Expression of the Parathyroid Hormone-Related Peptide Gene in Retinoic Acid-Induced Differentiation: Involvement of ETS and Sp1
Marcel Karperien,
Hetty Farih-Sips,
Clemens W.G.M. Löwik,
Siegfried W. de Laat,
Johannes Boonstra and
Libert H.K. Defize
Department of Endocrinology (M.K., H.F.-S., C.W.G.M.L.), Leiden
University, 2300 RC Leiden, The Netherlands,
Netherlands
Institute for Developmental Biology (M.K., S.W.d.L., L.H.K.D), 3584
CT Utrecht, The Netherlands,
Department of Molecular Cell
Biology (M.K., J.B.), University of Utrecht, 3584 CH Utrecht, The
Netherlands
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ABSTRACT
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Differentiation of P19 embryonal carcinoma (EC)
and embryonal stem (ES)-5 cells with retinoic acid (RA) induces
expression of PTH-related peptide (PTHrP) mRNA. In this study we have
characterized a region between nucleotide (nt) -88 and -58 relative
to the transcription start site in the murine PTHrP gene that was
involved in this expression. Sequence analysis identified two partially
overlapping binding sites for the Ets family of transcription factors
and an inverted Sp1-binding site. Two major specific bands were
detected in a bandshift assay using an oligonucleotide spanning nt -88
and -58 as a probe and nuclear extracts from both undifferentiated and
RA-differentiated P19 EC cells. The lower complex consisted of
Ets-binding proteins as demonstrated by competition with consensus
Ets-binding sites, while the upper complex contained Sp1-binding
activity as demonstrated by competition with consensus Sp1-binding
sites. The observed bandshift patterns using nuclear extracts of
undifferentiated or RA-differentiated P19 cells were indistinguishable,
suggesting that the differentiation-mediated expression was not caused
by the induction of expression of new transcription factors. Mutations
in either of the Ets-binding sites or the Sp1-binding site completely
abolished RA-induced expression of PTHrP promoter reporter constructs,
indicating that the RA effect was dependent on the simultaneous action
of both Ets- and Sp1-like activities. Furthermore, these mutations also
abolished promoter activity in cells that constitutively expressed
PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of
transcription factors in the expression regulation of the mouse PTHrP
gene.
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INTRODUCTION
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The PTH-related peptide (PTHrP) was first identified as the major
cause of increased serum calcium levels in patients suffering from
humoral hypercalcemia of malignancy (1, 2, 3). PTHrP is expressed in a
broad range of embryonal and adult tissues (4, 5, 6, 7, 8). It is implicated in
the regulation of several growth and differentiation processes such as
keratinocyte differentiation (9), chondrocyte differentiation (10), and
the formation of extraembryonic endoderm during mouse embryogenesis
(11, 12, 13) where it most likely acts in a para- or autocrine fashion.
The cDNAs for human, mouse, and rat PTHrP have been cloned, and the
genomic structure of the corresponding genes has been unraveled (4, 14, 15). Compared with the mouse and rat genes, which are highly
homologous, the human PTHrP gene is more complex. It contains two
TATA-box driven promoters, P1 and P2, respectively, and one GC-rich
TATA-box less promoter (16). The mouse and rat gene are driven by a
single promoter, which is the homolog of the human P2 promoter. Due to
differential splicing at the 3'-end of the human gene, three different
mature PTHrP peptides, which consist of 139, 141, or 173 amino acids,
have been identified. In mouse and rat no reports have been published
on differential splicing, and it is believed that both species express
a single messenger that encodes a 139-amino acid peptide (4, 14).
PTHrP mRNA regulation by growth factors, cytokines, and peptide and
steroid hormones, both in vitro and in vivo, has
been extensively studied (17, 18, 19, 20). In spite of these efforts, not much
is known about the transcription factors involved in the regulation of
PTHrP expression. Recently, it was shown that the human gene is using
cell- and tissue-specific promoters (16). These promoters are, like the
rat PTHrP promoter, regulated by multiple positive and negative
regulatory cis-acting elements (16, 21). In papers by
Dittmer et al., (22, 23) binding sites for members of the
Ets and Sp1 families of transcription factors that mediate the PTHrP
expression in HTLV-1-infected T cells have been identified in the human
P2 promoter.
In this study we set out to identify regulatory elements involved in
the induction of PTHrP expression during all-trans-retinoic
acid (RA)-mediated differentiation of murine embryonal carcinoma (EC)
and embryonal stem (ES) cells. Recently, we and others have shown that
during the RA-mediated differentiation of F9 EC and ES-5 cells toward
extraembryonic parietal endoderm, PTHrP mRNA and protein are induced
(11, 12). Here we show that PTHrP mRNA expression was also induced
during the RA-mediated differentiation of P19 EC cells. To identify the
regulatory elements that were involved in the
differentiation-associated induction of the mouse PTHrP gene, we
constructed promoter reporter constructs and were able to pinpoint
positive regulatory elements to a region between nucleotide (nt) -88
to -58 relative to the transcription start site. The region between nt
-88 to -58 encoded two partially overlapping binding sites for
Ets-related transcription factors and an inverted Sp1-binding site.
Using bandshift analyses, we detected two major specifically retarded
complexes in nuclear extracts of both RA- and undifferentiated P19 EC
cells. The lower complex consisted of Ets-binding proteins as
demonstrated by competition with consensus Ets-binding sites, while the
upper complex contained Sp1-binding activity. We identified the
sequence CCAGCCC as the most likely inverted Sp1-binding site. This
site is shifted 4 bp more upstream compared with the proposed
Sp1-binding site (sequence CCCACC) identified in the human PTHrP P2
promoter (23). The two complexes detected in nuclear extracts of
undifferentiated P19 cells were indistinguishable from the complexes
found using extracts of RA-differentiated cells. This suggested that
the RA differentiation-induced expression of the PTHrP gene might be
caused by posttranslational processing of preexisting transcription
factors rather than by the de novo synthesis of
transcription factors. Mutations in either the Ets- or Sp1-binding
sites completely abolished RA differentiation-induced expression of
PTHrP promoter reporter constructs.
Additionally, we show that the Ets- and Sp1-binding sites were also
involved in the expression of PTHrP promoter reporter constructs in
cells that constitutively expressed PTHrP, suggesting that the Ets
family of transcription factors, in cooperation with Sp1, play a
central role in the transcriptional regulation of the murine PTHrP
gene.
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RESULTS
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PTHrP mRNA Is Induced in RA-Differentiated P19 EC Cells
Recently, we and others have shown that during RA-mediated
differentiation of F9 EC and ES-5 cells, PTHrP expression is induced at
the mRNA and protein level (11, 12). To study the effect of RA
treatment on PTHrP mRNA expression in another EC cell line, the P19 EC
cell line, cells were seeded and cultured for 3 and 5 days in the
presence of 10-6 M RA, respectively, after
which total RNA was isolated. PTHrP mRNA concentrations were measured
by a RNAse protection assay and standardized using an antisense
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. As shown in
Fig. 1
, undifferentiated P19 EC cells did
not express PTHrP mRNA in contrast to P19 cells treated for 3 days with
RA (lanes marked -). Prolonged treatment with RA for 5 days resulted
in a further increase of PTHrP mRNA levels. Pretreatment of the cells
with cycloheximide resulted in an induction of PTHrP mRNA in
undifferentiated P19 EC cells and in a further up-regulation of the
message in the RA-treated cell cultures. These findings were in
agreement with previous studies in other cell lines showing that
cycloheximide was able to both induce and up-regulate PTHrP mRNA
expression (24). Similar results were found in RA-differentiated ES-5
and F9 EC cells (data not shown).

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Figure 1. PTHrP mRNA Is Induced during RA-Mediated
Differentiation of P19 EC Cells and by Cycloheximide Treatment
A, P19 EC cells were cultured in the absence (P19) or in the presence
of 10-6 M RA for 3 (P19 3dRA) or 5 days (P19
5dRA). Before total RNA was isolated, cultures were treated without
(-) or with 20 µg cycloheximide/ml for 1 h. PTHrP mRNA
expression was measured using a RNAse protection assay. GAPDH was used
to correct for equal amounts of RNA in each hybridization mix.
Exposures times: PTHrP, 2 weeks, -80 C; GAPDH, 1 day, -80 C. B,
PhosphorImager quantification of the gel shown in Fig. 1A using
ImageQuant software (Molecular Dynamics). Fold induction was related to
the value of untreated P19 EC cells, which was set to 1 and was
corrected for GAPDH.
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RA Differentiation-Induced PTHrP Expression Is Mediated by
Sequences Located between nt -185 and -53
To identify elements in the mouse PTHrP promoter that mediate the
RA differentiation-induced expression of the PTHrP gene, a panel of
progressive promoter deletion constructs was made and cloned in front
of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene
in the promoterless pKT CAT vector (Fig. 2A
). In agreement with the mRNA data,
none of the promoter reporter constructs showed activity in
undifferentiated EC and ES cells (data not shown). To localize the
differentiation-responsive elements, the promoter reporter constructs
were transiently transfected in P19 and ES-5 cells treated for 3 days
with 10-6 or 10-7 M RA,
respectively. After the medium was refreshed at day 4, cells were
harvested at day 5, and CAT activity was determined. CAT activity was
corrected for transfection efficiency and related to the activity of
the empty pKT CAT vector as fold induction (Fig. 2
, B and C). A
promoter reporter construct containing only the TATA box and
transcription start site did not induce promoter activity in either
cell line (pKT-49). Increasing the promoter fragment in front of the
reporter gene with 136 nt resulted in a 7-fold induction of CAT
activity in RA-differentiated P19 cells and a 3-fold induction in
RA-differentiated ES-5 cells (pKT -185), indicating that the
RA-induced expression of the PTHrP gene was, at least in part, mediated
by sequences located between nt -185 and -49 relative to the
transcription start site. The TATA box and transcription start site
were required for this induction as was demonstrated by construct pKT
delT in which these sites were deleted. An increase in promoter
sequences with 100 nt resulted in a further enhancement of the
RA-induced PTHrP expression (compare pKT -185 with pKT -285).
Constructs pKT -382 and -490 containing further increases in
5'-promoter sequences had activities comparable with or lower than the
activity of construct pKT -185. An additional increase in promoter
sequence with about 350 nt resulted in a decline in promoter activity
(compare constructs pKT -490 with pKT -853), suggesting the presence
of negative regulatory elements in this region. Increasing the promoter
fragment with an additional 300 nt did not significantly modify the
transactivation potential of the promoter (compare construct pKT -1145
and pKT -853). To determine the influence of intronic sequences
present between exon 1 coding for the untranslated leader and exon 2
coding for the translation start site and the signal peptide,
constructs pKT S-490 and pKT S-1145 were made and tested by transient
transfection. The intronic sequences did not significantly influence
promoter activity (compare the activity of pKT S-490 with pKT -490 and
the activity of pKT S-1145 with pKT -1145).

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Figure 2. Transient Transfection of Mouse PTHrP Promoter
Reporter Constructs in Different Cell Lines
A, Schematic representation of the 5'-organization of the mouse PTHrP
gene and the promoter reporter constructs used. The
arrows represent the transcription start site in front
of exon 1 and the ATG start codon in exon 2, respectively.
Abbreviations and positions of restriction sites: H,
HindIII (-1145); X, XbaI (-853); S,
SmaI (-490); A, AvaII (+220); He,
HaeII (-285; +80); P, PvuII (-185); *,
restriction sites introduced by PCR; X*, XbaI (-382);
H*, HindIII (+494). B, 10 µg PTHrP promoter reporter
constructs were transiently transfected into P19 cells treated for 5
days with RA. (see Materials and Methods for detailed
protocol). CAT activity was quantified using the PhosphorImager and
ImageQuant software and depicted relative to the activity of the empty
pKT CAT vector after correction for transfection efficiency. All
transfections were repeated at least three times, and a representative
experiment is shown. C, PTHrP promoter reporter constructs were
transiently transfected in ES-5 cells treated for 5 days with RA
(methods as in panel B). D, PTHrP promoter reporter constructs
transiently transfected in BHK cells (methods as in panel B). E, PTHrP
promoter reporter constructs transiently transfected in Mes-1 cells
(methods as in panel B).
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In summary, we concluded that the RA differentiation-induced expression
of PTHrP mRNA was, at least in part, mediated by sequences between nt
-185 and -49, with a minor contribution of sequences between nt -285
and -185. The activity of these regions was down-regulated by elements
located further upstream between nt -853 and -490.
Sequences Located between nt -185 and -49 Are Also Required for
Constitutive PTHrP Expression
To test whether the identified regulatory elements in the mouse
PTHrP promoter were specific for RA differentiation-induced PTHrP
expression or reflected a more general mechanism of regulating PTHrP
gene transcription, the promoter reporter constructs were also tested
in two other cell lines that constitutively expressed PTHrP: 1) Baby
hamster kidney fibroblasts (BHK) and 2) Mes-1 cells. Mes-1 is a cell
line with mesodermal characteristics and is derived from
RA-differentiated P19 EC cells (25). This cell line expresses PTHrP
mRNA (M. Karperien, unpublished observation). The results of the
transient transfection assays were comparable with the results observed
in the RA-differentiated EC and ES cells (compare Fig. 2
, D and E, with
Fig. 2
, B and C). Again major positive regulatory elements were located
between nt -185 and -49, while upstream from position -490 sequences
were located that down-regulated PTHrP transcription. Additional
negative regulatory elements were present between positions -490 and
-382 in Mes-1 cells (see construct pKT -490 in Fig. 2E
). These
results suggested that identical promoter regions were involved in the
regulation of both RA differentiation-induced gene expression in EC and
ES cells and constitutive PTHrP expression in Mes-1 and BHK cells.
The Region between nt -185 and -49 Contains Binding Sites for
Ets-Related Transcription Factors and an Inverted Sp1-Binding Site
Sequence analysis of the region between nt -185 and -49 did not
reveal homology to RA-responsive elements that mediate the induction of
genes that are under direct control of RA (26). This was not
unexpected, given the slow time course of PTHrP mRNA induction by RA
(Fig. 1
), which suggested that the appearance of PTHrP mRNA was a
secondary effect caused by RA-activated differentiation programs rather
than a direct effect of RA. Comparison of the nucleotide sequence of
the mouse, rat, and human P2 PTHrP promoters revealed a striking degree
of homology, especially between nt -94 and -62, which were completely
conserved in all three promoters (Fig. 3A
). This region contains two partially
overlapping binding sites for the Ets family of transcription factors:
one present at the sense (EBSI) and the other present at the antisense
strand (EBSII), respectively (Fig. 3B
). Furthermore, the CCCACC
sequence has been identified as an inverted Sp1-binding site in the
human PTHrP P2 promoter (23) (Fig. 3B
).

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Figure 3. Sequence of the Mouse PTHrP Promoter
A, Homology comparison between the mouse, rat, and human P2 PTHrP
promoters. The region between nt -94 and -62 is conserved in all
three promoters and is double underlined. B, Sequence of
the region between nt -88 to -58. The consensus Ets-binding site on
the sense strand, EBSI, is single underlined. The
consensus Ets-binding site on the antisense strand, EBSII, is
double underlined. The inverted Sp1-binding site is
depicted in italics.
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To test whether the Ets-binding sites were functional and were involved
in the RA differentiation-induced expression of the PTHrP gene, we
first cotransfected PTHrP promoter reporter constructs with
ets-1 and ets-2 expression vectors.
Ets-1 and ets-2 are differentially regulated by
RA in P19 EC cells. Ets-1 expression is up-regulated by RA treatment
while Ets-2 is constitutively expressed in undifferentiated and
RA-differentiated P19 EC cells at the mRNA level (27). As shown in Fig. 4A
, cotransfection with a chicken
ets-1 expression vector resulted in a 6-fold induction of
PTHrP promoter activity in undifferentiated P19 EC cells using pKT
-185 as reporter. In contrast, cotransfected Ets-2 was not able to
transactivate the PTHrP promoter, even when the amount of cotransfected
expression vector was increased from 2 to 5 µg DNA (Fig. 4A
, not
shown). As a positive control for the ets-1 and
ets-2 expression vectors, construct 63 was used, which
contained the ets-binding sites from the HTLV-1 long terminal repeat in
front of the CAT reporter gene. Cotransfection of both Ets expression
vectors induced promoter activity, although Ets-2 was less potent than
Ets-1 (Fig. 4A
). These results indicated that Ets-2, in contrast to
Ets-1, was not able to induce PTHrP promoter activity in
undifferentiated P19 EC cells.

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Figure 4. Ets-1 and Ets-2 Are Able to Transactivate PTHrP
Promoter Reporter Constructs Depending on the Differentiation State of
P19 EC Cells
A, Reporter (10 µg) was cotransfected with 2 µg empty expression
vector (pSG5), 2 µg chicken ets-1 expression vector,
or 2 µg human ets-2 expression vector, respectively,
in undifferentiated P19 EC cells. Fold induction was expressed relative
to the promoterless pKT CAT vector and corrected for transfection
efficiency. All transfections were repeated at least three times and a
representative experiment is shown. B, Same as in panel A except that
the P19 EC cells were differentiated for 5 days with RA.
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Similar experiments were performed in P19 EC cells differentiated for 5
days with RA (Fig. 4B
). Surprisingly, under these conditions both Ets-1
and Ets-2 were able to transactivate the PTHrP promoter, suggesting
that the RA-mediated differentiation created an environment in which
Ets-2 was now able to transactivate the PTHrP promoter.
Two Major Complexes Bind to the Region between nt -88 and -58
We next performed bandshift analyses using nuclear extracts
of PTHrP-expressing and nonexpressing cells. As a probe, a
double-stranded oligonucleotide spanning the region from nt -88 to
-58 (PLP wt) was used that encoded the Ets and inverted Sp1-binding
sites (Table 1
). As shown in Fig. 5
, two major complexes were specifically
retarded in a bandshift assay using nuclear extracts of
RA-differentiated P19 EC cells. These complexes were specific, since
they were competed with a 25-fold excess of nonlabeled PLP wt
oligonucleotide while no competition was observed with a 50-fold excess
of nonspecific DNA.

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Figure 5. Nuclear Extracts of Different Cell Lines Contain
Two Major Protein Complexes Binding to the Region between nt -88 and
-58
Nuclear extracts of P19 EC cells differentiated for 5 days with RA (P19
5dRA) were incubated with a radioactively labeled oligonucleotide probe
coding for nt -88 to -58 (PLP wt). Specific competition was performed
using a 25-fold excess of unlabeled probe. Nonspecific competition was
performed using a 50-fold excess of unlabeled pUC18 DNA. The sequence
of the oligonucleotides that were used as competitors are depicted in
Table 1 . The two major specific bands are indicated with the
bold arrows.
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To identify the proteins present in the shifted complexes,
bandshift analyses were performed in which mutated PLP wt
oligonucleotides and consensus binding sites for Ets and Sp1 were used
as competitors. These oligonucleotides are described in Table 1
. As
shown in Fig. 5
, oligonucleotide PLP mtETS, in which both Ets-binding
sites were inactivated by mutation, efficiently competed the upper
complex, suggesting that the lower complex was formed by proteins
binding to the Ets sites. This was confirmed by competition with a
consensus Ets-binding site (PEA3) and oligonucleotide Ets wt, which
efficiently competed the lower but not the upper complex.
Oligonucleotide PLP mtINT, in which the two nucleotides directly
downstream of the EBSI site were mutated (Table 1
), efficiently
competed both complexes, suggesting that the mutation did not interfere
with protein binding. Oligonucleotide PLP mtSp1, in which the inverted
Sp1-binding site was mutated, competed the lower Ets-containing complex
but not the upper complex. The presence of Sp1-like binding activity in
the upper complex was demonstrated by the efficient competition with
two different Sp1 consensus-binding sites (pSV2 Sp1, Sp1 con).
Furthermore, the upper shift was totally dependent on the presence of
Zn++ in the binding buffer, indicating that this complex
contained a zinc finger protein, such as Sp1 (data not shown).
Surprisingly, oligonucleotide Sp1a, encoding the CCCACC-inverted
Sp1-binding site identified in the human PTHrP P2 promoter (23), was
less efficient in competing the upper complex than oligonucleotides
INTa and INTmtG>T. Compared with Sp1a, these oligonucleotides encoded
5'-nucleotides located in between the Ets and CCCACC core sequence,
suggesting the involvement of these nucleotides in binding of the
Sp1-like activity. The change of G into T did not interfere with
protein binding in oligonucleotide INTmtG>T. Finally, mutation of both
Ets- and Sp1-binding sites completely abolished protein binding of
oligonucleotide PLP mtETS/Sp1.
Similar results were obtained using nuclear extracts of P19 EC
cells, which do not express PTHrP and BHK cells that constitutively
express PTHrP (data not shown). The two complexes found in a bandshift
assay using nuclear extracts of undifferentiated P19 EC cells were
indistinguishable from the complexes found when extracts of
RA-differentiated P19 EC were used. This suggested that the
differentiation-induced expression of the PTHrP gene was not caused by
the induction of expression of a new transcription factors, although
the possibility that the retarded bands consisted of different proteins
with the same electromobility could not be excluded (not shown).
Both Ets-Binding Sites Are Involved in Protein Binding
In the next experiment, the involvement of the individual
Ets-binding sites, EBSI and EBSII, in protein binding to the PLP wt
oligonucleotide was determined using nuclear extracts of
RA-differentiated P19 EC cells. Again, two major specific complexes
were observed in a bandshift assay of which the lower consisted of Ets
domain-containing transcription factors (Fig. 6A
). Increasing the concentration of the
consensus Ets-binding site PEA3 (from 5- to 25-fold excess) as a
competitor efficiently competed the lower complex. A 25-fold excess of
unlabeled competitor was sufficient to compete all bandshifts
containing Ets-binding proteins (Fig. 6A
). Similar results were found
using oligonucleotide Ets wt as competitor. This oligonucleotide
encodes both Ets-binding sites of the PTHrP promoter (nt -92 to -67).
None of the shifts was competed using oligonucleotide Ets mtI in which
both the EBSI- and EBSII-binding sites were inactivated by mutation
(see Table 1
and Fig. 6A
). Oligonucleotide Ets mtII, in which the
Ets-binding site at the antisense strand was mutated, leaving a
functional EBSI site, and oligonucleotide Ets mtIII, in which the Ets
binding site at the sense strand was mutated, leaving an intact EBSII
site, were equally potent in competing with the PLP wt oligonucleotide
for binding of Ets domain-containing proteins (Fig. 6A
). This suggested
that the EBSI and EBSII sites were both able to bind Ets
domain-containing transcription factors. Identical results were
obtained when nuclear extracts of undifferentiated P19 EC and BHK cells
were used (data not shown).

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Figure 6. Characterization of Ets-Binding Proteins in
Undifferentiated and RA-Differentiated P19 Cells
A, Nuclear extracts (10 µg) of P19 EC cells differentiated for 5 days
with RA were incubated with a radioactive labeled oligonucleotide probe
coding for nt -88 to -58 of the PTHrP promoter (PLP wt). Competition
was performed using a 25-fold excess of unlabeled oligonucleotides
except for oligonucleotides PEA3, Ets mtII, and Ets mtIII of which a
5-, 10-, and 25-fold excess was used. As nonspecific competitor a
50-fold excess of pUC 18 DNA was used. The sequence of the
oligonucleotides is depicted in Table 1 . The two major specific bands
are indicated by a bold arrow. In between the two major
bands, a nonspecific complex was observed (as). B, Nuclear extracts (10
µg) from undifferentiated P19 EC cells (left panel)
and from RA-differentiated P19 cells (right panel) were
incubated with a radiolabeled consensus Ets-binding site (PEA3).
Competition was performed using a 25-fold excess of specific
oligonucleotides and a 50-fold excess of nonspecific pUC18 DNA. The
sequence of the oligonucleo-tides used as competitors is depicted in Table 1 . The major
bandshift is indicated by a bold arrow, while the minor
shifts are indicated by thin arrows.
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We subsequently used the consensus Ets-binding site PEA3 as a probe in
a bandshift assay using nuclear extracts of undifferentiated P19 EC
cells, which do not express PTHrP, and RA-differentiated P19 EC cells,
which do. One major and several minor complexes were found using
nuclear extracts of both cell types (Fig. 6B
; major complex is
indicated by bold arrow). These complexes were specific,
since they were competed with a 25-fold excess of unlabeled probe but
not with a 50-fold excess of nonspecific competitor. The pattern of
complexes using the extracts of non-PTHrP-expressing (P19 EC, Fig. 6B
, left) and PTHrP-expressing cells (P19 cells treated with RA,
Fig. 6B
, right) were indistinguishable, suggesting that the
same Ets-binding proteins were present in both cell types. However, it
cannot be excluded that, although the mobility of the complexes was
identical, the retarded bands consisted of different proteins.
Surprisingly, we did not observe an up-regulation of any of the shifts
containing the PEA3 oligonucleotide, although RA treatment causes an
up-regulation of the expression of Ets-1 mRNA (27). All complexes were
efficiently competed by a 25-fold excess of oligonucleotides Ets wt,
Ets mtII, and Ets mtIII, encoding two or one functional Ets-binding
sites derived from the PTHrP promoter (Table 1
), respectively, but not
by oligonucleotide Ets mtI in which both Ets- binding sites were
inactivated by mutation. In contrast to the bandshift experiments in
which the oligonucleotide PLP wt was used as a probe, now the Ets mtIII
oligonucleotide, encoding a functional EBSII site (Table 1
) was a more
efficient competitor than oligonucleotide Ets mtII, encoding a
functional EBSI site (compare Fig. 6A
and 6B
). This difference in
affinity was confirmed by dose-response analysis (data not shown) and
suggested that the EBSII site had a somewhat higher affinity than the
EBSI site for Ets-binding proteins present in the nuclear extracts of
undifferentiated and RA-differentiated P19 cells.
The Inverted CCAGCCC Sequence Behaves as an Sp1-Binding Site
The competition experiments shown in Fig. 5
suggested that
nucleotides between the Ets-binding site and the sequence CCCACC, which
was identified as an inverted Sp1-binding site in the human PTHrP P2
promoter (23), were involved in protein binding. We therefore designed
oligonucleotides Sp1b, coding for the CCCACC sequence, and INTb, which
encoded the region between the Ets and CCCACC sequence. Both
oligonucleotides were used as probes in bandshift analysis using
nuclear extracts of BHK cells and RA-differentiated and
undifferentiated P19 cells. Two major specific bands were retarded
using extracts of these three cell lines and oligonucleotide INTb as a
probe (Fig. 7A
; data for BHK and
RA-differentiated P19 cells not shown). These bands were specific,
since they were efficiently competed by a 25-fold excess of the PLP wt
oligonucleotide, but not with a 50-fold excess of nonspecific DNA.
Increasing amounts of oligonucleotides INTa and INTmtG>T (5-, 10-,
25-fold excess) efficiently competed both complexes. The mutation of
one nucleotide in INTmtG>T did not significantly interfere with
protein binding. The most efficient competition was observed using
oligonucleotides pSV2 Sp1 and Sp1 con, which encoded consensus
Sp1-binding sites. The two shifts were totally dependent on the
presence of Zn++ in the binding buffer, which was further
evidence for the presence of Sp1-like binding activity in both
complexes (data not shown). Surprisingly, oligonucleotides Sp1a and
Sp1b, coding for the CCCACC sequence, did not compete with protein
binding (Fig. 7A
), suggesting that this sequence was not critically
involved in binding of the Sp1-like activity to the PTHrP promoter. In
our view, the results suggested that the Sp1-binding site is located
slightly more upstream, closer to the Ets-binding sites. This was
supported by the reversed experiment in which the Sp1b oligonucleotide
was used as a probe. In this experiment only faint bandshifts were
observed (Fig. 7B
). These results suggested that the sequence CCAGCCC
was the most likely inverted binding site for the Sp1-like binding
activity. This sequence matches the Sp1 consensus binding site
(G/AGGCG/TG/AG/A,
31 and is flanked by the nucleotides mutated in the PLP
mtINT oligonucleotide at its 5'-end and includes the nucleotides
mutated in the PLP mtSp1 oligonucleotide at its 3'-end.

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Figure 7. Sp1 Binds to the Inverted CCAGCCC Sequence
A, Nuclear extracts (20 µg) of undifferentiated P19 EC cells were
incubated with the radiolabeled oligonucleotide probe Intb in a
bandshift assay. Nonspecific competition was performed with a 50-fold
excess unlabeled oligonucleotide PEA3, while specific competition was
performed with a 5-, 10-, or 25-fold excess of unlabeled
oligonucleotides Inta, IntmtG>T, pSV2 Sp1, Sp1 con, and Sp1a or a
25-fold excess of the PLP wt or Sp1b oligonucleotides. B, Same as in
panel A, except that oligonucleotide Sp1b was used as a radiolabeled
probe. Specific competition was performed with a 25-fold excess of
unlabeled oligonucleotides PLP wt, Sp1a, and Sp1 con, while nonspecific
competition was performed with a 50-fold excess of oligonucleotide
PEA3.
|
|
Functional Ets- and Sp1-Binding Sites Are Required for RA
Differentiation-Induced and Constitutive PTHrP Expression
To define the role of the Ets- and Sp1-binding sites,
binding-perturbing mutations were introduced in one or both binding
sites. The same mutations as in the oligonucleotides used in the
bandshift assays were introduced by site-directed mutagenesis in
promoter reporter construct pKT -185 (Table 1
). The effect of the
mutations on promoter activity was subsequently tested by perfoming
transient transfections in RA-differentiated P19 cells. As shown in
Fig. 8A
, mutation of both Ets-binding
sites abolished PTHrP promoter activity in RA-differentiated P19 cells
(pKT Ets mtI). Promoter activity was also abolished in the constructs
in which the individual Ets-binding sites were mutated (pKT mt EtsII
and EtsIII). This suggested that either Ets-binding site must be
functional for transcriptional activation and that both sites
contributed to the activation of the promoter reporter construct.
Likewise, mutation of the inverted-Sp1 binding site in promoter
reporter construct pKT mtSp1 abolished reporter activity. These results
indicated that both functional Ets- and Sp1-binding sites were required
for transcription of the PTHrP promoter reporter construct pKT -185
and strongly pointed to a synergistic interaction between Ets and Sp1
in the regulation of PTHrP gene transcription, most likely by the
formation of a ternary complex. This was furthermore supported by
mutation of the two nucleotides in the gap between the Ets- and
Sp1-binding sites (pKT mtInt). Although this mutation did not interfere
with protein binding to the individual Ets- and Sp1-binding sites in a
bandshift assay (Fig. 6
), it caused a strong decline in promoter
reporter activity. Most likely, the mutation interfered with the
interaction between Ets and Sp1 by a change in DNA structure.

View larger version (17K):
[in this window]
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|
Figure 8. Mutations in Either the Ets- or Sp1-Binding Site
Strongly Diminish PTHrP Promoter Activity
A, Mutations were made in PTHrP promoter reporter construct pKT -185
(see Fig. 2A ). The mutated nucleotides are described in Table 1 . Mutant
promoter reporter constructs (10 µg) were transiently transfected in
P19 EC cells differentiated for 5 days with RA (see Materials
and Methods). Fold induction was relative to the activity of
the promoterless pKT CAT reporter construct and corrected for
transfection efficiency. All transfections were repeated at least three
times and a representative experiment is shown. B, Same as in panel A,
except that transfections were performed in BHK cells.
|
|
Promoter activity of the mutant promoter constructs was also tested in
BHK cells, which constitutively express PTHrP. All mutants strongly
diminished promoter activity, although the inhibition of promoter
activity was less than in RA-differentiated P19 EC cells (an average of
70% compared with an average of 85% inhibition of transcription
activity, respectively) (Fig. 8B
). The results indicated that
functional Ets- and Sp1-binding sites were required for both RA-induced
and constitutive PTHrP expression in P19 EC and BHK cells,
respectively.
 |
DISCUSSION
|
---|
Recently, we and others have provided evidence for a role of PTHrP
and its receptor in the formation of extraembryonic endoderm of the
parietal yolk sac during mouse embryogenesis (11, 12). Using F9 and ES
cell lines as in vitro model systems, we have shown that
during the RA-mediated differentiation toward parietal endoderm, PTHrP
mRNA and protein are induced. In this study we set out to identify
regulatory elements of the murine PTHrP promoter that mediate the RA
differentiation-induced expression of PTHrP in EC and ES cells. Using
promoter reporter constructs, we showed that the RA-mediated induction
of PTHrP-gene expression was mediated by a region located between nt
-88 to -58 relative to the transcription start site. In addition, we
showed that this region was also involved in the expression regulation
of cells that constitutively express PTHrP mRNA.
Using nuclear extracts of RA-differentiated and undifferentiated P19
cells, two specific protein complexes binding to the region between nt
-88 and -58 were identified. The lower complex consisted of Ets
domain-containing transcription factors as suggested by competition
with a consensus Ets-binding site, while the upper complex contained
Sp1-like activity as suggested by competition with Sp1
consensus-binding sites and its dependency on Zn++ in the
bandshift buffer. The identity of the Ets-binding factors is not yet
known and is the subject of further investigations. However, it seems
likely that Ets-1 and Ets-2 are constituents of the lower complex,
since 1) Ets-1 and Ets-2 are expressed in undifferentiated and
RA-differentiated P19 cells (27), and 2) both cotransfected
ets-1 and ets-2 are able to transactivate PTHrP
promoter reporter constructs in transient transfection assays. However,
it cannot be excluded that other members of the Ets-family of
transcription factors also bind to the PTHrP promoter in RA- and
undifferentiated P19 cells.
The sequence between nt -88 and -58 is completely conserved in the
mouse, rat, and human P2 PTHrP promoters in sequence and location
relative to the transcription start site. Dittmer and co-workers (22, 23) have recently shown that this region mediates the expression of the
human PTHrP gene in HTLV-1 infected T cells. They identified two
partially overlapping Ets-binding sites, EBSI and EBSII, and an
inverted Sp1-binding site in this region by showing efficient binding
of recombinant Ets-1 and Sp1 to their respective binding sites. In
contrast to Dittmer and co-workers (23), who identified the sequence
CCCACC as an inverted Sp1-binding site in the human PTHrP P2
promoter, our data suggested that the sequence CCAGCCC was the
most likely inverted Sp1-binding site. This site is located 4 bp
further upstream compared with the site identified by Dittmer and
associates (23), decreasing the gap between the Ets- and Sp1-binding
site to 2 bp. This sequence matches the published Sp1 consensus binding
site (31). Furthermore, a gap of 2 bp, instead of 6 bp, between Ets-
and Sp1-binding sites is also found in the promoter/enhancer regions of
various other genes in which cooperative interactions between Ets and
Sp1 are observed, such as the HTLV-1 long terminal repeat (28) and the
P4 promoter of mouse Minute Virus (30). Optimal PTHrP expression in
HTLV-1-infected T cells required the presence of functional binding
sites for either Ets or Sp1 (23). In fact, it was shown that Sp1 and
Ets-1 worked cooperatively in the regulation of PTHrP expression. Such
a cooperative interaction has also been described for other promoters
(28, 30, 32, 34) and is mediated by the formation of a ternary complex.
Dittmer and co-workers (23) demonstrated the formation of such a
ternary complex, using recombinant Ets-1 and Sp1 and the PTHrP promoter
as a probe in a bandshift assay (23). In our bandshift assays we did
not observe ternary complex formation. In contrast to Dittmer and
co-workers (23), we used crude nuclear extracts instead of recombinant
or partially purified Ets and Sp1, which might explain the absence of
ternary complex formation in our hands. More recently, it was shown
that the formation of a ternary complex between Ets and Sp1 in the
human PTHrP P2 promoter is facilitated by the HTLV-1 Tax protein (35).
This suggests that ternary complex formation might be stimulated by an
as yet unknown cellular protein.
Binding-perturbing mutations in one or both Ets-binding sites or the
Sp1-binding site abolished the RA differentiation-induced expresssion
of PTHrP promoter reporter constructs. This was in concordance with
findings of Dittmer and co-workers (22, 23), except that they showed
that mutations in the EBSI binding site were more effective in reducing
promoter reporter activity in HTLV-1-infected T cells than mutations in
the EBSII-binding site. Likewise, they showed that the EBSI site was
more effective in binding of recombinant Ets1 than the EBSII site. In
contrast, we showed that binding affinity of the EBSII site for Ets
domain-containing transcription factors present in the nuclear extracts
of RA-differentiated and undifferentiated P19 cells was equal to or
even somewhat higher than the EBSI site. Therefore, our data suggest
that both the EBSI and EBSII site might be occupied by Ets
domain-containing transcription factors for efficient transactivating
of PTHrP promoter reporter constructs. It is also possible that the
differences between our data and the observations made by Dittmer and
co-workers (22, 23) might be explained by the use of other mutated
nucleotides [CTTTCCAGAAGC vs.
CTTTCCGGTTGC (this study) for the EBSI site and
CTTTCAGGAAGC vs. CTAACCGGAAGC (this
study) for the EBSII site] and by the use of recombinant proteins in
the study of Dittmer vs. nuclear extracts in our study. The
absence of reporter activity in cells transfected with constructs in
which only one transcription factor-binding site was mutated indicated
the necessity of a synergistic interaction between the transcription
factors binding to the Ets- and Sp1-binding sites for the regulation of
gene transcription. Mutation of the two nucleotides between the Ets-
and Sp1-binding sites in PTHrP promoter reporter construct (pKT mtInt)
also abolished reporter activity. Although the mutation did not
interfere with protein binding in a bandshift assay, the change in DNA
structure apparently inhibited the formation of a ternary complex
in vivo. This might explain the absence of reporter activity
in transfection assays. The mutations that abolished the activity of
PTHrP promoter reporter constructs in RA-differentiated P19 cells also
strongly decreased reporter activity in cells that constitutively
expressed PTHrP mRNA. These findings, combined with the observations by
Dittmer et al. (22, 23), strongly point to a central role
for the Ets and Sp1 family of transcription factors as transcriptional
regulators of PTHrP gene expression.
The bandshift patterns observed by using nuclear extracts of
undifferentiated or RA-differentiated P19 cells and the consensus
Ets-binding site as a probe were indistinguishable, suggesting that the
same Ets domain-containing transcription factors were present in
undifferentiated and RA-differentiated P19 cells. Likewise, there were
no differences in the two shifts obtained by using the inverted Sp1
site as a probe and nuclear extracts of undifferentiated and
RA-differentiated P19 cells. This suggested that the induction of PTHrP
expression by RA differentiation was not caused by the induction of
expression of a novel Ets-related transcription factor or by the
induction of Sp-like activity, which in turn transactivated the PTHrP
promoter, although it could not be excluded that the retarded bands
consisted of different proteins with identical electromobility or that
the differentiation resulted in the induction of transcription factors
that could not be detected by the bandshift assays. However, we propose
that the induction of PTHrP expression by RA-mediated differentiation
was caused by posttranslational modifications of already existing
protein complexes. This hypothesis was supported by the observation
that cycloheximide induced the expression of PTHrP in undifferentiated
P19 EC cells, indicating that all factors required for the transciption
of the PTHrP gene were already present in cells that normally do not
express detectable levels of PTHrP. In addition, we showed that
cotransfected Ets-2 was able to transactivate PTHrP promoter reporter
constructs only in RA-differentiated P19 cells, in spite of the fact
that Ets-2 was also expressed in undifferentiated P19 cells (27). This
indicated that the RA-mediated differentiation created an environment
in which Ets-2 was now able to transactive the PTHrP promoter,
e.g. by posttranslational modifications. Several members of
the Ets family of transcription factors are subject to
posttranslational modifications such as phosphorylation. For example,
phosphorylation of Elk-1, an Ets domain-containing transcription factor
binding to the serum response element, dramatically enhances gene
transcription of the c-fos promoter (32, 34).
The activity of the transcription factors binding to the region between
nt -88 to -58 is down-modulated by negative regulatory elements
located more upstream in the PTHrP promoter. The nature of this
down-modulation is at present unclear and is the subject of further
investigations. The presence of negative regulatory elements in the
murine PTHrP promoter was not surprising given the observations that
the expression of both human and rat PTHrP genes is regulated by
multiple negative cis-acting elements (16, 21). These
elements might explain why PTHrP mRNA is difficult to detect in
RA-differentiated P19 EC cells. The message was only detectable using
the sensitive RNAse protection assay and 100 µg total RNA. PTHrP mRNA
is also hard to detect in other murine cell lines and tissues (4),
which might be attributed to the presence of negative regulatory
elements. These elements might play an important role in controlling
the expression of PTHrP, since overexpression of the gene in certain
human tumors is associated with development of humoral hypercalcemia of
malignancy (1, 2, 3).
In summary, we conclude that the region between nt -88 and -58 of the
PTHrP promoter plays a central role in the control of expression of the
murine PTHrP gene. This is accomplished by a synergistic interaction of
transcription factors belonging to the Ets- and Sp1 families of
transcription factors. The precise nature of the protein interactions
leading to PTHrP gene transcription and the role in this process of
posttranslational modifications are the subjects of further
studies.
 |
MATERIALS AND METHODS
|
---|
PTHrP Promoter Reporter Constructs, Plasmids, and
Oligonucleotides
A genomic clone coding for the mouse PTHrP gene was kindly
provided by Dr. A. Broadus. From this clone, a
HindIII-KpnI fragment of 1.8 kb encoding exon 2,
exon 1, and about 1150 nt upstream promoter sequences was subcloned in
SK-bluescript (Stratagene, La Jolla, CA) and was used to generate
promoter reporter constructs in front of the bacterial chloramphenicol
acetyl transferase gene. The promoter reporter constructs contained the
following restriction fragments: pKT -49; AccI
(-49)AvaII (+220), pKT -185; PvuII
(-185)AvaII (+220), pKT -285; HaeII
(-285)HaeII (+80), pKT -382: XbaI (-382)
AvaII (+220), pKT -490; SmaI
(-490)AvaII (+220), pKT -853; XbaI (-853)
AvaII (+220), pKT -1145; HindIII
(-1145) AvaII (+220), pKT S-490; SmaI
(-490)HindIII (+494), pKT S-1145; HindIII
(-1145) -HindIII (+494), pKT del T; PvuII
(-185)AvaII (+220) with a deletion between the
AccI (-49) and PvuII (+25) restrictions sites.
The XbaI and HindIII restriction sites at
position -382 and +494 were introduced by PCR using the following
oligonucleotides; 5'-AATCTAGACTGGGGTGGGGCTCCGT-3' in combination with a
T7 promoter primer or 5'-ATAAGCTTGCCGCTCGCTGGCTCTGG-3' in combination
with a T3 promoter primer, respectively, and the 1.8-kb promoter
subclone in SK-bluescript as template using a standard PCR reaction.
PCR products were subcloned and controlled by sequencing and
restriction enzyme analysis.
Site directed-mutagenesis was performed using the pSelect vector and
the Altered Sites cloning kit according to the manufacturers manual
(Promega, Madison, WI). After introduction of the mutations, the
constructs were controlled by sequencing. Expression plasmids for
chicken c-Ets-1, human c-Ets-2, and the HTLV-1 LTR reporter construct
63 have been previously described (36). Oligonucleotides were
synthesized by a Cyclone Plus DNA synthesizer (Millipore, Bedford, MA)
(see Table 1
for sequences).
Cells, RA, Cycloheximide, mRNA Isolation, and RNase
Protection
BHK cells were cultured in bicarbonate-buffered DMEM (DMEM-bic)
supplemented with 7.5% FCS. P19 EC and Mes-1 cells were cultured on
gelatinized surfaces in a 50%/50% mixture of Hams F12 and DMEM-bic
supplemented with 7.5% FCS as described by Mummery et al.
(25). Mes-1 is a stable cell line derived from differentiated P19 cells
that has mesodermal characteristics (25). Embryonic stem cells (ES-5)
were cultured as previously described (12). RA was dissolved in
dimethylsulfoxide as a 10-2 M stock and stored
in liquid nitrogen. RA was used at a concentration of 10-6
or 10-7 M for the differentiation of P19 EC
and ES-5 cells, respectively. Cycloheximide was purchased from Sigma
(St. Louis, MO) and was used at a concentration of 20 µg/ml. Total
cellular RNA was isolated using guanine isothiocyanate, followed by
phenol chloroform extraction and ethanol precipitation as described by
Chomczinsky and Sacchi (37). To generate PTHrP- and GAPDH-specific
riboprobes, a 345-nt mouse PTHrP genomic fragment cloned in pGEM
(kindly provided by Dr A. Broadus (Yale University, Hartford, CT) and a
110-nt mouse GAPDH cDNA fragment cloned in SK-Bluescript (38) were used
as templates. After linearization, antisense riboprobes were generated
in the presence of [
32P]UTP (Amersham, Arlington,
Heights, IL) and T7 RNA polymerase (BRL, Rockville, MD). Probes were
gel purified, counted, and diluted to a concentration of 100,000 cpm
per µl. Hybridization was carried out overnight at 56 C in a total
volume of 45 µl. The hybridization mix consisted of 100 µg total
RNA, 100,000 cpm PTHrP riboprobe, 50,000 cpm GAPDH riboprobe, 80%
deionized formamide, 400 mM NaCl, 40 mM
1,4-piperazine diethanesulfonic acid, pH 6.4, and 1 mM
EDTA. After RNase A treatment and phenol/chloroform extraction,
templates were precipitated and seperated by gel electrophoresis under
denaturating conditions. The gel was dried and signal was visualized by
autoradiography using Kodak XRX films and intensifying screens.
Quantification was performed using the PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Transfections and CAT Assays
Undifferentiated P19 EC, Mes-1, and BHK cells were seeded in a
six- well plate at day 1. The following day cells were transfected
using calcium phosphate precipitation as previously described (39). In
all transfections 1 µg pSV2LacZ plasmid was included to correct for
transfection efficiencies. After 16 h of incubation, the medium
was refreshed and the cells were incubated for an additional 24 h,
after which the cells were harvested in 1 ml PBS, 25 mM
EDTA. To study RA-induced PTHrP expression, P19 EC and ES-5 cells were
seeded at day 1 in the presence of RA in six-well plates at a density
of 100,000 or 70,000 cells per well, respectively. The cells were
transfected at day 3. The next day the medium was refreshed and fresh
RA was added. At day 5, cells were harvested as described above. The
cells were pelleted and dissolved in 4080 µl 250 mM
Tris, 10 mM EDTA, pH 7.5, and repeatedly freeze-thawed to
release the CAT enzyme. After the cell debris had been pelleted, 1040
µl supernatant were used in a CAT assay as previously described (40).
Samples were reassayed when the conversion was out of the linear range
of CAT enzyme activity. After separation of acetylated and
nonacetylated chloramphenicol by TLC, plates were exposed to
PhosphorImager screens and subsequently analyzed and quantified using
the PhosphorImager and ImageQuant software. LacZ staining was measured
at 420 nm using 10 µl supernatant as previously described and used to
correct for transfection efficiencies (41). Experiments were only
analyzed when difference in transfection efficiency between separate
wells was less than 2-fold. All transfection experiments were repeated
at least three times.
Nuclear Extracts and Bandshift Assays
To isolate nuclear extracts, confluent 150-cm2
dishes were washed with ice-cold PBS, and cells were subsequenty
scraped in PBS and pelleted. The pellet was dissolved in 200 µl
buffer C containing 10% glycerol, 20 mM Tris-HCl, pH 7.2,
50 mM KCL, 2 mM dithiothreitol, 0.1
mM EDTA, 0.15 mM spermine, 0.5 mM
spermidine, 10 µg leupeptin/ml, and 10 µg aprotinin/ml. The cell
membranes were disrupted by freeze-thawing, and nuclei were pelleted by
a 15-sec spin in an Eppendorf centrifuge at 5500 rpm. After washing the
nuclei in 200 µl buffer C, the pellet was dissolved in 200 µl
buffer C containing 600 mM KCl and incubated at 4 C for 10
min. Cell debris was pelleted by a 15-sec spin at 16,000 x
g at 4 C. The supernatant was diluted 3-fold using buffer C
without KCl. The protein concentration was measured using a protein
assay (Bio-Rad, Richmond, CA).
Bandshift reactions were performed in a total volume of 20 µl
containing 20 µg nuclear extract, 0.5 µg
polydeoxyinosinic-deoxycytidylic acid, 10 µl BSA, 10% glycerol, 10
mM Tris-HCl, pH 7.2, 25 mM KCl, 0.25
mM dithiothreitol, 0.25 mM ZnCl2.
The reaction mix was preincubated on ice for 10 sec in the absence or
presence of competitor before the probe was added. Oligonucleotide
probes were labeled by filling in the sticky ends with
[
32P]dCTP (Amersham) using the klenow fragment of
DNA-polymerase (BRL). The reaction proceeded for another 20 sec on ice
before the samples were loaded on a 4% TBE polyacrylamide gel that had
been prerun in TBE buffer for 30 sec. TBE buffer contained 25
mM Tris, 25 mM boric acid, and 0.5
mM EDTA, pH 8.3. Gels were run at room temperature for
2 h at 200 V.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. A. E. Broadus (Yale University,
Hartford, CT) for kindly providing us with the mouse PTHrP promoter and
cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marcel Karperien, Department of Endocrinology and Metabolic Disease, University Hopital Leiden, Building 1, C4-R, Albinusdreef 2, 2333 AALeiden, The Netherlands.
Received for publication May 28, 1996.
Revision received June 18, 1997.
Accepted for publication June 24, 1997.
 |
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