Identification of a Retinoic Acid-Inducible Element in the Murine PTH/PTHrP (Parathyroid Hormone/Parathyroid Hormone-Related Peptide) Receptor Gene

Marcel Karperien, Hetty Farih-Sips, Jeanine A.A. Hendriks, Beate Lanske1, Socrates E. Papapoulos, Abdul-Badi Abou-Samra, Clemens W.G.M. Löwik and Libert H.K. Defize

Department of Endocrinology (M.K., H.F.-S., S.E.P., C.W.G.M.L.) and Department of Pediatrics (M.K.) Leiden University Medical Center 2300 RC Leiden, The Netherlands
Netherlands Institute for Developmental Biology (J.A.A.H., L.H.K.D.) 3584 CT Utrecht, The Netherlands
Endocrine Unit (B.L., A.-B.A.-S.) Massachusetts General Hospital Boston, Massachusetts 02114


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-{alpha} (RAR{alpha})-retinoid X receptor-{alpha} (RXR{alpha}) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXR{alpha} heterodimers and RXR{alpha} and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a bandshift assay. This is caused by increased expression of RXR ({alpha}, ß, or {gamma}) and by the induction of expression of RARß and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR ({alpha}, ß, or {gamma}) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PTH/PTH-related Peptide (PTHrP) receptor binds N-terminal fragments of PTH and PTHrP with equal affinity (1). This receptor has been cloned from various species, including human (2), rat (1), mouse (3), and opossum (4), and belongs to a subfamily of G protein-coupled receptors that includes receptors for various peptide hormones (i.e. secretin, calcitonin, vasoactive intestinal polypeptide), which all share a high degree of sequence conservation, especially in the last three membrane-spanning domains and in the initial portion of the C-terminal tail (5).

Recently, we have isolated and characterized overlapping genomic clones encoding the human, mouse, and rat PTH/PTHrP-receptor genes (6). The genomic organization of the PTH/PTHrP-receptor gene is complex, since the protein is encoded by 14 exons. Furthermore, the PTH/PTHrP-receptor gene uses at least two transcription start sites in a tissue-specific manner, resulting in several alternative spliced transcripts with unique tissue distribution (7, 8). The upstream transcription start site P1 in front of the untranslated exon U1 is solely used in kidney and ovary, while the downstream P2 transcription start site in front of exon U3 is ubiquitously used (7–9; nomenclature according to Ref. 8).

Cloning of the receptor cDNA enabled the study of the regulation of PTH/PTHrP receptor mRNA expression in vitro and in vivo. In addition to bone and kidney, PTH/PTHrP-receptor mRNA is expressed in virtually all embryonal and adult tissues (3, 10). Furthermore, regulation of PTH/PTHrP receptor expression by various hormones, growth factors, and cytokines in vitro has been well documented (11, 12, 13, 14). However, detailed studies of regulation of PTH/PTHrP-receptor expression at the promoter level are lacking, partly due to the complex organization of the receptor gene at its 5'-end and to the cell-specific regulation of receptor mRNA expression by hormones and growth factors. For example, treatment of rat osteosarcoma and renal tubular cells with N-terminal fragments of PTH or PTHrP results in down-regulation of PTH/PTHrP-receptor mRNA in marked contrast to the effect of adding these hormones to retinoic acid (RA)-differentiated F9 embryonal carcinoma (EC) and embryonal stem (ES) cells, which results in up-regulation of receptor mRNA expression (3, 11, 14).

Previously, we have shown that PTH/PTHrP receptor mRNA becomes expressed very early in murine embryogenesis, i.e. in the extraembryonic endoderm of the parietal and visceral yolk sac in early postimplantation embryos (3). The formation of extraembryonic endoderm can be studied in vitro by using EC and ES cells (14, 15). Culturing these cells in the presence of RA in monolayer induces the formation of extraembryonic endoderm-like cell types and concomitant PTH/PTHrP-receptor mRNA expression (3, 14, 15). In this study, we have characterized promoter regions of the murine PTH/PTHrP-receptor gene that are involved in this expression at the transcriptional level. We show that the RA-induced expression is mediated by the downstream P2 promoter, which is activated by a hormone response element consisting of a direct repeat separated by an interspacing of 1 bp (R-DR1) located 2.7 kb upstream of the P2 transcription start site. We show that extracts of RA-differentiated P19 cells contain three protein complexes specifically binding to the R-DR1 in a bandshift assay. These complexes contain among others retinoid X receptor (RXR) ({alpha}, ß or {gamma}), retinoid acid receptor-ß (RARß), and the orphan receptor chicken ovalbumin upstream promoter/transcription factor I/II (COUP TFI/II), but not RAR{alpha}. Finally, we show that the promoter region conferring positive transcriptional regulation of the PTH/PTHrP-receptor gene in RA-differentiated P19 cells is also active in the proper location in vivo, since it induces expression of a reporter gene in extraembryonic endoderm of 7.5-day-old mouse embryos (3).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Downstream P2 Promoter Is Involved in RA-Induced Expression of the PTH/PTHrP-Receptor Gene
Treatment of P19 EC cells with all-trans-RA (10-6 M) for 3 days resulted in a strong induction of PTH/PTHrP-receptor mRNA expression (Fig. 1AGo). To investigate which of the PTH/PTHrP-receptor promoters (or both) was (were) involved in this RA-induced expression, two promoter reporter constructs were made in front of the promoterless bacterial chloramphenicol acetyltransferase (CAT) gene. Construct P1 5.0 CAT contained about 5 kb of the sequences upstream of exon U1 including the P1 promoter and transcription start site. Construct P2 3.3 CAT contained about 3.3 kb of the sequences upstream of exon U3 including the downstream P2 promoter and transcription start site. Subsequently, transient transfections were performed in undifferentiated P19 EC cells and P19 cells treated for 3 days with RA. Neither of the constructs induced reporter activity in undifferentiated P19 EC cells (data not shown). In marked contrast, P2 3.3 CAT, but not P1 5.0 CAT, induced reporter activity approximately 10-fold in RA-treated cells when compared with the promoterless pCAT vector (Fig. 1BGo), indicating that at least part of the RA-induced expression of the PTH/PTHrP-receptor gene was mediated by the downstream P2 promoter.



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Figure 1. The Downstream P2 Promoter Is Involved in RA-Induced Expression of the PTH/PTHrP-Receptor Gene

A, Northern blot loaded with 15 µg total RNA from undifferentiated P19 EC cells and P19 cells treated for 3 days with 10-6 M all-trans-RA hybridized with a mouse PTH/PTHrP-receptor cDNA probe (PTH-R). RA differentiation induces a marked expression of PTH/PTHrP-receptor mRNA. Hybridization with a probe for 28 S RNA was used as a control for RNA loading. B, Transient transfection of PTH/PTHrP-receptor promoter constructs in P19 cells differentiated for 3 days with 10-6 M RA. A reporter construct driven by the downstream P2 promoter (P2 3.3 CAT) induced CAT activity in RA-differentiated P19 cells only. Reporter activity is expressed as fold induction relative to the activity of the control vector (pCAT), is corrected for transfection efficiency, and represents the mean of three independent experiments ± SEM.

 
To identify RA-responsive sequences in the PTH/PTHrP-receptor gene, a series of progressive promoter deletion constructs was made in front of the more sensitive luciferase reporter gene (Fig. 2AGo). All the constructs contained the downstream P2 promoter and transcription start site. Transient transfections were performed in undifferentiated and RA-differentiated P19 cells, after which luciferase activity was determined. Luciferase activity was expressed as fold induction relative to the activity of the promoterless luciferase vector pLuc. As shown in Fig. 2BGo, construct U3 0.1 containing the minimal P2 promoter showed increased reporter activity in RA-treated P19 cells. Reporter activity did not change significantly by increasing the length of the promoter fragment from 0.1 kb to 1.4 kb (compare construct U3 0.1 with U3 0.5 and U3 1.4), except for the activity of construct U3 0.9, which was decreased. Important RA-responsive sequences were located between 1.4 and 2.8 kb upstream of the P2 transcription start site (compare U3 2.8 with U3 1.4). Further increase in length of the promoter fragments (constructs U3 5.8 and U3 7.8) resulted in a slight decrease in reporter activity, indicating that these sequences did not positively contribute to the RA-induced expression of the PTH/PTHrP-receptor gene. Inclusion of the 1.0-kb intron between exon U3 and S in the promoter reporter constructs resulted in enhanced luciferase activity when compared with similar constructs lacking the intronic sequences (construct U3 0.9 vs. S 1.9 and U3 1.4 vs. S 2.4). This suggested that the intronic sequences contributed to the RA-induced expression. Analysis of constructs S 3.0 and S 3.8 revealed the presence of an RA-responsive region between 2.0 and 2.8 kb upstream of the P2 transcription start site.



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Figure 2. Three Promoter Regions Are Involved in RA-Induced Expression of the PTH/PTHrP-Receptor Gene

A, Schematic representation of the organization of the murine PTH/PTHrP-receptor gene and the location of the used promoter reporter constructs cloned in front of the luciferase reporter gene. The position of the untranslated exons, U1, U2, and U3, are indicated as well as the position of exon S, which codes for the ATG. The promoters in front of exon U1 (P1) and exon U3 (P2) are depicted (arrows). B, The promoter reporter constructs were transiently transfected in undifferentiated P19 EC cells (P19 EC) and in P19 cells treated for 3 days with 10-6 M RA (P19 3 days RA) as described in Materials and Methods. Luciferase activity of the promoter reporter constructs in the undifferentiated P19 EC cells was corrected for transfection efficiency and was expressed as fold induction relative to the activity of the promoterless pLuc vector in the undifferentiated cells, which was set to 1. Similarly, the luciferase activity of the promoter reporter constructs in the RA-differentiated cells was corrected for transfection efficiency and was expressed as fold induction relative to the activity of the promoterless pLuc vector in the RA-differentiated cells, which was set to 1. Fold inductions represent the mean of three to four independent duplicate experiments ± SEM.

 
Similar experiments were performed using 10-6 M 9-cis-RA or 13-cis-RA instead of all-trans-RA as differentiation-inducing agents. Compared with all-trans-RA, the induction of luciferase activity by 9-cis-RA was approximately 1.5-fold higher for all constructs tested, while inductions by 13-cis-RA were approximately 2-fold lower (data not shown). This indicated that at least part of the inductive effect of all-trans RA on PTH/PTHrP-receptor mRNA expression was caused by conversion of all-trans-RA into 9-cis-RA.

In summary, at least two regions positively contributed to RA-induced expression mediated by the downstream P2 promoter: 1) the intronic sequences between exon U3 and S and 2) sequences between 2.0 and 2.8 kb upstream of the P2 transcription start site.

The Upstream RA-Responsive Region Contains a Direct Repeat
To test whether the intronic sequence between exon U3 and S contained an RA-responsive element, it was removed from its natural context and subcloned in front of the minimal P2 promoter in either orientation (constructs U3 0.1 + S and U3 0.1 + S rev, Fig. 3AGo). Subsequently, transient transfections were performed in RA-differentiated P19 cells. In line with the results in Fig. 2Go, construct U3 0.1, which contains the minimal P2 promoter only, already displayed increased reporter activity in RA-treated P19 cells (Fig. 3BGo). The presence of intronic sequences in front of promoter reporter construct U3 0.1 did not further increase RA-induced reporter activity (Fig. 3BGo). In fact, a small reduction in luciferase activity was observed when the intron was cloned in the reversed orientation (U3 0.1 + S rev). This made it unlikely that the intron contained RA-responsive enhancers, which was furthermore supported by the absence of homology to binding sites known to be responsive to RA as demonstrated by sequence analysis (data not shown).



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Figure 3. RA-Responsive Sequences Are Located between 2.8 and 2.5 kb Upstream of the P2 Promoter

A, Schematic representation of the 5'-organization of the PTH/PTHrP-receptor gene with the exons U1, U2, U3, and S and the location of the various restriction fragments cloned in front of a heterologous promoter. The P1 and P2 promoters are marked by arrows. H, HindIII; P2, BamHI-site introduced by PCR; P1, BamHI-site introduced by PCR; B, BglII; Xh, XhoI; K, KpnI; X, XbaI; S, SmaI. B, Luciferase reporter constructs in which the intronic sequences between exon U3 and S were recloned in front of the minimal P2 promoter (U3 0.1) were transiently transfected in P19 cells treated for 3 days with 10-6 M RA as described in Materials and Methods. Luciferase activity is expressed as fold induction relative to the promoterless pLuc vector of which the activity was set to 1. Values represent the mean of three independent duplicate experiments ± SEM. C, PTH/PTHrP-receptor promoter fragments were cloned in front of the luciferase reporter gene driven by the heterologous TK-promoter and transiently transfected in P19 cells treated for 3 days with 10-6 M RA as described in Materials and Methods. Luciferase activity is expressed as fold induction relative to the promoterless pTK Luc vector of which the activity was set to 1. Values represent the mean of three independent duplicate experiments ± SEM.

 
We then examined the upstream RA-responsive region for enhancer activity by cloning various fragments in front of the heterologous thymidine kinase promoter coupled to the luciferase reporter gene (pTK Luc). None of the fragments located between 2.0 and 0.5 kb upstream of the P2 transcription start site were able to activate the pTK Luc construct in RA-differentiated P19 cells (Fig. 3Go, A and C). Instead, a slight decrease in reporter activity was observed (constructs pTK 0.9–0.5, pTK 1.4–0.9, pTK 1.6–0.9, and pTK 2.0–1.4). In marked contrast, construct pTK 2.8–1.4 caused an approximately 4.5-fold induction of reporter activity, suggesting the presence of an RA-responsive sequence in this region, in line with the enhanced reporter activity of construct U3 2.8 vs. U3 1.4 (Fig. 2BGo). By testing three additional deletion constructs of pTK 2.8–1.4, the RA-responsive region was confined to a 300-bp fragment located between 2.8 and 2.5 kb upstream of the P2 transcription start site (constructs pTK 2.8–2.0, 2.8–2.3, and 2.8–2.5). The presence of an RA-responsive enhancer in this region was in agreement with the data of the promoter deletion constructs presented in Fig. 2BGo. Compared with constructs pTK 2.8–2.5 and pTK 2.8–2.3, reporter activity of construct pTK 2.8–2.0 was approximately 2-fold lower. This suggested that in the region between 2.3 and 2.0 kb upstream of the P2 transcription start site, elements were located that could down-modulate RA-induced expression of the enhancer region located further upstream.

Sequence analysis of the region between 2.8 and 2.5 kb upstream of the P2 promoter revealed the presence of a direct repeat with an interspacing of 1 bp (DR1) between nucleotide (nt) -2714 and -2702 (Fig. 4AGo) relative to the P2 transcription start site. This sequence matches the consensus DR1 (Fig. 4BGo), which is known to bind various members of the nuclear hormone receptor family including retinoid receptors (16, 17). Comparing the mouse PTH/PTHrP-receptor DR1 (R-DR1) with the sequence of the rat PTH/PTHrP-receptor gene demonstrated that the R-DR1 was conserved in the rat gene in location and sequence except for a nucleotide change in the 3'-repeat in which an adenosine was replaced by a guanidine (AGTTCA in mouse vs. GGTTCA in rat, Fig. 4BGo). The rat DR1, however, also matches the consensus DR1 sequence.



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Figure 4. The Upstream RA-Responsive Region Contains a Direct Repeat

A, Sequence of the region between 2.8 and 2.5 kb upstream of the P2 promoter which is responsive to RA. A direct repeat with an interspacing of 1 bp is depicted in uppercase letters and is underlined. B, The sequence of the DR1 in the mouse PTH/PTHrP-receptor gene (R-DR1) fits the consensus sequence for a DR1 (Pu, purine) and is conserved in the rat gene (direct repeat is depicted in uppercase letters). The sequence of the mouse R-DR1 is used as a probe in bandshift assays. Also depicted is the sequence of a mutant R-DR1 (mtR-DR1) oligonucleotide that is used in bandshift assays. The mutant nucleotides are depicted in bold lowercase letters and are underlined.

 
To determine whether the R-DR1 was able to bind members of the nuclear hormone receptor family such as the human retinoic acid receptor {alpha} (hRAR{alpha}), the human retinoid X receptor {alpha} (hRXR{alpha}), and the orphan receptor COUP-TFI, bandshift analyses were performed. For this, whole-cell extracts (WCE) of COS-7 cells transiently transfected with an empty expression vector, or a hRAR{alpha}, a hRXR{alpha}, or a COUP-TFI expression vector were used in a bandshift assay with the R-DR1 as probe (Fig. 5Go; see Fig. 4bGo for sequence of R-DR1 oligonucleotide). As shown in lane 1, extracts of mock transfected COS-7 cells did not contain binding activity to the R-DR1. hRAR{alpha}-containing extracts only faintly displayed binding activity to the R-DR1, in agreement with data indicating that homodimers of hRAR{alpha} cannot efficiently bind to DNA (Ref. 16 ; Fig. 5Go, lane 2). In marked contrast, extracts of hRXR{alpha}- and COUP-TFI-transfected cells contained binding activity indicating that these receptors were able to bind the R-DR1 as a homodimer (Refs. 16, 17 ; Fig. 5Go, lanes 4 and 5). Mixing hRAR{alpha}- and hRXR{alpha}-containing extracts resulted in strong binding to the R-DR1, probably due to heterodimerization (Fig. 5Go, lane 3). Also mixing of hRXR{alpha}- and COUP-TFI-containing extracts resulted in an increased binding activity, suggesting that heterodimerization between hRXR{alpha} and COUP-TFI might occur at the R-DR1 (Fig. 5Go, lane 6). Addition of hRAR{alpha} to COUP-TFI-containing extracts did not influence binding of COUP-TFI as a homodimer (Fig. 5Go, lane 7). Binding of the various nuclear hormone receptors either as homo- or as heterodimer suggested that the R-DR1 could behave as a classic hormone response element.



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Figure 5. The Direct Repeat Binds Members of the Nuclear Hormone Receptor Family

WCE of COS-7 cells transiently transfected with an empty expression vector, a hRAR{alpha}, a hRXR{alpha}, and a hCOUP-TFI expression vector were used in a bandshift assay with the R-DR1 oligonucleotide (see Fig. 4BGo for sequence) as a probe. Lane 1, 4 µg WCE of mock transfected cells (WCE-mock); lane 2, 2 µg WCE-mock mixed with 2 µg WCE of hRAR{alpha}-transfected cells (WCE-RAR); lane 3, 2 µg of WCE-RAR mixed with WCE of hRXR{alpha}-transfected cells (WCE-RXR); lane 4, 2 µg of WCE-mock mixed with 2 µg of WCE-RXR; lane 5, 2 µg of WCE-mock mixed with 2 µg of WCE of hCOUP-TFI-transfected cells (WCE-COUP); lane 6, 2 µg of WCE-RXR mixed with 2 µg of WCE-COUP; lane 7, 2 µg of WCE-RAR mixed with 2 µg of WCE-COUP.

 
RA Differentiation Induces Protein Binding to the PTH/PTHrP Receptor DR1
Having established the identity of the R-DR1 as a putative hormone response element, we determined whether WCE of undifferentiated and RA-differentiated P19 cells contained binding activity to the R-DR1 in a bandshift assay. As shown in Fig. 6AGo, three major complexes bound to the R-DR1 using extracts of undifferentiated P19 EC cells (I, II, and III, lane marked -). Complex II specifically bound to the direct repeat since it was efficiently competed by a 25-fold excess of unlabeled R-DR1 oligonucleotide and by a consensus DR1 (con DR1) binding site. Weak competition was observed with a consensus DR5 (direct repeat with interspacing of 5 bp, con DR5) binding site, while the complex was not competed by a 25-fold excess of an unlabeled nonspecific competitor or by a mutant (mt) R-DR1 binding site (See Fig. 4BGo for sequence). Complex I also bound to the direct repeat although with less affinity than complex II. This complex was efficiently competed by a 25-fold excess of unlabeled consensus DR1 oligonucleotide, while no competition was observed with the other competitors. Competition was found, however, when the concentration of the unlabeled R-DR1 oligonucleotide was raised to 100-fold excess (data not shown). Complex III most likely bound to the flanking sequence of the R-DR1 and mtR-DR1 oligonucleotide, since it was efficiently competed by a 25-fold excess of unlabeled R-DR1 and mtR-DR1 oligonucleotides, while no or only faint competition was observed using the nonspecific competitor and the consensus DR5- and DR1-binding sites, respectively. Using a supershifting antibody cross-reacting with RXR{alpha}, ß and {gamma}, it was shown that the protein complexes contained RXR. Likewise, it was shown that in undifferentiated P19 EC cells RAR{alpha}, but not RARß, PPARy, or COUP TFI or TFII, bound to the R-DR1 (Fig. 6AGo).



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Figure 6. RA-Differentiation Induces de NovoProtein Complexes Binding to the R-DR1

Four micrograms of WCE of undifferentiated P19 EC cells (panel A) and 4 µg of WCE of P19 EC cells (panel B) treated for 3 days with 10-6 M RA were used in a bandshift assay with the R-DR1 oligonucleotide as a probe. Lanes are labeled as follows: -, no competition; nonspecific, competition with a 25-fold excess of nonspecific oligonucleotide; R1-DR1, competition with 25-fold excess of nonlabeled R-DR1 oligonucleotide; mtR1-DR1, competition with 25-fold excess of nonlabeled mtR-DR1 oligonucleotide; con DR1, competition with 25-fold excess of a consensus DR1 oligonucleotide; con DR5, competition with 25-fold excess of a consensus DR5 oligonucleotide; anti-RXR{alpha}/ß/{gamma}, incubation with a supershifting antibody cross-reacting with RXR{alpha}, -ß, and -{gamma}. The arrowhead indicates the supershift; lanes marked anti RAR{alpha} represent incubation with a supershifting antibody for RAR{alpha}. The arrowhead indicates the supershift; lanes marked anti-RARß show incubation with a supershifting antibody for RARß. The small arrow indicates a nonspecific complex, while the arrowhead indicates the specific shift; lanes marked anti PPAR{gamma} depict incubation with a supershifting antibody for PPAR{gamma}; no shift was observed. Lanes marked anti COUP-TFI/II show incubation with a supershifting antibody cross-reacting with COUP-TFI/TFII. The arrowhead indicates the specific shift. The bold arrows in the margins of the figure indicate the four major specific complexes (I, II, III, and IV). Note the difference in exposure time, which for panel A is 3 days and for panel B is 1 day.

 
RA differentiation of P19 EC cells strongly increased protein binding to the R-DR1 (Fig. 6BGo). This was even more dramatic when the prolonged exposure time of 3 days for the undifferentiated cells compared with the 1-day exposure time of the RA-differentiated cells was taken into consideration. The enhanced protein binding was caused by strongly increased binding of complex I and the induction of expression of a novel complex (IV, Fig. 6BGo), while binding of complex II changed only marginally. Complex III was not present in the RA-differentiated P19 cells. The complexes I, II, and IV were specifically binding to the direct repeat, since they were efficiently competed by the unlabeled R-DR1 oligonucleotide and the consensus DR1- and DR5-binding sites, while no competition was observed using nonspecific and mtR-DR1 oligonucleotides. As for the undifferentiated P19 EC cells, complex I was most efficiently competed by the consensus DR1 binding site compared with the partial competition by the R-DR1 and consensus DR5 oligonucleotides. When the concentration of the latter oligonucleotides was increased to a 100-fold excess, however, complete competition was observed (data not shown). Using various antibodies for nuclear hormone receptors, shifts were observed using an antibody for RXR ({alpha}, ß, or {gamma}) and COUP TFI/TFII, and a very weak shift was observed using an antibody for RARß. No specific shifts were observed using an antibody for RAR{alpha} or PPAR{gamma} (Fig. 6BGo).

Mutation of the R-DR1 Strongly Impairs RA-Induced Expression of PTH/PTHrP Receptor Promoter Reporter Constructs
To determine the importance of a functional R-DR1 for RA-induced expression, the R-DR1 was mutated in various receptor promoter reporter constructs, and transient transfections were performed in RA-differentiated P19 cells. As shown in Fig. 7AGo, mutation of the R-DR1 in construct pTK 2.8–2.3 (see Fig. 3AGo for map) resulted in a marked reduction of reporter activity, indicating that a functional R-DR1 was a prerequisite for RA-induced expression. The reporter activity of the mutant construct (pTK 2.8–2.3 mtDR1) was reproducibly slightly higher than the empty pTK vector, suggesting that the mutation did not completely abolish binding of transcription factors to the R-DR1 or that RA-induced expression was not completely mediated by the R-DR1. The latter possibility was supported by construct pTK DR1 in which one copy of the R-DR1 was cloned in front of the thymidine kinase promoter. In contrast to its mutated counterpart (pTK mtDR1), this construct was able to confer RA-induced reporter activity but not as efficiently as construct pTK 2.8–2.3, suggesting a role for the flanking sequences of the R-DR1.



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Figure 7. Mutation of the R-DR1 Impairs RA-Induced Expression of PTH/PTHrP-Receptor Promoter Reporter Constructs

A, Transient transfections of promoter fragments of the PTH/PTHrP-receptor promoter cloned in front of the luciferase reporter gene driven by the heterologous TK-promoter in P19 cells treated for 3 days with 10-6 M RA. See Fig. 3AGo for location of the promoter fragments. Luciferase activity is expressed as fold induction relative to the activity of the empty pTK Luc vector of which the activity was set to 1. Values are expressed as mean of three independent duplicate experiments ± SEM. B, Same as in panel A except that now promoter fragments were cloned in front of the autologous P2 promoter (see Fig. 2AGo for map). Luciferase activity is expressed as fold induction relative to the empty pLuc vector of which the activity was set to 1. Values are expressed as mean of three independent duplicate experiments ± SEM.

 
In addition, mutation of the R-DR1 in two constructs, i.e. U3 0.5 + 2.8–2.3, in which the upstream RA-responsive region was cloned in front of a 500-bp fragment coding for the downstream P2 promoter and transcription start site (U3 0.5 +2.8–2.3 mtDR1), and U3 2.8D, which was similar to construct U3 2.8 (see Fig. 2AGo for map) except for an internal deletion between nt -2.3 and -2.1 kb upstream of the P2 transcription start site, reduced reporter activity in RA-differentiated P19 cells 2.5- to 3-fold (Fig. 7BGo). Since the RA-induced reporter activity was not completely abolished by mutation of the R-DR1, it was again suggested that the flanking sequences of the R-DR1 located between 2.8 and 2.3 kb upstream of the P2 transcription start site were also involved in RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs.

A PTH/PTHrP Receptor Promoter LacZ Construct Induces ß-Galactosidase Activity in the Extraembryonic Endoderm of 7.5-Day-Old Mouse Embryos
Previously we have demonstrated that PTH/PTHrP-receptor mRNA is expressed in the parietal and visceral endoderm of early postimplantation embryos (3). One of the results of RA-induced differentiation of P19 cells in monolayer is the appearance of an extraembryonic visceral endoderm-like cell type and concomitant PTH/PTHrP-receptor expression (3, 18, 19). We therefore tested whether promoter fragments that were able to induce reporter gene expression in RA-differentiated P19 cells were also able to induce expression of a reporter gene in the extraembryonic endoderm of 7.5-day-old mouse embryos. For this, two PTH/PTHrP-receptor promoter fragments were cloned in front of the bacterial ß-galactosidase reporter gene. The reporter constructs were subsequently injected into fertilized mouse oocytes, after which they were reimplanted into the uterus of pseudopregnant foster mothers. After 8.5 days, embryos were isolated and assayed for ß-galactosidase activity. As the injection resulted in a delayed development, these embryos represented 7.5-day-old normal embryos. Injection of a promoterless construct did not induce ß-galactosidase activity in any of the analyzed embryos (control; Table 1Go). In addition, injection of construct S1.9 LacZ (see Fig. 2AGo for map) did not induce ß-galactosidase staining in any of the embryos. This was in contrast to the comparable luciferase construct, which induced some reporter activity in RA-treated cells in vitro (Fig. 2Go). Construct S8.5 LacZ, which contained 7.5 kb of sequences upstream of the P2 transcription start site, as well as the intron between exon U3 and S, induced ß-galactosidase activity in 40% of the injected embryos. This construct contained the R-DR1 and efficiently induced PTH/PTHrP-receptor expression in RA-treated P19 cells. As shown in Fig. 8Go, the ß-galactosidase activity was confined to the extraembryonic endoderm of the parietal yolk sac and to the visceral endoderm, an expression pattern that exactly matches the endogenous expression pattern obtained by in situ hybridization (3). The absence of ß-galactosidase in the remaining embryos injected with S8.5 LacZ might be explained by either mosaicism and/or by integration of the injected DNA fragments in transcriptionally inactive DNA.


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Table 1. PTH/PTHrP-Receptor Promoter Constructs Induce ß-Galactosidase Activity in 7.5-Day-Old Mouse Embryos

 


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Figure 8. A Promoter Fragment of 8.5 kb Induces Expression of a Transgene in Extraembryonic Endoderm of Day 7.5 Postcoitum Mouse Embryos

Fertilized oocytes were injected with two promoter fragments of the mouse PTH/PTHrP-receptor gene cloned in front of the ß-galactosidase gene. After reimplantation of the injected oocytes in pseudopregnant foster mothers, normal pregnancy was allowed to occur. Embryos were isolated at day 8.5 after reimplantation, which at this time represents 7.5-day-old normal mouse embryos. Embryos were separated from the surrounding tissue, but the parietal and visceral yolk sac were left intact. Embryos were stained for ß-galactosidase activity for 2 days as described in Materials and Methods. A, Embryo injected with construct S 1.9 LacZ, containing 0.9 kb of sequences upstream of the P2 promoter and the intron between exon U3 and S. B, Embryo injected with construct S 8.5 LacZ containing 7.5 kb of sequences upstream of the P2 promoter and the intron between exon U3 and S. Specific staining is observed in extraembryonic endoderm and is marked with arrowheads. Staining as a result of the photographic procedure is indicated by small arrows. epc, Ectoplacental cone.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we have identified promoter elements in the murine PTH/PTHrP-receptor gene that are involved in RA-induced receptor expression in P19 EC cells. These elements include the downstream P2 promoter, the intron between exon U3 and S, and a hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The involvement of the downstream P2 promoter in the RA-induced expression of the PTH/PTHrP-receptor was not unexpected, since this promoter ubiquitously drives mRNA expression in most tissues and cell lines expressing the receptor (7, 8). Furthermore, we have cloned a PTH/PTHrP-receptor cDNA from RA-treated P19 cells containing part of the P2-specific exon U3 (3).

Sequence analysis and cloning of the intron between exon U3 and S in front of the minimal PTH/PTHrP-receptor promoter did not result in the identification of RA-responsive cis-acting elements. Since it has been described that the presence of an intron in the untranslated leader can contribute to either increased mRNA stability or translation efficiency, this may well explain enhanced reporter activity (20, 21). Another explanation was recently provided by Bettoun et al. (22). They identified a third promoter P3 in the human PTH/PTHrP-receptor gene that is located in the intron between exon U3 and S directly in front of the downstream exon and was mainly active in kidney. However, at present, there is no evidence that such a promoter also exists in mouse.

Testing of a panel of promoter reporter constructs with increasing length in P19 cells treated with RA, combined with sequence analysis, has led to the identification of a hormone response element consisting of a direct repeat separated by 1 bp (DR1). The PTH/PTHrP-receptor DR1 (R-DR1) was conserved in the rat gene in location and sequence. We confirmed the identity of the R-DR1 as a putative hormone response element by showing that various nuclear hormone receptors were efficiently able to bind to this sequence as a hetero- or homodimer in a bandshift assay. Factors binding to the R-DR1 include heterodimers between RAR{alpha} and RXR{alpha} and between COUP-TFI and RXR{alpha} as well as homodimers of RXR{alpha} and COUP-TFI, while RAR{alpha} homodimers displayed only minor binding activity to this sequence. These data are in agreement with previously reported binding characteristics of a DR1 (16, 17). Furthermore, DR1s have been identified in the promoters of various RA-responsive genes, e.g. the mouse cellular retinoic acid binding protein II (CRABPII) (23), {alpha}-fetoprotein (24), lactoferrin (25), and the human RXR{gamma}2 genes (26), and are involved in RA-induced gene expression. Mutation analysis of the R-DR1 unequivocally demonstrated the importance of the R-DR1 for RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. However, since the RA-induced expression was not completely abolished, we suggest that other sequences are also involved in the RA effect. This is currently under investigation.

In bandshift assays, extracts of undifferentiated and RA-differentiated P19 cells contained various protein complexes binding to the R-DR1. Compared with extracts of undifferentiated P19 cells, protein binding to the R-DR1 was markedly increased in RA-treated cells. This was in agreement with previous observations (23, 27). The increased binding was caused by both up-regulation of preexisting protein complexes as well as by induction of expression of a novel protein complex. Nuclear hormone receptors that bound to the direct repeat in extracts of undifferentiated P19 cells included RXR ({alpha}, ß, or {gamma}) and RAR{alpha}, while binding of RARß, COUP TFI/II, or PPAR{gamma} was not detected. In RA-differentiated cells, RXR ({alpha}, ß, or {gamma}), COUP TFI/II, and RARß bound to the R-DR1 while binding of RAR{alpha} and PPAR{gamma} was not detectable.

RA differentiation of P19 cells is known to induce various hormone receptors that can bind to a DR1 either as homodimer or as a heterodimer with the obligate partner RXR. These factors include RARß (28) and the orphan receptors, COUP-TFI and COUP-TFII/ARP-1 (27). In agreement with these studies, we showed that binding of COUP TFI/TFII and weak binding of RARß to the R-DR1 was induced during RA-mediated differentiation of P19 EC cells. However, as COUP-related transcription factors are generally considered to be negative regulators of RA-induced gene expression (29) and heterodimers of RARß and RXR generally repress transcription when bound to a DR1 (16), it does not seem likely that these de novo factors were responsible for RA-induced expression of the PTH/PTHrP-receptor gene. RA-mediated differentiation of P19 EC cells is also known to increase the expression of RXR{alpha} especially, whereas expression of RXRß is somewhat down-regulated particularly after prolonged treatment with RA (27, 28, 30). Binding of RXR family members was observed in undifferentiated and RA-differentiated P19 cells using a supershifting antibody cross-reacting with RXR{alpha}, -ß, and -{gamma}. Compared with undifferentiated cells, the expression of RXR was up-regulated in the RA-differentiated P19 cells, suggesting that at least part of the up-regulated protein binding of the preexisting complexes binding to the R-DR1 was caused by increased RXR expression. RXR homodimers are potent activators of gene transcription when bound to a DR1 and have been implicated in RA-induced expression of various RA-responsive genes (16, 17, 24, 26). RXR homodimers are therefore likely to be involved in the RA-induced expression of the PTH/PTHrP-receptor gene. This was also supported by our observation that the selective RXR ligand 9-cis-RA caused a more pronounced induction of PTH/PTHrP-receptor mRNA expression compared with all-trans-RA. Furthermore, 9-cis-RA activated PTH/PTHrP-receptor promoter reporter constructs more efficiently than all-trans-RA (data not shown), suggesting that at least part of the induction of PTH/PTHrP-receptor expression was caused by the conversion of all-trans-RA into 9-cis-RA. Such conversion is known to occur especially when cells are treated with high dosis of all-trans-RA as in this study (23). A role for RXR in regulation of PTH/PTHrP-receptor mRNA expression is also suggested by Sneddon and co-workers (44). They proposed that the up-regulated expression of PTH/PTHrP-receptor mRNA by 1,25-(OH)2D3 in renal tubuli cells occurred in a manner consistent with binding of heterodimers of RXR and the 1,25-(OH)2D3 receptor to a vitamin D response element in the PTH/PTHrP-receptor gene. In addition, they showed that 9-cis-RA alone was also able to increase PTH/PTHrP-receptor mRNA expression. The simultaneous presence of nuclear hormone receptors in extracts of RA-differentiated P19 cells that can either repress (e.g. COUP TFI/TFII, RARß/RXR heterodimers) or stimulate transcription (e.g. RXR homodimers) via a DR1 is in line with a model in which the relative expression of receptors with binding capacity to a DR1 in a given cell type eventually determines whether a DR1 acts as an activator or a repressor of transcription (16, 31).

Based upon our bandshift analysis we cannot exclude that other nuclear hormone or orphan receptors with binding affinity to a DR1 were also induced during RA-mediated differentiation of P19 EC cells and were involved in the RA-induced expression of the PTH/PTHrP-receptor gene. One such factor might be the orphan receptor HNF4{alpha}, since this receptor is a potent constitutive activator of transcription via a DR1, and HNF4{alpha} and the PTH/PTHrP-receptor are coexpressed in the visceral endoderm of early mouse embryos (3, 32). Furthermore, upon RA-mediated differentiation of F9 EC cells toward a visceral endoderm-like cell type, HNF4{alpha} as well as PTH/PTHrP-receptor mRNA expression are induced (32). Presently, we are testing whether RA also induces the expression of HNF4{alpha} in P19 EC cells and, if so, whether this receptor is involved in the RA-induced expression of the PTH/PTHrP-receptor gene.

Interestingly, undifferentiated P19 EC cells also expressed a protein complex binding to the flanking sequence of the R-DR1 oligonucleotide. This was based on the observation that this complex could not, or only faintly, be competed by a nonspecific oligonucleotide and consensus DR5 and DR1 binding sites, respectively, while efficient competition was observed with the R-DR1 and the mtR-DR1 oligonucleotide. The latter harbored mutations in the direct repeat only. This complex was specifically down-regulated by RA differentiation, suggesting that down-regulation of this complex could be a prerequisite for RA-induced expression of the PTH/PTHrP-receptor gene. Down-regulation of this complex would facilitate increased protein binding of RXR-homodimers, for example, to the R-DR1 that subsequently transactivate transcription of the PTH/PTHrP-receptor gene. The binding of RXR homodimers can be further enhanced by the increased expression of RXR{alpha}, in particular, during RA-mediated differentiation of P19 EC cells (30). Such a mechanism might well explain why induction of PTH/PTHrP-receptor mRNA expression by RA is delayed. Treatment of P19 cells with RA for 1 or 2 days results only in faint inductions of PTH/PTHrP-receptor mRNA expression, while the expression peaks after 3 days of RA treatment (3). Alternatively, the delayed expression could be caused by an indirect effect of RA requiring the synthesis of new factors that, in turn, are involved in the RA-induced expression of PTH/PTHrP-receptor mRNA. Thus far, computer analysis of the flanking sequences of the R-DR1 did not reveal homology to previously identified transcription factor-binding sites. Partial overlapping binding sites for distinct classes of transcription factors, including the nuclear hormone receptor family, have been described in various promoters, e.g. the composite glucocorticoid receptor and AP1 responsive element of the mouse proliferin gene (33) and the human papilloma virus type 16-regulatory region (34), and the overlap of the RA- and estrogen-responsive elements in the lactoferrin gene (25). The presence of partially overlapping transcription factor-binding sites enables cross-talk between multiple signal transduction cascades and greatly contributes to differential regulation of gene expression. Currently, we are trying to identify the binding site of this complex and its constituents in more detail.

As RA differentiation of P19 EC cells is an established model system to study early differentiation processes in mouse embryogenesis (18, 19), we tested whether promoter fragments of the PTH/PTHrP-receptor gene that were able to drive expression in RA-differentiated P19 cells were also able to induce expression of a reporter gene in early postimplantation mouse embryos. We have shown previously that PTH/PTHrP-receptor mRNA is highly expressed in the extraembryonic endoderm of early postimplantation embryos from day 5.5 post coitum and onward (3). Indeed, 8.5 kb of sequences upstream of the P2 promoter were sufficient to drive reporter gene expression in a spatio- and temporal fashion, which corresponded very well with the expression of PTH/PTHrP-receptor mRNA in a 7.5-day-old mouse embryo (3). Interestingly, this promoter fragment efficiently induced reporter activity in RA-differentiated P19 cells and contained the R-DR1. In marked contrast, a construct that contained 0.9 kb of sequence upstream of the P2 promoter as well as the intron between exon U3 and S did not induce reporter activity in any of the injected embryos, although this fragment induced some reporter activity in RA-differentiated P19 cells. This indicated that the minimal P2 promoter as well as the downstream intron were not sufficient for driving PTH/PTHrP-receptor expression in vivo. Apparently, this sequence did not contain enhancers that by themselves were sufficient for induction of reporter expression in extraembryonic endoderm in vivo. This observation provided indirect support for our conclusions that these regions did not contain cis-acting elements involved in the RA effect, but that the increased expression of these reporter constructs was caused by indirect mechanisms such as increased mRNA stability or translation efficiency. Experiments to further define the elements involved in regulation of receptor expression in vivo and the significance of the R-DR1 are currently underway.

At present, it is unclear whether the R-DR1 is important for regulation of PTH/PTHrP-receptor expression in the classic target tissues of PTH, i.e. kidney and bone. However, given the profound effects of retinoids on chondrogenesis and osteogenesis (35, 36), a role for the R-DR1 in regulation of PTH/PTHrP-receptor expression in osteoblasts or chondrocytes would not be unexpected. Interestingly, treatment of UMR106 osteoblast-like cells with RA causes a down-regulation of PTH/PTHrP-receptor expression (13). Although this effect is contrary to the observations in EC and ES cells, it might very well be that this effect is also mediated by the R-DR1. There is cumulating evidence that the relative expression of various hormone and orphan receptors, all competing for binding to a DR1 in a given cell type, eventually determines whether a DR1 acts as an activator or a repressor of transcription (16, 31). Such a mechanism might be responsible for the cell type-dependent regulation of PTH/PTHrP-receptor mRNA expression by various growth factors and hormones in which opposite effects are often found (11, 14). Currently, we are performing studies in osteoblasts and chondrocytes to test whether the R-DR1 is of general importance for PTH/PTHrP-receptor expression during bone formation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTH/PTHrP-Receptor Promoter Constructs, Expression Vectors, and Oligonucleotides
Isolation of a genomic clone encoding the 5'-region of the mouse PTH/PTHrP-receptor gene has been described elsewhere (6, 7). Double-stranded sequence analysis was either performed manually using the double-stranded DNA sequencing kit (Pharmacia Biotech, Piscataway, NJ) or using an automated sequencer (PE Applied Biosystems, Foster City, CA). All promoter reporter constructs were made according to standard DNA cloning techniques. The position of the restriction sites used for the construction of the promoter reporter constructs are all relative to the P2 promoter transcription start site. P1 and P2 promoter reporter constructs were cloned in front of the promoterless bacterial CAT gene: construct P1 5.0 CAT; XbaI (-8334)–XbaI (-3330). This site was introduced by PCR in exon U1. P2 3.3 CAT; PstI (-3166)–BamHI (+122). The following promoter fragments were cloned in front of the promoterless luciferase reporter gene (43): U3 0.1, SmaI (-32)–BamHI (+122); U3 0.5, XhoI (-445)–BamHI (+122); U3 0.9, XbaI (-847)–BamHI (+122); U3 1.4, KpnI (-1403)–BamHI (+122); U3 2.8, HindIII (-2783)–BamHI (+122), U3 5.8, BamHI (-5866)–BamHI (+122); U3 7.8 XmnI (-7803)–BamHI (+122); S 1.9, XbaI (-847)–XbaI (+1354, this site is introduced by PCR in exon S); S 2.4, KpnI (-1403)–XbaI (+1354); S 3.0, BglII (-1915)–XbaI (+1354); S 3.8, HindIII (-2783)–XbaI (+1354). The SmaI-restriction fragment coding for the intron between exon U3 and S was cloned in front of U3 0.1 (SmaI (+154)–SmaI (+1252). The following restriction fragments were cloned in front of the thymidine kinase promoter-driven luciferase reporter gene (41): pTK 0.9–0.5 [XbaI (-847)–XhoI (-445)]; pTK 1.4–0.9 [KpnI (-1403)–XbaI (-847)]; pTK 1.6–0.9 [XhoI (-1597)–XbaI (-847)]; pTK 2.0–1.4 [BglII (-1915)–KpnI (-1403)]; pTK 2.8–2.0 (HindIII (-2783)–BglII (-1915)]; pTK 2.8–2.3 [HindIII (-2783)–BamHI (-2282, introduced by PCR)]; pTK 2.8–2.5 [HindIII (-2783)–BamHI (-2513, introduced by PCR)]. Construct U3 0.5 + 2.8–2.3 and U3 2.8D are similar to construct U3 2.8 except for an internal deletion between nt -2326 and -2147, respectively. Mutations in the R-DR1 were introduced by PCR. All promoter reporter constructs were controlled by restriction fragment analysis and partial sequencing.

Expression vectors for hRAR{alpha} and hRXR{alpha} were derived from Dr. Pierre Chambon and Dr. R. Evans, respectively. A COUP-TFI expression vector was derived from Dr. S. Tsai. A polyclonal antibody cross-reacting with COUP-TFI/TFII was kindly provided by Dr. M. Parker (37). The antibodies 4RX1D12 (anti-RXR{alpha}, ß, {gamma}), 9{alpha} (anti-RAR{alpha}), and 6ß-2A10 (anti-RARß) were derived from Dr. P. Chambon (38), and a polyclonal antibody for PPAR{gamma} was derived from Dr. J. Auwerx.

Oligonucleotides for site-directed mutagenesis and bandshift analysis were provided by Eurogentec (Seraing, Belgium): R-DR1-sense, 5'-AGC TTC AGC CCA AGG TCA GAG TTC AGC CAC CGG TTG-3'; R-DR1-antisense, 5'-GAT CCA ACC GGT GGC TGA ACT CTG ACC TTG GGC TGA-3'; mtR-DR1-sense, 5'-AGC TTC AGC CCA AGG TCT AGA GCT CAG CCA CCG GTT G-3'; mtR-DR1-antisense, 5'-GAT CCA ACC GGT GGC TGA GCT CTA GAC CTT GGG CTG A-3'; consensus DR1-sense (29), 5'-AGC TGG AGG TCA CAG GTC ACA; consensus DR1-antisense, 5'-TCG AAC ACT GGA CAC TGG AGG; consensus DR5-sense (29), 5'-AGC TGG AGG TCA CTG TCA GGT CAC A; consensus DR5-antisense, 5'-TCG AAC ACT GGA CTG TCA CTG GAG G; nonspecific oligonucleotide sense, 5'-AGC TAT CAG AAA AAC CAC ACA GGG GTG-3'; antisense, 5'-GAT CCA CCC CTG TGT GGT TTT TCT GAT-3'.

Cells, RA, mRNA Isolation, and Northern Blot
P19 EC cells and COS-7 cells were cultured as described previously (39, 40). All-trans, 9-cis, and 13-cis-retinoic acid (RA) were obtained from Sigma Chemical Co. (St. Louis, MO) and stored under light-protected conditions as a 10-2 M stock solution in dimethylsulfoxide at -20 C. Total RNA was isolated by seeding 1.5 x 106 cells in a 56-cm2 tissue culture disk in the presence of 10-6 M RA or vehicle. Cells were cultured for 3 days, after which total RNA was isolated by the method of Chomczinsky and Sacchi (41) as described previously. Northern blotting and the mouse PTH/PTHrP-receptor cDNA probe have been described elsewhere (3).

Transient Transfections and Reporter Assays
For transient transfections of undifferentiated P19 cells, 12,500 cells/cm2 were seeded in a 12-well tissue culture disk in 1 ml culture medium at day 0. At day 2, cells were transfected with 0.5 µg (TK Luc) or 1.25 µg (Luc) reporter construct using DOTAP (Boehringer Mannheim, Indianapolis, IN) according the manufacturer’s protocol. In short, DNA was dissolved in 12.5 µl 20 mM HEPES (pH 7.4) and mixed with an 1:4 dilution of DOTAP in 20 mM HEPES (pH 7.4). After 10 min incubation at room temperature, DNA was mixed with 1 ml of prewarmed fresh medium and applied to cells from which the old medium had been removed. After 6 h of incubation, the transfection mix was replaced by fresh medium. For transient transfection of RA-treated cells, 15,000 cells/cm2 instead of 12,500 cells/cm2 were seeded. At day 1, all-trans-RA was added to a final concentration of 10-6 M. At day 2, cells were transfected using DOTAP as above except that after replacement of the transfection mix, fresh RA (10-6 M) was added. At day 5, cells were harvested and reporter assays were performed. To correct for transfection efficiency, 0.25 µg pSV2LacZ was included in all transfections.

CAT and ß-galactosidase assays were performed as described previously (39). For luciferase assays, cells were washed twice with ice-cold PBS. Cells were incubated with 500 µl Triton lysis buffer (1% Triton X-100, 25 mM Glycylglycin, pH 7.8, 15 mM MgSO4, 4 mM EGTA, pH 8.0, and 1 mM dithiothreitol) for 10 min at 4 C. Lysates were transferred to reaction tubes and centrifuged for 10 min at 13,000 rpm at 4 C. The supernatant was used for ß-galactosidase and luciferase assays. For luciferase assays, 75 µl of the supernatant were added to 250 µl sample buffer (4 mM ATP, pH 8.0, 36 mM Glycylglycin, pH 7.8, 22 mM MgSO4). Luciferase activity was started by injection of 100 µl 0.2 mM D-luciferin (Boehringer Mannheim) dissolved in 0.1 M KPO4, pH 7.8. Light output was measured for 10 sec at room temperature with a luminometer (Tropix, Inc., Bedford, MA). All luciferase samples were measured in duplicate. Values were corrected for transfection efficiency by measuring ß-galactosidase activity. Promoter activity was expressed as fold induction relative to the value of the control vector (pCAT, pLuc, or pTK Luc) of which the activity was set to 1. Values are expressed as mean of several duplicate or triplicate experiments ± SEM.

WCE and Bandshift Assays
WCE of P19 cells and transiently transfected COS-7 cells were prepared as described previously (40). COS-7 cells, cultured in a 56-cm2 tissue culture plate, were transiently transfected with 5 µg expression vector using DOTAP. Double-stranded oligonucleotide probes for bandshift analysis were labeled by filling in 5'-overhangs using {alpha}32P-dCTP (Amersham, Arlington, Heights, IL) and the Klenow fragment of DNA polymerase I (BRL, Rockville, MD). WCE (4 µg) was preincubated in a final volume of 19 µl containing 25 mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 2 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM ZnCl2, 10 µg BSA (fraction V, Sigma Chemical Co.), and 1.5 µg poly dIdC (Boehringer Mannheim) in the presence or absence of competitor DNA for 10 min on ice before 1 µl labeled probe (30,000 cpm) was added. After 20 min incubation on ice, samples were loaded on a 4% polyacrylamide gel that had been prerun in TBE buffer for 30 min. TBE buffer contained 25 mM Tris-HCl, 25 mM boric acid and 0.5 mM EDTA, pH 8.3. Gels were run at room temperature for 2 h at 200 V, after which gels were fixed in a mixture of methanol (10%) and acetic acid (10%) and dried. Signals were visualized by autoradiography using Kodak X-Omat AR films (Eastman Kodak Co., Rochester, NY) and intensifying screens at -80 C for 1–3 days.

In Vivo Injection Studies of PTH/PTHrP-Receptor Promoter Constructs
For in vivo promoter studies a receptor promoter fragment spanning from nt -8336 to +1354 was cloned in front of the promoterless bacterial ß-galactosidase gene in vector pSDK LacZ pA, which was kindly provided by Dr. J. Rossant (42). In this vector, the prokaryotic Kozac sequence has been changed to an eukaryotic sequence. The vector was linearized using the following restriction enzymes: XmnI for construct S 8.5 LacZ and the empty control vector and a double digestion with XbaI and XmnI for S 1.9 LacZ. The appropriate restriction fragments were gel purified and dissolved in 10 mM Tris, pH 7.6, 0.1 mM EDTA in a final concentration of 2 ng/ml.

Injection studies were performed essentially as described by Vogels et al. (42); 8.5 days after reimplantation the embryos were isolated. At this time of development, injected embryos represent normal embryos of day 7.5 post coitum due to a delay in development as a consequence of manipulations. Embryos were separated from the surrounding decidua and trophoblast cells, but the parietal and visceral yolk sac were left intact. After washing in PBS, embryos were fixed for 15 min in 1% formalin, 0.2% glutaraldehyde, 0.02% NP40 in PBS, pH 7.4, at 4 C. After two washes with PBS, embryos were stained in 1 mg/ml X-Gal (BRL), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 in PBS for 2 days at 30 C to avoid staining by endogenous ß-galactosidase. Blue staining was scored by visual examination.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. G. E. Folkers (Hubrecht Laboratory, Utrecht, The Netherlands) for helpful discussions and to Dr. P. Chambon (INSERM, Illkirch/Cedex France) for providing antibodies for RXR({alpha}, ß, or {gamma}), RAR{alpha}, and RARß, and a hRAR{alpha} expression vector; Dr. R. Evans (The Salk Institute, La Jolla, CA) for providing a hRXR{alpha} expression vector; Dr. S. Tsai (Baylor College of Medicine, Houston, TX) for providing a hCOUP-TFI expression vector; Dr. M. Parker (Imperial Cancer Research Fund, London, UK) for providing an antibody cross-reacting with COUP-TFI/TFII; Dr. J. Rossant (Mount Sinai Hospital, Toronto, Canada) for providing the vector pSDK LacZ pA; Dr. J. Auwerx (Institute Pasteur, Lille, France) for providing a PPAR{gamma}2 antibody; and Mark Reijnen for excellent technical support with embryo injection studies.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marcel Karperien, Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Building 1, C4-R Albinusdreef 2 POB 9600, Leiden, The Netherlands 2333 ZA.

1 Present address: Max-Planck Institut für Biochemie, Münich, Germany. Back

Received for publication July 23, 1998. Revision received March 19, 1999. Accepted for publication March 30, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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