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
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ABSTRACT
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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-
(RAR
)-retinoid X receptor-
(RXR
) and chicken ovalbumin
upstream promoter (COUP)-transcription factor I (TFI)-RXR
heterodimers and RXR
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 (
, ß, or
) 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 (
, ß, or
) 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.
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INTRODUCTION
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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 (79; 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) (
, ß or
), retinoid acid receptor-ß (RARß), and the orphan receptor
chicken ovalbumin upstream promoter/transcription factor I/II (COUP
TFI/II), but not RAR
. 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).
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RESULTS
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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. 1A
). 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. 1B
), 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.
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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. 2A
). 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. 2B
, 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.
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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. 3A
). Subsequently, transient transfections
were performed in RA-differentiated P19 cells. In line with the results
in Fig. 2
, construct U3 0.1, which contains the minimal P2 promoter
only, already displayed increased reporter activity in RA-treated P19
cells (Fig. 3B
). The presence of intronic sequences in front of
promoter reporter construct U3 0.1 did not further increase RA-induced
reporter activity (Fig. 3B
). 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.
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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. 3
, A and C). Instead, a
slight decrease in reporter activity was observed (constructs pTK
0.90.5, pTK 1.40.9, pTK 1.60.9, and pTK 2.01.4). In marked
contrast, construct pTK 2.81.4 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. 2B
).
By testing three additional deletion constructs of pTK 2.81.4, 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 2.82.0, 2.82.3, and 2.82.5). The presence of an RA-responsive
enhancer in this region was in agreement with the data of the promoter
deletion constructs presented in Fig. 2B
. Compared with constructs pTK
2.82.5 and pTK 2.82.3, reporter activity of construct pTK 2.82.0
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. 4A
) relative to the P2 transcription
start site. This sequence matches the consensus DR1 (Fig. 4B
), 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. 4B
). 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.
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To determine whether the R-DR1 was able to bind members of the nuclear
hormone receptor family such as the human retinoic acid receptor
(hRAR
), the human retinoid X receptor
(hRXR
), 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
, a hRXR
, or a COUP-TFI
expression vector were used in a bandshift assay with the R-DR1 as
probe (Fig. 5
; see Fig. 4b
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
-containing extracts only faintly displayed binding activity to
the R-DR1, in agreement with data indicating that homodimers of hRAR
cannot efficiently bind to DNA (Ref. 16 ; Fig. 5
, lane 2). In
marked contrast, extracts of hRXR
- 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. 5
, lanes 4 and 5).
Mixing hRAR
- and hRXR
-containing extracts resulted in strong
binding to the R-DR1, probably due to heterodimerization (Fig. 5
, lane
3). Also mixing of hRXR
- and COUP-TFI-containing extracts resulted
in an increased binding activity, suggesting that heterodimerization
between hRXR
and COUP-TFI might occur at the R-DR1 (Fig. 5
, lane 6).
Addition of hRAR
to COUP-TFI-containing extracts did not influence
binding of COUP-TFI as a homodimer (Fig. 5
, 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 , a hRXR , and a hCOUP-TFI expression vector were
used in a bandshift assay with the R-DR1 oligonucleotide (see Fig. 4B
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 -transfected cells (WCE-RAR); lane 3, 2 µg of WCE-RAR mixed
with WCE of hRXR -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.
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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. 6A
, 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. 4B
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
, ß and
, it was shown that the protein complexes
contained RXR. Likewise, it was shown that in undifferentiated P19 EC
cells RAR
, but not RARß, PPARy, or COUP TFI or TFII, bound to the
R-DR1 (Fig. 6A
).

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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 /ß/ , incubation with a supershifting antibody
cross-reacting with RXR , -ß, and - . The
arrowhead indicates the supershift; lanes marked anti
RAR represent incubation with a supershifting antibody for RAR .
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 depict incubation with a supershifting antibody for
PPAR ; 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.
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RA differentiation of P19 EC cells strongly increased protein binding
to the R-DR1 (Fig. 6B
). 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. 6B
), 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 (
, ß, or
) 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
or PPAR
(Fig. 6B
).
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. 7A
, mutation of the R-DR1 in construct pTK
2.82.3 (see Fig. 3A
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 2.82.3 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 2.82.3, 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. 3A 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. 2A 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 + 2.82.3, 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 +2.82.3 mtDR1), and U3
2.8D, which was similar to construct U3 2.8 (see Fig. 2A
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. 7B
). 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 1
). In addition, injection of construct S1.9 LacZ
(see Fig. 2A
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. 2
). 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. 8
, 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.

View larger version (101K):
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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
|
---|
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
and RXR
and between COUP-TFI and RXR
as
well as homodimers of RXR
and COUP-TFI, while RAR
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),
-fetoprotein (24),
lactoferrin (25), and the human RXR
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 (
, ß, or
) and RAR
,
while binding of RARß, COUP TFI/II, or PPAR
was not detected. In
RA-differentiated cells, RXR (
, ß, or
), COUP TFI/II, and
RARß bound to the R-DR1 while binding of RAR
and PPAR
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
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
, -ß, and -
. 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
, since this
receptor is a potent constitutive activator of transcription via a DR1,
and HNF4
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
as well as PTH/PTHrP-receptor mRNA expression are induced
(32). Presently, we are testing whether RA also induces the expression
of HNF4
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
, 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
|
---|
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 0.90.5 [XbaI
(-847)XhoI (-445)]; pTK 1.40.9 [KpnI
(-1403)XbaI (-847)]; pTK 1.60.9 [XhoI
(-1597)XbaI (-847)]; pTK 2.01.4 [BglII
(-1915)KpnI (-1403)]; pTK 2.82.0 (HindIII
(-2783)BglII (-1915)]; pTK 2.82.3
[HindIII (-2783)BamHI (-2282, introduced by
PCR)]; pTK 2.82.5 [HindIII (-2783)BamHI
(-2513, introduced by PCR)]. Construct U3 0.5 + 2.82.3 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
and hRXR
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
, ß,
), 9
(anti-RAR
), and 6ß-2A10
(anti-RARß) were derived from Dr. P. Chambon (38), and a polyclonal
antibody for PPAR
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 manufacturers 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
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 13
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(
,
ß, or
), RAR
, and RARß, and a hRAR
expression vector; Dr.
R. Evans (The Salk Institute, La Jolla, CA) for providing a hRXR
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
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. 
Received for publication July 23, 1998.
Revision received March 19, 1999.
Accepted for publication March 30, 1999.
 |
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