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Diabetes Branch (C.O., D.L.), National Institute of Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1770; Department of Molecular Genetics and Cell Biology (N.K.), Oncology Drug Discovery, Bristol-Myers Squid Pharmaceutical Research Institute, Princeton, New Jersey 08540; and Department of Clinical Biochemistry (H.W.), Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
Address all correspondence and requests for reprints to: Claes Ohlsson, M.D., Ph.D., Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 8S235A, 10 Center Drive, MCS-1770, Bethesda, Maryland 20892-1770. E-mail: claes{at}ss.gu.se
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Abstract |
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In conclusion, p53 regulates IGF-IR expression, as reflected by a reduction in IGF-IR protein and a parallel reduction in IGF-I-induced tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma cell line. These data indicate that the IGF-I receptor is a physiological target for p53 in osteosarcoma cells. Furthermore, data supporting an interaction between p53 and Sp1 in the regulation of the promoter activity of IGF-IR are presented.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several important pieces of evidence have been presented indicating that the IGF-IR is involved in tumorigenesis: 1) the IGF-IR is overexpressed in many tumors and cancer cell lines (2); 2) cells lacking the IGF-IR cannot be transformed by SV 40 large T antigen, Ha-ras, v-src, Raf, and bovine papilloma virus (3, 4, 5); 3) addition of IGF-I prevents apoptosis (6, 7, 8); 4) a reduction in IGF-IR function induces apoptosis in tumor cells (9, 10, 11); and 5) overexpression of the IGF-IR protects cells from apoptosis (10, 12). The molecular mechanism(s) for these tumorigenic effects of the IGF-IR is currently under investigation.
The IGF-IR gene promoter has been previously characterized as lacking TATA or CAAT elements (13, 14, 15, 16). Transcription is initiated from a single start site within an initiator motif, which directs accurate transcription, in the absence of a TATA-box (17). Like many TATA-less promoters, the proximal 5‘-flanking region of the IGF-IR gene is highly GC rich, and it contains multiple Sp1 and early growth response consensus-binding sequences in both the 5'-flanking region and the 5'-untranslated region (5'-UTR (13)). We have demonstrated earlier that Sp1 increases IGF-IR promoter activity by acting both on GC boxes in the 5'-flanking region of the promoter and on one homopurine/homopyrimidine motif (CT element) in the 5'-UTR (18). Sp1 is a member of a multigene family of zinc-finger transcription factors. It activates transcription in mammalian cells, primarily via interaction with GC box elements. Because no common regulatory features have been identified among the promoters bearing actual or putative Sp1 binding sites, it has been suggested that Sp1 provides a basal level of transcription that is subsequently modulated by its interaction with other regulatory factors (19). It has been demonstrated earlier, in different cell-lines, that Sp1 and p53 interact in the regulation of different promoters (19).
p53 is believed to be involved in the etiology of many human tumors, and mutations of the p53 gene are the most frequent mutations in human cancers (20, 21). The p53 protein is a transcription factor that can bind specifically to DNA sequences in various promoters and stimulate their transcriptional activity (22, 23). It also can function as a transcriptional repressor of many growth-regulated genes (24, 25). We have recently demonstrated that wild-type p53 expression vectors suppress IGF-IR promoter activity in a dose-dependent manner (26). This effect of p53 is mediated at the level of transcription, and it involves interaction with TBP, the TATA box-binding component of TFIID. p53 precludes binding of TBP to the promoter region and, as a result, TBP is no longer able to assemble a functional transcription initiation complex. On the other hand, mutant forms of p53 stimulated the activity of the IGF-IR promoter (26).
The aim of the present study was to investigate whether the IGF-IR is a physiological target for p53 in osteogenic tumors. Therefore, we studied whether p53 regulates IGF-IR expression and function in an osteosarcoma cell line lacking endogenous p53. Furthermore, a possible interaction between p53 and Sp1 in the regulation of the IGF-IR promoter in these cells was studied.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture, plasmids, and DNA transfection in transient
transfection experiments
Saos-2 cells were grown in DMEM supplemented with 10% FBS.
Drosophila Schneider cells were kindly provided by Carl Wu (National
Cancer Institute, Bethesda, MD). Schneider cells, which lack endogenous
Sp1, were cultured in HyQ-CCM3 serum-free medium (Hyclone Laboratories,
Inc, Logan UT).
The effect of Sp1 and/or p53 on IGF-IR promoter activity was studied by transient transfection assays of different expression vectors together with a fragment of the IGF-IR promoter (-476/+640) fused to a firefly luciferase reporter gene [p0LUC; (18)]. Wild-type p53 (inserted into a cytomegalovirus (CMV)-driven expression plasmid (pCB6, (26)], and Sp1 [inserted into an actin driven plasmid (pPacSp1)], generously provided by R. Tjian, University of California, Berkeley, CA (18), were used as expression vectors in experiments in Saos-2 cells.
Schneider cells were transfected with wild-type p53 (pC53-SN3) and a mutant p53 (pC53-273H) expression vector, kindly provided by Edward Mercer (Thomas Jefferson University, Philadelphia, PA). pC53-SN3 encodes wild-type p53 in the pCMV-Neo-Bam vector (29). pC53-273H is a mutant p53 in which an Arg residue at position 273 is mutated to His. Sp1, inserted into an alcohol dehydrogenase driven plasmid (pADHSp1), also was used as expression vector in transient transfection of Schneider cells.
Saos-2 cells were transfected by the calcium phosphate method using a kit from 5 Prime-3 Prime, Inc. (Boulder, CO); each 60-mm dish received 4 µg of reporter plasmid and 1.1 µg of expression vectors. Schneider cells were transfected by using the lipofectin method (Lipofectin Reagent, Gibco BRL, Life Technologies, Inc., Gaithersburg, MD); each 60-mm dish received 2 µg of reporter plasmid and 1.5 µg of expression vectors. Cells were harvested 48 h (Saos-2) or 60 h (Schneider cells) after transfection, and luciferase activities were measured as described earlier (16). In preliminary experiments, cells were cotransfected with a CMV-ß gal vector (ß-gal, ß-galactosidase), but because expression from the CMV-promoter was found to be affected by p53, subsequent experiments were normalized to total protein, which was measured using the Bradford-Lowry reaction (Bio-Rad, Hercules, CA). In pilot studies of the effect of p53 and/or Sp1 on IGF-IR promoter activity in Saos-2 cells, normalization for transfection efficiency was performed using a RAS-ß-gal plasmid (30), kindly provided by Ronald Evans (The Salk Institute, San Diego, CA). The levels of ß-gal generated by this plasmid were not affected by p53, and the results obtained were essentially the same as those obtained using protein normalization.
Immunoprecipitations
Cells were washed twice with ice-cold PBS and harvested in a
lysis buffer containing 50 mM NaCl, 4 mM sodium
pyrophosphate, 200 mM EDTA, 10 mM NaF, 1
mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotonin, and
1% Triton X-100. Lysates were incubated for 1 h at 4 C, then
centrifuged at 10,000 x g for 30 min at 4 C to remove
Triton-insoluble material. Protein content of the lysates was
determined by the Bio-Rad method. Protein (150 µg) from each dish was
immunoprecipitated overnight at 4 C with a primary antibody (Sp1,
sc-59-G, 1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; or
IGF-IR, sc-713, 1:1000, Santa Cruz Biotechnology), followed by
adsorption to 50 µl of 10% protein A-sepharose beads (for IGF-IR;
Pharmacia Biotech, Inc., Piscataway, NJ or protein G-sepharose beads
(for Sp1; GammaBindG Sepharose, Pharmacia) for 5 h at 4 C.
Immunoprecipitates were washed 3 times with ice-cold
immunoprecipitation buffer containing 10 mM Tris (pH 7.4),
150 mM NaCl, 0.2 mM sodium orthovanadate, 1
mM EDTA, 1 mM EGTA, 0.2 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, and 0.5% Nonidet
P-40. The entire immunoprecipitated samples were then boiled for 4 min
in sample buffer containing 50 mM Tris (pH 6.7), 2% SDS,
2% ß-mercaptoethanol, and bromphenol blue as a marker. Samples were
then run on 7.5% SDS-PAGE gels and transferred to nitrocellulose
membranes using standard electrophoresis and electroblotting
procedures.
Preparation of nuclear extracts
Nuclear extracts were prepared as described by Andrews and
Faller (19, 31). Cells were washed in cold PBS, and the cell pellet was
then suspended in 10 mM HEPES-KOH (pH 7,9), 1.5
mM MgCl2, 10 mM KCl, 0,5
mM DTT, and 0.2 mM phenylmethylsulfonyl
fluoride; kept on ice for 10 min; mixed on a vortex mixer for 10 sec;
and centrifuged for 10 sec at 14,000 x g. The pellet
was resuspended in 20 mM HEPES-KOH (pH 7.9), 25% glycerol,
420 mM NaCl, 1.5 mM MgCl2, 0.2
mM EDTA, 0.5 mM dithiothreitol, and 0.2
mM phenylmethylsulfonyl fluoride; incubated on ice for 20
min; and centrifuged for 2 min at 14,000 x g. The
supernatant was divided into aliquots and stored at -70 C. Protein
determinations and immunoprecipitations were then performed as
described above.
Immunoblotting
Nitrocellulose membranes were blocked with either 3%
insulin-free BSA (for phosphotyrosine blotting) or 3% nonfat dry milk
in a PBST buffer containing 10 mM sodium phosphate (pH
7.2), 140 mM NaCl, and 0.1% Tween 20. Blots were then
immunolabeled overnight at 4 C for phosphotyrosine (05–321, 1:1000,
Upstate Biotechnology, Incorporated), IRS-1 (06–248, 1:1000, Upstate
Biotechnology, Incorporated), IGF-IR (sc-713, 1:1000 Santa Cruz
Biotechnology), p53 (DO-1, sc-126, 1:000, Santa Cruz Biotechnology).
After washing in PBST, the filters were incubated with HRP-conjugated
secondary antibodies for 1 h at 4 C, followed by extensive washes
in PBST plus 0.1% Triton X-100. Proteins were detected using
chemiluminescence (ECL, Amersham, Arlington Heights, IL) according to
the manufacture‘s conditions. Some blots were stripped and reprobed
with a different antibody. Blots were stripped by incubation for 1
h at 50 C in a solution containing 62.5 mM Tris-HCl (pH
6.7), 2% SDS, and 0,7% ß-mercaptoethanol. Blots were then washed
for 1 h in several changes of PBST at room temperature and probed
with ECL to confirm that antibodies had been completely removed. Blots
were then reblocked and immunolabeled as described above.
IGF-I binding assay
Binding to IGF-binding proteins was excluded by using an IGF-I
analog, des(1, 2, 3)IGF-I, which binds exclusively to the IGF-IR. To
determine the level of IGF-IR expression, cells (
200,000) on 12-well
plates were washed with PBS and incubated with 1 ml ligand-binding
buffer (100 mM HEPES, pH 7.9, 120 mM NaCl, 5
mM KCl, 1.2 mM MgCl2, 1
mM EDTA, and 5 mg/ml BSA) containing 50,000 cpm of
125I-des(1, 2, 3)IGF-I and different concentrations of cold
ligand for 4 h at 4 C. After washing three times with PBS, cells
were lysed at 37 C for 1 h using 0.4 ml of 0.2 N NaOH; and the
total radioactivity, as absorbed to a filter, was counted by a 
counter (Gamma Trac, Tm Analytic, Elk Grove Village, IL). Cell numbers
were determined for wells treated simultaneously with the experimental
wells. Data representing specific binding were analyzed according to
the method of Scatchard (32).
Statistical analysis
Values are given as means ± SEM. The
statistical significance of differences between means were calculated
by ANOVA, followed by Student-Neuman-Keul’s multiple range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To avoid temperature-induced effects
on IGF-IR expression and IGF-I responsiveness, the effects of
p53-induction were always related to control cultures kept at the same
temperature as the p53-induced cultures (30 C for wild-type p53 and 37
C for mutated p53). Western immunoblots, using an antibody specific for
the IGF-IR, demonstrated that expression of wild-type p53 for 5 days
decreased (69 ± 8% of controls at 30 C), whereas mutated p53
increased (128 ± 3% of controls at 37 C), IGF-IR expression,
compared with cells not expressing p53 (Fig. 2, A and B). Thus, cells expressing
mutated p53 expressed 85% more IGF-IR protein, compared with cells
expressing wild-type p53 (Fig. 2B).
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). There was no significant difference
in the number of IGF-IRs per cell in cells with no p53 expressed
(cultured at 30 C), compared with those cultured at 37 C. Expression of
wild-type p53 decreased IGF-IRs to 32,000/cell, whereas cells
expressing mutated p53 expressed 55,000 receptors/cell. Thus, cells
expressing mutated p53 had 72% more IGF-IRs than cells expressing
wild-type p53. Neither wild-type p53 nor mutated p53 modulated the
affinity of the IGF-IR.
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). IGF-I-induced tyrosine
phosphorylation of the IGF-IR was decreased by wild-type p53 and
increased by mutated p53 (wild-type p53: 42 ± 14%, compared with
cells that do not express p53 cultured at 30 C; mutated p53: 146
± 18% of control cells cultured at 37 C; Fig. 4, A and B). Thus,
cells expressing mutated p53 demonstrated 246% higher IGF-I-induced
tyrosine phosphorylation of IGF-IR, compared with cells expressing
wild-type p53. Because we have shown that cells expressing mutated p53
express more IGF-IR protein than cells expressing wild-type p53, it is
likely that the increase in IGF-I-induced tyrosine phosphorylation of
the IGF-IR is primarily the result of increased numbers of
IGF-IRs available for phosphorylation.
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, A
and B). No effect on IGF-I-induced tyrosine phosphorylation of IRS-1
was seen by mutated p53. Because the amount of IRS-1 protein was not
regulated by wild-type p53 (data not shown), the reduced IGF-I-induced
tyrosine phosphorylation of IRS-1 in cells expressing wild-type p53 was
most likely the result of the reduction in IGF-IR expression and
tyrosine phosphorylation.
Functional and physical interactions between Sp1 and p53 in the
regulation of the IGF-IR promoter
Sp1 increases IGF-IR promoter activity in cells without endogenous
Sp1 (Schneider cells) (18), whereas p53 decreases IGF-IR promoter
activity in cells without endogenous p53 (Saos-2 cells) (26). Because
both Sp1 and p53 regulate IGF-IR promoter activity, a direct
interaction between these two factors in this regulation was studied.
The functional interaction between p53 and Sp1 was first studied in the
osteogenic sarcoma-derived cell line, Saos-2. Sp1 and/or p53 expression
vectors were transiently transfected into Saos-2 cells, together with
the IGF-IR promoter fused to a luciferase reporter gene. Luciferase
activity was measured after 48 h. p53 decreased promoter activity
by 59% in the absence of Sp1 (Fig. 5A).
No effect on promoter activity was seen by Sp1 by itself. However, Sp1
counteracted the inhibitory effect of p53 on promoter activity in a
dose-dependent manner (Fig. 5A). This effect of Sp1 was not caused by
inhibition of p53 expression, because p53 expression (as determined
with immunoblotting) was not affected by Sp1 (Fig. 5B). A functional
interaction between p53 and Sp1 also was investigated in Schneider
cells, which lack endogenous Sp1. Sp1 strongly increases IGF-IR
promoter activity in these cells (Fig. 6A). Wild-type p53 or mutated p53 was
expressed in Schneider cells using transient transfection (Fig. 6B).
p53, by itself, did not regulate promoter activity in Schneider cells
(Fig. 6A). However, 60% of the Sp1-induced increase in IGF-IR promoter
activity was counteracted by a maximum dose of p53 (Fig. 6A).
Surprisingly, both wild-type p53 and mutated p53 partially counteracted
the Sp1-induced increase in promoter activity (Fig. 6A). To test
whether the functional interaction between p53 and Sp1 was caused by a
physical interaction between these two factors, nuclear extracts of
Saos-2 cells, transfected with Sp1 in the absence of p53 or in the
presence of wild-type p53 or mutated p53, were immunoprecipitated with
an Sp1-specific antiserum, followed by immunoblotting for p53.
Wild-type and mutated p53 were coimmunoprecipitated with Sp1 (Fig. 5C).
Coimmunoprecipitation of p53 and Sp1 also was found in Schneider cells
transfected with p53 and Sp1 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of wild-type p53 and mutated p53 on IGF-I-induced tyrosine phosphorylation of IGF-IR is explained, in part, by the finding that cells expressing mutated p53 express more IGF-IRs than cells expressing wild-type p53. The potential mechanism(s) for the difference in the magnitude of change between IGF-I-induced autophosphorylation of the IGFI-R and the expression of the IGFI-R protein, remains to be determined. In contrast, IRS-1 expression was not regulated by p53, and the reduction of IGF-I-induced tyrosine phosphorylation of IRS-1 seen in cells expressing wild-type p53 may be explained by the reduced number of activated IGF-IRs.
Our results, showing that p53 regulates the numbers of IGF-IR and IGF-I-induced tyrosine phosphorylation of the IGF-IR and IRS-1, together with the results of Prisco et al. (12), showing that p53 counteracts IGF-I inhibition of apoptosis, indicate that either wild-type p53 or IGF-IR antisense may be an effective treatment strategy for p53-negative osteogenic tumors expressing high levels of IGF-IRs.
p53 is not unique in regulating IGF-IR expression. The Wilms tumor supressor gene (WT1) also suppresses IGF-IR promoter activity (17, 35), and overexpression of WT1 decreases the endogenous levels of IGF-IR and reduces IGF-I-mediated cellular proliferation (36).
Two other components of the IGF system, besides the IGF-IR, are regulated by p53. IGF-binding protein-3 (IGFBP-3) has been shown to be induced by p53 (37), whereas promoter activity of IGF-II is reduced by wild-type p53 (38). IGFBP-3 is commonly an inhibitor of the mitogenic signaling by IGFs. Thus, p53 regulates the IGF system at the level of expression of one of its ligands (IGF-II), the bioavailability of ligands (IGFBP-3 expression), and at the expression of the IGF-IR. The fact that p53 induced regulation on all three levels of the IGF system was demonstrated using the Saos-2 osteosarcoma cells lacking p53.
In addition to the IGF-I receptor, two other genes, bcl-2 and MAP4, have been shown to be down-regulated by p53 (39, 40). The functional role of MAP4 is not clear. Thus, the bcl-2 and the IGF-IR genes are the first genes shown to be down-regulated by p53 and known to play a direct role in the process of cellular transformation.
Interestingly, Sp1 has been shown to be a target for p53 in a erythroleukemia cell line (19). In these cells, it was found that GM-CSF-inducible DNA-binding complexes contained both Sp1 and p53 and that these heterocomplexes bound both p53- and Sp1-binding sequences with high affinity. Immunoprecipitation of nuclear extracts indicated that Sp1 was associated, as a heterocomplex, with p53. The functional effect of such an interaction was, however, not investigated. p53 and Sp1 also interact and cooperate in tumor necrosis factor (TNF)-induced transcriptional activation of the HIV-1 Long-terminal repeat (41). A physical interaction between p53 and Sp1 was seen after TNF stimulation. Our result, that Sp1 and p53 were coimmunoprecipitated in Saos-2 cells, supports the notion that Sp1 and p53 interact in a complex. Furthermore, in the present study, a functional interaction between Sp1 and wild-type p53, in the regulation of IGF-IR promoter activity, was found in Saos-2 cells. This is supported by our finding in Schneider cells that wild-type p53 counteract the stimulatory effect of Sp-1 on IGF-IR promoter activity. Surprisingly, in Schneider cells, the same effect was seen with mutated p53. The physiology of and mechanism behind this inhibitory effect of mutated p53 in Schneider cells remains to be elucidated. It has been suggested that Sp1 and p53 can form distinct associations that exhibit different DNA binding affinities, perhaps involving other nuclear proteins. Both Sp1 and p53 have the propensity to form large oligomeric structures in solution, with subsequent changes in their DNA binding activity (42, 43). Heterocomplexes between Sp1 and p53 could exhibit selectivity in binding to Sp1 or p53 regulatory elements or a change in their trans-activating activity. The sequestering of Sp1 by p53 also could directly affect transcription from Sp1-regulated promoters. A precedent for such a regulatory mechanism has been reported for the tumor suppresser Rb, which was found to affect transcription of target genes through interactions with Sp1 (44). p53-induced stimulation of transcription is thought to occur when p53 binds to promoter sequences directly, whereas repression is thought to be via protein-protein interactions between p53 and other transcription factors. We are currently investigating the molecular mechanisms for the interaction between Sp1 and p53 in the regulation of the promoter activity of the IGF-IR.
In conclusion, p53 regulates IGF-IR expression and tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma cell-line. Furthermore, data indicating an interaction between p53 and Sp1 in the regulation of the promoter activity of IGF-IR are presented.
Received September 25, 1997.
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), in the presence
of tetracycline at 37 C (no p53, 
), or in the absence of
tetracycline at 37 C (mutated p53, 
). Determination of IGF-I binding
is described under Materials and Methods. B/F,
bound/free. Each experiment was carried out in triplicate cultures. One
representative experiment is shown in Fig 3
