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Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900
Address all correspondence and requests for reprints to: Lee-Chuan C. Yeh, Ph.D., Department of Biochemistry (Mail Code 7760), University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: carolyeh{at}biochem.uthscsa.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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)-like element, and a c-Myb or E-box-like motif.
Mutations in the CAAT-like sequence reduced the promoter activity in
both control and OP-1-treated cells, but did not abrogate the
OP-1-induced down-regulation. Mutations in the C/EBP-like element
reduced the promoter activity in both control and OP-1-treated cells
without significantly affecting the extent of down-regulation.
Mutations in the putative c-Myb or E-box-like motif reduced the
promoter activity in both the OP-1-treated and control cells and
completely abolished the inhibitory effect of OP-1 on the IGFBP-5
promoter activity. Gel mobility shift analyses further showed specific
interaction between nuclear protein(s) in FRC cells and the 21-bp
region. OP-1 down-regulates the nuclear regulatory protein interaction
with the 21-bp region by reducing either the cellular concentration of
the regulatory protein(s) or the affinity of the regulatory protein(s)
for the OP-1 responsive element. In conclusion, we identified an OP-1
response region in the rat IGFBP-5 promoter and further showed that
OP-1 down-regulates the nuclear protein interaction with the response
element(s). |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OP-1 also stimulates the synthesis of several growth factors, such as the insulin-like growth factors (IGFs), in cultured osteoblastic cells. IGF-I and IGF-II show mitogenic activity for bone cells and enhance osteoblasts differentiation (15, 16, 17). The biological activity of the IGFs can be affected by their binding proteins, the IGF-binding proteins (IGFBPs) (18). For example, IGFBP-4 inhibits bone formation, but IGFBP-5 can be either stimulatory or inhibitory of the IGF-I activity on bone cell (18, 19, 20, 21). Synthesis of IGFBP-5 by osteoblastic cells appears to be tightly controlled (22) and can be affected by numerous factors (20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). For example, IGF-I (20, 24, 36), IL-6 with its soluble receptor (31), PGE2 (27, 28, 34, 35), PTH fragments (26), and retinoic acid (24, 36) stimulate IGFBP-5 messenger RNA (mRNA) expression in osteoblastic cells. In contrast, basic fibroblast growth factor, cortisol, dexamethasone, platelet-derived growth factor, and transforming growth factor-ß inhibit IGFBP-5 mRNA expression (20, 23, 30, 32, 37). In fetal rat calvaria (FRC) cells, OP-1 decreases the steady state IGFBP-5 mRNA level by reducing transcription of the IGFBP-5 gene. The stability of the IGFBP-5 mRNA is unaffected (25). We further show that exogenous human IGFBP-5 protein inhibits osteoblast differentiation induced by OP-1, as determined by the measurement of a differentiation biochemical marker, alkaline phosphatase activity (37). Taken together, these data suggest that IGFBP-5 plays a critical role in OP-1-induced bone cell differentiation.
To investigate the molecular basis for the OP-1-induced down-regulation of IGFBP-5 gene expression, a series of plasmid constructs that contain the rat IGFBP-5 promoter fused to a reporter gene were generated. These plasmids were used to identify OP-1-response elements in transient transfection experiments in primary cultures of FRC cells. We found that OP-1 down-regulates rat IGFBP-5 promoter activity via a 21-bp region. The region contains several sequences analogous to certain highly conserved transcriptional regulatory elements. Mutation studies showed that sequences within the E-box-like motif are involved in the OP-1-induced down-regulation of the rat IGFBP-5 promoter. Gel mobility shift analyses further showed that OP-1 decreases the interaction between the 21-bp region and a trans-acting factor(s).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of rat IGFBP-5 promoter-luciferase genes
A 1-kb DNA fragment, comprising nucleotides from -888 bp
upstream to +114 bp downstream from the transcription start site (+1)
of the rat IGFBP-5 gene was generated by PCR using genomic DNA isolated
from rat liver. The sense primer is 5'-AGG ATC TGC CTG CCC TGT-3', and
the antisense primer is 5'-ACC GAG GAG GGG GAT AAC-3'. The reaction was
performed for 25 cycles at 94 C for 30 sec, 55 C for 30 sec, and 68 C
for 60 sec, with a final extension at 68 C for 10 min. The PCR product
was directly cloned into the pCRII-TOPO vector
(Invitrogen, Carlsbad, CA). The clone containing the sense
(positive) orientation of the IGFBP-5 promoter was confirmed by
restriction enzyme mapping and double stranded (ds) DNA sequencing. The
IGFBP-5 promoter fragment was then subcloned into the SacI
and XhoI sites of the pGL2-Basic vector (Promega Corp.) containing the promoterless luciferase reporter gene
(Luc). The antisense fragment of the IGFBP-5 promoter was ligated into
the XhoI and HindIII sites of the pGL2-Basic
vector. All plasmids were checked for purity on 1% agarose gels. Only
the ultrapure DNA preparations were used for transfection studies.
Generation of rat IGFBP-5 promoter mutants
Deletions in the 5'-end of the rat IGFBP-5 promoter were
generated by digestion of the plasmid containing the 1-kb promoter
fragment with unique restriction enzymes or by PCR (Fig. 1
). Constructs with 5'-end beginning at
positions -390, -297, -151, -131, -71, and +32 were generated by
digestion of the parent plasmid with AccI, AlwNI,
StuI, DraIII, SacI, and
DraI, respectively. Constructs with the 5'-end beginning at
positions -50 and -33 were generated by PCR using sense primers
5'-TGG CAG CCA GGG GCC GTC-3' and 5'-CTA TTT AAA AGC GCC TGC-3',
respectively. PCR conditions were 35 cycles at 94 C for 60 sec, 50 C
for 60 sec, 72 C for 60 sec, and a final extension at 72 C for 10 min.
The resultant DNA fragments were subcloned into the pGL2-Basic vector.
The internal mutations within the 21-bp sequence of the promoter were
generated by PCR with sequence-specific oligonucleotide primers (Fig. 3B) and wild-type DNA template (-71/+114) as described above.
Alternatively, the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA) was used. The -390/+114 mutant
clones containing specific mutations in the CAAT,
CCAAT/enhancer-binding protein (C/EBP), or E-box sequence
were constructed by replacement of the wild-type sequence with the
corresponding mutant sequence. The DNA products were sequenced in their
entirety to ensure the absence of unintended mutations.
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MEM plus serum and were
transiently transfected with promoter constructs by the calcium
phosphate-DNA coprecipitation method (38). Plasmid DNA (12 µg) was
used for each six-well plate. The medium was removed after 3 h of
transfection, and the cells were treated with 15% glycerol/1 x
HEPES-buffered saline for 2 min at room temperature. Transfected FRC
cells were incubated overnight in fresh complete MEM, followed
by treatment with OP-1 (300 ng/ml) or vehicle in serum-free MEM for
24 h. The IGFBP-5 promoter activity was determined by measuring
the luciferase reporter gene activity as previously described (25).
Briefly, cells were lysed, and an aliquot (20 µl) was taken to
measure luciferase activity using a luciferase assay kit (Promega Corp.) and the OPTOCOMP I luminometer ILA911 (Tropix). Another
aliquot (10 µl) was used to determine transfection efficiency by
measuring ß-galactosidase activity using the Galacto-Light kit
(Tropix). Luciferase activity was normalized to ß-galactosidase
activity. In some experiments the Dual-Light Reporter System was used.
The system is a chemiluminescent reporter gene assay system for the
combined luciferase and ß-galactosidase, using as substrates
luciferin and Galacton-Plus, respectively. The values obtained were
comparable to those using the single enzyme system.
Gel mobility shift assay
Proteins for the gel mobility shift assays were extracted from
control or OP-1-treated confluent FRC cells basically as previously
described (39). Protein extracts were stored as aliquots at -80 C
until use. The protein concentration was determined using the Bradford
method (40). The gel mobility shift experiments were carried out using
the BandShift kit. Briefly, radiolabeled dsDNAs were produced by
annealing complementary oligonucleotides (consisting of the sequence
from -71 to -51 and its complementary sequence) followed by treatment
with the T4 polynucleotide kinase in the presence of
[
-32P]ATP. Varying concentrations of protein
extracts were incubated with a fixed amount of radiolabeled DNA probe
(5500 cpm) for 20 min at room temperature. Samples were analyzed on 5%
nondenaturing polyacrylamide gels in 1 x TBE. Gels were dried,
and the radioactive bands were detected and quantified using a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA)
and ImageQuant software (Becton Dickinson and Co.,
Mountain View, CA). For the competition experiments, an excess amount
(50- to 200-fold) of unlabeled DNA probe or unrelated EBNA-1
or Oct-1 DNA was included in the reaction.
Statistical analysis
Multiple means were compared by one-way ANOVA, followed by
Student’s t test for paired comparison with the control.
The ANOVA and Student’s t test programs in the PSI-Plot
(Ploy Software International, Salt Lake City, UT) for personal
computers were used for the analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These DNA constructs were
used in transient transfection studies of subconfluent FRC cells to
define the OP-1-responsive element.
Figure 2A shows the reporter gene
activity of the various clones in FRC cells treated with OP-1 or
vehicle. The promoter activity for the pGL2-Basic vector in transfected
FRC cells treated with vehicle was very low and was not affected by
OP-1. In control cells, the 1-kb promoter construct (-888/+114) was
about 110-fold more active than the parent pGL2-Basic (Fig. 2A).
Sequential removal of the 5'-end sequence beginning from -888 resulted
in a stepwise reduction of the basal promoter activity. Deletion of the
500 nucleotides from the 5'-end (the -390/+114 construct) led to an
approximately 40% decline in the reporter activity compared with the
-888/+114 construct. Additional deletion of the promoter sequence to
position -131 led to a further 30% decline in promoter activity. The
promoter containing the -71/+114 sequence retained about 15% of the
most active promoter (the -888/+114 construct) activity and was about
13-fold more active than the pGL2-Basic. The promoter construct with
only 50 nucleotides of the 5'-flanking sequence and the first 114 bp of
exon 1 (the -50/+114 construct) retained about 2–3% of the activity
exhibited by the -888/+114 construct and was about 2- to 3-fold more
active than the parent pGL2-Basic.
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40%) of
reduction in IGFBP-5 promoter activity by OP-1 (Fig. 2B). These
promoter constructs retained significant levels of residual promoter
activity compared with the pGL2-Basic, e.g. the promoter
containing the -71/+114 sequence displayed about 8-fold higher
activity than the pGL2-Basic vector control. In contrast, deletions
beyond -71 (e.g. the -50/+114 and -33/+114 constructs)
resulted in promoters that did not show any OP-1-induced reduction in
IGFBP-5 promoter activity (Fig. 2B). However, these promoters still
displayed about 2-fold higher activity than pGL2-Basic. These
observations strongly suggest that the element(s) responsible for
down-regulation of the IGFBP-5 promoter by OP-1 is located in a 21-bp
region (-71 to -51). The deletion construct of +32 to +114 did not
show any promoter activity and was not responsive to OP-1. The reverse
orientation of the largest construct containing -936 to +114 exhibited
little promoter activity and was not responsive to OP-1 (Fig. 2, A and
B).
The 21-bp region contains several putative regulatory transcriptional
elements: a CAAT-like sequence, a C/EBP-like (ATTGCAGC) element, and
a c-Myb-like (T/CAACG/TG) or E-box-like (CANNTG) motif (41, 42, 43, 44). The
first two elements are also present in the human (43) and mouse (41)
IGFBP-5 promoters (Fig. 3A). The E-box
element is present in the mouse and rat IGFBP-5 promoters, but an extra
A residue is present in the human sequence (43). To further delineate
which one or more of these putative regulatory elements are responsible
for the down-regulation of the rat IGFBP-5 by OP-1, each element was
mutated, and their sequences are shown (Fig. 3B). Mutational analyses
were performed using either the shorter -71/+114 promoter sequence or
the longer -390/+114 promoter sequence. The shorter construct did not
contain any flanking sequence 5' of the CAAT box and exhibited about
15% of the basal promoter activity. The longer construct
exhibited a much higher activity (64% of the basal promoter
activity of the 1-kb construct). The promoter activity of each mutant
was measured after transfection of FRC cells. Figure 4A shows the relative luciferase
activities of the various shorter mutant promoter clones in FRC cells
treated with OP-1 or vehicle. In agreement with the results shown in
Fig. 2, OP-1 dramatically reduced (by 47%) the activity of the
IGFBP-5 promoter construct containing the wild-type sequence from -71
to +114. Mutations in the CAAT-like sequence reduced the promoter
activity in both control and OP-1-treated cells. The extent of
reduction in promoter activity induced by OP-1 was essentially
unchanged (40%). A single T to C mutation or a 4-base mutation
within the C/EBP-like element also reduced the IGFBP-5 promoter
activity in both control and OP-1-treated cells without affecting OP-1
responsiveness. Mutations in the E-box-like motif reduced promoter
activity in both control and OP-1-treated cells and completely
abolished the effect of OP-1 (Fig. 4A). Additionally, to substantiate
these observations obtained with the shorter mutant promoter
constructs, these analyses were conducted with the longer mutant
constructs. Figure 4B shows the relative luciferase activities of the
various longer mutant promoter clones. Similar to the results obtained
with the shorter clones, the CAAT mutant promoter constructs showed
reduced activity in both control and OP-1-treated cells, and the extent
of the OP-1-induced reduction in promoter activity was similar to that
of the wild-type (46% vs. 40%). Mutations in C/EBP also
reduced promoter activity in both control and OP-1-treated cells. The
extent of the OP-1-induced reduction remained unchanged. Mutations in
the putative E-box-like motif completely abolished down-regulation of
promoter activity by OP-1. These observations suggest that the putative
E-box-like motif (or at least the CAAC sequence within it) contains
cis-acting elements responsible for the down-regulation of
IGFBP-5 transcription by OP-1.
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shows results of a
representative gel shift assay. One major shifted band with a slower
mobility in both control and OP-1-treated samples was observed. The
intensity of the band was proportional to the protein concentration.
However, at the same protein concentration, the band intensity was
consistently lower in the OP-1-treated sample (by 40–50%) than in
the control sample (Fig. 5B). The decrease was readily observable
24 h after OP-1 treatment of FRC cells and was not changed further
at 48 h. The radioactive probe in the shifted band was competed
out by increasing concentrations of the nonradioactive, homologous
oligonucleotide and was also competed out by the mutant oligonucleotide
containing the E-box mutations (Fig. 6).
The latter oligonucleotide was not as efficient in competing with the
wild-type sequence. Unrelated oligonucleotides, EBNA-1 and
Oct-1, did not compete with the wild-type oligonucleotide
(Fig. 6). The finding suggests that the nuclear protein-DNA interaction
is specific.
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, and E-box) within the 21-bp sequence in the
OP-1-induced down-regulation of IGFBP-5, the ability of mutant
oligomers (Fig. 3B) consisting of mutations in each of the conserved
element to bind nuclear protein(s) was examined. Gel mobility shift
assays with radiolabeled wild-type and mutant oligonucleotide probes
and proteins from control or OP-1-treated cells were conducted. In
agreement with the results described above, OP-1 caused a decrease in
the interaction between the nuclear protein(s) and the wild-type ds
oligonucleotide probe (Fig. 7, lanes 1
and 2). The ds oligonucleotide probe containing mutations in the CAAT
element only without changing the C/EBP element and the E-box motif
formed a DNA-protein complex. Although one would expect that the mutant
DNA-protein complex might be smaller than the wild-type DNA-protein
complex due to the failure of binding of the CAAT-box-binding protein
to the mutant complex, the apparent electrophoretic mobility of the
mutant DNA-protein complex was not significantly dissimilar to that of
the wild-type DNA-protein complex. A possible reason is that the gel
system might not be sufficiently sensitive to detect small changes in
the number of proteins bound to the complex. The band intensity of the
complex was lower in the OP-1-treated sample than in the control (Fig. 7, lanes 3 and 4). Hence, the OP-1 induced decrease in protein binding
to the wild-type 21-bp sequence was probably not due to a change in the
CAAT-binding protein(s). Similarly, the ds oligonucleotide probe
containing mutations in the C/EBP element only without changes in
the CAAT element and E-box motif still displayed a decrease in band
intensity in the OP-1 sample compared with the control (Fig. 7, lanes 5
and 6). The observation suggests that OP-1 did not change the
C/EBP-binding protein(s). However, the ds oligonucleotide containing
mutations in the E-box motif only without changing the CAAT and
C/EBP elements no longer displayed the OP-1-induced decrease in band
intensity (Fig. 7, lanes 7 and 8). The observation suggests that OP-1
altered either the E-box-binding protein(s) or a protein(s) that binds
to the E-box-binding protein(s).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study also shows that the rat DNA fragment with 151 bp of the 5'-flanking sequence and the first 114 bp of exon 1 retained about 40% of the maximal promoter activity displayed by the -888/+114 promoter sequence in fetal rat calvaria cells. The DNA fragment with only the -50/+114 sequence retained about 2–3% of the maximal promoter activity. By comparison, Kou et al. (41) reported that the mouse DNA fragment with only 156 bp of the 5'-flanking sequence and the first 120 bp of exon 1 confers about 61% of the promoter activity in human Hep G2 cells. Maximal promoter activity was defined as that exhibited by the promoter containing the first 1 kb 5' upstream of the transcription start site of the mouse IGFBP-5 gene cloned 5' to the luciferase reporter gene. In addition, the mouse promoter fragment containing the -51/+120 sequence shows about 23% of the maximal promoter activity. Only deletion to position -31 leads to a marked decrease in promoter activity to about 6%. The reasons for this apparent difference between the two sets of data are not clear. One might speculate that the species difference in the promoter and the cell types used in the transfection studies might contribute to this dissimilarity. It is unlikely that the difference in activity lies in the two promoters (mouse vs. rat), because their base sequences are almost identical. It is possible that the difference in cell origin (human vs. rat and liver vs. calvaria) and the physiological state of the cells (transformed vs. nontransformed) used in these studies may contribute to the disparity. The published study was performed in a heterologous system with the mouse promoter in human hepatoma cells and the present study was performed in a homologous system with the rat promoter in rat calvaria cells.
In the present study we identified a 21-bp promoter element that
mediates the down-regulation of IGFBP-5 by OP-1. The element is located
between -71 and -51 in the rat IGFBP-5 promoter and is the first
reported negative OP-1-responsive element that causes repression of the
IGFBP-5 gene. The 21-bp OP-1-responsive element contains a CAAT-like
sequence, a C/EBP-like element, and a c-Myb-like or E-box-like
motif. Mutations of the CAAT-like sequence and the C/EBP-like
elements reduced the overall transcription activity of the promoter,
but did not eliminate the OP-1 response specifically. Thus, it is
unlikely that this cis-element plays an important role in
the OP-1-induced down-regulation of IGFBP-5 gene expression. Mutation
studies further allowed identification of the putative E-box or c-Myb
motif as part of the OP-1-responsive element.
In addition, our results on gel mobility shift assays with the 21-bp sequence and nuclear extract from control and OP-1-treated cells suggest that the OP-1 reduces either the concentration or the affinity of the nuclear protein(s) that interacted with this promoter region in FRC cells, resulting in a decrease in transcription. Such an interpretation is supported by our prior observation that OP-1 down-regulates IGFBP-5 transcription (17). However, the present data could not distinguish the two possibilities. Furthermore, the data on gel mobility shift assays using oligonucleotides with mutations in each of the three conserved elements indicated that an E-box-binding protein(s) or an ancillary protein(s) that binds to the E-box-binding protein(s) may play an important role, through the IGF-IGFBP-axis, in mediating at least in part the effects of OP-1 in inducing bone cell differentiation. Alternations in the ancillary protein may result in a change in the DNA binding affinity of the E-box-binding protein through protein-protein interaction. Although the molecular basis for the decrease in binding remains to be defined, phosphorylation/dephosphorylation of the trans-acting factor may be involved. Such a posttranslational modification, in turn, can alter the binding affinity of a regulatory protein.
These conserved elements in the IGFBP-5 proximal promoter are probably involved in the regulation of IGFBP-5 transcription by various agents. For example, previous reports showed that cortisol inhibits IGFBP-5 gene expression in osteoblasts (30). Deletion studies of the IGFBP-5 promoter further showed that the region responsive to cortisol lies between -70 to +22 bp of the promoter, and that the putative E-box or c-Myb motif is required for basal transcription and cortisol-mediated transcriptional repression. Although this DNA region appears to be responsive to both OP-1 and cortisol, whether an identical base sequence is responsive to both agents is not clear at present. However, the actions of OP-1 and cortisol on bone cells are different; thus, it is unlikely that the two agents use an identical regulatory mechanism(s) for the down-regulation of the IGFBP-5 promoter. It is conceivable that different trans-acting factors might be involved. Knowledge of the mechanism(s) of transcriptional down-regulation by OP-1 and cortisol should be informative.
A minimal DNA sequence, spanning -69 to -35 bp, for PGE2 stimulation of IGFBP-5 promoter activity has been reported recently (28). The sequence contains E box, C/EBP, nuclear factor-1, and activating protein-2 (AP-2) like elements. Mutation of the E box-like element reduced the basal promoter activity by 50% and eliminated the stimulatory effect of PGE2. Mutations in the C/EBP or nuclear factor-1 like elements reduced the basal promoter activity without affecting the PGE2 effects. An earlier report revealed two PGE2-responsive regions, located between -2695 to -1470 and -989 to -332 bp (35).
Progesterone also stimulates IGFBP-5 gene transcription. Recently, a progesterone-responsive region, spanning from -162 to -124 bp in the human IGFBP-5 has been identified. The region contains two tandem CACCC box sequences, and mutation of the proximal CACCC box eliminated PG activation (27).
The base sequence of the negative OP-1-responsive element that
participates in the OP-1-induced down-regulation of the IGFBP-5
promoter reported here differs from that of the positive
OP-1-responsive elements reported previously (45, 46). For example,
OP-1 stimulates mouse collagen type X promoter activity involving a
33-bp region consisting of nucleotides from -310 to -278 (45).
The sequence is TTAAAAATAAAAAGGGTGAATCATCATTCCATC. The sequence
contains a myocyte-specific enhancer binding factor (MEF)-2-like
(TTAAAAATAAAAA) sequence and an AP-1-like (TGAATCATCA) sequence. Both
elements are necessary for the OP-1 effect. In rat calvaria-derived
chondrogenic cells, OP-1 induced nuclear protein interaction with the
MEF-2-like sequence, but not with the AP-1-like sequence. Additionally,
a 316-bp BMP-responsive region in the chicken collagen type X promoter
has been reported (46). The region was more responsive to BMP-4 than to
BMP-2 and -7. Although the BMP-responsive element contains numerous
putative regulatory protein-binding sites (such as Inf-, NF-E1,
Ets-1/PEA-3, HNF-5, CBF, AP-2, MBF-1, GATA-1, and c-Mos), it does
not contain the MEF-2-like sequence or the AP-1-like sequence reported
for the OP-1-responsive element in the mouse collagen type X promoter.
The region also does not contain the E-box/c-Myb-like sequence reported
here for the negative OP-1-responsive element in the rat IGFBP-5
promoter.
In summary, the present study demonstrates that the gene element responsible for the down-regulation of IGFBP-5 by OP-1 is located between -71 and -51 in the rat IGFBP-5 promoter. The E-box/c-Myb-like motif is necessary for the OP-1 effect. The results also show the presence of a nuclear protein(s) in FRC cells that binds to the DNA region. The observed decrease in the concentration or activity of this protein(s) in OP-1-treated FRC cells further suggests that a change in the nuclear protein might be the mechanism for the down-regulation of IGFBP-5 in OP-1-treated FRC cells.
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Acknowledgments |
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Footnotes |
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Received January 20, 2000.
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References |
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