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*Breast Cancer
Endocrinology Vol. 142, No. 9 3817-3827
Copyright © 2001 by The Endocrine Society

ARTICLES

Butyrate, a Histone Deacetylase Inhibitor, Activates the Human IGF Binding Protein-3 Promoter in Breast Cancer Cells: Molecular Mechanism Involves an Sp1/Sp3 Multiprotein Complex

Gillian E. Walker, Elizabeth M. Wilson, David Powell and Youngman Oh

Department of Pediatrics (G.E.W., E.M.W., Y.O.), Oregon Health Sciences University, Portland, Oregon 97201; and Baylor College of Medicine (D.P.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Youngman Oh, Ph.D., Department of Pediatrics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: ohy{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Specific cell growth stimulators and inhibitors regulate IGF binding protein-3 (IGFBP-3), where in turn IGFBP-3 mediates their biological effects. The molecular mechanism(s) by which these factors regulate IGFBP-3 are unknown. Sodium butyrate, a histone deacetylase inhibitor causing growth arrest and differentiation, increases IGFBP-3 expression. We investigated the molecular mechanism of this induction using an IGFBP-3 promoter reporter system in MCF-7 and Hs578T breast cancer cells. IGFBP-3 promoter activity was induced up to 40-fold following a 24-h treatment with sodium butyrate and 46-fold in cells treated with trichostatin A, a pure histone deacetylase inhibitor. Deletion analysis of the IGFBP-3 promoter identified key sodium butyrate-responsive element(s) to a 45-bp region containing consensus binding sites for Sp1 and activating protein-2. Sp1 binding to the Sp1 site and Sp3 to the activating protein-2/GA-box played a functional role in sodium butyrate’s activation of the IGFBP-3 promoter, however, with no change in binding direct sodium butyrate regulation was attributed to cofactors. The histone acetyltransferase p300 and histone deacetylase-1 were identified in multiprotein complexes containing DNA bound Sp1 and Sp3, with p300 accumulating following sodium butyrate treatment. Taken together, these data suggest that sodium butyrate increases IGFBP-3 expression by activating the IGFBP-3 promoter via an Sp1/Sp3 multiprotein complex, a mechanism that may be important for other key regulators of IGFBP-3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF FAMILY is a critical modulator of cellular growth, differentiation, transformation, and apoptosis in most tissue (1). Both in the circulation and tissues, IGF-I and -II are regulated by a family of six high affinity IGF-binding proteins (IGFBP-1 to -6). These IGFBPs serve to extend the half-life as well as transport and modulate the biological actions of IGFs on target cells (reviewed by Ref. 2). Certain members of the IGFBP family, IGFBP-1, -3, and -5, have also been shown to possess biological actions independent of their ability to modulate IGF bioactivity, thus diversifying the biological role of the IGF family (3, 4, 5).

IGFBP-3 is the major circulating IGFBP, binding to >75% of serum IGFs in a complex with the acid-labile subunit (6). IGFBP-3 is synthesized by the liver and released into the circulation, where it is postulated to be the major transporter of the IGF ligands as well as protecting them from degradation (7). Besides its hepatic synthesis, many tissues synthesize IGFBP-3, including the mammary tissue (8). It has been shown that IGFBP-3 both potentiates IGF actions (9, 10), as well as functions as a cell growth inhibitor and/or promoter of apoptosis (4, 11). Evidence has been provided that IGFBP-3 cell growth inhibition/apoptotic effects may occur by sequestering and inhibiting IGF binding to its cognate receptor (12). Alternatively, this also occurs in an IGF-independent fashion. Several mechanisms have been proposed, and these include the direct interaction of IGFBP-3 with a cell-surface associated protein (4, 11), its interaction with the RXR-{alpha} (13), IGFBP-3 binding to TGF-ß type V receptor (14), or IGFBP-3 translocation to the nucleus via the importin ß subunit (15).

The significance of IGFBP-3’s role in modulating cellular functions is further supported by evidence that not only is IGFBP-3 expression regulated by specific growth promoters and inhibitors, but IGFBP-3, in turn, mediates their mitogenic or growth inhibitory effects (16, 17, 18). Agents that inhibit breast cancer cell proliferation in vitro via IGFBP-3 mechanisms include TGF-ß (16, 17), RA (17) vitamin D (19), TNF-{alpha} (20) and antiestrogen compounds such as tamoxifen and ICI 182,780 (21, 22). These factors, as well as p53, have been reported to increase the synthesis and secretion of IGFBP-3 (17, 19, 20, 21, 23). Additionally, our laboratory has recently demonstrated that sodium butyrate (NaB) and trichostatin A (TSA), antiproliferative agents of breast cells in vitro, also up-regulate IGFBP-3 synthesis and secretion up to 13-fold before the induction of growth arrest and apoptosis (24). Moreover, a recent investigation also identified TSA increases IGFBP-3 expression in Hep3B cells, an hepatocellular carcinoma cell line (25).

Of the many agents that up-regulate IGFBP-3, the molecular mechanism by which they achieve this regulation is unknown. Forskolin and IGF-I, two proliferative agents, have been shown to stimulate IGFBP-3 synthesis in bovine mammary epithelial cells up to 8- and 15-fold, respectively (26). This stimulation was found to be associated with moderate increases in IGFBP-3 promoter activity. In contrast, p53 is the only antiproliferative agent that has been shown to up-regulate IGFBP-3 synthesis in vitro via direct protein/gene interaction (23). The purpose of the present study was to investigate the molecular regulation of IGFBP-3 by elucidating the mechanism(s) by which NaB increased the synthesis of IGFBP-3 in breast cancer cells, a mechanism which may be important for other key regulators of IGFBP-3 synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The human breast cancer cell lines MCF-7 and Hs578T, as well as the osteosarcoma cell line SaOS-2 and the Drosophila Schneider SL 2 cell line, were purchased from American Type Tissue Collection (ATCC, Manassas, VA). MCF-7, Hs578T and SaOS-2 cells were cultured in DMEM (Life Technologies, Inc., Rockville, MD), supplemented with 4.5 g/liter glucose, 110 mg/liter sodium pyruvate and 10% FBS (Life Technologies, Inc.). Cells were maintained at 37 C in a humidified atmosphere of 5% CO2. Drosophila Schneider SL 2 cells were cultured at room temperature (RT) in Schneider’s Drosophila medium (Life Technologies, Inc.) supplemented with 10% FBS.

Plasmid preparation
A 1.9-kb IGFBP-3 promoter (-1936 to -64; 27) was subcloned to the BglII site of the luciferase reporter vector pGL2-Basic (Promega Corp., Madison, WI) to create pGL2-IGFBP-3. The nomenclature adopted for the following luciferase expression plasmids refers to the number of nucleotides removed from the 5 prime (5') end of pGL2-IGFBP-3 (see Fig. 3Go). To generate pGL2-{Delta}650, pGL2-IGFBP-3 was digested with MluI and SacII and blunt ligated to the SacII site of pGL2-Basic. pGL2-{Delta}1100 and pGL2-{Delta}1600 were prepared by digesting with SspI and BglII or AccI and BglII, respectively, and ligating into the SmaI/BglII of pGL2-Basic. pGL2-{Delta}1675 were created by digesting pGL2-{Delta}1600 with SacI and subcloning to the SacI site of pGL2-Basic. pGL2-{Delta}1755 was prepared by using an oligo from -206 to -64 of the IGFBP-3 promoter, which included SacI and HindIII restriction enzyme sites either end. This oligo was subcloned into SacI and BglII sites of pGL2-Basic. The plasmid pGL2-{Delta}1708 was generated by digesting with ApaI and BglII and ligating into the SmaI/BglII sites of pGL2-Basic. pGL2-{Delta}1795 was prepared by digesting pGL2-{Delta}1675 with SmaI and religating. pGL2-Sp1/GC-rich and pGL2-activating protein-2 (AP2)/GA-box mutants were generated using site-directed mutagenesis as described (28). In brief, pGL2-{Delta}1708 was used as the single stranded DNA template and mutant oligos 5'-GTGGGGGTTGGGAGGGGGTTTTGTCGGCGCAGGCAGGGCT-3' and 5'-GCGCCCAGGAGTGGGGGTTGGTTTTGGGGCGGGTCGGCGCAGGCAC-3' were used to generate pGL2-Sp1/GC-rich and pGL2-AP2/GA-box mutant expression plasmids respectively. pPacSp1 and pPacSp3 expression plasmids were kind gifts from Dr. Charles T. Roberts, Jr. (Oregon Health Sciences University, Portland, OR).



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  		Figure 3. A key NaB-responsive element (NaB-RE) is located to a 45-bp region in IGFBP-3 promoter from position -206 to -251. The estrogen-responsive MCF-7, estrogen-nonresponsive Hs578T breast cancer cells were transiently transfected with pGL2-IGFBP-3, -{Delta}650, -{Delta}1100, -{Delta}1600, -{Delta}1675, -{Delta}1708, -{Delta}1755, and -{Delta}1795 luciferase reporter plasmids. Luciferase activity was measured following incubations for 24 h with or without 5 mM NaB. Data are shown as means of fold-induction relative to untreated transfected cells for four separate experiments (bars, SEM). *, P < 0.05.

 
Transient transfection
MCF-7, Hs578T, and SaOS-2 cells were transiently transfected using Fugene6 transfection reagent according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Each cell line was seeded at a density of 1 x 105 cells/well in 12-well plates. At 50–60% confluency, cells were transfected with 0.5 µg/well of reporter plasmid DNA in serum-containing medium. Drosophila Schneider SL 2 cells were plated at 1 x 106 cells/well in 12-well plates. The following day, SL 2 cells were transiently transfected with 0.5 µg/well of reporter plasmid DNA in serum-containing medium using the calcium phosphate transfection system (Life Technologies, Inc.). Twenty-four hours post transfection, cells were washed twice with serum-free medium (SFM) and treated with either 5 mM NaB (Sigma, St. Louis, MO), 100 nM TSA (Sigma), or controls (1x PBS or ethanol) in SFM for an additional 24 h. Cell lysates were then collected for luciferase assays. Transfections were performed in duplicate and experiments were performed at minimum in triplicate.

Luciferase assay
Luciferase activities of cell lysates were measured according to the manufacturer’s instructions (Promega Corp.). Luciferase activities were normalized for total protein determined using the Bradford Assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Isolation of nuclear and cytoplasmic proteins
Nuclear and cytoplasmic extracts from MCF-7 and Hs578T cells treated with 5 mM NaB or 1x PBS in SFM for 24 h, were prepared as previously described (29), with modifications of the procedure. In brief, cells were scraped and incubated in 10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; and 0.5 mM dithiothreitol (DTT) for 15 min on ice. Cell membranes were disrupted by repeatedly plunging through a 22-gauge blunt needle. Following a 20 sec centrifugation at 4 C, the supernatant/cytoplasmic extract (CE) was removed and the nuclei were resuspended in 20 mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM DTT; and 0.5 mM phenylmethylsulfonyl fluoride. The nuclei were incubated with stirring at 4 C for 30 min and then centrifuged at 14,000 rpm for 15 min at 4 C. The supernatant was recovered as nuclear extract (NE) and was dialyzed against 20 mM HEPES, pH 7.9; 20% glycerol; 0.1 M KCl; 0.2 mM EDTA; 0.5 mM phenylmethylsulfonyl fluoride; and 0.5 mM DTT at 4 C overnight (O/N). Protein concentrations were determined using the Bradford assay.

Western immunoblot (WIB)
For the detection of Sp1, Sp3, and AP2, 15 µg of nuclear and cytoplasmic proteins were size-fractionated on a 12.5% SDS-PAGE gel under reducing conditions. Proteins were electrotransferred onto immunoblot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.) and blocked with 5% nonfat dry milk/Tris-buffered saline-Tween (20 mM Tris-Cl, pH 7.6; 150 mM NaCl; 0.1% Tween-20) for 1 h at RT. Membranes were incubated with primary antibodies (polyclonal anti-Sp1, -Sp3, and -AP2, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) O/N at 4 C and followed with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc., Piscataway, NJ) for 1 h at RT. Immunoreactive proteins were detected using enhanced chemiluminescence (NEN Life Science Products, Boston, MA) and autoradiography (Kodak X-Omat Blue XB-1, Eastman Kodak Co., Rochester, NY).

EMSA and supershift assays
Five prime fluorescein (FITC) end-labeled and unlabeled sense and antisense oligonucleotides from -234 to -214 within the IGFBP-3 promoter, were custom-made by Life Technologies, Inc. Five prime FITC end-labeled sense and antisense oligonucleotides from -234 to -214 mutating either the Sp1/GC-rich site (5'-GCCGACAAAACCCCCTCCCAA) or AP2/GA-box (5'-GCCGACCCGCCCCAAAACCAA), were also custom-made by Life Technologies, Inc. The oligonucleotides were annealed and gel purified by resolving on a 12% PAGE gel. The FITC-labeled double-stranded (ds) oligos were excised, incubated at -20 C in water O/N and centrifuged through a Spin-X column (Costar, Corning, NY) at 14,000 rpm for 15 min at RT. EMSAs were performed by preincubating 7.5 µg of NEs in 60 mM KCl; 25 mM HEPES, pH 7.6; 5 mM MgCl2; 7.5% glycerol; 0.1 mM EDTA; and 1 mM DTT on ice for 30 min. Ten to 20 nM FITC-labeled ds oligo was added to the preincubation and incubated on ice for a further 30 min. DNA/protein complexes were resolved on a 6% PAGE gel and analyzed with the Quantity One software on a Molecular Imager FX (Bio-Rad Laboratories, Inc.). Competition EMSAs and supershift assays were performed by incorporating increasing concentrations (50–500 nM) of unlabeled ds oligo or 0.2 µg of antibody respectively, in the preincubation step of the assay.

Immunoprecipitation (IP)
IPs were performed by incubating 25 µg of NEs with 0.3 µg of the appropriate IP antibody in S. Aureus Cowan I (SACI) buffer (10 mM Tris, pH 8.0; 150 mM NaCl; and 0.5% NP-40) O/N at 4 C with gentle mixing. After the addition of protein A Sepharose (Amersham Pharmacia Biotech) or protein G agarose (Sigma), the incubations were left for an additional 24 h. Precipitates were then washed four times with SAC I buffer and resolved on a 7.5% SDS-PAGE gel under reducing conditions. Coimmunoprecipitated proteins were examined by WIB using polyclonal anti-Sp1 and -Sp3 antibodies (Santa Cruz), a monoclonal anti-histone deacetylase (HDAC)I (Santa Cruz), a monoclonal anti-p300 (Upstate Biotechnology, Inc., Lake Placid, NY) and a polyclonal anti-ZBP-89 antibody (Transduction Laboratories, Lexington, KY).

DNA affinity precipitation assay (DAPA)
5' biotin end-labeled and unlabeled sense and antisense oligonucleotides from -234 to -214 within the IGFBP-3 promoter were custom-made by Life Technologies, Inc. The oligos were annealed and gel purified by resolving on a 12% PAGE gel. Twenty micrograms of NE was preincubated on ice for 30 min in DAPA buffer (60 mM KCl; 25 mM HEPES, pH 7.6; 5 mM MgCl2; 7.5% glycerol; 0.1 mM EDTA; 1 mM DTT; and 0.25% Triton X-100). One micromolar of biotin-labeled ds oligo was added to the preincubation and further incubated on ice for 45 min. The DNA/protein complexes were then incubated with neutravidin coated agarose beads (Pierce Chemical Co., Rockford, IL) preequilibrated in DAPA buffer at 4 C for 1 h with gentle agitation. Complexing proteins were resolved on a 7.5% SDS-PAGE gel and examined by WIB with polyclonal anti-Sp1 and -Sp3 antibodies, monoclonal anti-p300, monoclonal anti-HDACI, and a polyclonal anti-ZBP-89 antibody.

Statistical analysis
Data are expressed as mean ± SEM. Differences between two groups were evaluated using an unpaired t test, where P < 0.05 was used to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NaB and TSA stimulate IGFBP-3 promoter activity
To address the molecular mechanism of the NaB activation of IGFBP-3 expression in breast cell lines (24), we first investigated whether NaB and TSA could activate the promoter of the IGFBP-3 gene under the same conditions. The estrogen-responsive MCF-7 and estrogen-nonresponsive Hs578T breast cancer cell lines, as well as the p53 negative osteosarcoma cell line SaOS-2, were transiently transfected with the pGL2-IGFBP-3 luciferase expression vector containing 1.9 kb of the human IGFBP-3 promoter or pGL2 alone. Transfected cells were incubated in the presence or absence of 5 mM NaB for 24 h. Transfected MCF-7 cells were also treated with or without 100 nM TSA for 24 h. Although both NaB and TSA failed to induce cells transfected with pGL2 (data not shown), NaB induced IGFBP-3 promoter activity 30- to 70-fold in the cell lines examined when compared with untreated transfected controls, whereas TSA induced the IGFBP-3 promoter 46-fold in MCF-7 cells (Fig. 1Go). These results suggest that both NaB and TSA treatment induce the expression of IGFBP-3 through IGFBP-3 promoter interactions.



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  		Figure 1. NaB and TSA stimulate IGFBP-3 promoter activity. The estrogen-responsive MCF-7, estrogen-nonresponsive Hs578T breast cancer cells and the p53 negative SaOS-2 osteosarcoma cell lines were transiently transfected with pGL2-IGFBP-3 luciferase reporter plasmid. Luciferase activity was measured following incubations for 24 h with or without 5 mM NaB, or with and without 100 nM TSA for MCF-7 transfected cells. Data are shown as means of fold-induction relative to untreated transfected cells from four separate experiments (bars, SEM). *, P < 0.05.

 
Analysis of NaB-responsive elements in IGFBP-3 promoter
The 1.9-kb human IGFBP-3 promoter includes a cluster of 11 p53 consensus binding sites for potential transcriptional regulation by p53, and five consensus Yin and Yang-1 (YY1) sites. In addition, it contains a cluster comprised of an Sp1/GC-rich site, two consensus independent AP2 sites overlapping a GA-box and a putative p300 DNA-binding site, all 5' to a TATA box (Fig. 2Go). The osteosarcoma cell line SaOS-2 does not express endogenous p53, yet the transiently transfected pGL2-IGFBP-3 expression vector was induced 68-fold in the presence of NaB for 24 h, suggesting that p53 is not required for the transcriptional activation of IGFBP-3 by NaB (Fig. 1Go).



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  		Figure 2. Schematic diagram depicting the human IGFBP-3 promoter. The approximate 1.9-kb portion of the human IGFBP-3 promoter includes a cluster of 11 p53 consensus binding sites and 5 consensus Yin and Yang-1 (YY1) sites. It also has a sequence cluster approximately 100 bp 5' to a TATA box. This cluster is comprised of a GC-rich/Sp1 site and 2 consensus AP2 sites with the more 3' AP2 site overlapping a GA-box. Immediately 3' to the GA-box is a p300 DNA binding site. Numbering is relative to the translation start site designated +1.

 
To determine the NaB responsive region(s) (NaB-RE) within the IGFBP-3 promoter, a series of 5' deletion constructs of pGL2-IGFBP-3 were generated (see Materials and Methods). The resulting expression vectors were transiently transfected into MCF-7 and Hs578T cells and the luciferase activity was analyzed following treatment with or without 5 mM NaB for 24 h. In both MCF-7 and Hs578T cells the pGL2-{Delta}650, -{Delta}1100, -{Delta}1600, -{Delta}1675, and -{Delta}1708 promoter luciferase expression vectors, which progressively lost the YY1 consensus sites and the p53 clusters through 5' stepwise deletions, were consistently activated with NaB treatment (Fig. 3Go). These data provide further evidence to show that the cluster of p53 binding sites, as well as the five YY1 binding sites, are not essential for NaB activation of the IGFBP-3 promoter. In contrast, transient transfection with the pGL2-{Delta}1755 and -{Delta}1795 luciferase expression vectors resulted in up to a 10-fold loss of IGFBP-3 promoter activity when compared with the 1.9-kb IGFBP-3 promoter (Fig. 3Go). It was noted that the loss of IGFBP-3 promoter activity was more significant for Hs578T than MCF-7 cells, suggesting that there may be additional regulation that is cell-type specific or related to the estrogen sensitivity of these cells. We also observed a 5-fold loss of promoter activity in MCF-7 cells transfected with pGL2-{Delta}1755, when compared with pGL2-{Delta}1708 transfected cells following a 24 h treatment with 100 nM TSA (data not shown). Overall, the significant loss in promoter activity between the pGL2-{Delta}1755 and -{Delta}1795 expression vectors suggests that a primary NaB-RE is located within a 45-bp region between -206 and -251 upstream of the transcription start site. Activation by NaB could also involve the putative p300 DNA-binding site that is disrupted by the generation of the pGL2-{Delta}1755 expression vector.

Identification of transcription factors that bind the major NaB-RE within the IGFBP-3 promoter
The 45-bp NaB-RE was analyzed for consensus sequences of known transcription factors. Within this region the Sp1/GC-rich site, two AP2 binding sites and an overlapping GA-box were identified (Fig. 4AGo). To address whether associated transcription factors could potentially participate in the NaB induction of the IGFBP-3 promoter, we analyzed their cellular localization and levels of expression following treatment with 5 mM NaB for 24 h by WIB analyses of both MCF-7 and Hs578T cytoplasmic and NEs (Fig. 4BGo). The transcription factor Sp1 and its close relative Sp3, which is reported to bind GC-rich and GA-box sequences and to collaborate with Sp1 in the activation of a number of promoters (reviewed by Ref. 30), were present in the nuclear compartment of both MCF-7 and Hs578T cells. The overall levels of these transcription factors were reduced in Hs578T cells, when compared with MCF-7 cells. However, their expression levels were generally unchanged in both cell lines with a small decrease in Sp1 expression observed in Hs578T cells following NaB treatment. Interestingly, AP2 was not detectable in either the nuclear or cytoplasmic compartment of MCF-7 cells and was mainly present in the cytoplasmic compartment of Hs578T cells, therefore suggesting that AP2 may not have a functional role in NaB activation of the IGFBP-3 promoter.



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  		Figure 4. The transcription factors Sp1 and Sp3, but not AP2, are located in the nucleus with no alteration in the pattern of expression following NaB treatment. A, Schematic showing the 45-bp region from -251 to -206 of the human IGFBP-3 promoter that contains a potential major NaB-RE. The sequence contains a GC-rich putative Sp1 site (boxed), two adjacent AP2 sequences (bracketed) with the more 3' AP2 site overlapping a GA-box (underlined). Portion of a putative p300 binding domain is represented (dotted box). Asterisks delineate the 21 mer oligonucleotide probe used in EMSA and DAPA analyses. B, MCF-7 and Hs578T breast cancer cells were each treated with 5 mM NaB (+) or 1x PBS (-) for 24 h upon which nuclear (NE) and cytoplasmic (CE) proteins were isolated. A total of 20 µg of protein from each cellular compartment was run on a 12.5% PAGE gel under reducing conditions and analyzed by WIB with polyclonal anti-Sp1, -AP2, and -Sp3 antibodies. Specific proteins are marked (*). Results are representative of three experiments.

 
To establish whether Sp1 and/or Sp3 are able to bind the major NaB-RE containing the Sp1/GC-rich and AP2/GA-box sequences and whether there is any altered pattern of binding, EMSAs were performed. A wild-type (WT) 21-mer oligonucleotide from -214 to -234 of the IGFBP-3 promoter was incubated with NEs isolated from MCF-7 and Hs578T cells grown in the presence or absence of 5 mM NaB for 24 h (Fig. 5AGo). As shown in the representative experiment performed in MCF-7 cells, two major DNA/protein complexes were observed in both cell lines, and no alteration in this pattern could be observed with NaB treatment (Fig. 5BGo). Specificity of binding was verified as each complex was successfully competed away with increasing concentrations of unlabelled oligonucletides (data not shown). To establish whether the identified DNA/protein complexes represented the binding of Sp1 and Sp3, supershift experiments were performed using polyclonal antibodies specific for Sp1 and Sp3. As shown in the representative experiment in MCF-7 cells, NEs preincubated with an Sp1 antibody resulted in a supershift of the upper complex in both cell lines, whereas with an Sp3 antibody the upper complex was diminished, whereas the lower complex was lost (Fig. 5BGo). Preincubation with a combination of both antibodies confirmed these results (Fig. 5BGo). Supershift experiments with an AP2 antibody had no effect on either of the complexes (data not shown). To establish the specificity of binding, WT 21-mer oligonucleotides with either the Sp1/GC-rich site or AP2/GA-box mutated were preincubated with NEs from MCF-7 cells treated in the absence of NaB for 24 h (Fig. 5AGo). In the representative gel, results show clearly that mutation of the Sp1/GC-rich site causes a significant but not complete loss of the upper complex, whereas the lower complex is lost (Fig. 5CGo). Mutation of the AP2/GA-box causes a decrease in the intensity of the upper complex with no loss of the lower complex (Fig. 5CGo). In combination with supershift analyses, these results suggest that both Sp1 and Sp3 bind the Sp1/GC-rich site, whereas Sp3 binds the AP2-GA-box. Overall, these results show that Sp1 and Sp3 bind DNA containing the consensus sequences within the proposed NaB-RE, but with no change in their binding pattern with NaB treatment no functional relevance could be proposed.



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  		Figure 5. The transcription factors Sp1 and Sp3 bind within the putative NaB-RE with no alteration in their pattern of expression following NaB treatment. A, Schematic showing WT FITC-labeled 21-mer oligonucleotide from -243 to -214 of the IGFBP-3 promoter containing the consensus binding sequences for Sp1 and AP2/GA-box as well as FITC-labeled Sp1/GC-rich and AP2/GA-box mutant probes used in EMSAs. The GA-box is underlined and mutations are highlighted in bold. B, EMSAs were performed using NEs isolated from MCF-7 cells treated with either 5 mM NaB (+) or 1x PBS (-) for 24 h. The WT 21-mer FITC-labeled oligonucleotide (P) was incubated with the NEs. Polyclonal anti-Sp1 ({alpha}Sp1) and anti-Sp3 ({alpha}Sp3) antibodies were used in supershift experiments as indicated. C, EMSAs were performed using NEs isolated from MCF-7 cells not treated with 5 mM NaB (-) for 24 h. The WT 21-mer FITC-labeled oligonucleotide (P), Sp1/GC-rich (P-Sp1m) or AP2/GA-box (P-AP2m) mutant probes were incubated with NE. Identification of the DNA/protein complexes and supershifted (SS) complexes are shown on the right of each panel. Results are representative of three experiments for each cell line.

 
Identification that Sp1 and Sp3 have a functional role in the NaB-activation of the IGFBP-3 promoter
To identify whether Sp1 and Sp3 have a functional role in the NaB activation of the IGFBP-3 promoter, the pGL2-{Delta}1708 luciferase expression vector was mutated at the Sp1 site (pGL2-{Delta}1708-Sp1/AP2 mutant) and the AP2/GA-box (pGL2-{Delta}1708-AP2/GA-box mutant; Fig. 6AGo). The putative p300 DNA-binding site was not altered. Each expression vector was cotransfected with pPacSp1 or pPacSp3 expression plasmids in Drosophila SL 2 cells, a cell line that lacks mammalian Sp proteins and then treated in the presence or absence of 5 mM NaB for 24 h. It was observed that Sp1 was able to transactivate the pGL2-{Delta}1708 expression vector 28-fold, which was further enhanced to 139-fold in the presence of 5 mM NaB (Fig. 6BGo). Mutation of the Sp1 site significantly reduced the NaB activation of pGL2-{Delta}1708 in the presence of Sp1 to 13-fold; conversely, the AP2/GA-box mutant had no effect on the activation. As for Sp1, Sp3 was able to modestly transactivate the pGL2-{Delta}1708 expression vector 2.2-fold, which was further amplified to 18-fold in the presence of 5 mM NaB. In contrast to Sp1, the AP2/GA-box mutant reduced this effect to 4-fold, whereas the Sp1/GC-rich mutant did not appear to effect the transacting capabilities of Sp3. Overall, these results suggest that Sp1 and Sp3 are key transcriptional regulators for the NaB activation of the IGFBP-3 promoter through their binding to Sp1/GC-rich and AP2/GA-box binding sites, respectively.



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  		Figure 6. The transcription factors Sp1 and Sp3 have a functional role in the NaB-activation of the IGFBP-3 promoter. A, Schematic diagram depicting the wild-type and mutant pGL2-{Delta}1708 luciferase expression plasmids. The pGL2 -{Delta}1708 luciferase expression plasmid from position -228 to -225 containing the GC-rich/Sp1 site and overlapping 5' AP2 site was mutated to adenines to create the pGL2-Sp1/AP2 mutant expression plasmid. The 3' AP2 site and overlapping GA-box from position -221 to -218 were mutated to adenines to generate the pGL2-AP2/GA-box mutant. B, Drosophila Schneider SL 2 cells were transiently transfected with either pGL2-{Delta}1708, pGL2-Sp1/AP2 mutant or pGL2-AP2/GA-box mutant luciferase expression plasmids, along with either pPacSp1 or pPacSp3. Luciferase activity was measured following incubations for 24 h with or without 5 mM NaB. Data are shown as means of fold-induction relative to untreated transfected cells for four independent experiments (bars, SEM). *, P < 0.05.

 
Complexing proteins to Sp1 and Sp3 may regulate the NaB-activation of the IGFBP-3 promoter
Sp1 and Sp3 have a key functional role in the NaB activation of the IGFBP-3 promoter, but with no alteration in their binding following NaB treatment we proceeded to investigate whether this regulation could depend on associating proteins. This was addressed firstly by using IP studies to establish proteins that could complex with Sp1 and Sp3 within the nucleus and secondly with a DAPA to establish protein complexes present on the proposed NaB-RE. Nuclear extracts isolated from MCF-7 and Hs578T cells grown in the presence or absence of 5 mM NaB were immunoprecipitated using polyclonal antibodies specific to Sp1 and Sp3 (Fig. 7Go). As expected, Sp1 and Sp3 levels did not change with NaB treatment. However, we identified that Sp1 and Sp3 were able to complex, a complex that disassociated following NaB treatment. As NaB is proposed to inhibit HDAC activity resulting in the hyperacetylation of core histones, we then examined whether p300, a key high molecular weight protein with histone acetylase activity, and HDAC1, a key protein with deacetylase activity, were able to complex to Sp1 and Sp3. We also analyzed the participation of the transcription factor ZBP-89, a protein shown recently to collaborate with Sp1 and p300 in the transactivation of the p21Waf1/Cip1 promoter (31). Supershift analyses using antibodies specific to p300 and HDAC1 did not alter the pattern of expression for the two major DNA/protein complexes identified (data not shown). IP experiments demonstrated that p300 could associate with both Sp1 and Sp3, associations that were significantly decreased 38.5 ± 2.6% for Sp1 and 98.8 ± 1.2% for Sp3 following treatment with NaB (Fig. 7Go). In contrast, both HDAC1 (Fig. 7Go) and ZBP-89 (data not shown) could not be detected in either the Sp1 or Sp3 IP experiments. A reverse IP experiment, using a monoclonal antibody to HDAC1, showed that HDAC1 was able to associate with both Sp1 and Sp3, with no alteration in the binding pattern with NaB treatment (Fig. 7Go). We did not rule out the participation of HDAC1 in this process because NaB is an inhibitor of HDAC activity and may not influence binding. Overall, these results suggest the existence of protein complexes in the nucleus of breast cancer cells between Sp1, Sp3, p300, and HDAC1, complexes that in the case of Sp1, Sp3, and p300 disassociate in the presence of NaB.



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  		Figure 7. The cofactor p300 complexes with Sp1 and Sp3 in the nucleus, complexes that disassociate with NaB-treatment of breast cancer cells. Co-IPs were performed incubating polyclonal antibodies specific to Sp1 ({alpha}Sp1), Sp3 ({alpha}Sp3), or a monoclonal HDAC1 ({alpha}HDAC1) antibody with 25 µg of nuclear proteins from MCF-7 cells treated with 5 mM NaB (+) or without (-) for 24 h. The straight NE, and immunoprecipitated samples: NE, antibodies alone (ab), and antibodies plus NE with and without NaB treatment, were resolved on a 7.5% PAGE gel under reducing conditions. Complexes were analyzed by WIB with polyclonal anti-Sp1 and -Sp3 antibodies and monoclonal anti-p300 and -HDAC1 antibodies, as indicated. Molecular weight markers are indicated on the left. Results are representative of three experiments for MCF-7 and Hs578T cells.

 
To assess whether p300, HDAC1, and ZBP-89 could complex with Sp1 and Sp3 on the IGFBP-3 promoter, we performed a DAPA. A 5'-biotin labeled WT 21-mer oligonucleotide from -214 to -234 of the IGFBP-3 promoter described earlier was incubated with NEs from MCF-7 and Hs578T cells grown in the presence or absence of 5 mM NaB. After separating the DNA bound protein complexes on a 7.5% SDS-PAGE gel under reducing conditions, complexing proteins were then analyzed by WIB. As expected, there was no alteration in Sp1 or Sp3 binding to the 21-mer oligonucleotide whether cells were treated with or without 5 mM NaB (Fig. 8Go). Interestingly, the cofactor p300 associated in the presence of IGFBP-3 DNA, an association that increased 40.3 ± 3.2% when MCF-7 cells were treated with NaB. HDAC1 was also able to bind in the presence of DNA, but there was no alteration of HDAC1 binding with NaB treatment, whereas the binding of ZBP-89 could not be detected (data not shown). These results suggest that p300 and HDAC1 may participate with Sp1 and Sp3 in a multiprotein complex bound to the IGFBP-3 promoter, with p300 binding increasing following NaB treatment.



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  		Figure 8. The cofactor p300 complexes to the DNA bound Sp1 and Sp3 and may regulate the NaB-activation of the IGFBP-3 promoter. DAPAs were performed using a 21-mer biotin end-labeled oligonucleotide containing the sequence of the proposed main NaB responsive element from -234 to -214 of the IGFBP-3 promoter. A total of 25 µg of NEs from MCF-7 breast cancer cells treated with either 5 mM NaB (+) or 1x PBS (-) for 24 h, were incubated with the biotinylated oligonucleotide (DNA). DNA/protein complexes, positive control NE, the biotin-labeled oligonucleotide (DNA) and NE taken though the procedure without DNA (NE), were resolved on a 7.5% PAGE gel under reducing conditions. The presence of Sp1, Sp3, p300, and HDAC1 were analyzed by WIB using polyclonal and monoclonal antibodies specific to these proteins as described previously. Molecular weight markers are indicated on the left. Results are representative of three experiments for MCF-7 and two Hs578T-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBP-3 is a protein with clearly divergent functions. It both potentiates and inhibits cell proliferation and does so through IGF-dependent and -independent mechanisms in different cell systems (4, 9, 10, 11, 12). IGFBP-3 is also tightly regulated by a large variety of cell growth stimulators and inhibitors where in turn IGFBP-3 mediates their mitogenic or antiproliferative effects (16, 17, 18, 21, 22). The molecular mechanism(s) by which these factors regulate IGFBP-3 is largely unknown. Therefore, the purpose of the present investigation was to explore the molecular regulation of IGFBP-3 synthesis by NaB in breast cancer cells. It has shown previously that inhibitors of HDAC activity, NaB and TSA, increase the synthesis and secretion of IGFBP-3 in breast cell lines and an hepatocellular carcinoma cell line (24, 25) with the NaB-induced up-regulation occurring before the induction of growth arrest and apoptosis (24). Here, we show evidence that this up-regulation involves the strong induction of the IGFBP-3 promoter, regulated at a key NaB-RE. We will also show that this activation may involve multiprotein complexing of the DNA bound transcription factors Sp1 and Sp3, with HDAC1 and the acetyltransferase p300.

NaB is a nontoxic, four-carbon fatty acid synthesized naturally by the anaerobic fermentation of carbohydrates in the colon (32). Physiological concentrations of butyrate induce potent cellular effects in vitro in a range of cell lines, including growth arrest and cellular differentiation (33, 34, 35). The molecular mechanism(s) of this action are not clear. Like TSA, NaB induces a number of nuclear changes that include the hyperacetylation of core histones through HDAC inhibition (36), the methylation of DNA (37) and the dephosphorylation of the retinoblastoma (Rb) protein (38). Such alterations can lead to the selective regulation of gene transcription (39, 40), as observed for IGFBP-3 (24). The NaB-regulated increase of IGFBP-3 synthesis before growth arrest and apoptosis suggests that the regulation of IGFBP-3 expression could be another mechanism for NaB action, rather than an effect of treatment. This has also been suggested for other cell cycle-specific genes regulated by NaB and TSA including p21Waf1/Cip1, a cyclin cdk-inhibitor (41), p16INK4 (38), p27Kip1 (42), Rb protein (43), cyclin-D1 (44), Bcl-2 and Bax (45, 46). It is apparent that the mechanism of action for NaB and TSA is multifactorial and may also include the regulation of IGFBP-3 synthesis and secretion.

As described, NaB and TSA are HDAC inhibitors. HDAC inhibition and the hyperacetylation of core histones by histine acetylases can lead to the decondensation of local chromatin (39, 40). In turn, this allows access for transcription factors and the RNA polymerase complex, thus resulting in the activation of gene transcription (39, 40). The NaB induced up-regulation of IGFBP-3 synthesis in breast cell lines was found to be correlated to an increase in the hyperacetylation of the core histones H3 and H4 (24). Based on these observations, we investigated whether this up-regulation occurred through interactions with the IGFBP-3 promoter. Both the NaB and TSA induction of IGFBP-3 expression involved the strong activation of the IGFBP-3 promoter. Butyrate/promoter interactions have also been identified as being responsible for the regulation of the cyclin cdk-inhibitor, p21Waf1/Cip1 (47) and cyclin-D1 genes (44). In the case of IGFBP-3, regulation via promoter interactions has only been proposed for IGF-I and forskolin, although the evidence for this type of regulation is modest, a 1.57- and 1.59-fold induction, respectively, and 2.25-fold when IGF-I and forskolin are combined (26).

The first 1.9 kb of the human IGFBP-3 promoter contains a cluster of eleven-p53 consensus binding sites for potential regulation by p53. When activated, p53 stimulates the expression of a broad range of genes including IGFBP-3, whose protein products are associated to cell cycle progression, DNA repair and apoptosis (23). The cluster of p53-binding sites within the IGFBP-3 promoter was excluded as the NaB-RE as SaOS-2 cells, which do not express endogenous p53, were able to significantly induce the IGFBP-3 promoter following NaB treatment. To assess the involvement of identified consensus sequences within the IGFBP-3 promoter, truncated IGFBP-3 promoters were assessed for their ability to be induced by NaB in the breast cancer cell lines. A significant loss in IGFBP-3 promoter activity localized a key NaB-RE and potentially a TSA-responsive element to a 45-bp region that contained an Sp1/GC-rich and adjacent AP2 sites with the overlapping a GA-box. NaB induction was not completely abolished by the loss of this responsive region in MCF-7 cells, therefore suggesting that NaB activation may involve minor cell specific or estrogen responsive regulation at more 3' DNA binding sites.

GC-rich sequences have been implicated in the regulation of a number of promoters by external stimuli. The cell cycle protein p21Waf1/Cip1 is regulated by NaB (47), TSA (48), nerve growth factor (NGF; 49) and TGF-ß (50) via GC-rich sequences within its promoter. Similarly, the closely related cell cycle protein p15INK4B is regulated by TGF-ß at a GC-rich sequence within its promoter (51). The function of GC-rich sequences usually involves the ubiquitously expressed Sp family of proteins, which are DNA-bound transcription factors that bind GC-rich and related GA- and GT-boxes (reviewed by Ref. 30) and regulate several constitutively active or inducible genes. We established that Sp1 and Sp3 bind to the Sp1/GC-rich and AP2/GA-box within the putative NaB-RE of the IGFBP-3 promoter, with each having a functional role in the NaB activation of the IGFBP-3 promoter. In the presence of NaB, Sp1 more potently transactivated the IGFBP-3 promoter, whereas Sp3 appeared to independently transactivate the IGFBP-3 promoter, but to levels that were significantly lower than Sp1, confirming studies in other model systems (52). It was also observed that mutants of the consensus sequences did not completely abolish NaB induction in the presence of the Sp proteins, thus suggesting a minor role for more 3' DNA binding sites. The previously proposed intrinsic DNA binding by p300 was largely ruled out as the major NaB-RE, due to the transacting effects of Sp1 and Sp3 in the Drosophila SL2 cells (53). However, it cannot be ruled out that intrinsic p300 binding plays a secondary role, one that requires further investigation.

The Sp-family of proteins, in particular Sp1, is necessary for the transcription of TATA-less genes and genes that possess a TATA-box, collaborating with other proteins to provide gene-specific regulation (reviewed by Ref. 30). Both Sp1 and Sp3 have been shown to functionally interact with Rb to superactivate gene transcription (54). Likewise, TGF-ß regulation of the cell cycle inhibitor p21Waf1/Cip1 in hepatic cells was mediated through Sp1 interactions with Smad proteins (55). Cooperation between Sp1 and the transcriptional coactivators CBP/p300 have been shown to regulate NGF-, progesterone- and TSA-mediated induction of p21Waf1/Cip1 (56, 57, 58). The evidence that DNA bound Sp1 and Sp3 played an important role in the NaB up-regulation of IGFBP-3, without being directly regulated themselves, suggested that NaB regulation may depend on proteins that can complex and influence Sp1 and/or Sp3-mediated transcription. In the present investigation, cooperation between Sp1, Sp3, and the transcriptional coactivator p300 were identified within the nucleus; however, following NaB treatment there was a significant decrease in the presence of these associations, suggesting regulation of protein complexes by NaB. Interestingly, we identified that p300 specifically accumulated in complexes on the IGFBP-3 promoter following NaB treatment; inversely, HDAC1 was present but did not change with treatment.

The protein p300 is a mammalian histone acetyltransferase, whereas HDAC1 is a histone deacetyltransferase, proteins that are fundamental to transcriptional activation and suppression, respectively. p300 was originally cloned as an adenoviral-transforming protein E1A associated protein (59). Since its isolation, p300 has been identified as a coactivator for a number of transcriptional activators including p53, c-Jun, MyoD, and Sp1 (58, 60, 61, 62), whereas HDAC1 has been shown to collaborate with Sp1 and repress transcription through competitive means (63). With NaB treatment, p300 accumulation at the putative NaB-RE in the IGFBP-3 promoter was evident. p300 accumulation has also been observed for the p21Waf1/Cip1 promoter following NGF treatment (56). As proposed for this study, the progressive accumulation of p300 following NaB treatment may be the means for NaB regulation of IGFBP-3 expression. Functional studies are required to show p300 accumulation participates in the NaB induction of the IGFBP-3 promoter.

Transcriptional regulation is dependent on the fine equilibrium between histone acetylation and deacetylation (39). From the results obtained in the present investigation, a hypothetical model has been proposed for the NaB transcriptional regulation of IGFBP-3, a mechanism that may be important for other key regulators of IGFBP-3 expression (Fig. 9Go). Treatment with NaB leads to a significant induction of the IGFBP-3 promoter involving a putative NaB-RE upstream of the TATA box. Sp1 and Sp3 bind to the putative NaB-RE and participate in the NaB activation of the IGFBP-3 promoter, but there is no alteration in the pattern of their binding with treatment. Within the nuclear compartment the coactivator p300 both complexes with Sp1 and Sp3 either directly or via other unknown cofactors. In the presence of NaB, nuclear Sp1/Sp3/p300 complexes disassociate. HDAC1 and p300 participate in complex formation at the putative NaB-RE within the IGFBP-3 promoter but p300 accumulation itself increases following NaB treatment. It is, therefore, tempting to speculate that NaB activates the IGFBP-3 promoter by inhibiting HDAC1 activity, disassociating the nuclear Sp1/Sp3/p300 complexes and allowing p300 to complex with Sp1 and Sp3 bound to the IGFBP-3 promoter. This, subsequently, leads to histone acetylation and transcriptional activation of the IGFBP-3 promoter.



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  		Figure 9. Schematic diagram depicting the hypothetical up-regulation of the human IGFBP-3 promoter by NaB. Combining the results from this study we propose the following model for the transcriptional up-regulation of IGFBP-3 by NaB in breast cancer cells. Within the nucleus, numerous protein associations exist including p300 complexing with Sp1 and Sp3 either directly or via yet to be determined cofactors. In the presence of 5 mM NaB, these complexes disassociate releasing these proteins. Sp1 and Sp3 bind to consensus Sp1/GC-rich and GA-box sequences within the IGFBP-3 promoter. They also directly participate in the NaB activation of the IGFBP-3 promoter but there is no change in the pattern of their binding with NaB treatment. The cofactors HDAC1 and p300 participate directly or indirectly in the multiprotein complex formation at the NaB-RE on the IGFBP-3 promoter, but it is p300 accumulation that increases following NaB treatment. Overall this suggests that with NaB treatment, HDAC1 activity is inhibited and disassociated p300 is acquired for complex formation with Sp1 and Sp3 on the IGFBP-3 promoter, leading to histone acetylation and the activation of gene transcription.

 


   Acknowledgments
 
We would like to thank Dr. Julia Billiard for helpful advice with the EMSA studies and Drs. Peter Rotwein and Paolo Marzullo for critical manuscript reading.


   Footnotes
 
This study was supported by an American Cancer Society Grant RPG-99-103-01-TBE.

Abbreviations: AP2, Activating protein-2; CE, cytoplasmic extract; DAPA, DNA affinity precipitation assay; ds, double-stranded; DTT, dithiothreitol; HDAC, histone deacetylase; IGFBP, IGF binding protein; IP, immunoprecipitation; NaB, sodium butyrate; NE, nuclear extract; NGF, nerve growth factor; O/N, overnight; Rb, retinoblastoma; RT, room temperature; SAC I, S. Aureus Cowan I; SFM, serum-free medium; TSA, trichostatin A; WIB, Western immunoblot; WT, wild-type.

Received February 15, 2001.

Accepted for publication May 15, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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