J Biol Chem, Vol. 274, Issue 37, 26431-26438, September 10, 1999

Progesterone Stimulation of Human Insulin-like Growth Factor-binding Protein-5 Gene Transcription in Human Osteoblasts Is Mediated by a CACCC Sequence in the Proximal Promoter^*

Viroj Boonyaratanakornkit^§, Donna D. Strong^¶, Suburraman Mohan^**, David J. Baylink, Candice A. Beck, and Thomas A. Linkhart^§§¶¶

From the J. L. Pettis Veterans Affairs Medical Center and the Departments of  Biochemistry, ^¶ Microbiology and Molecular Genetics, ^§§ Pediatrics, ^** Physiology, and  Medicine, Loma Linda University, Loma Linda, California 92357 and the  University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulin-like growth factor-binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates insulin-like growth factor mitogenic stimulation in osteoblast cell cultures. Progesterone (PG) increased IGFBP-5 expression in normal human osteoblasts and increased IGFBP-5 transcription in U2 human osteosarcoma cells. We developed a chloramphenicol acetyltransferase reporter construct containing the human IGFBP-5 proximal promoter sequence, which includes TATA and CAAT boxes, and five putative PG response element half-sites. 10⁸ M PG increased promoter activity of this construct in U2 cells co-transfected with a PG receptor isoform A (PR_A) expression vector. Analysis of 5' deletion constructs indicates that PG transactivation of IGFBP-5 promoter activity does not require the PG response element half-sites but does require the region 162 to 124 containing two tandem CACCC box sequences. Mutation of the proximal CACCC box at 139 eliminated PG transactivation. Gel shift assays using a 162 to 124 DNA fragment, U2 cell nuclear extracts, and purified PR_A protein indicate that nuclear factors bind to a CACCC sequence at 139 and that PR_A alters the pattern of transcription factor interaction with the CACCC sequence. Using a luciferase reporter construct containing base pairs 252 to +24 of the IGFBP-5 promoter, we found that both PR_A and PR_B isoforms mediated PG stimulation of promoter activity. These results suggest that PG may stimulate IGFBP-5 gene transcription via a novel mechanism involving PR and CACCC-binding factors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulin-like growth factor-binding protein-5 (IGFBP-5)¹ belongs to a family of six structurally related proteins that are unrelated to the IGF receptors and that bind IGF-I and IGF-II with high affinity (1-3). IGFBPs modulate the mitogenic and metabolic activities of the IGFs in vitro, suggesting an important role in IGF physiology in vivo. Of the various IGFBPs, IGFBP-5 is unique in that it stimulates IGF-induced osteoblastic cell proliferation when added simultaneously with IGF I or II to serum-free osteoblast-like cell cultures (4-7). Recent evidence suggests that IGFBP-5 may stimulate cell proliferation by an IGF-independent mechanism involving IGFBP-5 specific cell surface binding sites (7, 8). In addition, IGFBP-5 binds with high affinity to hydroxyapatite and to various extracellular matrix proteins, properties that are consistent with a role for this binding protein in fixing IGFs to extracellular matrices including mineralized bone (7, 9, 10). IGFBP-5 may be a more potent enhancer of IGF activities when bound to extracellular matrix than in solution (10). Coding sequences and 5'-flanking regions of IGFBP-5 genes are highly conserved in human, rat, and mouse (4, 5, 11-13), and the mouse and human 5'-flanking regions have been found to impart basal promoter activity to reporter gene constructs in several cell types, including osteoblasts (12-16).

IGFBP-5 is expressed by a number of cell types including fibroblasts, myoblasts, and osteoblasts, and production of IGFBP-5 is regulated by growth factors, cytokines, and systemic hormones (15-21). Among the nuclear receptor activating hormones, glucocorticoids and retinoic acid have been shown to alter IGFBP-5 mRNA and protein levels in human and rat osteoblast-like cells (16, 22-24). However, effects of progesterone on IGFBP-5 expression have not been previously reported.

Pertinent to this study, we found that progesterone (PG) stimulated human osteoblast cell proliferation (25, 26) and increased IGFBP-5 mRNA levels (27). However, the molecular mechanism by which PG increased IGFBP-5 mRNA levels is not known. As a first step toward understanding how bone cell production of IGFBP-5 is regulated by PG, we isolated the 5'-flanking region of the human IGFBP-5 gene, linked the IGFBP-5 proximal promoter to a chloramphenicol acetyltransferase (CAT) reporter, and measured basal and progesterone-inducible promoter activity in transient transfection assays with U2 human osteosarcoma cells. We also examined the roles of progesterone receptor isoforms PR_A and PR_B in mediating PG induction of IGFBP-5 transcription. PRs are expressed in various cells as two different isoforms that are transcribed from separate promoters in the PR gene (28, 29). The B-receptor isoform (PR_B) is 933 amino acids in length, and the A-receptor isoform (PR_A) lacks 164 amino acids at the N terminus.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Progesterone acetate was from Sigma, and R5020 (promegestone) was from NEN Life Science Products. U-2 OS (U2) human osteogenic sarcoma cells (HTB96) were from the American Type Culture Collection (Manassas, VA). Dulbecco's modified Eagle's medium (DMEM) was from Mediatech, Inc. (Herndon, VA). Iron-supplemented bovine calf serum (CS) and charcoal-dextran-treated fetal bovine serum (CD-FBS) were from Hyclone (Logan, UT). Penicillin, streptomycin, and trypsin were from Life Technologies, Inc. Bovine serum albumin, crystallized, and guanidine isothiocyanate were from Fluka (Ronkonkoma, NH). The RNA polymerase inhibitor 5,6-dichlorobenzimidazole-1--D-ribofuranoside was from Calbiochem (San Diego, CA). Recombinant human IGFBP-5 cDNA was the generous gift of Drs. C. Dony and K. Lang of Roche Molecular Biochemicals Therapeutics (Penzberg, Germany). PR mammalian expression vectors phPR-A and phPR-B (30) were provided by Dr. B. O'Malley (Baylor College of Medicine, Houston, TX). Purified PR_A and a specific monoclonal antibody (AB52) that recognizes PR_A (31) were provided by Dr. D. P. Edwards (University of Colorado Health Sciences Center, Denver, CO). Mouse mammary tumor virus (MMTV) long terminal repeat promoter constructs pMSG-CAT and pMMTV-Luc were from Amersham Pharmacia Biotech and Dr. Ron Evans (Salk Institute, San Diego, CA), respectively. D-threo-1,2-[¹⁴C]Chloramphenicol, [-³²P]dCTP, and [-³²P]ATP were from ICN Biochemical, Inc. (Irvine, CA). [-³²P]UTP was from NEN Life Science Products. All other molecular biology grade chemicals were obtained from U. S. Biochemical Corp., Sigma, and Calbiochem.

Oligodeoxynucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). For gel shift assays complimentary molecules were annealed to produce double-stranded probes. The names and upper strand sequences of these oligodeoxynucleotides are: BP5-PDRE, 5'-CCTCTCCCCACCCCCACCCCGTGTG-3' (wild type sequence); BP5-PDREm1 5'-CCTCTCaaaACCaaaACCCCGTGTG-3'; BP5-PDREm2, 5'-CCTCTCaaaACCCCCACCCCGTGTG-3'; BP5-PDREm3, 5'-CCTCTCCCaACCCCaACCCCGTGTG-3'; BP5-PDREm4, 5'-CCTCTCCCaACCCCCACCCCGTGTG-3'; BP5-PDREm5, 5'-CCTCTCCCCACCCCaACCCCGTGTG-3' (mutations in lowercase); c-fos RCE, 5'-CGCGCCACCCCTCTGGCGCCACCGTG-3' from the human c-fos promoter (32); TGF- RCE, 5'-CGCCCCCGGCCCCACCCCAGGAAG-3' from the human TGF promoter (33); MNF, 5'-ACGCACAACCACCCCACCCCCTGTG-3' from the human myoglobin promoter (34); AP-2, 5'-GATCGAACTGACCGCCCGCGGCCCGT-3' from the human metallothionein-IIA promoter (35); Sp1, 5'-AATCGATCGGGGCGGGGCGAGC-3' from the SV40 early promoter (36); and MMTV-PRE, 5'-TTTGGTTACAAACTGTTCTTAAAACGAG-3' from the mouse mammary tumor virus long terminal repeat (37).

Cell Culture-- U2 cells were maintained in DMEM supplemented with 10% CS at 37 °C in a humidified atmosphere composed of 95% air and 5% CO₂. In most experiments, cells were changed to phenol red-free DMEM and CD-FBS (described below). PG was dissolved in ethanol at 10² M and was further diluted with serum-free DMEM containing 0.1% bovine serum albumin before adding to the cells. Control cultures were treated with ethanol diluted to the same extent as for PG treatment.

Northern Blot Analysis-- Total RNA was extracted from cell cultures by a single-step acid guanidinium thiocyanate-phenol-chloroform method (38). 20 µg of total RNA was subjected to Northern analysis as described previously (23), using Magnagraph hybridization membranes (MSI, Westboro, MA). The human IGFBP-5 cDNA was a 0.35-kilobase fragment containing the coding region of exon 1 and was labeled with ³²P and hybridized as described (23). Relative abundance of each mRNA species was quantitated by laser densitometry (Biomed Instruments, Fullerton, CA) and corrected as described (39) for the amounts of RNA transferred to the hybridization membrane by dividing the densities of 28 S rRNA bands that were stained with methylene blue before hybridization. Relative mRNA levels are presented as the ratio of treated to control.

Nuclear Run-on Analysis-- The effect of PG on the rate of IGFBP-5 gene transcription was determined with a modified nuclear run-on analysis as described (40). Briefly, U2 cells were treated with 10 nM PG or with vehicle for 1 and 4 h. The cells were rinsed and scraped into ice-cold phosphate-buffered saline, nuclei were isolated, and then nuclei were incubated in transcription buffer consisting of 0.6 M KCl, 12.5 mM MgCl₂, 2.5 mM of ATP, CTP, and GTP, and 100 µCi of [³²P]UTP (800 Ci/mmol). RNA was isolated and equal amounts (2 × 10⁶ dpm) were hybridized to 10 µg of alkali-denatured plasmid DNAs (IGFBP-5 cDNA, -actin cDNA, and pBluescript DNA) that had been immobilized to Hybond-N filters. The filters were hybridized in a solution containing 10 mM TES, pH 7.4, 10 mM EDTA, 0.2% SDS, and 0.6 M NaCl at 68 °C for 48 h. After hybridization, the filters were washed twice with 6× SSPE, 0.1% SDS at 42 °C for 30 min and once with 1× SSPE, 0.1% SDS at 42 °C for 30 min and autoradiographed with two DuPont Cronex III intensifying screens for 3 days. The relative intensity of each band on the autoradiographs was quantitated by laser densitometry (Biomed Instruments, Fullerton, CA).

Isolation of IGFBP-5 Genomic Clones-- A mixture of two oligodeoxynucleotides, 5'-CCCAGGTAAGAGGAGAGGAA-3' and 5'-CTGGCTAGAGGAGGAGACA-3', corresponding to the 3'-untranslated region of the human IGFBP-5 gene, was labeled with [-³²P] ATP using T4 polynucleotide Kinase and was used to screen 6 × 10⁶ clones of a human cosmid (pWE15) library derived from placental DNA (CLONTECH, Palo Alto, CA). Positive clones were rescreened with a human IGFBP-5 full-length coding region cDNA probe. DNA from a selected clone was digested with restriction enzymes EcoRI, PstI, SacI, BamHI, and HindIII, and analyzed by Southern blot using a ³²P-labeled oligodeoxynucleotide, 5'-ACTCTCGCTCTCCTGCCCCA-3', corresponding to the 5'-untranslated exon 1 region of human IGFBP-5. A 4.6-kilobase EcoRI fragment was gel purified and subcloned into pGEM3Zf() (Promega, Madison, WI). Both DNA strands of the fragment were sequenced with the dideoxy chain termination method (41). It contained 1390 bp of 5'-flanking region, all of exon 1, and 2.9 kilobases of intron 1. Primer extension analysis was carried out as described (42).

Reporter Plasmid Construction-- Seven IGFBP-5 promoter-reporter constructs containing 753, 461, 345, 325, 252, 162, and 124 bp of IGFBP-5 5'-flanking region were prepared by inserting selected restriction enzyme fragments or polymerase chain reaction products into a CAT reporter gene vector pJFCAT1 (43). For pCAT162 CACCC box mutants, double-stranded oligodeoxynucleotides with the sequence corresponding to BP5-PDREm1 flanked by SphI sites were synthesized, annealed and inserted in the sense orientation into the SphI site of pCAT124 upstream of the IGFBP-5 5'-flanking region. The IGFBP-5 promoter fragment from 252 to +24 bp was subcloned to pGEM7 and then to pGL3 (Promega, Madison, WI) to create the luciferase reporter construct pLUC-252. The sequence and orientation of all promoter constructs used were confirmed using the dideoxy method.

DNA Transfection-- To determine basal promoter activities of the IGFBP-5 promoter CAT constructs, 3 × 10⁵ U2 cells were seeded in 60-mm dishes in DMEM supplemented with 10% CS. After 18 h of incubation, cells were rinsed once with DMEM and 1.6 ml of DMEM containing 12 µl of LipofectAMINE (Life Technologies, Inc.), and 6 µg of Qiagen purified DNA (5 µg of CAT construct + 1 µg of pCMV-gal) was added to the cells. The cells were incubated for 4 h at 37 °C, in 95% air, 5% CO₂. Medium was then replaced with DMEM supplemented with 10% CS and cells were incubated for 48 h before -gal and CAT assay. To evaluate the effect of PG on promoter activity, U2 cells were grown in phenol red-free DMEM supplemented with 5% CD-FBS for 72 h before transfection. U2 cells were plated at 2 × 10⁵/dish and, in most experiments, were transfected with 2 µg of phPR-A plasmid (44), 3 µg of IGFBP-5 promoter construct, and 1 µg of pCMV--gal. After transfection, cells were incubated in phenol red-free DMEM supplemented with 2% CD-FBS for 24 h. Medium was then replaced with phenol red-free DMEM supplemented with 2% CD-FBS, plus 10 nM PG or solvent, for another 24 h. Cells were lysed with reporter lysis buffer (Promega, Madison, WI) as recommended by the manufacturer. CAT and -galactosidase activities were determined as described (45, 46), and CAT activity was expressed per unit of -galactosidase activity. To evaluate transactivation of IGFBP-5 promoter activity in a luciferase reporter construct, cells were plated in 6-well plates at 120,000/well. All incubation, transfection, and treatment procedures were as described for the CAT reporter construct studies except that amounts of pLUC-252 and pCMV--gal were 0.375 and 0.125 µg/well, respectively, phPR-A (and phPR-B) were tested at 0.0625-0.50 µg/well, and total DNA added was kept constant at 1.0 µg/well by adding pGEM7 plasmid DNA. Luciferase activities were determined using the Enhanced Luciferase Assay Kit (Pharmingen, San Diegao, CA). Because this assay was less complex to perform than the CAT assay, experiments were done with 6 replicate wells/group. Statistical analysis of differences between groups by analysis of variance was done using the Systat computer program (Systat Inc., Evanstan, IL).

Electrophoretic Mobility Shift Assay-- U2 cells were plated at 5 × 10⁵ cells per 100 mm dish in 5% CD-FBS for 18 h. The medium was replaced with 2% CD-FBS and incubated for 48 h. Nuclear extracts were prepared as described (47). For the EMSA, the binding reaction contained 4% glycerol, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05 mg/ml poly(dI-dC), 1.5 µl (3 µg of protein) of nuclear extract, and 1 µl (50,000 cpm) of ³²P-labeled double-stranded oligodeoxynucleotides in a total volume of 20 µl. For cold competition experiments, nuclear extract was preincubated with unlabeled double-stranded oligodeoxynucleotides for 10 min at room temperature before the addition of labeled double-stranded oligodeoxynucleotides. The oligodeoxynucleotides used for competition with the labeled IGFBP-5 CACCC box sequence are described under "Materials." The mouse mammary tumor virus PRE oligodeoxynucleotide was a double-stranded 28-mer based on the sequence 189-162 bp 5' of the transcription start site and was described previously (37).

Purified PR_A was obtained from a baculovirus expression system and purified as described (31). To evaluate PR interactions with U2 cell nuclear factors, different concentrations of PR proteins were preincubated with 5 µg of U2 cell nuclear extract protein before the addition of labeled double-stranded oligodeoxynucleotides. The reaction mixture was incubated at room temperature for 20 min and analyzed by electrophoresis using a 5% polyacrylamide-0.125% bis-acrylamide gel in 0.25 × TBE. For supershift experiments, 1 µg of anti-PR antibody PR52 (41) was added to the reaction and incubated on ice for an additional 15 min before loading onto the gel. After electrophoresis, the gel was dried and autoradiographed using Kodak X-OMAT or Biomax MS film and Cronex III screens.

Nucleotide Sequence Accession Number-- The GenBank^TM data base accession number of the human IGFBP-5 5'-flanking region determined in this study is U20271.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of PG and RU486 on IGFBP-5 Levels in Normal Human Osteoblasts-- Previously we found that PG increased IGFBP-5 mRNA levels in normal human osteoblasts and in MG63 and U2-OS osteosarcoma cells (27). In the present study we replicated this earlier finding with normal human vertebral osteoblasts and determined whether the progestin antagonist RU486 would affect PG induction of IGFBP-5 expression. Treatment with 10⁸ M PG for 4 h increased IGFBP-5 mRNA levels to 190% of control (Fig. 1). 10⁶ M RU486 by itself did not affect IGFBP-5 mRNA levels and prevented the increase in IGFBP-5 mRNA levels induced by PG. These results support the conclusion that PG increases IGFBP-5 expression by a PR-dependent mechanism.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   PG increases IGFBP-5 mRNA levels in normal human osteoblasts. Osteoblast-like cells from human calvaria bone were changed to serum-free medium with 1 mg/ml bovine serum albumin and treated with vehicle control, 106 M RU486, 108 M PG, or RU486 + PG for 4 h. Northern blot of 20 µg of total RNA/lane shows IGFBP-5 mRNA and 28 S rRNA bands. Average relative levels of IGFBP-5 mRNA in two separate experiments, determined by laser densitometry and normalized to 28 S rRNA levels, were 1.0, 0.9, 1.9, and 1.1, respectively.

Effect of PG on IGFBP-5 Transcription-- U2 cells were selected to investigate the mechanism of PG action in the present study because both IGFBP-5 expression and transfection efficiency were higher in this cell type than in other human osteoblast-like cell lines. PG treatment for 1 h increased IGFBP-5 gene transcription determined by nuclear run-on analysis to 334% of control (p < 0.002), whereas the rate of -actin gene transcription was not significantly altered (Fig. 2). After 4 h of PG treatment, the IGFBP-5 transcription rates in both the treated and control groups were elevated such that the difference between PG-treated and control groups was less compared with that found after 1 h of PG treatment (data not shown). To determine whether PG effected IGFBP-5 mRNA stability, U2 cells were treated with PG for 4 h, and thereafter RNA polymerase II-mediated transcription was inhibited with 4 × 10⁵ M 5,6-dichlorobenzimidazole-1--D-ribofuranoside. IGFBP-5 mRNA levels in control and PG-treated cultures were determined at 5-24 h by Northern analysis. In these experiments, IGFBP-5 mRNA half-life was estimated to be approximately 24 h and was not affected by PG (data not shown). The long IGFBP-5 mRNA half-life and absence of a PG effect on half-life are consistent with the results of nuclear run-on analysis, suggesting that PG rapidly increased IGFBP-5 mRNA levels in human osteoblasts primarily by a transcriptional mechanism.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   PG increases IGFBP-5 gene transcription in U2 cells. U2 cells were treated for 1 h with 108 M PG or vehicle control (Con), and nuclei were isolated. Transcription in 107 nuclei/group was continued in vitro in the presence of [32P]UTP. 32P-Labeled RNA was hybridized to immobilized -actin cDNA, IGFBP-5 cDNA, and pBluescript vector DNA as described under "Experimental Procedures." Autoradiographs from three preparations are shown. Labeled RNA binding to target DNAs were measured by laser densitometry of the autoradiographs and are expressed as percentages of solvent-treated control ± S.E. -actin, 130 ± 66%; IGFBP-5, 334 ± 32%, p < 0.01.

Functional Analysis of the IGFBP-5 Gene-- We isolated a pWE15 human genomic cosmid clone containing the IGFBP-5 gene and then isolated a 4.6-kilobase EcoRI fragment containing the 5'-flanking region. The nucleotide sequence from position 461 through the first exon was identical to that reported previously (12). The proximal 5'-flanking region contains a TATA box, a CAAT box, and several putative response elements. Primer extension analysis in U2 cells confirmed a single transcription start site 33 nucleotides from the TATA box (data not shown) that was reported previously in a study of human breast cancer cells (12).

To determine whether the putative IGFBP-5 promoter functioned in human osteoblasts, CAT reporter constructs were made containing a PstI fragment from 753 to +23 (pCAT753) and a 484-bp HindIII/PstI fragment from 461 to +23 (pCAT461). U2 cells were transiently transfected with sense reporter constructs, antisense constructs, or the pJFCAT1 promoterless control vector and then incubated for 48 h in medium with 10% CS. CAT activity in cells that were transfected with pCAT753 or pCAT461 sense constructs was 620 and 730%, respectively, of CAT activity in cells transfected with pJFCAT1. In contrast, CAT activity did not increase in U2 cells transfected with antisense promoter constructs.

Identification of IGFBP-5 Promoter Sequences That Confer PG Responsiveness-- In experiments for assessing effects of PG treatment, concentrations of steroids and other factors in serum were minimized by maintaining U2 cells for 72 h in phenol red-free DMEM plus 5% CD-FBS and then plating the cells in 5% CD-FBS at two-thirds of the cell density used in the basal promoter expression experiments. Cells were co-transfected with pCAT753 and pCMV--gal, plus PR expression vector phPR-A (44), or a PR control vector from which the PR coding region was removed. Cells were incubated 48 h in phenol red-free DMEM supplemented with 2% CD-FBS in the absence or presence of 10 nM PG. In these conditions basal promoter activity from pCAT753 was 285% of activity from the promoterless control pJFCAT1. The reduction of basal promoter activity in lower density U2 cells maintained in 2% CD-FBS compared with 10% CS suggests that serum factors and cell density-dependent signals may increase promoter activity.

When U2 cells were co-transfected with the expression vector phPR-A, along with pCMV--gal and pCAT753, PG reproducibly increased IGFBP-5 promoter activity to 200-300% of control (p < 0.001, n = 5). Similar results were obtained after treatment of co-transfected cells with promegestone (R5020), a nonmetabolizable progestin analog (data not shown). Transfection with the PR control vector did not impart PG responsiveness, suggesting that the effects of phPR-A were mediated by PR_A and not by the expression vector backbone. PG increased activity of the MMTV promoter pMSG-CAT to 170% of solvent-treated control in cells co-transfected with phPR-A (data not shown) (48).

Although the human IGFBP-5 promoter sequence does not contain a classic palindromic PRE consensus sequence (49), five PRE half-site motifs were identified at positions 528, 392, 333, 215, and 193. Clusters of similar PRE half-sites mediated PG transcriptional activation of other genes (49-51). We made a series of 5' deletions that contained 753, 461, 345, 325, 252, 162, and 124 bp of the IGFBP-5 5'-flanking region with 5, 4, 3, 2, 2, 0, and 0 putative PRE half-sites, respectively (see Fig. 4). Basal activities determined in cultures maintained in DMEM plus 10% CS were highest in the 345 and 325 bp constructs, nearly 20-fold higher than promoterless pJFCAT1.

PG responsiveness of each deletion construct was determined in cells precultured in DMEM + 5% CD-FBS, co-transfected with phPR_A, pCMV-gal and the deletion construct, and then changed to DMEM + 2% CD-FBS with either solvent or 10 nM PG. In multiple experiments, PG reproducibly increased promoter activity 200-300% in all constructs containing from 753 to 162 bp of proximal promoter (Fig. 3). PG did not increase CAT activity in cells transfected with pCAT124, which contained only the sequence from the CAAT box to the start of transcription, although pCAT124 and pCAT162 had similar levels of basal promoter activity in the same experiments. These data suggest that none of the PRE half-sites were required for PG-dependent transactivation of the IGFBP-5 promoter and that elements between 162 and 124 that contain two tandem CACCC box sequences, 5'-CCCCACCCCCACCC-3', may impart PG responsiveness to the IGFBP-5 promoter.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Analysis of IGFBP-5 promoter deletion constructs. A schematic representation of the IGFBP-5 proximal promoter-reporter construct indicates relative positions of putative PRE half-sites (solid bars), CACCC box sequences (hatched box), CAAT box (oval), TATA box (open box), and the start of transcription (+1). Constructs shown below contain 753, 461, 345, 325, 252, 162, and 124 bp of IGFBP-5 5'-flanking region + 23 bp of untranslated Exon 1 inserted into pJFCAT1. Basal promoter activities in cultures co-transfected with each construct and pCMV--gal and then maintained in 10% CS are shown in the first column, expressed as percentages of promoterless pJFCAT1 activity. Progesterone inductions of promoter activity in cultures co-transfected with each IGFBP-5 promoter construct, phPR-A and pCMV--gal, and then changed to CD-FBS and treated with solvent or 108 M PG are shown in the second column, expressed as percentages of the respective solvent control group. All data are based on normalizing CAT to -galactosidase activities, and are means ± S.E. of at least three independent experiments for each construct. There was no significant difference in fold induction of CAT expression by PG among groups pCAT753-pCAT162 as determined by analysis of variance. PG induction of CAT expression in the pCAT124 group was not significant and was significantly lower than in all other groups (p < .001). Basal promoter activities (minus PG) in pCAT162 and pCAT124 were the same, approximately 3-fold of pJFCAT1 control.

The CACCC box is an important enhancer element that binds several different transcription factors (32, 33, 35, 52-58). CACCC-binding proteins can either bind directly to the CACCC box or interact with other transcription factors and bind as a complex (58). To determine whether CACCC sequences between positions 162 and 124 are required for PG stimulation of IGFBP-5 promoter activity, mutations were made in the distal (pCAT162mDi), proximal (pCAT162mPr), or both (pCAT162mut) CACCC sequences in pCAT162 (changing CCCACCC to aaaACCC). In multiple experiments, PG increased pCAT162 and pCAT162mDi promoter activities but failed to increase pCAT162mut and pCAT162mPr promoter activities (Fig. 4). Thus mutation of the proximal CACCC box at position 139 abolished PG transactivation of hIGFBP-5 promoter activity. These data provide strong evidence that the proximal CACCC box at position 139 is required for PG-dependent transactivation of IGFBP-5 promoter activity.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Mutation of CACCC boxes eliminates PG induction of IGFBP-5 promoter activity. U2 cells were transfected as described under "Experimental Procedures" with phPR-A, with pCMV-gal, and with pCAT124, wild type pCAT162, or mutant pCAT162 promoter-reporter constructs. In pCAT162mut both CACCC boxes at positions 147 to 134 were mutated from CCCACCCCCACCCC to aaaACCaaaACCCC. Only the distal CACCC box was mutated to aaaACCCCCACCCC in pCAT162mDi, and only the proximal CACCC box was mutated to CCCACCaaaACCCC in pCAT162mPr. Transfected cells were incubated in 2% CD-FBS in the presence or absence of 108 M PG. CAT activity was normalized to -galactosidase activity and expressed as a percentage of respective control group (transfected cells incubated without PG), mean ± S.E. of three independent experiments. PG significantly increased CAT activity in pCAT162- and pCAT162mDi-transfected cells (p < 0.001) but did not significantly affect CAT activities in pCAT162mut, pCAT162mPr, or pCAT124-transfected cells.

Interaction of IGFBP-5 Promoter CACCC Box Sequences with Nuclear Extract Proteins-- To determine whether U2 cell nuclear proteins interact with the CACCC box motif of the IGFBP-5 promoter, EMSA were performed. ³²P-Labeled double-stranded synthetic oligodeoxynucleotides corresponding to the wild type IGFBP-5 promoter sequence from 155 to 128, designated the IGFBP-5 progesterone-dependent responsive element (BP5-PDRE), were incubated with U2 cell nuclear extract proteins. Analysis by EMSA resulted in a reproducible set of shifted bands (Fig. 5A). Unlabeled BP5-PDRE oligodeoxynucleotide or different oligodeoxynucleotides containing CACCC box core motif sequences from enhancers of other promoters were used to compete for binding to labeled BP5-PDRE. These enhancers were RCE sequences from the TGF- and c-fos promoters (32, 33) and the MNF-binding site sequence from the myoglobin promoter (34). Other competitors tested were the consensus AP-2-binding site sequence from the metallothionein-IIA promoter (35) and the consensus Sp1-binding site sequence from Simian virus 40 (36) because Sp1 and AP2 have been shown to bind to nonconsensus sequences containing CACCC boxes (52, 57, 59). As shown in Fig. 5A, 50-fold excess unlabeled BP5-PDRE specifically competed with labeled BP5-PDRE for binding to U2 cell nuclear proteins and eliminated bands 1-4. At a 50-fold excess molar ratio of unlabeled DNA to labeled BP5-PDRE DNA, consensus AP-2, Sp-1, MNF, and the RCE sequence from the TGF- promoter competed as effectively as unlabeled BP5-PDRE for binding in complexes 1, 3, and 4 (Fig. 5A). AP-2, Sp-1, and c-fos RCE sequences did not compete as effectively as BP5-PDRE, MNF, or TGF- RCE sequences for protein binding in complex 2. Competition with 25-, 50-, and 100-fold molar excess of BP5-PDRE and MNF-binding sequences are compared in Fig. 5B. Competition for binding to band 5 was variable and may be nonspecific. These results suggest that nuclear protein(s) in the band 2 complex are specific for the CCCCACCCC sequence, which is identical in the BP5-PDRE, MNF, and TGF- RCE sequences and different in the other sequences that were tested. Binding of nuclear protein(s) in bands 1, 3, and 4 may be more specific for sequences adjacent to CCCCACCCC.

View larger version (67K): [in this window] [in a new window]   Fig. 5.   EMSA of BP5-PDRE binding to U2 cell nuclear factors. All oligodeoxynucleotide sequences used for cold competition experiments with 32P-labeled BP5-PDRE are listed under "Experimental Procedures." A, 32P-labeled BP5-PDRE (155 to 128 of human IGFBP-5 promoter) was incubated with 3 µg of U2 cell nuclear extract as described under "Experimental Procedures" in the absence (lane b) or presence of a 50-fold molar excess of unlabeled BP5-PDRE (lane c), BP5-PDREm1 mutant with six altered nucleotides in the CACCC boxes (lane d), consensus AP-2 (lane e) and Sp1 (lane f) sequences, MNF-binding sequence (lane g), and RCE sequences from TGF and fos promoters (lane h and i, respectively). Lane a shows the 32P-end labeled BP5-PDRE in the absence of nuclear extract. B, 32P- labeled BP5-PDRE was incubated with 3 µg of U2 cell nuclear extract in the absence (lane b) or the presence of 25-, 50-, and 100-fold molar excess of unlabeled BP5-PDRE (lanes c, d, and e, respectively), or with 25-, 50-, and 100-fold molar excess of CACCC core sequence-containing MNF-binding sequence from the human myoglobin promoter (lanes f, g, and h, respectively). Lanes a in A and B show 32P-labeled BP5-PDRE without added proteins.

When CCCACCC sequences at positions 147 and 139 were mutated to aaaACCC (BP5-PDREm1), this mutant oligodeoxynucleotide did not compete with labeled BP5-PDRE oligodeoxynucleotides (Fig. 6). However, when the distal CACCC box alone was mutated from CCCACCC to aaaACCC (BP5-PDREm2), the unlabeled BP5-PDREm2 competed very effectively with labeled BP5-PDRE, supporting the conclusion that only the proximal CACCC box at 139 is required for PG-dependent transactivation of the IGFBP-5 promoter. To further determine which of the tandem CACCC boxes in the IGFBP-5 promoter is required for binding to nuclear factors, single base pair mutations from CCCACCC to CCaACCC were made. Mutation of both distal and the proximal CACCC sequences or mutation of only the proximal CACCC sequence (BP5-PDREm3 and BP5-PDREm5) eliminated the ability of the sequence to compete with wild type labeled BP5-PDRE for nuclear factor binding (Fig. 6). However, when the distal CACCC sequence alone was mutated (BP5-PRDEm4), this oligodeoxynucleotide competed very effectively with labeled BP5-PDRE for nuclear factor binding. These results suggest that the distal CACCC sequence at 147 may not be important for forming the complexes observed by EMSA. Furthermore, these data provide evidence that the cytosine at position 139 in the proximal CACCC box is important for the binding of U2 cell nuclear protein(s).

View larger version (129K): [in this window] [in a new window]   Fig. 6.   Cold competition EMSA of labeled BP5-PDRE with mutated BP5-PDRE deoxyoligonucleotides. All mutated oligonucleotide sequences used for cold competition experiments with 32P-labeled BP5-PDRE are shown under "Experimental Procedures." 32P-Labeled BP5-PDRE was incubated with 3 µg of U2 cell nuclear extract as described under "Experimental Procedures" in the absence (lane b) or presence of 50-fold molar excess of wild type unlabeled BP5-PDRE (lane c), BP5-PDREm1 (lane d), BP5-PDREm2 (lane e), BP5-PDREm3 (lane f), BP5-PDREm4 (lane g), and BP5-PDREm5 (lane h). Lane a contains 32P-labeled BP5-PDRE without added proteins.

PR_A Interation with Nuclear BP5-PDRE-binding Protein(s)-- Deletion of the region 162 to 124 and mutation of the CACCC sequences within this region abolished PG responsiveness of the IGFBP-5 promoter, and the BP5-PDRE oligodeoxynucleotide corresponding to this region of the promoter bound to U2 cell nuclear factors. These results suggested that PR_A could either directly bind to the BP5-PDRE or interact with nuclear protein(s) that bind to the BP5-PDRE. To determine whether PR_A could bind to the IGFBP-5-PDRE sequence at 139 we used EMSA to examine binding of baculovirus-expressed, purified human PR_A to labeled BP5-PDRE oligodeoxynucleotide. As a positive control the PR_A preparation was first tested with a consensus progesterone response element oligodeoxynucleotide (MMTV-PRE) from the mouse mammary tumor virus promoter (30) and the anti-PR monoclonal antibody, AB52 (31). Purified PR_A binding to labeled MMTV-PRE was dependent on the presence of nuclear extract proteins, as observed previously (31), and produced a strong mobility shift band, which was supershifted by incubating the protein-DNA complexes with the PR antibody (Fig. 7A). Purified PR_A protein alone did not bind the BP5-PDRE oligodeoxynucleotide, which is similar to the result with the MMTV-PRE. Incubation of labeled BP5-PDRE with 5 µg of U2 cell nuclear protein alone produced multiple shifted bands (Fig. 7B), as in Fig. 4. When increasing amounts of PR_A were preincubated with nuclear extract proteins and then mixed with labeled BP5-PDRE, the intensity of complexes 1 and 3 progressively decreased, whereas the intensity of complex 4 progressively increased (Fig. 7B). Addition of anti-PR antibody together with PR_A and nuclear extract proteins did not alter the gel shift pattern of labeled BP5-PDRE, suggesting that either PR_A did not participate in direct binding to BP5-PDRE or that the PR_A epitope to which the monoclonal antibody binds was masked by the nuclear proteins. As a negative control for the effects of PR_A on the BP5-PDRE gel shift pattern, PR_A was added with nuclear extract to labeled Sp1 and TGF- RCE oligodeoxynucleotides, which have similar CACCC box sequences. PR_A had no effect on the gel shift patterns with these sequences (data not shown).

View larger version (81K): [in this window] [in a new window]   Fig. 7.   PRA interaction with nuclear BP5-PDRE-binding protein(s). A, labeled consensus PRE from the MMTV promoter (37) was incubated with 5 fmol of purified PRA (lane a) or with 1 µg of U2 cell nuclear extract (lane b) or combination of nuclear extract and PRA (lane c) prior to analysis by EMSA. Lane d was the same as lane c except 1 µg of PR antibody AB-52 was added as described under "Experimental Procedures." Lane e was labeled BP5-PDRE incubated with 5 fmol of purified PRA. These data demonstrate that the PRA protein preparation used in panel B is active. B, labeled BP5-PDRE was incubated with 5 µg of U2 cell nuclear extract (lane a) in the presence of increasing amounts of PRA (lanes b-f). Numbers above each lane indicate fmol of PRA. 1 µg of anti-PR antibody AB-52 was added to the mixture run in lane f.

PR_A and PR_B Mediate PG Induction of IGFBP-5 Promoter Activity-- Initial experiments comparing abilities of PR_A and PR_B isoforms to transactivate the IGFBP-5 promoter in CAT reporter constructs suggested that PR_B was inactive (data not shown), so experiments to define the regions of the promoter that mediate transactivation were limited to PR_A. This apparent specificity for PR_A could not be confirmed, however, using different phPR-B plasmid preparations and a luciferase reporter construct containing 252 bp of IGFBP-5 5'-flanking region. U2-OS cells were co-transfected with 0.0625-0.50 µg/well of PR expression plasmids phPR-A and phPR-B together with pCMV--gal and pLUC-252. With this luciferase reporter construct, both PR_A and PR_B transactivated IGFBP-5 promoter activity in the presence of PG, and PR_B was more effective at higher transfected DNA levels (Fig. 8). PG-dependent PR_B transactivation of the MMTV promoter was much higher than PR_A transactivation of this promoter (data not shown), as described previously (48). We also found that RU486 inhibited PR_A and PR_B transactivation of the IGFBP-5 promoter, which correlates with the effects of RU486 to decrease IGFBP-5 mRNA levels in normal human osteoblast cultures.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   PRA and PRB mediate PG stimulation of IGFBP-5 promoter activity. A, U2-OS cells were plated in 6-well plates and co-transfected with pLUC-252, pCMV--gal, and different amounts of phPR-A (open bars) or phPR-B (hatched bars) expression vectors as described under "Experimental Procedures." The cells were treated with solvent control or 108 M PG. for 24 h. Promoter activities were calculated as luciferase light units (photons)/-gal activities and are expressed as percentages of the respective control (0 progesterone) for each group (n = 6). B, cells were transfected with pLUC-252, pCMV--gal, and 0.25 µg/well of phPR-A (open bars) or phPR-B (hatched bars) and analyzed as in A. Cells were treated with PG, 106 M RU486, or both. a indicates significantly different from respective solvent control (p < 0.001). b indicates significant inhibition by RU486 (p < 0.001) as determined by analysis of variance.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human IGFBP-5 promoter does not contain classic consensus palindromic progesterone or glucocorticoid response elements (GRE/PRE), defined as two hexameric half-sites with a 3-bp spacer (60). However the IGFBP-5 promoter contains five putative GRE/PRE half-sites that have strong sequence similarity to the hexanucleotide motifs 5'-TGTTCT-3' of the MMTV promoter and 5'-TGTTCA-3' of the uteroglobin gene promoter (50, 60). Although GR and PR do not bind to GRE/PRE half-sites as effectively as to the palindromic GRE/PRE consensus sequence, clusters of GRE/PRE half-site sequences in MMTV, uteroglobin, and other promoters have been shown to bind the receptors and effectively mediate ligand-dependent transactivation through synergistic interactions between the multiple half-sites (50, 60). Furthermore, PR as well as other steroid hormones receptors affect transcription of many genes without directly binding to consensus hormone response elements (61-66). Many of these actions of nuclear receptors involve interactions with co-activators and co-repressors (67, 68). Although the presence of PRE half-sites in the IGFBP-5 promoter suggested the possibility that they might mediate PG transactivation, our results demonstrate that this is not the case. Rather, results of the series of reporter construct IGFBP-5 promoter deletions suggest that a repeated CACCC box motif (BP5-PDRE) proximal to the PRE half-sites mediates PG transactivation.

The CACCC box is an important promoter element required for efficient and accurate gene expression in the TGF-, c-fos, c-jun, and IGF I genes (32, 33, 52, 59). In addition, a number of transcription factors including the RCE-binding protein, MNF, and AP2 bind to CACCC box sequences and effect transcription (35, 54). CACCC-binding proteins can either bind directly to the CACCC box sequence or interact with other transcription factors and bind as a complex (58). Duan and Clemmons (14) reported that human IGFBP-5 promoter activity is increased by AP-2. They found that the two overlapping AP-2-binding sites in the CACCC box motif at positions 162 to 124 were not required for AP-2 stimulation of promoter activity in human hepatoma cells and skin fibroblasts. Rather, an alternate consensus AP-2-binding sequence 5'-GCCNNNGGC-3' at positions 52 to 35, adjacent to the TATA box, mediated AP-2 transactivation. Both of the sequences bound purified AP-2 protein in EMSA experiments even though only the sequence adjacent to the TATA box was involved in AP-2 transactivation of promoter activity. In contrast, we found that PR_A transactivation of the IGFBP-5 promoter in osteoblastic cells required the AP-2-binding sequence at position 139. This suggests that AP-2 and PR_A transactivate the IGFBP-5 promoter by different mechanisms.

Although EMSA analysis suggested that purified PR_A did not bind to BP5-PDRE directly, addition of PR_A altered the gel shift band pattern produced by binding of nuclear extract proteins to the BP5-PDRE DNA sequence. This result suggests that PR_A interacted with BP5-PDRE-binding proteins, although the identities of the binding proteins are not known. Of the CACCC box sequences tested in competition EMSA experiments, the RCE and MNF-binding sites of the TGF- and myoglobin promoters (33, 34) are most similar to the BP5-PDRE sequence, and the RCE and MNF sequences most effectively competed with the BP5-PDRE in binding nuclear extract proteins. These results, although not definitive, are consistent with the possibility that RCE, MNF, or related proteins may be involved in binding BP5-PDRE.

Our results indicate that both PR_A and PR_B can mediate PG induction of hIGFBP-5 promoter activity. For many genes that are induced by progestins, human PR_B is more active than human PR_A (44, 69, 70), although PR_A has been reported to be a stronger transactivator than PRB for chicken ovalbumin and human tyrosine aminotransferase genes (44, 48). In many PG-inducible genes that have been examined, activated PR_B increases transcription by binding to a PRE within the gene promoter, and PR_A functions as a negative regulator without directly binding to a PRE (44, 69). It remains to be established whether PR_B mediates transactivation of the IGFBP-5 promoter by a mechanism that is similar to or different from that of PR_A (70).

PR_A and PR_B are differentially expressed in a cell type- and species-specific manner (71, 72). Depending on the cell type, either both isoforms are induced by estrogen or PR_B is differentially induced (73-75). In human osteoblasts both isoforms of PR are expressed, and estrogen induced both A and B promoters (74). Progestins have been found to stimulate osteoblast proliferation, differentiation, and growth factor expression (25, 26, 76-80) and to increase bone formation (81, 82). In ovariectomized dogs, PG increased bone formation (83) and in postmenopausal women progestins combined with estrogen increased bone density to a greater extent than estrogen alone (84, 85). Because IGFBP-5 increases the mitogenic and anabolic activities of IGFs and IGF activities are important for bone formation, one could speculate that PG induction of IGFBP-5 expression in osteoblasts contributes to the positive effects of PG on bone formation.

	ACKNOWLEDGEMENTS

We thank Dr. N. Weigel and Dr. B. O'Malley (Baylor College of Medicine) and Dr. R. Evans (Salk Institute), respectively, for supplying PR and GR expression constructs. We gratefully acknowledge Jerry L. Pettis Veterans Affairs Medical Center Medical Media Department for photography. We acknowledge and thank Sylvia Morales and Connie Zastrow for excellent technical assistance and Dr. Kuk-Wha Lee for helping with the nuclear run-on assay.

	FOOTNOTES

^* This work was supported by grants from the Veterans Administration (to D. J. B., D. D. S, and T. A. L.), National Institutes of Health Grant AR 31062, funds from the Loma Linda University Medical School Research Seed Grant Committee, and grants from the Departments of Pediatrics and Medicine, Loma Linda University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a Predoctoral grant from the Department of Biochemistry, Loma Linda University.

^¶¶ To whom correspondence should be addressed: Musculoskeletal Disease Center, Research Service (151), J. L. Pettis VAMC, 11201 Benton St., Loma Linda, CA 92357. E-mail: linkhart@llvamc.va.gov.

	ABBREVIATIONS

The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; IGF, insulin-like growth factor; PG, progesterone; PR, progesterone receptor; CAT, chloramphenicol acetyltransferase; bp, base pairs; -gal, -galactosidase; DMEM, Dulbecco's modified Eagle's medium; CS, calf serum; CD-FBS, charcoal-dextran-treated fetal bovine serum; PRE, PG response element; MMTV, mouse mammary tumor virus; TES, 2-{[tris-(hydroxymethyl)]-methylamino}-ethanesulfonic acid; EMSA, electrophoretic mobility shift assay(s); RCE, retinoblastoma control element; MNF, myocyte nuclear factor; GRE, glucocorticoid response element.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES