Expression of the Parathyroid Hormone-Related Peptide Gene in Retinoic Acid-Induced Differentiation: Involvement of ETS and Sp1

Marcel Karperien, Hetty Farih-Sips, Clemens W.G.M. Löwik, Siegfried W. de Laat, Johannes Boonstra and Libert H.K. Defize

Department of Endocrinology (M.K., H.F.-S., C.W.G.M.L.), Leiden University, 2300 RC Leiden, The Netherlands,
Netherlands Institute for Developmental Biology (M.K., S.W.d.L., L.H.K.D), 3584 CT Utrecht, The Netherlands,
Department of Molecular Cell Biology (M.K., J.B.), University of Utrecht, 3584 CH Utrecht, The Netherlands


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PTH-related peptide (PTHrP) was first identified as the major cause of increased serum calcium levels in patients suffering from humoral hypercalcemia of malignancy (1, 2, 3). PTHrP is expressed in a broad range of embryonal and adult tissues (4, 5, 6, 7, 8). It is implicated in the regulation of several growth and differentiation processes such as keratinocyte differentiation (9), chondrocyte differentiation (10), and the formation of extraembryonic endoderm during mouse embryogenesis (11, 12, 13) where it most likely acts in a para- or autocrine fashion.

The cDNAs for human, mouse, and rat PTHrP have been cloned, and the genomic structure of the corresponding genes has been unraveled (4, 14, 15). Compared with the mouse and rat genes, which are highly homologous, the human PTHrP gene is more complex. It contains two TATA-box driven promoters, P1 and P2, respectively, and one GC-rich TATA-box less promoter (16). The mouse and rat gene are driven by a single promoter, which is the homolog of the human P2 promoter. Due to differential splicing at the 3'-end of the human gene, three different mature PTHrP peptides, which consist of 139, 141, or 173 amino acids, have been identified. In mouse and rat no reports have been published on differential splicing, and it is believed that both species express a single messenger that encodes a 139-amino acid peptide (4, 14).

PTHrP mRNA regulation by growth factors, cytokines, and peptide and steroid hormones, both in vitro and in vivo, has been extensively studied (17, 18, 19, 20). In spite of these efforts, not much is known about the transcription factors involved in the regulation of PTHrP expression. Recently, it was shown that the human gene is using cell- and tissue-specific promoters (16). These promoters are, like the rat PTHrP promoter, regulated by multiple positive and negative regulatory cis-acting elements (16, 21). In papers by Dittmer et al., (22, 23) binding sites for members of the Ets and Sp1 families of transcription factors that mediate the PTHrP expression in HTLV-1-infected T cells have been identified in the human P2 promoter.

In this study we set out to identify regulatory elements involved in the induction of PTHrP expression during all-trans-retinoic acid (RA)-mediated differentiation of murine embryonal carcinoma (EC) and embryonal stem (ES) cells. Recently, we and others have shown that during the RA-mediated differentiation of F9 EC and ES-5 cells toward extraembryonic parietal endoderm, PTHrP mRNA and protein are induced (11, 12). Here we show that PTHrP mRNA expression was also induced during the RA-mediated differentiation of P19 EC cells. To identify the regulatory elements that were involved in the differentiation-associated induction of the mouse PTHrP gene, we constructed promoter reporter constructs and were able to pinpoint positive regulatory elements to a region between nucleotide (nt) -88 to -58 relative to the transcription start site. The region between nt -88 to -58 encoded two partially overlapping binding sites for Ets-related transcription factors and an inverted Sp1-binding site. Using bandshift analyses, we detected two major specifically retarded complexes in nuclear extracts of both RA- and undifferentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity. We identified the sequence CCAGCCC as the most likely inverted Sp1-binding site. This site is shifted 4 bp more upstream compared with the proposed Sp1-binding site (sequence CCCACC) identified in the human PTHrP P2 promoter (23). The two complexes detected in nuclear extracts of undifferentiated P19 cells were indistinguishable from the complexes found using extracts of RA-differentiated cells. This suggested that the RA differentiation-induced expression of the PTHrP gene might be caused by posttranslational processing of preexisting transcription factors rather than by the de novo synthesis of transcription factors. Mutations in either the Ets- or Sp1-binding sites completely abolished RA differentiation-induced expression of PTHrP promoter reporter constructs.

Additionally, we show that the Ets- and Sp1-binding sites were also involved in the expression of PTHrP promoter reporter constructs in cells that constitutively expressed PTHrP, suggesting that the Ets family of transcription factors, in cooperation with Sp1, play a central role in the transcriptional regulation of the murine PTHrP gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTHrP mRNA Is Induced in RA-Differentiated P19 EC Cells
Recently, we and others have shown that during RA-mediated differentiation of F9 EC and ES-5 cells, PTHrP expression is induced at the mRNA and protein level (11, 12). To study the effect of RA treatment on PTHrP mRNA expression in another EC cell line, the P19 EC cell line, cells were seeded and cultured for 3 and 5 days in the presence of 10-6 M RA, respectively, after which total RNA was isolated. PTHrP mRNA concentrations were measured by a RNAse protection assay and standardized using an antisense glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. As shown in Fig. 1Go, undifferentiated P19 EC cells did not express PTHrP mRNA in contrast to P19 cells treated for 3 days with RA (lanes marked -). Prolonged treatment with RA for 5 days resulted in a further increase of PTHrP mRNA levels. Pretreatment of the cells with cycloheximide resulted in an induction of PTHrP mRNA in undifferentiated P19 EC cells and in a further up-regulation of the message in the RA-treated cell cultures. These findings were in agreement with previous studies in other cell lines showing that cycloheximide was able to both induce and up-regulate PTHrP mRNA expression (24). Similar results were found in RA-differentiated ES-5 and F9 EC cells (data not shown).



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Figure 1. PTHrP mRNA Is Induced during RA-Mediated Differentiation of P19 EC Cells and by Cycloheximide Treatment

A, P19 EC cells were cultured in the absence (P19) or in the presence of 10-6 M RA for 3 (P19 3dRA) or 5 days (P19 5dRA). Before total RNA was isolated, cultures were treated without (-) or with 20 µg cycloheximide/ml for 1 h. PTHrP mRNA expression was measured using a RNAse protection assay. GAPDH was used to correct for equal amounts of RNA in each hybridization mix. Exposures times: PTHrP, 2 weeks, -80 C; GAPDH, 1 day, -80 C. B, PhosphorImager quantification of the gel shown in Fig. 1AGo using ImageQuant software (Molecular Dynamics). Fold induction was related to the value of untreated P19 EC cells, which was set to 1 and was corrected for GAPDH.

 
RA Differentiation-Induced PTHrP Expression Is Mediated by Sequences Located between nt -185 and -53
To identify elements in the mouse PTHrP promoter that mediate the RA differentiation-induced expression of the PTHrP gene, a panel of progressive promoter deletion constructs was made and cloned in front of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless pKT CAT vector (Fig. 2AGo). In agreement with the mRNA data, none of the promoter reporter constructs showed activity in undifferentiated EC and ES cells (data not shown). To localize the differentiation-responsive elements, the promoter reporter constructs were transiently transfected in P19 and ES-5 cells treated for 3 days with 10-6 or 10-7 M RA, respectively. After the medium was refreshed at day 4, cells were harvested at day 5, and CAT activity was determined. CAT activity was corrected for transfection efficiency and related to the activity of the empty pKT CAT vector as fold induction (Fig. 2Go, B and C). A promoter reporter construct containing only the TATA box and transcription start site did not induce promoter activity in either cell line (pKT-49). Increasing the promoter fragment in front of the reporter gene with 136 nt resulted in a 7-fold induction of CAT activity in RA-differentiated P19 cells and a 3-fold induction in RA-differentiated ES-5 cells (pKT -185), indicating that the RA-induced expression of the PTHrP gene was, at least in part, mediated by sequences located between nt -185 and -49 relative to the transcription start site. The TATA box and transcription start site were required for this induction as was demonstrated by construct pKT delT in which these sites were deleted. An increase in promoter sequences with 100 nt resulted in a further enhancement of the RA-induced PTHrP expression (compare pKT -185 with pKT -285). Constructs pKT -382 and -490 containing further increases in 5'-promoter sequences had activities comparable with or lower than the activity of construct pKT -185. An additional increase in promoter sequence with about 350 nt resulted in a decline in promoter activity (compare constructs pKT -490 with pKT -853), suggesting the presence of negative regulatory elements in this region. Increasing the promoter fragment with an additional 300 nt did not significantly modify the transactivation potential of the promoter (compare construct pKT -1145 and pKT -853). To determine the influence of intronic sequences present between exon 1 coding for the untranslated leader and exon 2 coding for the translation start site and the signal peptide, constructs pKT S-490 and pKT S-1145 were made and tested by transient transfection. The intronic sequences did not significantly influence promoter activity (compare the activity of pKT S-490 with pKT -490 and the activity of pKT S-1145 with pKT -1145).



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Figure 2. Transient Transfection of Mouse PTHrP Promoter Reporter Constructs in Different Cell Lines

A, Schematic representation of the 5'-organization of the mouse PTHrP gene and the promoter reporter constructs used. The arrows represent the transcription start site in front of exon 1 and the ATG start codon in exon 2, respectively. Abbreviations and positions of restriction sites: H, HindIII (-1145); X, XbaI (-853); S, SmaI (-490); A, AvaII (+220); He, HaeII (-285; +80); P, PvuII (-185); *, restriction sites introduced by PCR; X*, XbaI (-382); H*, HindIII (+494). B, 10 µg PTHrP promoter reporter constructs were transiently transfected into P19 cells treated for 5 days with RA. (see Materials and Methods for detailed protocol). CAT activity was quantified using the PhosphorImager and ImageQuant software and depicted relative to the activity of the empty pKT CAT vector after correction for transfection efficiency. All transfections were repeated at least three times, and a representative experiment is shown. C, PTHrP promoter reporter constructs were transiently transfected in ES-5 cells treated for 5 days with RA (methods as in panel B). D, PTHrP promoter reporter constructs transiently transfected in BHK cells (methods as in panel B). E, PTHrP promoter reporter constructs transiently transfected in Mes-1 cells (methods as in panel B).

 
In summary, we concluded that the RA differentiation-induced expression of PTHrP mRNA was, at least in part, mediated by sequences between nt -185 and -49, with a minor contribution of sequences between nt -285 and -185. The activity of these regions was down-regulated by elements located further upstream between nt -853 and -490.

Sequences Located between nt -185 and -49 Are Also Required for Constitutive PTHrP Expression
To test whether the identified regulatory elements in the mouse PTHrP promoter were specific for RA differentiation-induced PTHrP expression or reflected a more general mechanism of regulating PTHrP gene transcription, the promoter reporter constructs were also tested in two other cell lines that constitutively expressed PTHrP: 1) Baby hamster kidney fibroblasts (BHK) and 2) Mes-1 cells. Mes-1 is a cell line with mesodermal characteristics and is derived from RA-differentiated P19 EC cells (25). This cell line expresses PTHrP mRNA (M. Karperien, unpublished observation). The results of the transient transfection assays were comparable with the results observed in the RA-differentiated EC and ES cells (compare Fig. 2Go, D and E, with Fig. 2Go, B and C). Again major positive regulatory elements were located between nt -185 and -49, while upstream from position -490 sequences were located that down-regulated PTHrP transcription. Additional negative regulatory elements were present between positions -490 and -382 in Mes-1 cells (see construct pKT -490 in Fig. 2EGo). These results suggested that identical promoter regions were involved in the regulation of both RA differentiation-induced gene expression in EC and ES cells and constitutive PTHrP expression in Mes-1 and BHK cells.

The Region between nt -185 and -49 Contains Binding Sites for Ets-Related Transcription Factors and an Inverted Sp1-Binding Site
Sequence analysis of the region between nt -185 and -49 did not reveal homology to RA-responsive elements that mediate the induction of genes that are under direct control of RA (26). This was not unexpected, given the slow time course of PTHrP mRNA induction by RA (Fig. 1Go), which suggested that the appearance of PTHrP mRNA was a secondary effect caused by RA-activated differentiation programs rather than a direct effect of RA. Comparison of the nucleotide sequence of the mouse, rat, and human P2 PTHrP promoters revealed a striking degree of homology, especially between nt -94 and -62, which were completely conserved in all three promoters (Fig. 3AGo). This region contains two partially overlapping binding sites for the Ets family of transcription factors: one present at the sense (EBSI) and the other present at the antisense strand (EBSII), respectively (Fig. 3BGo). Furthermore, the CCCACC sequence has been identified as an inverted Sp1-binding site in the human PTHrP P2 promoter (23) (Fig. 3BGo).



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Figure 3. Sequence of the Mouse PTHrP Promoter

A, Homology comparison between the mouse, rat, and human P2 PTHrP promoters. The region between nt -94 and -62 is conserved in all three promoters and is double underlined. B, Sequence of the region between nt -88 to -58. The consensus Ets-binding site on the sense strand, EBSI, is single underlined. The consensus Ets-binding site on the antisense strand, EBSII, is double underlined. The inverted Sp1-binding site is depicted in italics.

 
To test whether the Ets-binding sites were functional and were involved in the RA differentiation-induced expression of the PTHrP gene, we first cotransfected PTHrP promoter reporter constructs with ets-1 and ets-2 expression vectors. Ets-1 and ets-2 are differentially regulated by RA in P19 EC cells. Ets-1 expression is up-regulated by RA treatment while Ets-2 is constitutively expressed in undifferentiated and RA-differentiated P19 EC cells at the mRNA level (27). As shown in Fig. 4AGo, cotransfection with a chicken ets-1 expression vector resulted in a 6-fold induction of PTHrP promoter activity in undifferentiated P19 EC cells using pKT -185 as reporter. In contrast, cotransfected Ets-2 was not able to transactivate the PTHrP promoter, even when the amount of cotransfected expression vector was increased from 2 to 5 µg DNA (Fig. 4AGo, not shown). As a positive control for the ets-1 and ets-2 expression vectors, construct 6–3 was used, which contained the ets-binding sites from the HTLV-1 long terminal repeat in front of the CAT reporter gene. Cotransfection of both Ets expression vectors induced promoter activity, although Ets-2 was less potent than Ets-1 (Fig. 4AGo). These results indicated that Ets-2, in contrast to Ets-1, was not able to induce PTHrP promoter activity in undifferentiated P19 EC cells.



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Figure 4. Ets-1 and Ets-2 Are Able to Transactivate PTHrP Promoter Reporter Constructs Depending on the Differentiation State of P19 EC Cells

A, Reporter (10 µg) was cotransfected with 2 µg empty expression vector (pSG5), 2 µg chicken ets-1 expression vector, or 2 µg human ets-2 expression vector, respectively, in undifferentiated P19 EC cells. Fold induction was expressed relative to the promoterless pKT CAT vector and corrected for transfection efficiency. All transfections were repeated at least three times and a representative experiment is shown. B, Same as in panel A except that the P19 EC cells were differentiated for 5 days with RA.

 
Similar experiments were performed in P19 EC cells differentiated for 5 days with RA (Fig. 4BGo). Surprisingly, under these conditions both Ets-1 and Ets-2 were able to transactivate the PTHrP promoter, suggesting that the RA-mediated differentiation created an environment in which Ets-2 was now able to transactivate the PTHrP promoter.

Two Major Complexes Bind to the Region between nt -88 and -58
We next performed bandshift analyses using nuclear extracts of PTHrP-expressing and nonexpressing cells. As a probe, a double-stranded oligonucleotide spanning the region from nt -88 to -58 (PLP wt) was used that encoded the Ets and inverted Sp1-binding sites (Table 1Go). As shown in Fig. 5Go, two major complexes were specifically retarded in a bandshift assay using nuclear extracts of RA-differentiated P19 EC cells. These complexes were specific, since they were competed with a 25-fold excess of nonlabeled PLP wt oligonucleotide while no competition was observed with a 50-fold excess of nonspecific DNA.


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Table 1. Oligonucleotides Used in Bandshift Assays and Site-Directed Mutagenesis

 


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Figure 5. Nuclear Extracts of Different Cell Lines Contain Two Major Protein Complexes Binding to the Region between nt -88 and -58

Nuclear extracts of P19 EC cells differentiated for 5 days with RA (P19 5dRA) were incubated with a radioactively labeled oligonucleotide probe coding for nt -88 to -58 (PLP wt). Specific competition was performed using a 25-fold excess of unlabeled probe. Nonspecific competition was performed using a 50-fold excess of unlabeled pUC18 DNA. The sequence of the oligonucleotides that were used as competitors are depicted in Table 1Go. The two major specific bands are indicated with the bold arrows.

 
To identify the proteins present in the shifted complexes, bandshift analyses were performed in which mutated PLP wt oligonucleotides and consensus binding sites for Ets and Sp1 were used as competitors. These oligonucleotides are described in Table 1Go. As shown in Fig. 5Go, oligonucleotide PLP mtETS, in which both Ets-binding sites were inactivated by mutation, efficiently competed the upper complex, suggesting that the lower complex was formed by proteins binding to the Ets sites. This was confirmed by competition with a consensus Ets-binding site (PEA3) and oligonucleotide Ets wt, which efficiently competed the lower but not the upper complex. Oligonucleotide PLP mtINT, in which the two nucleotides directly downstream of the EBSI site were mutated (Table 1Go), efficiently competed both complexes, suggesting that the mutation did not interfere with protein binding. Oligonucleotide PLP mtSp1, in which the inverted Sp1-binding site was mutated, competed the lower Ets-containing complex but not the upper complex. The presence of Sp1-like binding activity in the upper complex was demonstrated by the efficient competition with two different Sp1 consensus-binding sites (pSV2 Sp1, Sp1 con). Furthermore, the upper shift was totally dependent on the presence of Zn++ in the binding buffer, indicating that this complex contained a zinc finger protein, such as Sp1 (data not shown). Surprisingly, oligonucleotide Sp1a, encoding the CCCACC-inverted Sp1-binding site identified in the human PTHrP P2 promoter (23), was less efficient in competing the upper complex than oligonucleotides INTa and INTmtG>T. Compared with Sp1a, these oligonucleotides encoded 5'-nucleotides located in between the Ets and CCCACC core sequence, suggesting the involvement of these nucleotides in binding of the Sp1-like activity. The change of G into T did not interfere with protein binding in oligonucleotide INTmtG>T. Finally, mutation of both Ets- and Sp1-binding sites completely abolished protein binding of oligonucleotide PLP mtETS/Sp1.

Similar results were obtained using nuclear extracts of P19 EC cells, which do not express PTHrP and BHK cells that constitutively express PTHrP (data not shown). The two complexes found in a bandshift assay using nuclear extracts of undifferentiated P19 EC cells were indistinguishable from the complexes found when extracts of RA-differentiated P19 EC were used. This suggested that the differentiation-induced expression of the PTHrP gene was not caused by the induction of expression of a new transcription factors, although the possibility that the retarded bands consisted of different proteins with the same electromobility could not be excluded (not shown).

Both Ets-Binding Sites Are Involved in Protein Binding
In the next experiment, the involvement of the individual Ets-binding sites, EBSI and EBSII, in protein binding to the PLP wt oligonucleotide was determined using nuclear extracts of RA-differentiated P19 EC cells. Again, two major specific complexes were observed in a bandshift assay of which the lower consisted of Ets domain-containing transcription factors (Fig. 6AGo). Increasing the concentration of the consensus Ets-binding site PEA3 (from 5- to 25-fold excess) as a competitor efficiently competed the lower complex. A 25-fold excess of unlabeled competitor was sufficient to compete all bandshifts containing Ets-binding proteins (Fig. 6AGo). Similar results were found using oligonucleotide Ets wt as competitor. This oligonucleotide encodes both Ets-binding sites of the PTHrP promoter (nt -92 to -67). None of the shifts was competed using oligonucleotide Ets mtI in which both the EBSI- and EBSII-binding sites were inactivated by mutation (see Table 1Go and Fig. 6AGo). Oligonucleotide Ets mtII, in which the Ets-binding site at the antisense strand was mutated, leaving a functional EBSI site, and oligonucleotide Ets mtIII, in which the Ets binding site at the sense strand was mutated, leaving an intact EBSII site, were equally potent in competing with the PLP wt oligonucleotide for binding of Ets domain-containing proteins (Fig. 6AGo). This suggested that the EBSI and EBSII sites were both able to bind Ets domain-containing transcription factors. Identical results were obtained when nuclear extracts of undifferentiated P19 EC and BHK cells were used (data not shown).



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Figure 6. Characterization of Ets-Binding Proteins in Undifferentiated and RA-Differentiated P19 Cells

A, Nuclear extracts (10 µg) of P19 EC cells differentiated for 5 days with RA were incubated with a radioactive labeled oligonucleotide probe coding for nt -88 to -58 of the PTHrP promoter (PLP wt). Competition was performed using a 25-fold excess of unlabeled oligonucleotides except for oligonucleotides PEA3, Ets mtII, and Ets mtIII of which a 5-, 10-, and 25-fold excess was used. As nonspecific competitor a 50-fold excess of pUC 18 DNA was used. The sequence of the oligonucleotides is depicted in Table 1Go. The two major specific bands are indicated by a bold arrow. In between the two major bands, a nonspecific complex was observed (as). B, Nuclear extracts (10 µg) from undifferentiated P19 EC cells (left panel) and from RA-differentiated P19 cells (right panel) were incubated with a radiolabeled consensus Ets-binding site (PEA3). Competition was performed using a 25-fold excess of specific oligonucleotides and a 50-fold excess of nonspecific pUC18 DNA. The sequence of the oligonucleo-tides used as competitors is depicted in Table 1Go. The major bandshift is indicated by a bold arrow, while the minor shifts are indicated by thin arrows.

 
We subsequently used the consensus Ets-binding site PEA3 as a probe in a bandshift assay using nuclear extracts of undifferentiated P19 EC cells, which do not express PTHrP, and RA-differentiated P19 EC cells, which do. One major and several minor complexes were found using nuclear extracts of both cell types (Fig. 6BGo; major complex is indicated by bold arrow). These complexes were specific, since they were competed with a 25-fold excess of unlabeled probe but not with a 50-fold excess of nonspecific competitor. The pattern of complexes using the extracts of non-PTHrP-expressing (P19 EC, Fig. 6BGo, left) and PTHrP-expressing cells (P19 cells treated with RA, Fig. 6BGo, right) were indistinguishable, suggesting that the same Ets-binding proteins were present in both cell types. However, it cannot be excluded that, although the mobility of the complexes was identical, the retarded bands consisted of different proteins. Surprisingly, we did not observe an up-regulation of any of the shifts containing the PEA3 oligonucleotide, although RA treatment causes an up-regulation of the expression of Ets-1 mRNA (27). All complexes were efficiently competed by a 25-fold excess of oligonucleotides Ets wt, Ets mtII, and Ets mtIII, encoding two or one functional Ets-binding sites derived from the PTHrP promoter (Table 1Go), respectively, but not by oligonucleotide Ets mtI in which both Ets- binding sites were inactivated by mutation. In contrast to the bandshift experiments in which the oligonucleotide PLP wt was used as a probe, now the Ets mtIII oligonucleotide, encoding a functional EBSII site (Table 1Go) was a more efficient competitor than oligonucleotide Ets mtII, encoding a functional EBSI site (compare Fig. 6AGo and 6BGo). This difference in affinity was confirmed by dose-response analysis (data not shown) and suggested that the EBSII site had a somewhat higher affinity than the EBSI site for Ets-binding proteins present in the nuclear extracts of undifferentiated and RA-differentiated P19 cells.

The Inverted CCAGCCC Sequence Behaves as an Sp1-Binding Site
The competition experiments shown in Fig. 5Go suggested that nucleotides between the Ets-binding site and the sequence CCCACC, which was identified as an inverted Sp1-binding site in the human PTHrP P2 promoter (23), were involved in protein binding. We therefore designed oligonucleotides Sp1b, coding for the CCCACC sequence, and INTb, which encoded the region between the Ets and CCCACC sequence. Both oligonucleotides were used as probes in bandshift analysis using nuclear extracts of BHK cells and RA-differentiated and undifferentiated P19 cells. Two major specific bands were retarded using extracts of these three cell lines and oligonucleotide INTb as a probe (Fig. 7AGo; data for BHK and RA-differentiated P19 cells not shown). These bands were specific, since they were efficiently competed by a 25-fold excess of the PLP wt oligonucleotide, but not with a 50-fold excess of nonspecific DNA. Increasing amounts of oligonucleotides INTa and INTmtG>T (5-, 10-, 25-fold excess) efficiently competed both complexes. The mutation of one nucleotide in INTmtG>T did not significantly interfere with protein binding. The most efficient competition was observed using oligonucleotides pSV2 Sp1 and Sp1 con, which encoded consensus Sp1-binding sites. The two shifts were totally dependent on the presence of Zn++ in the binding buffer, which was further evidence for the presence of Sp1-like binding activity in both complexes (data not shown). Surprisingly, oligonucleotides Sp1a and Sp1b, coding for the CCCACC sequence, did not compete with protein binding (Fig. 7AGo), suggesting that this sequence was not critically involved in binding of the Sp1-like activity to the PTHrP promoter. In our view, the results suggested that the Sp1-binding site is located slightly more upstream, closer to the Ets-binding sites. This was supported by the reversed experiment in which the Sp1b oligonucleotide was used as a probe. In this experiment only faint bandshifts were observed (Fig. 7BGo). These results suggested that the sequence CCAGCCC was the most likely inverted binding site for the Sp1-like binding activity. This sequence matches the Sp1 consensus binding site (G/AGGCG/TG/AG/A, 31 and is flanked by the nucleotides mutated in the PLP mtINT oligonucleotide at its 5'-end and includes the nucleotides mutated in the PLP mtSp1 oligonucleotide at its 3'-end.



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Figure 7. Sp1 Binds to the Inverted CCAGCCC Sequence

A, Nuclear extracts (20 µg) of undifferentiated P19 EC cells were incubated with the radiolabeled oligonucleotide probe Intb in a bandshift assay. Nonspecific competition was performed with a 50-fold excess unlabeled oligonucleotide PEA3, while specific competition was performed with a 5-, 10-, or 25-fold excess of unlabeled oligonucleotides Inta, IntmtG>T, pSV2 Sp1, Sp1 con, and Sp1a or a 25-fold excess of the PLP wt or Sp1b oligonucleotides. B, Same as in panel A, except that oligonucleotide Sp1b was used as a radiolabeled probe. Specific competition was performed with a 25-fold excess of unlabeled oligonucleotides PLP wt, Sp1a, and Sp1 con, while nonspecific competition was performed with a 50-fold excess of oligonucleotide PEA3.

 
Functional Ets- and Sp1-Binding Sites Are Required for RA Differentiation-Induced and Constitutive PTHrP Expression
To define the role of the Ets- and Sp1-binding sites, binding-perturbing mutations were introduced in one or both binding sites. The same mutations as in the oligonucleotides used in the bandshift assays were introduced by site-directed mutagenesis in promoter reporter construct pKT -185 (Table 1Go). The effect of the mutations on promoter activity was subsequently tested by perfoming transient transfections in RA-differentiated P19 cells. As shown in Fig. 8AGo, mutation of both Ets-binding sites abolished PTHrP promoter activity in RA-differentiated P19 cells (pKT Ets mtI). Promoter activity was also abolished in the constructs in which the individual Ets-binding sites were mutated (pKT mt EtsII and EtsIII). This suggested that either Ets-binding site must be functional for transcriptional activation and that both sites contributed to the activation of the promoter reporter construct. Likewise, mutation of the inverted-Sp1 binding site in promoter reporter construct pKT mtSp1 abolished reporter activity. These results indicated that both functional Ets- and Sp1-binding sites were required for transcription of the PTHrP promoter reporter construct pKT -185 and strongly pointed to a synergistic interaction between Ets and Sp1 in the regulation of PTHrP gene transcription, most likely by the formation of a ternary complex. This was furthermore supported by mutation of the two nucleotides in the gap between the Ets- and Sp1-binding sites (pKT mtInt). Although this mutation did not interfere with protein binding to the individual Ets- and Sp1-binding sites in a bandshift assay (Fig. 6Go), it caused a strong decline in promoter reporter activity. Most likely, the mutation interfered with the interaction between Ets and Sp1 by a change in DNA structure.



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Figure 8. Mutations in Either the Ets- or Sp1-Binding Site Strongly Diminish PTHrP Promoter Activity

A, Mutations were made in PTHrP promoter reporter construct pKT -185 (see Fig. 2AGo). The mutated nucleotides are described in Table 1Go. Mutant promoter reporter constructs (10 µg) were transiently transfected in P19 EC cells differentiated for 5 days with RA (see Materials and Methods). Fold induction was relative to the activity of the promoterless pKT CAT reporter construct and corrected for transfection efficiency. All transfections were repeated at least three times and a representative experiment is shown. B, Same as in panel A, except that transfections were performed in BHK cells.

 
Promoter activity of the mutant promoter constructs was also tested in BHK cells, which constitutively express PTHrP. All mutants strongly diminished promoter activity, although the inhibition of promoter activity was less than in RA-differentiated P19 EC cells (an average of 70% compared with an average of 85% inhibition of transcription activity, respectively) (Fig. 8BGo). The results indicated that functional Ets- and Sp1-binding sites were required for both RA-induced and constitutive PTHrP expression in P19 EC and BHK cells, respectively.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently, we and others have provided evidence for a role of PTHrP and its receptor in the formation of extraembryonic endoderm of the parietal yolk sac during mouse embryogenesis (11, 12). Using F9 and ES cell lines as in vitro model systems, we have shown that during the RA-mediated differentiation toward parietal endoderm, PTHrP mRNA and protein are induced. In this study we set out to identify regulatory elements of the murine PTHrP promoter that mediate the RA differentiation-induced expression of PTHrP in EC and ES cells. Using promoter reporter constructs, we showed that the RA-mediated induction of PTHrP-gene expression was mediated by a region located between nt -88 to -58 relative to the transcription start site. In addition, we showed that this region was also involved in the expression regulation of cells that constitutively express PTHrP mRNA.

Using nuclear extracts of RA-differentiated and undifferentiated P19 cells, two specific protein complexes binding to the region between nt -88 and -58 were identified. The lower complex consisted of Ets domain-containing transcription factors as suggested by competition with a consensus Ets-binding site, while the upper complex contained Sp1-like activity as suggested by competition with Sp1 consensus-binding sites and its dependency on Zn++ in the bandshift buffer. The identity of the Ets-binding factors is not yet known and is the subject of further investigations. However, it seems likely that Ets-1 and Ets-2 are constituents of the lower complex, since 1) Ets-1 and Ets-2 are expressed in undifferentiated and RA-differentiated P19 cells (27), and 2) both cotransfected ets-1 and ets-2 are able to transactivate PTHrP promoter reporter constructs in transient transfection assays. However, it cannot be excluded that other members of the Ets-family of transcription factors also bind to the PTHrP promoter in RA- and undifferentiated P19 cells.

The sequence between nt -88 and -58 is completely conserved in the mouse, rat, and human P2 PTHrP promoters in sequence and location relative to the transcription start site. Dittmer and co-workers (22, 23) have recently shown that this region mediates the expression of the human PTHrP gene in HTLV-1 infected T cells. They identified two partially overlapping Ets-binding sites, EBSI and EBSII, and an inverted Sp1-binding site in this region by showing efficient binding of recombinant Ets-1 and Sp1 to their respective binding sites. In contrast to Dittmer and co-workers (23), who identified the sequence CCCACC as an inverted Sp1-binding site in the human PTHrP P2 promoter, our data suggested that the sequence CCAGCCC was the most likely inverted Sp1-binding site. This site is located 4 bp further upstream compared with the site identified by Dittmer and associates (23), decreasing the gap between the Ets- and Sp1-binding site to 2 bp. This sequence matches the published Sp1 consensus binding site (31). Furthermore, a gap of 2 bp, instead of 6 bp, between Ets- and Sp1-binding sites is also found in the promoter/enhancer regions of various other genes in which cooperative interactions between Ets and Sp1 are observed, such as the HTLV-1 long terminal repeat (28) and the P4 promoter of mouse Minute Virus (30). Optimal PTHrP expression in HTLV-1-infected T cells required the presence of functional binding sites for either Ets or Sp1 (23). In fact, it was shown that Sp1 and Ets-1 worked cooperatively in the regulation of PTHrP expression. Such a cooperative interaction has also been described for other promoters (28, 30, 32, 34) and is mediated by the formation of a ternary complex. Dittmer and co-workers (23) demonstrated the formation of such a ternary complex, using recombinant Ets-1 and Sp1 and the PTHrP promoter as a probe in a bandshift assay (23). In our bandshift assays we did not observe ternary complex formation. In contrast to Dittmer and co-workers (23), we used crude nuclear extracts instead of recombinant or partially purified Ets and Sp1, which might explain the absence of ternary complex formation in our hands. More recently, it was shown that the formation of a ternary complex between Ets and Sp1 in the human PTHrP P2 promoter is facilitated by the HTLV-1 Tax protein (35). This suggests that ternary complex formation might be stimulated by an as yet unknown cellular protein.

Binding-perturbing mutations in one or both Ets-binding sites or the Sp1-binding site abolished the RA differentiation-induced expresssion of PTHrP promoter reporter constructs. This was in concordance with findings of Dittmer and co-workers (22, 23), except that they showed that mutations in the EBSI binding site were more effective in reducing promoter reporter activity in HTLV-1-infected T cells than mutations in the EBSII-binding site. Likewise, they showed that the EBSI site was more effective in binding of recombinant Ets1 than the EBSII site. In contrast, we showed that binding affinity of the EBSII site for Ets domain-containing transcription factors present in the nuclear extracts of RA-differentiated and undifferentiated P19 cells was equal to or even somewhat higher than the EBSI site. Therefore, our data suggest that both the EBSI and EBSII site might be occupied by Ets domain-containing transcription factors for efficient transactivating of PTHrP promoter reporter constructs. It is also possible that the differences between our data and the observations made by Dittmer and co-workers (22, 23) might be explained by the use of other mutated nucleotides [CTTTCCAGAAGC vs. CTTTCCGGTTGC (this study) for the EBSI site and CTTTCAGGAAGC vs. CTAACCGGAAGC (this study) for the EBSII site] and by the use of recombinant proteins in the study of Dittmer vs. nuclear extracts in our study. The absence of reporter activity in cells transfected with constructs in which only one transcription factor-binding site was mutated indicated the necessity of a synergistic interaction between the transcription factors binding to the Ets- and Sp1-binding sites for the regulation of gene transcription. Mutation of the two nucleotides between the Ets- and Sp1-binding sites in PTHrP promoter reporter construct (pKT mtInt) also abolished reporter activity. Although the mutation did not interfere with protein binding in a bandshift assay, the change in DNA structure apparently inhibited the formation of a ternary complex in vivo. This might explain the absence of reporter activity in transfection assays. The mutations that abolished the activity of PTHrP promoter reporter constructs in RA-differentiated P19 cells also strongly decreased reporter activity in cells that constitutively expressed PTHrP mRNA. These findings, combined with the observations by Dittmer et al. (22, 23), strongly point to a central role for the Ets and Sp1 family of transcription factors as transcriptional regulators of PTHrP gene expression.

The bandshift patterns observed by using nuclear extracts of undifferentiated or RA-differentiated P19 cells and the consensus Ets-binding site as a probe were indistinguishable, suggesting that the same Ets domain-containing transcription factors were present in undifferentiated and RA-differentiated P19 cells. Likewise, there were no differences in the two shifts obtained by using the inverted Sp1 site as a probe and nuclear extracts of undifferentiated and RA-differentiated P19 cells. This suggested that the induction of PTHrP expression by RA differentiation was not caused by the induction of expression of a novel Ets-related transcription factor or by the induction of Sp-like activity, which in turn transactivated the PTHrP promoter, although it could not be excluded that the retarded bands consisted of different proteins with identical electromobility or that the differentiation resulted in the induction of transcription factors that could not be detected by the bandshift assays. However, we propose that the induction of PTHrP expression by RA-mediated differentiation was caused by posttranslational modifications of already existing protein complexes. This hypothesis was supported by the observation that cycloheximide induced the expression of PTHrP in undifferentiated P19 EC cells, indicating that all factors required for the transciption of the PTHrP gene were already present in cells that normally do not express detectable levels of PTHrP. In addition, we showed that cotransfected Ets-2 was able to transactivate PTHrP promoter reporter constructs only in RA-differentiated P19 cells, in spite of the fact that Ets-2 was also expressed in undifferentiated P19 cells (27). This indicated that the RA-mediated differentiation created an environment in which Ets-2 was now able to transactive the PTHrP promoter, e.g. by posttranslational modifications. Several members of the Ets family of transcription factors are subject to posttranslational modifications such as phosphorylation. For example, phosphorylation of Elk-1, an Ets domain-containing transcription factor binding to the serum response element, dramatically enhances gene transcription of the c-fos promoter (32, 34).

The activity of the transcription factors binding to the region between nt -88 to -58 is down-modulated by negative regulatory elements located more upstream in the PTHrP promoter. The nature of this down-modulation is at present unclear and is the subject of further investigations. The presence of negative regulatory elements in the murine PTHrP promoter was not surprising given the observations that the expression of both human and rat PTHrP genes is regulated by multiple negative cis-acting elements (16, 21). These elements might explain why PTHrP mRNA is difficult to detect in RA-differentiated P19 EC cells. The message was only detectable using the sensitive RNAse protection assay and 100 µg total RNA. PTHrP mRNA is also hard to detect in other murine cell lines and tissues (4), which might be attributed to the presence of negative regulatory elements. These elements might play an important role in controlling the expression of PTHrP, since overexpression of the gene in certain human tumors is associated with development of humoral hypercalcemia of malignancy (1, 2, 3).

In summary, we conclude that the region between nt -88 and -58 of the PTHrP promoter plays a central role in the control of expression of the murine PTHrP gene. This is accomplished by a synergistic interaction of transcription factors belonging to the Ets- and Sp1 families of transcription factors. The precise nature of the protein interactions leading to PTHrP gene transcription and the role in this process of posttranslational modifications are the subjects of further studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTHrP Promoter Reporter Constructs, Plasmids, and Oligonucleotides
A genomic clone coding for the mouse PTHrP gene was kindly provided by Dr. A. Broadus. From this clone, a HindIII-KpnI fragment of 1.8 kb encoding exon 2, exon 1, and about 1150 nt upstream promoter sequences was subcloned in SK-bluescript (Stratagene, La Jolla, CA) and was used to generate promoter reporter constructs in front of the bacterial chloramphenicol acetyl transferase gene. The promoter reporter constructs contained the following restriction fragments: pKT -49; AccI (-49)–AvaII (+220), pKT -185; PvuII (-185)–AvaII (+220), pKT -285; HaeII (-285)–HaeII (+80), pKT -382: XbaI (-382)– AvaII (+220), pKT -490; SmaI (-490)–AvaII (+220), pKT -853; XbaI (-853) –AvaII (+220), pKT -1145; HindIII (-1145)– AvaII (+220), pKT S-490; SmaI (-490)–HindIII (+494), pKT S-1145; HindIII (-1145) -HindIII (+494), pKT del T; PvuII (-185)–AvaII (+220) with a deletion between the AccI (-49) and PvuII (+25) restrictions sites. The XbaI and HindIII restriction sites at position -382 and +494 were introduced by PCR using the following oligonucleotides; 5'-AATCTAGACTGGGGTGGGGCTCCGT-3' in combination with a T7 promoter primer or 5'-ATAAGCTTGCCGCTCGCTGGCTCTGG-3' in combination with a T3 promoter primer, respectively, and the 1.8-kb promoter subclone in SK-bluescript as template using a standard PCR reaction. PCR products were subcloned and controlled by sequencing and restriction enzyme analysis.

Site directed-mutagenesis was performed using the pSelect vector and the Altered Sites cloning kit according to the manufacturers manual (Promega, Madison, WI). After introduction of the mutations, the constructs were controlled by sequencing. Expression plasmids for chicken c-Ets-1, human c-Ets-2, and the HTLV-1 LTR reporter construct 6–3 have been previously described (36). Oligonucleotides were synthesized by a Cyclone Plus DNA synthesizer (Millipore, Bedford, MA) (see Table 1Go for sequences).

Cells, RA, Cycloheximide, mRNA Isolation, and RNase Protection
BHK cells were cultured in bicarbonate-buffered DMEM (DMEM-bic) supplemented with 7.5% FCS. P19 EC and Mes-1 cells were cultured on gelatinized surfaces in a 50%/50% mixture of Ham’s F12 and DMEM-bic supplemented with 7.5% FCS as described by Mummery et al. (25). Mes-1 is a stable cell line derived from differentiated P19 cells that has mesodermal characteristics (25). Embryonic stem cells (ES-5) were cultured as previously described (12). RA was dissolved in dimethylsulfoxide as a 10-2 M stock and stored in liquid nitrogen. RA was used at a concentration of 10-6 or 10-7 M for the differentiation of P19 EC and ES-5 cells, respectively. Cycloheximide was purchased from Sigma (St. Louis, MO) and was used at a concentration of 20 µg/ml. Total cellular RNA was isolated using guanine isothiocyanate, followed by phenol chloroform extraction and ethanol precipitation as described by Chomczinsky and Sacchi (37). To generate PTHrP- and GAPDH-specific riboprobes, a 345-nt mouse PTHrP genomic fragment cloned in pGEM (kindly provided by Dr A. Broadus (Yale University, Hartford, CT) and a 110-nt mouse GAPDH cDNA fragment cloned in SK-Bluescript (38) were used as templates. After linearization, antisense riboprobes were generated in the presence of [{alpha}32P]UTP (Amersham, Arlington, Heights, IL) and T7 RNA polymerase (BRL, Rockville, MD). Probes were gel purified, counted, and diluted to a concentration of 100,000 cpm per µl. Hybridization was carried out overnight at 56 C in a total volume of 45 µl. The hybridization mix consisted of 100 µg total RNA, 100,000 cpm PTHrP riboprobe, 50,000 cpm GAPDH riboprobe, 80% deionized formamide, 400 mM NaCl, 40 mM 1,4-piperazine diethanesulfonic acid, pH 6.4, and 1 mM EDTA. After RNase A treatment and phenol/chloroform extraction, templates were precipitated and seperated by gel electrophoresis under denaturating conditions. The gel was dried and signal was visualized by autoradiography using Kodak XRX films and intensifying screens. Quantification was performed using the PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Transfections and CAT Assays
Undifferentiated P19 EC, Mes-1, and BHK cells were seeded in a six- well plate at day 1. The following day cells were transfected using calcium phosphate precipitation as previously described (39). In all transfections 1 µg pSV2LacZ plasmid was included to correct for transfection efficiencies. After 16 h of incubation, the medium was refreshed and the cells were incubated for an additional 24 h, after which the cells were harvested in 1 ml PBS, 25 mM EDTA. To study RA-induced PTHrP expression, P19 EC and ES-5 cells were seeded at day 1 in the presence of RA in six-well plates at a density of 100,000 or 70,000 cells per well, respectively. The cells were transfected at day 3. The next day the medium was refreshed and fresh RA was added. At day 5, cells were harvested as described above. The cells were pelleted and dissolved in 40–80 µl 250 mM Tris, 10 mM EDTA, pH 7.5, and repeatedly freeze-thawed to release the CAT enzyme. After the cell debris had been pelleted, 10–40 µl supernatant were used in a CAT assay as previously described (40). Samples were reassayed when the conversion was out of the linear range of CAT enzyme activity. After separation of acetylated and nonacetylated chloramphenicol by TLC, plates were exposed to PhosphorImager screens and subsequently analyzed and quantified using the PhosphorImager and ImageQuant software. LacZ staining was measured at 420 nm using 10 µl supernatant as previously described and used to correct for transfection efficiencies (41). Experiments were only analyzed when difference in transfection efficiency between separate wells was less than 2-fold. All transfection experiments were repeated at least three times.

Nuclear Extracts and Bandshift Assays
To isolate nuclear extracts, confluent 150-cm2 dishes were washed with ice-cold PBS, and cells were subsequenty scraped in PBS and pelleted. The pellet was dissolved in 200 µl buffer C containing 10% glycerol, 20 mM Tris-HCl, pH 7.2, 50 mM KCL, 2 mM dithiothreitol, 0.1 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 10 µg leupeptin/ml, and 10 µg aprotinin/ml. The cell membranes were disrupted by freeze-thawing, and nuclei were pelleted by a 15-sec spin in an Eppendorf centrifuge at 5500 rpm. After washing the nuclei in 200 µl buffer C, the pellet was dissolved in 200 µl buffer C containing 600 mM KCl and incubated at 4 C for 10 min. Cell debris was pelleted by a 15-sec spin at 16,000 x g at 4 C. The supernatant was diluted 3-fold using buffer C without KCl. The protein concentration was measured using a protein assay (Bio-Rad, Richmond, CA).

Bandshift reactions were performed in a total volume of 20 µl containing 20 µg nuclear extract, 0.5 µg polydeoxyinosinic-deoxycytidylic acid, 10 µl BSA, 10% glycerol, 10 mM Tris-HCl, pH 7.2, 25 mM KCl, 0.25 mM dithiothreitol, 0.25 mM ZnCl2. The reaction mix was preincubated on ice for 10 sec in the absence or presence of competitor before the probe was added. Oligonucleotide probes were labeled by filling in the sticky ends with [{alpha}32P]dCTP (Amersham) using the klenow fragment of DNA-polymerase (BRL). The reaction proceeded for another 20 sec on ice before the samples were loaded on a 4% TBE polyacrylamide gel that had been prerun in TBE buffer for 30 sec. TBE buffer contained 25 mM Tris, 25 mM boric acid, and 0.5 mM EDTA, pH 8.3. Gels were run at room temperature for 2 h at 200 V.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. A. E. Broadus (Yale University, Hartford, CT) for kindly providing us with the mouse PTHrP promoter and cDNA.


    FOOTNOTES
 
Address requests for reprints to: Marcel Karperien, Department of Endocrinology and Metabolic Disease, University Hopital Leiden, Building 1, C4-R, Albinusdreef 2, 2333 AALeiden, The Netherlands.

Received for publication May 28, 1996. Revision received June 18, 1997. Accepted for publication June 24, 1997.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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