not due to significantly altered levels of PR in the two cell lines after 3 h of treatment with R5020 (see Fig. 5BGo).



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Figure 5. Dose Response of Transiently and Constitutively Expressed PR

A, R5020 dose-response curves for transiently and constitutively expressed PR. Extracts were isolated from 3017.1 cells transfected with pLTRluc or 1470.2 cells transfected with pLTRluc and pcPRO that had been treated for 6 h with various concentrations of R5020 in the presence of charcoal/dextran-treated serum. Luciferase activities were normalized to protein and fold inductions were calculated relative to normalized luciferase activity in cells treated with 1 pM R5020. Curve fitting and calculation of EC50 values were carried out using Prism software (GraphPad Software, Inc., San Diego, CA). B, Analysis of PR levels in response to treatment with R5020 (30 nM) for 0, 3, or 6 h. In the case of 1470.2 cells, sorting of transfected cells by magnetic affinity was carried out before preparation of whole-cell extracts. Equal amounts of extract protein were subjected to SDS-PAGE and Western blotting with the PR22 antibody.

 
Although the PR is expressed at a slightly higher level than the GR in 3017.1 cells, it is not as efficient in activating the replicating MMTV template. Fig 1CGo shows average fold inductions by both hormones in 3017.1 cells. Constitutively expressed PR induces MMTV transcription from the replicating template to a level roughly half that induced by the endogenous GR. However, both receptors are equally efficient in activating a transiently transfected MMTV reporter, as shown in Figs. 4CGo and 6BGo. In support of our previous study (42 ), this observation indicates the existence of rate-limiting cofactors that are necessary for PR induction of the MMTV promoter only when incorporated into an ordered chromatin structure.



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Figure 4. PR Activity in Response to Serum

A, Whole-cell extracts were isolated from 3017.1 cells or sorted 1470.2 cells transfected with pcPRO and pCMVIL2R that had been cultured in either regular, untreated FBS, or charcoal-stripped (charcoal/Dextran-treated) serum. PR levels were detected with an antibody directed against both forms of the chicken PR after SDS-PAGE and Western transfer. B, 1470.2 cells were transfected with pLTRluc in the presence or absence of pcPRO and treated as shown for 6 h with concentrations of steroids described in Fig. 1Go. Cell extracts were assayed for luciferase activity, which was then normalized to total protein. Results from three independent experiments were subjected to statistical analysis and expressed as fold inductions relative to the untreated control grown in charcoal-stripped serum. C, 3017.1 cells were transfected with pLTRluc. Extracts were assayed and results were calculated as described for 1470.2 cells.

 


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Figure 6. PR Response to cAMP Signaling

A, 1470.2 cells were transfected with pLTRluc in the presence or absence of pcPRO. Cells were grown in the presence of charcoal/dextran-treated serum and treated as shown for 6 h. Final concentrations of Dex and R5020 used are described in Fig. 1Go. 8-Br-cAMP was used at a final concentration of 1 mM. Luciferase activities were normalized to protein. The data from five independent experiments are represented as fold inductions relative to untreated controls. B, 3017.1 cells transfected with pLTRluc were treated as described above. Data were analyzed as described also. C, Cells were transfected as described above except that the IL2R expression vector was included in the transfections of 1470.2 cells. Cells were treated with or without 8-Br-cAMP (1mM) for 6 h. Before preparation of whole-cell extracts, 1470.2 cells were subjected to sorting by magnetic affinity. Equal amounts of extract protein were separated by SDS-PAGE and subjected to Western blotting with the PR22 antibody.

 
Comparison of Mechanisms Used by GR and PR to Activate the Replicating MMTV Template
One possible model for the gain in PR function is that the nucleoprotein structure of the MMTV template changes over time to accommodate a PR-specific mechanism of action. The PR may cause these alterations through interaction with its binding sites in the MMTV promoter during DNA replication and subsequent reassembly of chromatin. Several previous studies (44 45 ) have shown that an integrated MMTV template in a human mammary adenocarcinoma cell line expressing endogenous PR has an altered nucleoprotein structure in which the chromatin is in a constitutively remodeled state.

We compared the mechanism of GR and PR activation of the MMTV template in chromatin by several criteria. The most obvious question is whether the proximal promoter region containing the hormone response elements (HREs) is in a constitutively open state in 3017.1 cells, or whether it undergoes remodeling in response to hormone treatment as it does in the parental 1470.2 cells. The remodeling event can be measured by SacI cleavage of the MMTV promoter in nuclei from treated cells (43 ). SacI cleaves the promoter in the area of the proximal set of HREs. The extent of cleavage changes approximately 10–20% upon dexamethasone (Dex) treatment in a variety of cell lines with replicating forms of the MMTV long terminal repeat (LTR) (51 ). Figure 2AGo shows the results of the SacI cleavage assay in both 1470.2 and 3017.1 cells. In 1470.2 cells Dex treatment induces a change in SacI cleavage of 11.6%, which is nearly identical to that induced in 3017.1 cells, as is shown graphically in Fig. 2BGo. Because the GR is clearly able to induce hypersensitivity in 3017.1 cells and the change in SacI cleavage observed in the two cell lines is of the same magnitude, we think it unlikely that MMTV chromatin in 3017.1 cells exists in a constitutively remodeled state. As shown in Fig. 2AGo, R5020 treatment also results in the induction of SacI cleavage in 3017.1 cells. The change in cleavage averages to 6% (Fig. 2BGo). Remarkably, the ratio of R5020- to Dex-induced cleavage is virtually identical to the same ratio applied to induction of RNA as shown in Fig. 1CGo, 0.53 to 0.52, respectively. This strongly suggests that the GR and PR activate transcription of the replicating MMTV template to the extent to which they can remodel the chromatin.



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Figure 2. Analysis of Chromatin Remodeling at the MMTV Promoter

A and B, SacI access analysis was carried out on nuclei that had been isolated from 1470.2 cells or 3017.1 cells treated as shown for 1 h at the concentrations described in the legend to Fig. 1Go. DNA from the digested nuclei was isolated and digested to completion with Dpn II. Digestion products were detected by multiple rounds of linear amplification with a labeled MMTV primer. Fractional cleavage by SacI in each sample was determined by calculating the ratio of SacI cleavage to total cleavage (DpnII plus SacI). A, A representative analysis of SacI cleavage in 1470.2 (left panel) and 3017.1 (right panel) nuclei is shown. B, The changes in fractional cleavage induced by either Dex or R5020 (relative to basal levels of cleavage in untreated cells) in three independent experiments with 3017.1 cells were averaged and subjected to statistical analysis. The R5020/Dex ratio was calculated as described in Fig. 1Go. C, Exonuclease analysis was carried out on nuclei isolated from 3017.1 cells treated as shown. Digestion products were measured by linear amplification as described above.

 
Another hallmark of GR-induced activation of the replicating MMTV template is the hormone-dependent binding of the ubiquitous transcription factor NF1 (38 39 ). In the absence of hormone, the repressive chromatin structure of the template excludes NF1 from accessing its binding site (43 ). The remodeling event induced by the binding of activated GR then allows NF1 to bind and participate in the activation of transcription. We therefore carried out exonuclease analysis on nuclei from 3017.1 cells treated as shown in Fig. 2CGo. The results show that both GR and PR can significantly induce the binding of NF1 to the replicating MMTV template, further indicating that the promoter is not in an open state in the absence of hormone. This result stands in contrast to the cell line in which PR holds MMTV chromatin in an open state; NF1 binding to the promoter was strong in the absence of ligand and did not increase upon activation of the PR (44 ).

Activation of MMTV transcription by the GR is transient, peaking at 1 h posttreatment and declining to near basal levels by 8–12 h even in the continued presence of hormone. This change in transcription correlates with a reversion of the nucleoprotein structure to a repressive state (52 53 ). This is a chromatin-specific effect because transiently transfected MMTV reporter constructs, which do not undergo hormone-induced remodeling, remain activated in the continued presence of hormone (39 ). We asked whether PR, after it initially remodels the chromatin and activates transcription, keeps the promoter active for an extended period of time. Figure 3Go shows the levels of MMTV mRNA generated from the replicating template for short-term and long-term hormone treatment of 3017.1 cells. It is clear that both receptors activate the template strongly over the short term, but after 24 h of hormone treatment, RNA levels have declined significantly. Thus, the constitutively expressed PR appears to follow the same transient kinetics of MMTV promoter activation as the endogenous GR.



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Figure 3. Time Course of Induction of MMTV mRNA by GR and PR in 3017.1 Cells

RNA was isolated from 3017.1 cells that had been treated as shown for 0, 3, or 24 h. Concentrations of Dex and R5020 used are described in Fig. 1Go. MMTV-CAT and actin mRNA levels were measured by S1 nuclease analysis.

 
By all the criteria we examined, once the PR becomes competent to activate the MMTV promoter in ordered chromatin, the mechanism by which it does so is very similar to that of the endogenous GR. The constitutively expressed PR has not changed the structure of the template in a major way, such as derepressing the proximal promoter region as reported in a different cell line (44 ). However, we cannot rule out more subtle changes in the nucleoprotein structure of the template such as a shift in the frequency of occupied nucleosomal frames.

Functional Comparison of Transiently and Constitutively Expressed PR
Another possible model for the change in the ability of the PR to activate the replicating MMTV template over time is based on intracellular receptor status rather than template structure. The PR may undergo a change in processing over time, which may have functional outcomes. This might be reflected by functional differences in PR activity at the transient MMTV template, which poses no barriers to activation. Therefore, we measured various aspects of PR function on a transiently transfected MMTV reporter construct in 3017.1 cells and also in 1470.2 cells cotransfected with the PR expression vector, pcPRO.

Typically, experiments done on cells transfected with various steroid receptor expression vectors are carried out in the presence of charcoal-stripped FBS since untreated serum contains levels of steroids that might result in activation of the receptors. However, our experience with 3017.1 cells and other PR-expressing lines indicated that untreated serum does not activate the PR. We therefore designed a set of experiments to systematically determine whether the transiently and constitutively expressed forms of the PR respond differently to serum. We transfected 1470.2 cells with pLTRluc, in the presence or absence of the PR expression vector, and then cultured the cells in medium containing charcoal-stripped serum or regular, untreated serum. The 3017.1 cells were transfected with pLTRluc alone and cultured in the same fashion. To ensure that equal levels of PR were being expressed in 3017.1 and in transfected 1470.2 cells grown in the two types of serum, we did a parallel experiment in which we included the IL-2 receptor expression vector and isolated the transfected 1470.2 cell population by magnetic affinity. Cytosols isolated from 3017.1 cells and sorted 1470.2 cells were examined for PR expression by immunoblotting. As shown in Fig. 4AGo, both PR isoforms, A and B, are expressed at similar levels in transfected 1470.2 and in 3017.1 cells, regardless of the type of serum in which they were cultured. Therefore, any observed differences in PR activity are not due to altered PR expression levels.

Figure 4BGo shows the results of analysis in 1470.2 cells transfected with pLTRluc in the presence or absence of the PR expression vector. The data are expressed as fold inductions relative to the untreated control grown in charcoal-stripped serum. In the absence of pcPRO, there was a relatively strong induction of luciferase activity by Dex treatment and, as expected, no induction by R5020. Growth of the transfected cells in untreated serum slightly boosted the level of luciferase activity generated under all conditions. In the presence of the PR expression vector, both Dex and R5020 treatment resulted in the induction of luciferase levels when the cells were grown in charcoal-stripped serum. However, in untreated serum the basal level of luciferase activity was elevated 5-fold relative to basal levels in stripped serum. Treatment with Dex or R5020 resulted in greater amounts of transcriptional activity but the inductions were small, around 2-fold. These observations indicate that growth of the transfected cells in untreated serum leads to the partial activation of the transfected PR without the addition of ligand. In contrast, neither the basal level of luciferase activity nor the hormone inductions were affected by growth of transfected 3017.1 cells in charcoal-stripped vs. untreated serum (Fig. 4CGo). Unlike the transiently expressed PR, the constitutively expressed PR is refractory to activation by serum.

There are two possible explanations for serum-induced activation of the transiently expressed PR. Either a signal transduction pathway is induced selectively by the untreated serum that leads to ligand-independent activation, or the concentration of progestins in the untreated serum is sufficient to cause partial activation of the PR. This latter possibility seems unlikely given that the constitutively expressed PR in 3017.1 cells was not activated at all by untreated serum. However, it has been shown recently that increasing amounts of transiently expressed GR can cause a left shift in the dose-response curve for activation of a glucocorticoid-inducible promoter (54 ), which indicates an increasing sensitivity to ligand.

Thus, we carried out an R5020 dose-response experiment on 1470.2 cells transfected with pcPRO and pLTRluc and also on 3017.1 cells transfected with the latter. Transfected cells were grown in charcoal-stripped serum to ensure maximal hormone inductions. As shown in Fig. 5AGo, the transiently expressed PR gave rise to a significantly left-shifted dose-response curve when compared with that generated by the constitutively expressed PR. In the case of the latter, the dose response could be considered typical for the PR, the EC50 (0.89 nM) being similar to the Kd (0.58 nM, see Table 1Go). For the transiently expressed PR, this was not the case. Its EC50 (0.056 nM) was more than 1 order of magnitude lower than its measured Kd of 0.4 nM (data not shown), which is in agreement with observations made by others (48 55 ) (see Discussion). Levels of PR were analyzed by Western blotting of whole-cell extracts made after 0, 3, and 6 h of R5020 treatment (Fig. 5BGo). Since receptor levels were comparable between the two cell lines at all time points tested, this cannot be the reason underlying the differences in EC50. Therefore, the two forms of the PR have the same affinity for R5020, but differ significantly in their sensitivity to this ligand.

Based on the progesterone content of the untreated serum provided by the manufacturer, we estimate that the progesterone concentration in our complete media is approximately 0.02–0.03 nM. According to the dose-response curves in Fig. 5Go, this amount of progesterone may result in a 2- to 3-fold activation of the transfected MMTV template by the transiently expressed PR but would not be sufficient to activate the constitutively expressed receptor. Transiently expressed PR has been shown to be partially activated by EGF treatment (4 ). We do not know the epidermal growth factor (EGF) content of our serums but serum mitogens are undoubtedly removed by charcoal-dextran treatment since most of our cell lines grow at a reduced rate in the stripped serum (C. L. Smith, unpublished observations). It is possible that the PR-dependent, 5-fold induction of the transfected MMTV promoter in untreated serum is due to a combination of endogenous progesterone and higher levels of serum mitogens.

In addition to EGF-induced signaling, it has been reported that the chicken PR is activated very well by cAMP signaling in a ligand-independent fashion (3 4 12 ). However, in each of these studies, the PR had been transiently expressed. We therefore tested PR activity in the presence of cAMP in our highly related cell lines. In transfected 1470.2 cells treatment with 8-Br-cAMP caused a PR-dependent induction of luciferase activity (Fig. 6AGo), which was, on average, greater than that generated by either Dex or R5020 alone, an observation also made in other studies. Once again, the behavior of the constitutively expressed PR was different. There was very little induction of luciferase activity by 8-Br-cAMP treatment in 3017.1 cells (Fig. 6BGo). The amount of induction observed is very similar to that induced by cAMP treatment in 1470.2 cells in the absence of transiently expressed PR. Analysis of PR expression levels in both cell lines indicates that they are unchanged by treatment of cAMP (Fig. 6CGo), which is in agreement with other studies (12 56 ). These results led to the conclusion that while the transiently expressed PR is easily activated by cAMP signaling the constitutively expressed form of the PR is refractory to this form of ligand-independent activation. Dopamine, which can activate the cAMP signaling pathway, has also been shown to activate PR in a ligand-independent fashion (7 ). Unfortunately, our cell lines do not appear to express the D1 dopamine receptor because they do not respond to dopamine at all (T. B. O’Neill and C. L. Smith, unpublished observations).

Localization and Nuclear Binding Properties of Transiently and Constitutively Expressed PR
Taken together, our data imply that transiently expressed and constitutively expressed PR respond differently to extracellular signals, the latter being more refractory to activation than the former. The dramatically enhanced sensitivity of the transiently expressed PR to activation suggests that it has greater access to both coactivators and its binding sites on the transfected MMTV template. One possible explanation for this could be an altered partitioning between the nucleus and cytoplasm relative to the constitutively expressed PR. A predominantly nuclear localization in the absence of ligand could result in greater accessibility to DNA and transcription complexes once the receptor is activated. Therefore, we carried out indirect immunofluorescence experiments on 3017.1 and transfected 1470.2 cells. Cells were either untreated or treated with 30 nM R5020 for 1 h before fixation. As shown in Fig. 7Go, the PR is predominantly localized to the nucleus regardless of whether it is transiently or constitutively expressed. In the absence of ligand there is a small fraction of fluorescence in the cytoplasm that appears to move into the nucleus upon treatment with ligand. These results are different from those reported for the transiently expressed human PR(B) (57 ). Its predominant localization pattern was a distribution between the nucleus and cytoplasm. Since those experiments were carried out in a cell line very similar to ours, the different localization pattern is probably due to species-specific differences between the chicken and human PR.



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Figure 7. PR Localization by Indirect Immunofluorescence

Transfected 1470.2 cells (A and B) or 3017.1 cells (C and D) were either not treated (A and C) or treated with R5020 (30 nM) for 1 h (B andD). Cells were fixed, permeabilized, and exposed sequentially to primary antibody against the B and A isoforms of the PR and Texas Red-conjugated secondary antibody.

 
Although receptors such as ER and PR reside in the nucleus even in the absence of ligand, they are not tightly bound to the nucleus until activated by ligand (58 59 ), presumably because they are transcriptionally engaged on chromatin. The nuclear binding properties of these receptors can be determined through salt dependence of extraction from the nucleus. Therefore, we made whole-cell, nuclear, and cytosolic extracts from either 3017.1 cells or the transfected population of 1470.2 cells in the presence or absence of R5020. Cytosolic extracts were made by lysing the cells in buffer containing no sodium chloride. Nuclei were then pelleted, washed, and exposed to buffer containing 250 mM NaCl. Higher NaCl concentrations did not result in greater amounts of PR extraction (data not shown). Immunoblot analysis of the PR extracted under the various conditions is shown in Fig. 8Go. Equal amounts of protein from whole-cell extracts (lanes 1 and 2) show not only that total PR levels are equivalent in the two cell lines, but also that the ratio of isoforms is very similar. Equal fractions of cytosolic and nuclear extracts were used to compare relative PR concentrations (lanes 3–10). In the absence of R5020, the partitioning of both isoforms is different in the two cell lines. In the transfected 1470.2 cells, the A form of the PR is found predominantly in the nuclear extract, while the B form is more evenly distributed (lanes 3 and 4). In contrast, both forms of the constitutively expressed PR are significantly enriched in the cytosol (lanes 7 and 8).



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Figure 8. Nuclear Binding Properties of Transiently and Constitutively Expressed PR

Cytosolic and nuclear extracts were isolated from transfected 1470.2 cells (sorted IL2R+ population) and 3017.1 cells as described in Materials and Methods. Treatment with R5020 was carried out for 1 h before harvest. Equal fractions of each extract were separated by SDS-PAGE and transferred to nitrocellulose. Both PR isoforms were detected with the use of the PR22 antibody. Signal detection was carried out by chemiluminesence.

 
In 1470.2 cells exposed to R5020, there is little redistribution of the transiently expressed PR between the nuclear extract and the cytosol. However, the small amount of A form that partitions with the cytosol in the absence of R5020 redistributes to the nuclear extract upon treatment (lanes 3–6). In the case of the constitutively expressed PR, the ligand-dependent repartitioning of the A form to the nuclear extract is dramatic (lanes 7–10). The B form also becomes more enriched in the nuclear extract relative to the uninduced state (compare lanes 8 and 10) but more evenly partitions between the cytosol and nuclear extracts. This analysis shows that a significant fraction of the transiently expressed PR is tightly bound to the nucleus in both the presence and absence of ligand. In contrast, the constitutively expressed PR shows significantly more redistribution upon activation by ligand.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
PR plays a crucial role in a variety of processes involved in reproduction including ovulation, uterine response to hormonal stimulation, mammary gland development, and some sexual behaviors (60 61 ). In many progesterone target tissues, PR expression is controlled by cyclic changes in other hormones, primarily estrogen. Our results show that the PR can exist in two functional states, raising the possibility of an alternate mechanism of PR regulation in vivo, or a pathway by which PR function could become abnormal in the neoplastic state.

We have shown previously that, when the PR is transiently expressed, it is a poor activator of the MMTV promoter, having an ordered and repressive chromatin structure (41 ). This deficiency in activation results from the inability of the PR to induce the chromatin remodeling event (42 ), which is an integral step in the transactivation of the MMTV promoter by GR (43 ). This form of the PR is not generally deficient in transcriptional activity, because it efficiently activates a transiently transfected form of the MMTV promoter (42 ), which does not have an ordered nucleoprotein structure (43 ). The action of the transiently expressed PR may therefore be restricted to endogenous target genes that are not in a repressed conformation and do not require a remodeling event characterized by the formation of a nuclease-hypersensitive site. Upon prolonged expression in our cell lines, the PR undergoes a dramatic increase in its ability to activate the stably replicating form of the MMTV promoter. Although the constitutively expressed PR is present at a level slightly higher than that of the endogenous GR, it is only about half as efficient as the GR in activating transcription from this promoter. This difference in transactivation potential is not observed at the transient MMTV template, which is activated by the constitutively expressed PR to approximately the same extent as by the endogenous GR (see Figs. 4BGo and 6BGo). This observation implies that PR-induced activation is limited by a feature of its interaction with the ordered chromatin, possibly specific rate-limiting cofactors that help PR bind to and/or remodel chromatin. In fact, the ability of the PR to induce the chromatin remodeling event in the MMTV promoter region is tightly linked to its transactivation potential, again being only half as effective as the endogenous GR. Therefore, the PR activates the promoter to the extent to which it can open the chromatin.

In this study we show that the PR does not appear to gain the ability to activate the MMTV promoter in ordered chromatin through major alterations in promoter structure. Such a change might be necessary if the PR activates the MMTV promoter by a different mechanism than the GR, as was implied by the studies of Archer and colleagues (44 45 ). In a human mammary adenocarcinoma cell line containing integrated MMTV templates and expressing endogenous PR, they found that the structure of the promoter was in a constitutively hypersensitive state and that NF1 binding was no longer hormone-dependent. In our PR-expressing cell line this is clearly not the case. The MMTV promoter does not exhibit constitutive hypersensitivity or NF1 binding; both events remain hormone dependent. This discrepancy may be due to cell type differences or to the fact that PR levels in 3017.1 cells are much lower than in the cells described above. When we examined various characteristics of GR induction of the stably replicating MMTV template, we did not find that the constitutively expressed PR behaved differently. It induced the same changes in nucleoprotein structure and demonstrated the same transient activation kinetics as the GR. Therefore, if constitutively expressed PR does cause changes in template structure, they must be more subtle and not necessarily related to a PR-specific mechanism of action.

Our previous work indicated that the difference in PR function between the transiently and constitutively expressed states occurred in the presence of ligand. Our current study extends those differences to the unliganded state, strongly indicating that the PR goes from one functional state to the other through intracellular processing rather than template remodeling. We demonstrate dramatic changes in PR function at a promoter which, unlike the stably replicating MMTV template, provides no barriers to activation. In the transiently expressed state, the PR can be activated in a ligand-independent fashion by cAMP signaling and possibly other signal transduction pathways induced by exposure to serum. It also has an altered sensitivity to its own ligand, becoming maximally active at doses at least 1 order of magnitude lower than would be expected based on its dissociation constant. In fact, these doses fall at the low end of the physiological range (62 ). Thus, in vivo, this form of the receptor would have the potential to be constitutively active. In sharp contrast, the constitutively expressed form of the PR is refractory to activation by ligand-independent mechanisms and has an appropriate sensitivity to progestins. In fact, the constitutively expressed PR functions in accordance with the classical model for steroid receptor action in that it is activated only in the presence of physiological doses of its specific ligand.

Our observations of altered sensitivity to ligand are supported by other studies on steroid receptors. Simons and colleagues (54 ) recently addressed this issue in a study in which they examined activity of GR transiently expressed in Hela cells. Although interpretation of their results is more complex because Hela cells also express GR endogenously, they observed leftward shifts in the dose-response curves with increasing amounts of GR expression vector transfected. Weigel and colleagues (48 55 ) have also observed a difference between the dissociation constant and EC50 of transfected chicken PR (48 55 ). The affinity of the transiently expressed PR for ligand is in the same range as that of the constitutively expressed PR, but its EC50 for transcriptional activation is about 10 times lower. This means that the transiently expressed PR is a much more effective transactivator in that far fewer occupied receptors (relative to the constitutively expressed state) are necessary for achieving the maximal transcriptional response.

One interpretation of this phenomenon would be that the transiently expressed PR has a greater affinity for coactivators than the constitutively expressed PR, perhaps by virtue of its access to those factors, since the two receptors are identical in amino acid sequence. Although the transiently and constitutively expressed forms of the PR are predominantly localized to the nucleus, their nuclear binding properties are significantly different. In the unoccupied state, a significant fraction of the transiently expressed receptor is tightly bound to the nucleus. Upon activation with ligand, there is little change in the partitioning between the cytosol and nuclear extracts. In contrast, the majority of constitutively expressed PR is not bound tightly to the nucleus and partitions with the cytosolic extract. Upon activation with ligand, there is a significant increase in the amount of both isoforms that become tightly bound to the nucleus. These results strongly suggest that the PR can be localized to two different nuclear compartments. The transiently expressed receptor, given the fact that it is tightly bound to the nucleus even in the unoccupied state, may be constitutively associated with target genes. This contention is supported by the observation that the nuclear binding properties of the transiently expressed PR in the absence of ligand closely resemble those of the constitutively expressed PR in the presence of ligand. The constitutively expressed receptor may be targeted to a different compartment of the nucleus where it is refractory to activation by other signaling pathways. It must then relocate to its target sites when activated by ligand. In contrast, the transiently expressed PR, if already associated with target genes in the absence of ligand, may be in an environment rich in transcriptional coactivators, prepoised for activation. This would provide an explanation for its enhanced sensitivity to ligand and its ability to be activated by other signaling pathways. In this sense the localization of the unoccupied PR in the nucleus may have important effects on its function.

In the absence of ligand, steroid receptors are thought to exist in complexes with various heat shock proteins and immunophilins even if they are localized to the nucleus (63 64 ). These complexes are thought to keep the receptor in a conformation able to bind ligand but unable to bind DNA or coactivators (65 ). The majority of studies done on PR processing have been carried out on in vitro assembled receptor complexes or on receptor complexes from tissues or cells in which the expression of the receptor is endogenous. It is possible that atypical receptor/chaperone complexes may be generated in a transfected cell that is suddenly flooded with a receptor it does not express. These atypical complexes may result in a receptor that is partially transformed and localized to accessible target genes in the nucleus. Association with hsp90 and p23 is necessary to confer efficient and stable hormone binding ability on GR (66 ) and PR (67 68 ). Since affinity for ligand is the same for both forms of the PR, these associations probably occur, but may be unstable or altered in the case of the transiently expressed PR, since it behaves like it is partially transformed. In addition to hsp90 and p23, mature receptor complexes also contain one of several large immunophilins, notably FKBP52, FKBP51/54, and Cyp40, which bind directly to hsp90 (65 ). The role of such proteins in these complexes has not been firmly established, but they are reported to be involved in stability of the untransformed receptor complex (69 70 ) and trafficking of the receptor between the cytoplasm and the nucleus (71 ). Thus, the immunophilins might play a role in receptor processing (folding, posttranslational modification, etc.) and localization.

Our study suggests that the processing of the PR can have important consequences for its ability to productively interact with target genes in repressed chromatin. Even though the transiently expressed PR is promiscuously activated by other signaling pathways and has increased sensitivity to ligand, it is a poor activator of the MMTV promoter in ordered, replicating chromatin. The complexes formed by the PR and its localization in the nucleus may be important for efficient interactions with chromatin remodeling machinery in vivo. In addition, achievement of a more repressed, unoccupied state allows the PR to ignore other intracellular signals and respond to the appropriate extracellular stimuli. Since the majority of studies on steroid receptor function are carried out with receptors that are transiently expressed, it is important to know whether regulation of these receptors is representative of that which would occur in vivo. Further studies will be directed at addressing this important issue.


    MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
Cell Culture, Transfection, Cell Sorting
Cell line 1470.2 was derived from C127 mouse mammary adenocarcinoma cells and contains stably replicating copies of BPV-MMTV LTR fusions (46 ). Cell line 3017.1 was derived as a single-cell clone from 1470.2 cells after stable transfection with pRSVneo, a neomycin resistance expression vector, and pcPRO, a chicken PR expression vector (72 ). Cells were cultured in DMEM containing either 10% FBS (Life Technologies, Inc., Gaithersburg, MD; Atlanta Biologicals, Inc., Norcross, GA) or 10% charcoal-dextran-treated FBS (HyClone Laboratories, Inc.. Logan, UT). Transfections were carried out by electroporation as described previously (42 ). Typically, 1470.2 cells were transfected with 1–3 µg pcPRO, 10 µg pLTRluc, an MMTV reporter construct (73 ), and, if necessary, 5 µg pCMVIL2R, an interleukin 2 receptor (IL-2R) subunit expression vector (74 ) used for cell sorting. Transfected 1470.2 cells were grown in the presence of DMEM/10% charcoal-dextran-treated FBS unless stated otherwise. In some cases, 3017.1 cells were transfected with 10 µg pLTRluc only. Cells were treated and harvested the day after electroporation. Cell sorting was carried out by a magnetic affinity method, as described previously (42 ). Briefly, transfected cells were exposed to magnetic beads coated with antibodies to the Tac subunit of the IL2R. Beaded cells were separated from nontransfected cells with the use of magnets.

Hormone Binding Analysis
GR and PR were extracted from either 3017.1 cells or sorted 1470.2 cells by the following method. Pelleted cells were resuspended in HEDM buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 2 mM dithiothreitol, 10 mM sodium molybdate) and lysed by Dounce homogenization (A pestle). Glycerol was then added to the lysate to a final concentration of 10%. Cytosols were isolated by subjecting the lysate to centrifugation at 100,000 x g. Before use, they were maintained in liquid nitrogen. For hormone binding analysis, cytosols were divided into aliquots containing equal quantities of total protein (250–300 µg) and allowed to incubate for 90 min with various concentrations of either [3H]promegestone (NEN Life Science Products, Boston, MA) for analysis of the PR or [3H]triamcinolone acetonide (NEN Life Science Products) for analysis of the GR, in the presence or absence of a 500-fold excess of the appropriate unlabeled ligand. Free steroid was removed by exposure of the cytosols to dextran-coated charcoal [3% Norit-A (ICN Biochemicals, Inc., Cleveland, OH), 0.6% Dextran T-70 (Sigma, St. Louis, MO) in 10 mM HEPES, pH 7.3], and bound steroid was assessed by liquid scintillation counting. Hormone binding data was then analyzed by the method of Scatchard.

Analysis of RNA and Luciferase
Total RNA was isolated from 3017.1 cells or sorted 1470.2 cells and subjected to S1 nuclease analysis as described previously (46 ). Probes used were specific for detection of MMTV-CAT and ß-actin mRNA. S1 digestion products were separated on 8% denaturing gels that were dried and exposed to phosphorimaging screens. Quantitation was carried out using Imagequant software (Molecular Dynamics, Inc., Sunnyvale, CA). For analysis of luciferase activity, transfected cells were harvested by scraping, pelleted, and resuspended in 0.1 M potassium phosphate pH 7.8, 1 mM dithiothreitol. Cells were lysed by three cycles of freezing and thawing; cellular debris was removed by centrifugation. After protein analysis, 5–10 µg extract protein were analyzed for luciferase activity. Luciferase values were normalized to total protein in each sample.

Analysis of Nucleoprotein Structure
Nuclei were isolated from 3017.1 and 1470.2 cells after hormone treatment for 1 h as described previously (42 ). SacI digestion of nuclei was carried out at a concentration of 5 U per µg DNA for 15 min at 30 C in buffer containing 50 mM NaCl, 50 mM Tris pH 8.0, 1 mM MgCl2, 1 mM ß-mercaptoethanol, and 2.5% glycerol. Reactions were stopped by the addition of 5 volumes 10 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS, and 100 µg/ml proteinase K. DNA was purified and subjected to digestion with DpnII. For exonuclease analysis, nuclei were digested at 37 C for 15 min with HaeIII (1000 U/ml) in the presence or absence of {lambda} exonuclease (100–200 U/ml). An antisense oligonucleotide containing MMTV sequences from +27 to +1 bp was end-labeled with {gamma}-32P-ATP and polynucleotide kinase and used in multiple rounds of linear amplification with Taq polymerase (Stratagene, La Jolla, CA) to detect specific digestion products, which were subsequently separated on 8% denaturing gels. Quantitation of radiolabeled digestion products was carried out using a Phosphorimager and Imagequant software (Molecular Dynamics, Inc.).

Extract Preparation and Immunoblotting
For comparison of PR levels in transfected, sorted 1470.2 cells vs. 3017.1 cells, whole-cell extracts were made as follows. Cells were harvested, washed with PBS, and resuspended in HEGDM buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 2 mM dithiothreitol, 10% glycerol, and 10 mM sodium molybdate) containing 250 mM NaCl and 0.1% NP40. After a 5-min lysis period on ice, cellular debris was removed by centrifugation at 12,000 x g for 5 min at 4 C. For comparison of PR partitioning, cytosols and nuclear extracts were made as follows. Harvested cells were resuspended in HEDM buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 2 mM dithiothreitol, 10 mM sodium molybdate) and lysed by Dounce homogenization (A pestle). Glycerol was then added to the lysate to a final concentration of 10%. Nuclei were pelleted by low-speed centrifugation for 5 min and the supernatant (cytosol) was removed. The nuclei were gently resuspended in 5–10 volumes in HEGDM buffer and centrifuged again. The supernatants were combined and subjected to centrifugation at 100,000 x g for 1 h to yield the cytosolic fraction. The nuclei were resuspended again in HEGDM buffer to which NaCl was added to a final concentration of 250 mM. After incubation on ice for 30 min, the samples were subjected to centrifugation at 30,000 x g for 20 min to generate nuclear extracts. All buffers used in making extracts from R5020-treated cells contained 30 nM R5020.

Extracted proteins (20–40 µg) were subjected to SDS-PAGE (3% stack, 7.5% separating) and transferred to nitrocellulose membranes (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) in Tris glycine buffer containing 20% methanol. Before immunoblotting, membranes were blocked in Tris-buffered saline (TBS)/2% nonfat dry milk. The PR was detected using antibody PR22 (kindly provided by D. Toft) as described previously (42 ). Detection of bound antibody was carried out using chemiluminescence kits (Amersham Pharmacia Biotech or Pierce Chemical Co., Rockford, IL).

Indirect Immunofluorescence.
Cells were plated in 35-mm wells containing glass coverslips. After the appropriate treatments, cells were fixed with 3.5% paraformaldehyde in PBS (without Ca+2 and Mg+2) and permeabilized with PBS (without Ca+2 and Mg+2) containing 0.5% Triton X-100. Coverslips were incubated with primary antibody (0.2 µg/ml) diluted in PBS/0.1% Tween-20/10% calf serum overnight at 4 C. After several washes in PBS/0.1% Tween-20/10% calf serum, coverslips were exposed to secondary antibody (1:500) for 1–2 h at room temperature. After several washes in PBS/0.1% Tween-20/10% calf serum, cells were rinsed in dH2O and mounted on slides. Images were collected using a TCS SP confocal microscope (Leica Corp., Deerfield, IL). The primary antibody was directed against the both isoforms of the PR (aPR-22, Affinity BioReagents, Inc., Golden, CO). The secondary antibody was Texas Red-conjugated goat antimouse (Calbiochem, La Jolla, CA).


    Congress: 37th SEMDSA (Society for Endocrinology, Metabolism and Diabetes of South Africa) and 10th Bone & Mineral Metabolism
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 
Dates: April 1–3, 2001 (Bone & Mineral Metabolism) April 4–6, 2001 (37th SEMDSA)

Venue: Holiday Inn Crowne Plaza, Sandton

Contact: Shelley Harris, SEMDSA (Congress Organiser) PO BOX 783155 SANDTON 2146 SOUTH AFRICA email: rsh@novo.dk phone: +27 11 8070794 fax: +27 11 8077989

Registration Fees: US$300.000 if before January 24, 2001 US$400.00 if after January 24, 2001


    ACKNOWLEDGMENTS
 
Our thanks are extended to members of the Smith and Hager laboratories for helpful discussion and analysis. We would also like to thank Drs. Chris Baumann (National Cancer Institute) and Mark Danielsen (Georgetown University) for critical evaluation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Catharine L. Smith, National Institutes of Health/National Cancer Institute, Laboratory of Receptor Biology and Gene Expression, Building 41, Room B600, 41 Library Drive, MSC 5055, Bethesda, Maryland 20892-5055.

Received for publication November 16, 1999. Revision received February 16, 2000. Accepted for publication March 16, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Congress: 37th SEMDSA (Society...
 REFERENCES
 

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