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Endocrinology, doi:10.1210/en.2003-1552
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Endocrinology Vol. 145, No. 4 1776-1785
Copyright © 2004 by The Endocrine Society

Insulin-Like Growth Factor-Binding Protein-3 in Porcine Ovarian Granulosa Cells: Gene Cloning, Promoter Mapping, and Follicle-Stimulating Hormone Regulation

E. Moige Ongeri, Qin Zhu, Michael F. Verderame and James M. Hammond

Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Dr. James M. Hammond, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: jhammond{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role and regulation of IGF-binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12,257 bp of the pig IGFBP-3 gene that includes 4,296 bp of the flanking promoter sequence. The porcine IGFBP-3 promoter sequence shares two highly conserved regions with the human and bovine IGFBP-3 promoters and a mouse DNA clone. The first is a 38 bp region between -1095 and -1058, whereas the second is a 73-bp region between -63 and +10 of the pig sequence. Projected translation of the open reading frame of our sequence gave a peptide sequence identical to that determined by peptide sequencing, but with 27 additional amino acids upstream of this sequence and is highly similar to the human, bovine, rat, and mouse IGFBP-3 peptides. Using RT-PCR we demonstrated that FSH regulates IGFBP-3 mRNA expression in a biphasic manner, with an early induction (maximal at 3 h) and an inhibition at 24 h after FSH treatment. The inhibition at 24 h was not due to changes in IGFBP-3 mRNA stability. A similar pattern of FSH modulation of the IGFBP-3 gene transcription was demonstrated by the reporter activity of granulosa cells transiently transfected with IGFBP-3 promoter constructs. The site for FSH stimulation of the IGFBP-3 gene was localized to the sequence between -61 and -48 relative to the transcription start site. Regulation of IGFBP-3 transcription by FSH suggests a role for IGFBP-3 in follicular development that may be independent of IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-BINDING PROTEINS (IGFBPs) regulate IGF signaling. IGFBP-3, a 40- to 45-kDa glycoprotein, is the most abundant IGFBP in the circulation and in ovarian follicular fluid. The binding affinity of IGFBP-3 for IGF peptides is higher than that of IGF receptors, implying that IGFBP-3 can modulate IGF binding to its receptor and thereby determine IGF biological action in the local environment. Although there remains some uncertainty regarding the origin of IGFBP-3 in follicular fluid, it has been shown that IGFBP-3 is expressed by porcine ovarian granulosa cells in vivo (1, 2) and in vitro (3, 4, 5). The local expression of IGFBP-3 demonstrated in the porcine ovary and cultured granulosa cells suggests an autocrine role of IGFBP-3 in the regulation of ovarian function. In various cells, the expression of IGFBP-3 is regulated by both growth factors and growth inhibitory factors. However, the mechanism of IGFBP-3 gene regulation is not fully understood. Ca2+ and cAMP have been implicated as stimulatory or inhibitory in different systems (4, 6, 7, 8). The gonadotropin FSH, a key regulator of follicular development and steroidogenesis, was shown to decrease IGFBP-3 in cultured porcine granulosa cells at 24 and 48 h (3, 9). However, the mechanism of FSH modulation of IGFBP-3 expression in ovarian granulosa cells has not been studied. Although porcine IGFBP-3 cDNA was among the first IGFBPs cloned, the gene has not been cloned nor has the promoter been mapped. As a result, there has been little functional mapping of regions of the pig IGFBP-3 gene that could mediate these responses. Sequence analysis of the promoter region could identify consensus binding sites for factors that induce transcription in response to stimulatory molecules such as 12-O-tetradecanoyl phorbol 13-acetate, cAMP, GH, IGF-I, FSH, retinoic acid, and glucocorticoids.

The objectives of this study were to evaluate the mechanism of FSH action on the expression of IGFBP-3 mRNA in cultured porcine ovarian granulosa cells. To accomplish this goal, we decided to clone the IGFBP-3 gene, map the promoter sequence, and identify putative consensus sites for transcription factors. The last objective was to generate promoter constructs for use in studying FSH and growth factor regulation of the IGFBP-3 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The following reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO): BioBond-Plus nylon membrane, deoxy-NTP mix, JumpStart Taq DNA polymerase, dichloro-ß-D-ribofuranosyl-benzimidazole (DRB) and dimethylsulfoxide. We purchased the following supplies from Life Technologies, Inc./Invitrogen (Grand Island, NY): fetal bovine serum, lipofectin, and gentamicin. Falcon brand cell culture dishes and flasks were purchased from Becton Dickinson LabWare (Franklin Lakes, NJ). The reporter lysis buffer, luciferase assay kit, and ß-galactosidase assay kits were purchased from BD Biosciences (Palo Alto, CA). FSH was purchased from the National Hormone and Peptide Program (Harbor-University of California-Los Angeles Medical Center, Torrance, CA). The following supplies were obtained from the Microbiology Department of Pennsylvania State Hershey Medical Center: 100x penicillin/streptomycin solution, DMEM, Ham’s F-10, trypsin, and PBS. The pLUC vector was a gift from Dr. Rich Day (University of Virginia, Charlottesville, VA). Primers for PCR and sequencing were either generated by the Hershey Medical Center Core Facility or purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Other materials used included formamide (Roche, Indianapolis, IN), formaldehyde (Fluka Chemicals, Buchs, Switzerland), Oligo(deoxythymidine) (PE Applied Biosystems, Foster City, CA), HEPES (Fisher Scientific, Fair Lawn, NJ), and herring sperm DNA (Promega Corp., Madison, WI).

Granulosa cell culture
Granulosa cells were harvested from slaughterhouse ovaries. Five- to 8-mm follicles were opened using a surgical scalpel, and cells were scrapped off as previously described (3, 4, 5) into a base wash medium of 50% DMEM and 50% Ham’s F-10 supplemented with penicillin/streptomycin, 0.026 M HEPES, 0.012 M NaOH, and 32 µg/ml bactrin. After five washes and centrifugations, the cells were dispersed and cultured in 250-ml Falcon flasks in the base medium supplemented with 10% horse serum. Cultures were maintained in a 5% CO2 incubator at 100% humidity until confluent. To establish passaged cell cultures, frozen cells were thawed and washed once in the base medium and seeded at a density of 0.5–1 x 106 cells in 60-mm Falcon cell culture dishes and cultured in 10% fetal bovine serum to 99% confluence. Cultures were then serum-starved overnight and incubated in serum-free medium with 100 ng/ml FSH or an equal volume of PBS (for controls) for 0, 1, 3, 6, 9, 12, or 24 h depending on the objective of the experiment.

Evaluation of IGFBP-3 mRNA expression
To evaluate the effect of FSH on the regulation of IGFBP-3 mRNA expression, we employed RT-PCR. Total RNA was extracted from the cultured granulose cells using the Qiagen (Valencia, CA) RNeasy total mRNA kit, according to the manufacturer’s instructions, and quantified using a spectrophotometer at 260 nm. To generate cDNA, 1 µg total RNA was reverse transcribed with 10 U Superscript II reverse transcriptase in a reaction mix containing 0.5 µg oligo(deoxythymidine) and 0.1 mM deoxy-NTP mix, first strand buffer, and 0.1 M dithiothreitol at 42 C for 1 h. The cDNA generated was then amplified by PCR using primers designed from the open reading frame sequence of the IGFBP3 gene (forward, 5'-TCC ACA TCC CTA ACT GCG AC-3'; reverse, 5'-TCA ATT CGC CAC AAG GAG AC-3') and Jumpstart Taq polymerase (Sigma-Aldrich Corp.). Briefly, 2 µl cDNA from each RT reaction were used as template for a 30-cycle PCR reaction with an initial denaturation temperature of 95 C for 1 min, amplifications at 94 C for 45 sec and 55 C for 45 sec, and a final incubation at 72 C for 1 min. The reaction was completed by a 5-min elongation step at 72 C at the end of the last cycle. As an internal control, primers for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; forward, 5'-ATG GCA AAT TCC ACG GCA C-3'; reverse, 5'-TCC ACG ACA TAC TCA GCA CCA G-3') were included for each PCR. The PCR amplification products were resolved by agarose gel electrophoresis, stained with ethidium bromide, and recorded using Polaroid 665 film (Sigma-Aldrich Corp.). The staining intensity of the DNA bands was quantified by densitometric scanning using a laser scanner (Molecular Dynamics, Sunnyvale, CA) and Quantity One of the PDI analysis software for biological data. The PCR conditions used were initially optimized for the primer sets used to ensure nonsaturation. The quantity of GAPDH cDNA was used to adjust the calculated amount of IGFBP-3 mRNA for each reaction. We also determined that FSH had no effect on the expression levels of GAPDH by the granulosa cells used.

Evaluation of mRNA stability
To evaluate whether FSH treatment has an effect on IGFBP-3 mRNA stability, a selective inhibitor of RNA polymerase II, DRB, which arrests gene transcription, was used. Cells were treated with FSH or PBS for 3 h (the optimal mRNA induction time), washed in serum-free medium, and then cultured in serum-free medium with 75 µM DRB. Total RNA was extracted at the indicated time points. The percentage of IGFBP-3 mRNA remaining at each time point was expressed as a percentage of the 0 h IGFBP-3 mRNA level.

Genomic DNA library screening for porcine IGFBP-3
PCR primers designed from the Sus scrofa exon 2 IGFBP-3 sequence (5'-AAA CGG CAG TGA GTC C-3' and 5'-TGA TCA CGT CCA TC-3') were used to generate a 300-bp product that was subcloned into a TA vector (Promega Corp.), sequenced to confirm its identity, and labeled with [32P]deoxy-ATP (NEN Life Science Products, Boston, MA) using the Prime-a-Gene labeling system (Promega Corp.). The specific activity of the probe was determined using a ß-counter. A porcine genomic DNA library from Clontech Laboratories (Palo Alto, CA) was screened using standard screening procedures (10, 11). Positive clones were harvested twice to isolate pure clones. The {lambda} DNA was purified from an initial eight plaques using the {lambda} Maxi Kit (Qiagen).

IGFBP-3 subcloning and sequence analysis
The IGFBP-3 {lambda} DNA was digested using BamHI, and resolved on 0.8% agarose/ethidium bromide gels. Southern blot hybridization with a porcine IGFBP-3 DNA probe generated by PCR with exon 1 primers (forward, 5'-CGG CTG CTG CCT GAC CTG-3'; reverse, 5'-CCG CAT CGC TCG GTG TAG AC-3') was used to identify the fragment with exon 1 sequence. This and all the other BamHI-cut DNA fragments (five in all) were subcloned into a pGEM-7z+ vector (Promega Corp.), and the sequence was determined by the Pennsylvania State Hershey Medical Center Core Facility using an ABI PRISM 377 DNA sequencer (PE Applied Biosystems). For each subcloned plasmid, the sequence was determined starting with vector-specific primers (T7 and SP6) and completed using a sequence walking strategy. Each segment of the sequence was independently sequenced at least twice. Software from the Genetics Computer Group (SeqWeb version 2 and Wisconsin Package version 10.3, Accelrys, Inc., San Diego, CA) was used to assemble the sequence of each subclone. The identity of the gene was validated by use of NCBI Blast program (12). To confirm the relative position of each BamHI-cut subclone in the IGFBP-3 gene, primers adjacent to the BamHI sites were designed and used to read the intact {lambda} DNA sequence across each subclone to the next. This strategy was important for reading the sequence of a very small fragment (47 bp) that was difficult to see on agarose gels and was therefore not subcloned, and a second fragment that was nearly the same size as one of those we subcloned (1023 bases) and was therefore difficult to tell apart on a standard agarose gel. To identify intron-exon boundaries, we compared our sequence to the pig cDNA sequence available in the GenBank database (13, 14). The classic splice site sequences were used to validate the boundaries. The end of the exon 5 sequence was predicted by the presence of a polyadenylation signal downstream of the stop codon at the end of the open reading frame.

Generation of IGFBP-3 promoter constructs
Using the Sus scrofa exon 1 sequence from the database (accession no. AF085482) (14), we identified the location of the promoter sequence. The presence of a TATA box and analysis of the sequence using PubMed’s Alibaba2 program helped us identify the predicted transcription start site and predict the putative transcription factor binding sites present in the promoter. To analyze promoter function, we used PCR to generate a series of progressively shorter promoter constructs sharing a common 3' end. The longest construct tested was from -1388/+47, and the shortest constructs were -47/+47 and -47/+9 (we arbitrarily defined the predicted transcription start site as nucleotide +1). A SalI recognition site was incorporated at the beginning of the reverse primer, and a BamHI site was incorporated at the beginning of the forward primers to facilitate cloning into a pLUC vector, which exhibits little or no response to FSH in our system. Because of the high GC content of the template used, 1.3 M betaine was included in the PCR reaction, and the annealing temperature was raised to 62 C.

Evaluating the effect of FSH on IGFBP-3 transcription activity
Triplicate sets of porcine granulosa cells cultured in 60 x 15-mm Falcon dishes were transiently transfected with 1 µg IGFBP-3 promoter DNA and 0.25 µg ß-galactosidase plasmid DNA/dish using the lipofectin method (Life Technologies, Inc./Invitrogen). After 6 h, the DNA was removed, and cells were cultured in 10% fetal bovine serum for 24 h. Cells were then serum-starved overnight before adding FSH (100 ng/ml) or an equal volume of PBS for control cells. Promega’s luciferase assay system (to measure IGFBP-3 promoter activity) was used in combination with Promega’s ß-galactosidase assay kit (to serve as an internal control). Both luciferase and ß-galactosidase activities were read in a Monolight 3010 luminometer (BD PharMingen, San Diego, CA). Controls of nontransfected cells and cells transfected with a promoterless pLUC vector exhibited reporter activity that was barely detectable. A time course of promoter activity after FSH treatment indicated that maximal response was obtained at 3 h. Accordingly, subsequent evaluations were performed 3 h after FSH treatment. Each assay was repeated at least three times.

Evaluating the effect of prolonged FSH exposure on IGFBP-3 transcription
We observed an increase in both IGFBP-3 mRNA and luciferase activity at 3 h. In contrast, there was an obvious reduction of IGFBP-3 mRNA and no significant change in luciferase activity at 24 h. We therefore sought to determine the mechanism of the divergent effect of prolonged FSH exposure on IGFBP-3 transcription activity. Simply extending the incubation time after transfection before luciferase assay would not accurately assess late promoter activity, as the amount of luciferase would be a combination of new transcription plus stable mRNA from earlier times. Thus, we took an alternative approach in which granulosa cells were cultured to 50–60% confluence, serum-starved for 6 h, and divided into three groups. For each group, triplicate sets of cells were maintained in serum-free medium supplemented with FSH (group 1) and PBS (groups 2 and 3) and transiently transfected with the -386/+47 IGFBP-3 promoter construct. At the end of the 3-h transfection, cells were cultured for another 3 h, with FSH being maintained in group 1 cells for the entire time and added to group 2 cells at the end of the transfection (to assess transcription early after FSH treatment). Group 3 cells (no FSH) served as the control. Table 1Go summarizes the experimental approach used. The relative luciferase activities of the three groups of cells were then assayed and compared.


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  		TABLE 1. Schematic presentation of the experimental approach used to evaluate the effect of prolonged FSH exposure of IGFBP-3 reporter activity

 
Statistical analysis
Each experiment was repeated at least three times, and data were analyzed using PRISM software (GraphPad, Inc., San Diego, CA) (15). Differences between groups were analyzed using paired t tests for mRNA expression and two-way ANOVA for reporter activity. The Bonferroni method was used to test for differences between various reporter constructs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FSH increases IGFBP-3 mRNA
We evaluated the effect of FSH treatment on IGFBP-3 mRNA expression by cultured porcine granulose cells at 0, 1, 3, 6, 9, 12, and 24 h after FSH treatment. Our data show a strong induction of IGFBP-3 mRNA levels that was significant 1 h after FSH addition, maximal at 3 h, and sustained through 9 h. At 12 h, IGFBP-3 mRNA level began to decline. Interestingly, by 24 h the mRNA levels in FSH-treated cells were lower than those in controls without FSH cultured for the same length of time (Fig. 1Go, A and B). At 3 h, FSH treatment induced a nearly 3-fold increase in relative IGFBP-3 mRNA (Fig. 1CGo). To test whether the changes in mRNA levels in FSH-treated cells were due to a reduction in IGFBP-3 mRNA half-life, we evaluated the stability of IGFBP-3 mRNA in cells treated with FSH compared with control cells without FSH. IGFBP-3 mRNA levels in cells treated with FSH for 3 h before arresting mRNA transcription by introducing the RNA polymerase inhibitor DRB were significantly higher than those in control cells without FSH (3-fold higher; P < 0.05). However, there was no significant difference in the rate of RNA decay over the 27-h period evaluated between the two groups of cells (Fig. 2Go).



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  		FIG. 1. Granulosa cell IGFBP-3 mRNA expression after FSH treatment. Cells were cultured in 10% serum to 95% confluence, then serum-starved. The medium was then changed, and culture was continued in serum-free medium with 100 ng/ml FSH. Initially, a time course was performed with IGFBP-3 mRNA extracted at 0 h used as the baseline (A and B). Subsequently, the 3 h point was used, and the experiment was repeated six times (C). IGFBP-3 mRNA was quantified using densitometric scanning, and GAPDH was used as an internal control to normalize IGFBP-3 mRNA levels. The bars in C represent the mean ± SE of six separate experiments. IGFBP-3 mRNA was significantly higher (P < 0.05) in cells treated with FSH compared with controls without FSH.

 


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  		FIG. 2. Effect of FSH treatment on IGFBP-3 mRNA stability. Granulosa cells were cultured as described in Fig. 1Go and treated with FSH for 3 h. The culture medium was then changed, and cells were kept in serum-free medium supplemented with 75 µM DRB. Total RNA was extracted, and RT-PCR was used to amplify IGFBP-3 mRNA. Using the IGFBP-3 mRNA at 0 h as 100%, the IGFBP-3 level at each time point was quantified and expressed as a percentage of the 0 h mRNA level. There was no significant difference in the rate of IGFBP-3 mRNA decay between FSH-treated cells and controls without FSH.

 
IGFBP-3 gene sequence
We have determined 12,257-bp sequence of the pig IGFBP-3 gene. This sequence has been submitted to the GenBank database (accession no. AY464121). The sequence includes 4296 bp of 5'-flanking promoter sequence, part of which is shown in Fig. 3Go, 543 bp of exon 1, 2943 bp of intron 1, 227 bp of exon 2, 808 bp of intron 2, 120 bp of exon 3, 1021 bp of intron 3, 139 bp of exon 4, 678 bp of intron 4, and 1362 bp of exon 5. The overall organization of the pig gene is similar to that of the human (16) and bovine (17) genes, with minor differences in intron length. A schematic presentation of this sequence and how it compares to that of the human and bovine is shown in Fig. 4Go. We were able to accurately estimate the size of the pig exon 5 sequence by making a prediction based on the presence of the polyadenylation signal downstream of the stop codon. The stop codon is at nucleotide 10,837, which corresponds to the downstream end of the cDNA sequence reported by Pampusch et al. (14). However, the Shimasaki cDNA sequence (13) has another 226 bp of cDNA sequence downstream of the stop codon, which is also present in our genomic sequence. The classic polyadenylation signal is found 1,252 bases downstream of the stop codon (12,097–12,102), which gives a total cDNA sequence of 2,391. Another 161 bp of sequence is found after the polyadenylation signal. This would give an IGFBP-3 mRNA close in size to that reported for Northern blot analysis (2.6 kDa) (13, 18, 19). Figure 6Go shows the peptide sequence obtained from projected translation of the open reading frame of our DNA sequence.



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  		FIG. 3. Porcine IGFBP-3 promoter sequence used to design the promoter constructs used in our studies. The predicted transcription start site is numbered +1. The currently available promoter sequence spans a total of 4296 bases and can be accessed in the GenBank database (accession no. AY464121).

 


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  		FIG. 4. Schematic representation of the 12,257-bp pig IGFBP-3 sequence and how it compares to the bovine and human IGFBP-3 gene sequences. The pig IGFBP-3 gene was cloned from a commercial porcine genomic library (Clontech) digested with BamHI. Resulting DNA fragments were subcloned into pGEM-7z+ vector, and sequence was determined using the walking strategy. Exons are indicated as boxes, and the promoter and introns are indicated as lines.

 


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  		FIG. 6. A comparison of the peptide sequence obtained from projected translation of the open reading frame of the pig genomic DNA sequence determined in the present study (porcine 2) with the peptide sequences of the human, bovine, rat, and mouse peptide sequences. Porcine 1 is the sequence determined from direct sequencing of the pig IGFBP-3 protein (13 ).

 
We compared our pig IGFBP-3 promoter sequence to those of the human (16) and bovine (17, 20) IGFBP-3 promoters and a mouse genomic clone from the GenBank database that included IGFBP-3 (clone RP-20C9 on chromosome 11; accession no. AL607124) (21), all of which were found to be similar by a Blast search. This comparison revealed two highly conserved regions in the promoter sequence of the pig, human, and bovine genes and in a corresponding region of the mouse DNA clone. The first, a 38-bp sequence, lies between -1095 and -1058 in the pig and is 100% and 95% identical to the bovine and human promoters, respectively, and 90% identical to the mouse DNA clone (Fig. 5AGo). The only transcription factor binding site predicted in this region is a partial GATA-1-binding site. The second conserved region is a 73-bp region that lies between -63 and +10 of our pig sequence, and is 95% identical to both the human and bovine IGFBP-3 promoters and 93% identical to the mouse DNA clone (Fig. 5BGo). This region includes the TATA box and the predicted transcription start site, suggesting that the transcription factor-binding sites within the region may be involved in the modulation of IGFBP-3 gene transcription. Transcription factor-binding sites predicted to be present in the conserved 73-bp region of the pig sequence are four Sp1 sites (three of which overlap over a 24-bp sequence), TATA binding protein, Oct-1, and activating protein-2{alpha} (AP-2{alpha}). Although these sites are present in the bovine and human promoters and the mouse DNA clone, there are some differences. In the human there is an additional Sp1-, an AP-2-, and a MIG1 (GC box-binding zinc finger protein that is a member of metabolic regulators in yeast)-binding site, whereas in the mouse only three Sp1-binding sites are present. However, there is an additional AP-2 site, a MIGI, a YYI, a Pit-1a, and a nuclear factor-1 site in the mouse clone. Transient transfection studies (see below) demonstrate that the -61/-48 sequence of the pig IGFBP-3 promoter is required for FSH induction of the gene.



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  		FIG. 5. A comparison of two regions in the pig IGFBP-3 promoter that appear to be highly conserved in the human and bovine IGFBP-3 promoters and a mouse cDNA clone (clone RP-20C9). The first region is 38 bp (-1095 to -1058) and is 100%, 95%, and 90% identical to the bovine, human, and mouse cDNA clones, respectively. The second, a 73-bp region between -63 and +10, is 95% identical to the bovine and human IGFBP-3 promoters and 93% identical to the mouse cDNA clone.

 
We also compared the coding sequence from our pig sequence to that of pig cDNA sequences in the GenBank database (13, 14). Our sequence adds 36 bases upstream (to the predicted transcription start site) of the cDNA sequence submitted by Pampusch et al. (14) and 245 bases upstream of the partial cDNA sequence submitted by Shimasaki et al. (13). Although the Shimasaki cDNA sequence is 100% identical to the corresponding regions of our genomic sequence, the porcine IGFBP-3 peptide sequence in the database (13) differs in one amino acid from the peptide sequence obtained from translating our sequence (Fig. 6Go). Additionally, translating our sequence adds a 27-amino acid sequence upstream of the Shimasaki IGBP-3 peptide sequence, making the porcine IGFBP-3 peptide comparable in size to the bovine, human, rat, and mouse peptides. Based on this outcome, we predict the translation start site to be at nucleotide +135 relative to the transcription start site. The high percent identity with the three species confirms the validity of our genomic DNA sequence.

FSH induces IGFBP-3 transcription
Transient transfection of granulosa cells with IGFBP-3 promoter vectors showed an induction of IGFBP-3 gene transcription by FSH. Granulosa cells transfected with the longest IGFBP-3 promoter construct tested (-1388/+47) had a significantly higher (P < 0.0001) luciferase activity compared with nontransfected cells or cells transfected with a promoterless pLUC vector (Fig. 7Go). FSH treatment of transfected cells significantly stimulated luciferase activity (>4-fold; P < 0.001). This induction was significant at 1 h and maximal at 3 h after FSH treatment. However, the luciferase activity began to decline by 12 h, and by 24 h the luciferase activity of the FSH-treated cells was not significantly different from that of controls without FSH (Fig. 8Go). The higher levels of luciferase activity after FSH treatment were also observed for cells transfected with shorter promoter constructs (-386/+47, -191/+47, -61/+47, and -61/+9), suggesting that sequence within the -61/+8 of the promoter contains critical elements required for FSH stimulation of the IGFBP-3 gene transcription (Fig. 9Go). Among the FSH-responsive promoter constructs tested, luciferase activity and the fold change in relative luciferase activity were significantly lower in cells transfected with the -61/+47 and -61/+9 promoter constructs (P < 0.01) compared with cells transfected with the promoter constructs with sequence upstream of -61 (Fig. 9Go). This suggests that sequence between the -191 and -62 regions may contain transcription factor sites that are important in enhancing basal and/or FSH-stimulated transcription of the IGFBP-3 gene. Indeed, a second region that is rich in transcription factor-binding sites is found between -102 and -65. The putative transcription factor binding sites present in this region are five overlapping Sp1 sites, two overlapping Krox-20 sites, an AP-2, a WT1, an AP-2{alpha}, and an Adf-1 site. Deletion of sequence between -61 and -48 not only abolished the FSH induction of IGFBP-3 promoter activity, but also reduced basal luciferase activity to the levels observed in control cells transfected with a promoterless pLUC vector construct. This suggests that the sequence in this region is required for both basal and FSH regulation of IGFBP-3 gene transcription. The only transcription factor-binding site predicted in this region is Sp1.



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  		FIG. 7. Reporter activity of porcine granulosa cells transiently transfected with a -1388/+47 IGFBP-3 promoter construct. Luciferase activity was assayed using a commercial assay kit (Promega Corp.) according to the manufacturer’s instructions and was read with a Monolight 3010 luminometer. Assay of ß-galactosidase activity was used as an internal control to normalize luciferase activity. Two negative controls were included, control 1 was made of cells transfected with a promoterless pLUC vector (vector) treated with FSH for 3 h, and control 2 was made of cells transfected with the -1388/+47 IGFBP-3 promoter construct, but treated with PBS. The luciferase activity for cells treated with FSH for 3 h was significantly higher than that for cells exposed to FSH for a total of 24 h and for those treated with PBS (P < 0.001).

 


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  		FIG. 8. A time course of IGFBP-3 reporter activity after transfection with a -1388/+47 IGFBP-3 promoter construct. Reporter activity was assayed at 0, 1, 3, 6, 9, 12, and 24 h post-FSH treatment. Based on these data, subsequent reporter activity was assayed at 3 h post-FSH treatment.

 


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  		FIG. 9. Reporter activity of granulosa cells transiently transfected with different IGFBP-3 promoter constructs. A schematic representation of the constructs tested is presented in A. Assays for ß-galactosidase activity were performed and used to normalize the luciferase activity levels. The fold change in C was determined by setting the value for control cells at 1. There was a significant reduction in luciferase activity for the -61/+47 construct compared with the other longer constructs tested. Deletion of the sequence between -61 and -48 led to loss of the FSH response and a significant reduction in basal luciferase activity.

 
It seemed important to determine whether the apparent decrease in FSH stimulation of IGFBP-3 transcription was caused by FSH or related to other changes during the 24 h of culture. Using the -386/+47 promoter construct and the experiment described in Table 1Go, we found no significant difference in luciferase activity between cells exposed to FSH for 24 h (with medium change and addition of fresh FSH) and control cells not exposed to FSH. On the contrary, cells exposed to FSH for only 3 h had significantly higher luciferase activity compared with controls without FSH or cells exposed to FSH for 24 h (Fig. 10Go). Thus, prolonged exposure to FSH (24 h) abolishes FSH induction of IGFBP-3 gene transcription.



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  		FIG. 10. Effect of prolonged exposure to FSH on IGFBP-3 gene transcription by porcine granulose cells. Group 1 cells were treated with FSH for 24 h (with medium change and fresh FSH addition); group 2 cells were exposed to FSH only for the 3 h before luciferase assay. Group 3 cells did not receive FSH (see Table 1Go for experimental protocol). Transfected granulosa cells treated with FSH had significantly higher (P < 0.01) reporter activity compared with controls without FSH and cells treated with FSH for 24 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBP-3 is known to regulate IGF bioavailability by forming heterodimeric complexes with IGF-I or IGF-II and the 85-kDa acid-labile subunit. Under normal physiological conditions, the IGF-IGFBP-3 complex acts as a storage pool for circulating IGFs and thus prolongs their half-life (22). As both IGFBP-3 and acid-labile subunit are expressed in the ovary (2), this reservoir function is probably important. In addition, there is growing evidence for IGF-independent actions of IGFBP-3 (23). The presence of cell surface IGFBP-3 receptors has been suggested (24, 25, 26), and an intracellular role of IGFBP-3 in the regulation of IGF action has been demonstrated. IGFBP-3 can be inhibitory to cell growth and survival even in the absence of IGF signaling (27). Furthermore, translocation of IGFBP-3 into the nucleus occurs in actively dividing cells (28, 29), and nuclear IGFBP-3 has been identified in a variety of cells, suggesting that IGFBP-3 may directly control gene expression. It has further been demonstrated that IGFBP-3 binds to the nuclear receptor, retinoic X receptor {alpha}, a key gene transcription regulator, and antiproliferative effects of retinoic acid and p53 protein have been shown to be mediated through increased expression of IGFBP-3 (30).

No uniform function has been established for IGFBP-3, and both stimulatory and inhibitory actions are possible even within one cell. The action of extracellular IGFBP-3 may differ from that of locally produced IGFBP-3. Thus, transfection with a plasmid vector containing IGFBP-3 cDNA made mammary cells more responsive to IGF-I in terms of cell growth. This response could not be elicited by addition of exogenous IGFBP-3 (31). Endogenous IGFBP-3 has further been shown to induce an increase in Akt phosphorylation and an increase in Akt enzyme activity (31). The role of endogenous IGFBP-3 in the pig ovary has not been settled. Several studies (32, 33), including some with pig granulosa cells (4, 5, 34), have shown inhibitory effects of exogenous IGFBP-3 on granulosa cell function. However, the effects of locally produced IGFBP-3 may be quite different. We consider mapping of the pig IGFBP-3 promoter a key step in our understanding of IGFBP-3 gene regulation; these studies seem essential to understanding the role of ovarian IGFBP-3.

In the present study we have cloned and sequenced 12,257 bp of the pig IGFBP-3 gene, which includes a 4,296-bp promoter sequence and a 2,492-bp coding sequence. As discussed in Results, our data add significantly to the coding sequence and provide the first information on the porcine promoter. The human, bovine, and pig promoters share two conserved regions: a 38-bp region between -1058 and -1095, and a 73-bp region between -63 and +10 relative to the transcription start site. Our studies failed to disclose a regulatory role for the 38-bp upstream conserved sequence in FSH-stimulated transcription of the IGFBP-3 gene. However, it may be important in vivo or in the modulation of the IGFBP-3 gene by other factors. The downstream conserved region includes the TATA box and transcription start site. Among the putative transcription factor-binding sites predicted in the 73-bp (-63/+10) conserved region of the pig promoter sequence is an AP-2{alpha} site. AP-2 recognition sites have been reported to mediate cAMP induction of IGFBP-3 mRNA (35). In hepatoma cells, the IGFBP-3 promoter can be activated by up-regulating Sp1 activity (36); hence, the Sp1-binding sites could be important in regulating the IGFBP-3 gene. Indeed, our truncated reporter genes have localized the dominant FSH-responsive elements to a region between -61 and -48, which contains a putative Sp1 site (see below). The presence of a site for the tumor suppressor, WT1 (at -93/-84) could indicate involvement of the IGFBP-3 gene in regulation of tumor development. Studies should be performed to determine whether the WT1-binding site is functional, because there is growing evidence for involvement of IGFBP-3 in apoptosis, a process that is critical in suppressing tumor growth and is the underlying mechanism for follicular atresia (24, 37, 38).

Various hormones and growth factors have been shown to regulate IGFBP-3. In general, the expression of the IGFBP-3 gene is up-regulated by growth inhibitory (and apoptosis-inducing) agents such as retinoic acid (37, 38, 39), vitamin D (40), and TGFß (37, 41, 42), antiestrogens such as tamoxifen, ICI 182,780 (43), IL-1 (41), TNF{alpha} (41, 44, 45), glucocorticoids (46), and the tumor suppressor gene p53 (47). In contrast, cytokines and growth factors such as epidermal growth factor (48) have been shown to down-regulate IGFBP-3 expression. Elevated calcium levels in cell culture medium caused a reduction in secreted IGFBP-3 protein in a human keratinocyte cell line, HaCaT (7), whereas cAMP reduced IGFBP-3 expression in a dose-dependent manner (8). However, in a separate study cAMP and agents that increase intracellular cAMP levels (such as forskolin) stimulated IGFBP3 mRNA via a protein kinase A-mediated process (6). IGFBP-3 may thus be a common downstream target and/or effector of many growth regulatory agents.

Only a few studies have evaluated IGFBP-3 expression and regulation in the ovary, and for most species they have emphasized the corpus luteum (19, 49, 50, 51, 52, 53, 54, 55, 56). In the pig, IGFBP-3 expression is higher in the porcine corpus luteum than in follicles (2, 18). As in other species (monkey and human) (55), endothelial cells in the corpus luteum are a major source of expression. Using in situ hybridization, Wandji et al. (2) also demonstrated follicle cell expression of IGFBP-3 mRNA, with the thecal and granulosa layers both contributing. Based on our culture studies (3, 4, 5), in situ hybridization (2), and assessment of follicular fluid levels (57), IGFBP-3 seems physiologically regulated in the pig ovary. In cultured porcine granulosa cells, TGFß and FSH inhibited, whereas epidermal growth factor and IGF-I enhanced, IGFBP-3 secretion (3, 4, 5). LH stimulated IGFBP-3 production by bovine thecal cells, but inhibited IGFBP-3 production by granulosa cells (58). FSH also inhibited binding of IGFBP-3 to IGF-I in human granulosa cells by stimulating IGFBP-3 proteolysis (59).

We sought to better understand the importance and mechanism of ovarian IGFBP-3 regulation. Using more sensitive RT-PCR and luciferase assay techniques, we discovered that the regulation of IGFBP-3 by FSH is more complex than previously documented. The fact that FSH stimulated IGFBP-3 gene transcription within 1 h suggests that FSH has a direct effect on IGFBP-3 gene transcription. However, this effect was biphasic, and prolonged FSH treatment actually inhibited the transcription of reporter genes and the expression of IGFBP-3 mRNA. One possibility for the early induction and later reduction in IGFBP-3 mRNA levels and transcription activity could be a reduction in the IGFBP-3 mRNA half-life by FSH treatment. In other cell types, cAMP changes IGFBP-3 mRNA stability (6, 8, 60). However, we did not find a significant change in IGFBP-3 mRNA stability between FSH-treated cells and controls without FSH. The inhibitory action was the dominant effect of FSH in our earlier studies of IGFBP-3 and its mRNA (3, 34), with measurements conducted after 24–48 h of FSH treatment in culture. However, we could demonstrate a stimulatory action by low FSH concentrations (4). FSH may thus regulate IGFBP-3 in a truly biphasic manner, with an early induction and later inhibition mediated by different signal transduction pathways. Further studies will be required to establish the mechanism of the inhibitory effect of FSH. It is possible that after the early induction of IGFBP-3 gene transcription, the protein levels of locally produced intracellular IGFBP-3 increase. The IGFBP-3 could then modulate other signal transduction pathways in a stimulatory or inhibitory manner, ultimately inhibiting FSH induction of IGFBP-3 gene transcription in a negative feedback-like loop. Regarding the stimulatory effect, the sequence within the -61/+9 region relative to the transcription start site was sufficient for the FSH response. Deletion of the sequence between -61 and -48 abolished FSH stimulation and basal gene transcription. This suggests that this sequence contains the FSH-responsive elements on the IGFBP-3 gene.

We have postulated that the IGF system plays an autocrine/paracrine role in ovarian function (61, 62, 63, 64, 65). However, it was not clear whether FSH activated a signal transduction pathway that directly stimulated IGFBP-3 gene transcription, or whether it caused the synthesis of intermediate protein molecules that then modulated IGFBP-3 gene transcription. In the present study we demonstrate that FSH directly stimulates IGFBP-3 gene transcription. This reinforces previous suggestions that FSH can directly modulate the secretion of endogenous IGFBP-3 by ovarian granulosa cells. To understand the significance of this pathway, the autocrine actions of IGFBP-3 will need to be clarified. The endogenously secreted IGFBP-3 could be transported into the nucleus and modulate the transcriptional activity of other genes that play key roles in ovarian function. As IGFBP-3 appears to be a common effector of many signaling pathways, including apoptosis, modulation of endogenous IGFBP-3 levels in granulosa cells can potentially regulate mechanisms that either enhance cell survival or induce apoptosis and thus lead to follicular atresia. Studies such as those employing overexpression vectors or inhibitory DNA and/or RNA constructs are needed to evaluate the role of endogenous IGFBP-3. The current studies will pave the way for efforts to manipulate endogenous IGFBP-3 expression in pig granulosa cells and thus better understand the role of IGFBP-3 in ovarian function.


   Acknowledgments
 
We thank Dr. Rich Day (University of Virginia) for his kind gift of the pLUC vector used for making our promoter constructs. Many thanks to Melissa Cunningham, an M.D./Ph.D. student in Dr. Hammond’s laboratory, for her advice and assistance with many aspects of the study.


   Footnotes
 
This work was supported by Grant HD-24564.

Abbreviations: AP-2, Activating protein-2; DRB, dichloro-ß-D-ribofuranosyl-benzimidazole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP-3, IGF-binding protein-3.

Received November 17, 2003.

Accepted for publication December 31, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. M. Ongeri, M. F. Verderame, and J. M. Hammond
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