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*Nutrition
Endocrinology Vol. 142, No. 11 4643-4651
Copyright © 2001 by The Endocrine Society

ARTICLES

Up-Regulation of Upstream Stimulatory Factors by Protein Malnutrition and Its Possible Role in Regulation of the IGF-Binding Protein-1 Gene

Tadashi Matsukawa1, Yoshihisa Inoue1, Yuichi Oishi, Hisanori Kato and Tadashi Noguchi

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Address all correspondence and requests for reprints to: Dr. Hisanori Kato, Department of Applied Biological Chemistry, The University of Tokyo Graduate School of Agricultural and Life Sciences, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: akatoq{at}mail.ecc.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein malnutrition drastically induces the expression of the IGF-binding protein-1 (IGFBP-1) gene. We have previously shown that the region between -77 and -112 bp upstream of the rat IGFBP-1 gene contributes to the response of this gene to amino acid limitation. In an attempt to elucidate the basis of the responsiveness of this putative amino acid response unit (AARU), we searched the nucleus of the rat liver for a trans-acting factor whose binding to AARU was dependent on protein nutrition. Liver nuclear extracts of rats fed a protein-free diet and of those fed a control diet were compared by EMSA using the AARU as probe. One of the protein-probe complexes underwent a drastic increase after dietary protein deprivation. Assays using specific antibodies and several competitor oligonucleotides led to identification of the protein composing the complex as upstream stimulatory factor-1 (USF) and USF-2. The binding site of the USF proteins in the AARU turned out to be a CACGGG sequence that was homologous to the consensus USF-binding sequence (E box; CANNTG). Further, Western blot analyses showed that a protein-free diet caused significant increases in USF-1 and USF-2 levels. Thus, elevated expression of the IGFBP-1 gene under protein malnutrition can be attributable to increased binding of USF to its promoter, which results from increased USF levels. The data suggest that the changes in these ubiquitously distributed transcription factors play an important role in the nutritional regulation of expression of mammalian genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEIN MALNUTRITION, resulting from an insufficient supply of protein or intake of proteins with poor nutritional value, remains a serious and prevalent problem in many countries. It leads to alterations in physiological and pathological status and can result in growth retardation, higher susceptibility to infectious diseases, emaciation, and sometimes death (1). Protein malnutrition is accompanied by changes in the expression of a number of genes (2, 3, 4). The altered expression of some of these genes causes the adverse conditions mentioned above, but may also serve as an adaptive response of the body to maintain homeostasis or simply to survive. Whereas many genes are suppressed by protein malnutrition, the expression of some genes is elevated. Among the latter, the gene encoding IGF-binding protein-1 (IGFBP-1) (1) is highly sensitive to nutritional status (3, 5, 6, 7).

IGFBP-1, which is mainly synthesized and secreted by the liver (8, 9), is considered to modify the activity of IGFs. The plasma level of IGFBP-1 is increased by fasting, diabetes, and malnutrition (10, 11, 12, 13, 14, 15). It has been shown that transcription of the IGFBP-1 gene is suppressed by insulin and calorie intake and is enhanced by glucocorticoids and fasting (16, 17, 18, 19). Analyses of the promoter region of the IGFBP-1 gene revealed putative binding sites of a number of transcription regulation factors, including hepatocyte nuclear factor-1 (HNF-1), GR, cAMP response element-binding protein, HNF-3, and insulin response element (IRE)-binding protein (20, 21, 22, 23, 24, 25). The mechanisms by which these factors regulate IGFBP-1 gene expression have been extensively studied. Insulin, for instance, suppresses IGFBP-1 gene expression by phosphorylating a forkhead transcription factor, FKHR, followed by exclusion of this factor from the nucleus (26, 27, 28, 29).

Expression of the IGFBP-1 gene is very sensitive to the status of protein nutrition. We have shown that protein nutrition regulates the expression of the IGFBP-1 gene at the transcriptional level in vivo and in vitro. In rats fed a protein-free diet for 1 wk, for example, a 3-fold increase in the transcription rate of the IGFBP-1 gene with a 5-fold elevation of its mRNA level were observed (7). Using cell culture systems, we further characterized the transcriptional regulation of the IGFBP-1 gene by amino acid limitation and identified a region that participates in the response of the IGFBP-1 promoter to amino acid availability (30). These studies suggest that this region, tentatively referred to as an AARU, participates in the response to protein restriction in vivo. This AARU corresponds to -77 to -112 bp upstream of the transcription start site of the IGFBP-1 gene and contains a glucocorticoid response element (GRE), an IRE, and an HNF-3-binding site. However, no information is yet available on the mechanism by which protein nutrition regulates IGFBP-1 promoter activity throughout this region.

The aim of the present study was to identify the transcription factor that plays a pivotal role in the stimulation of IGFBP-1 gene expression by protein malnutrition. We first investigated whether the nucleus of the rat liver contained any transcription factor for which the binding capacity to AARU of the IGFBP-I promoter was strongly affected by dietary protein deprivation. We could detect one of the candidate factors, and we identified it as a mixture of upstream stimulatory factor-1 (USF-1) and USF-2 and further revealed that the USFs themselves were regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[{gamma}-32P]ATP, antirabbit IgG-horseradish peroxidase, and the enhanced chemiluminescence detection system were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Anti-USF-1 antibody (C-20) and anti-USF-2 antibody (C-20) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Casein, ß-corn starch, cellulose powder, mineral mixture, and vitamin mixture were obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan). The mineral mixture and vitamin mixture, respectively, were prepared according to the methods described by Rogers and Harper (31). Soybean oil and choline chloride were obtained from Nacalai Tesque Co. (Kyoto, Japan).

Animals
Male Wistar rats, 6 wk old, were purchased from Charles River Laboratories, Inc. (Kanagawa, Japan). The rats were kept in a room maintained at 22 ± 1 C with a 12-h light-dark cycle (lights on at 0800 h). The animals were given a 12% casein diet (C) between 1000–1800 h for 3 d before being switched to an experimental diet [12% C or protein-free diet (PF)]. The experimental diets were given for 7 d on the same schedule, and water was available ad libitum. The composition of the experimental diets has been described previously (32). On d 8, rats were allowed access to food for 1.5 h, then they were anesthetized with pentobarbital, and blood was taken from the carotid artery. The liver was excised, quickly frozen in liquid nitrogen, and stored at -80 C until preparation of nuclear extract. All experiments were performed under the guidelines of the animal usage committee of the Faculty of Agriculture, The University of Tokyo (Tokyo, Japan).

Preparation of liver nuclear extract
The nuclear protein extract of the liver was prepared by sucrose gradient centrifugation. The samples were kept at 0-4 C throughout the procedure. Liver tissue (1.5 g) was washed three times with ice-cold PBS containing 0.5 mM MgCl2 (PBS) and once with buffer A [10 mM HEPES-KOH (pH 7.8), 10 mM KCl, 0.2 mM EDTA, 0.35 M sucrose, 0.5 mM phenylmethylsulfonylfluoride (PMSF), 14 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM benzamidine, 1 mM dithiothreitol (DTT), 2 mM Na2VO4, 10 mM NaF, 25 mM ß-glycerophosphate disodium salt, 0.15 mM spermine, and 0.5 mM spermidine]. The tissue was hashed with scissors in 8 ml buffer A and homogenized by 15 strokes of the Potter homogenizer. The homogenate was filtrated through gauze and centrifuged for 10 min at 550 x g. The pellet was resuspended in 6 ml buffer A, mixed by one stroke of the Dounce homogenizer (Kontes Co., Vineland, NJ), transferred into an equal volume of buffer B (buffer A with the addition of 0.5 M sucrose) and centrifuged at 1300 x g for 15 min. The pellet was resuspended in 2 ml buffer C (buffer A without sucrose) and centrifuged at 1300 x g for 10 min. The pellet was dissolved in approximately twice the volume of buffer D [20 mM HEPES-KOH (pH 7.8), 0.33 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.5 mM PMSF, 14 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM benzamidine, 1 mM DTT, 2 mM Na2VO4, 10 mM NaF, and 25 mM ß-glycerophosphate disodium salt]. By slowly adding 5 M NaCl, the final concentration of NaCl was gradually adjusted to 0.33 M. This solution was homogenized by 15 strokes of the Dounce homogenizer and gently mixed by rotating for 45 min. After centrifugation at 9000 x g for 15 min, the supernatant was stored at -80 C until use. The protein content of the nuclear extract was determined by the Bradford method (protein assay kit, Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as the standard.

EMSA
EMSA was performed according to the method of Frain et al. (33). The sense and antisense strand oligonucleotides were annealed and end labeled using a MEGALABEL kit (Takara Shuzo, Japan) and [{gamma}-32P]ATP. The reaction mixture of EMSA was composed of 20 mM HEPES-KOH (pH 7.8), 20 mM KCl, 1 mM DTT, 10% glycerol, 2 µg poly(dI-dC)/poly(dI-dC) (Amersham Pharmacia Biotech), and 4 µl of the extract containing 10 µg protein and 2 µl (50,000 cpm/µl) of the probe and had a final volume of 20 µl. The mixture without the probe was preincubated for 5 min at room temperature and, after addition of the probe, was further incubated for 10 min. The mixture was electrophoresed on a 6% polyacrylamide gel at 150 V for 2 h using Tris-glycine-EDTA buffer. After drying the gel, the bands were visualized by an imaging analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Competition assays were performed by adding an excess of unlabeled double strand DNA when starting the preincubation. In the supershift assay, 1 µl (0.2 µg/µl) anti-USF-1 or USF-2 antibody (Santa Cruz Biotechnology, Inc.) was included and preincubated for 30 min before the addition of probe.

Western immunoblot analysis
Equal amounts of nuclear protein were fractionated by SDS-PAGE (Mini-Protean III, Bio-Rad Laboratories, Inc.) on 10% gels or 5–20% gradient gels (Fig. 7BGo), then transferred to a nitrocellulose membrane (Optitran BA-S 85, Schleicher & Schuell, Inc., Keene, NH) using a semidry blotting unit (Transblot SD, Bio-Rad Laboratories, Inc.). The membrane was incubated in blocking solution [10 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1 mM EDTA, and 5% BSA] at room temperature for 1 h, then treated with one of the polyclonal anti-USF antibodies [USF-1 (C-20) or USF-2 (C-20), Santa Cruz Biotechnology, Inc.] diluted by the blocking solution at room temperature for 1 h. The membrane was washed with TBS-T [20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1 mM EDTA, and 0.1% Tween 20], treated with the second antibody (horseradish peroxidase-conjugated antirabbit IgG, Amersham Pharmacia Biotech) diluted in TBS-T for 1 h, followed by a wash with TBS-T. The dilutions of the first and second antibodies were 1:500 and 1:4000, respectively. USFs were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech) and were quantified with an LAS1000plus imaging system (Fuji Photo Film Co., Ltd.).



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  		Figure 7. Partial purification and characterization of the complex I protein. The complex I protein was crudely purified without the use of an immunological technique. The purification steps consisted of gel chromatographies of SP-Sepharose, diethylaminoethyl-Sepharose, heparin-Sepharose, and DNA affinity Sepharose using the complex I-forming activity as a biochemical assay. A, Coomassie brilliant blue-stained gel of the nuclear extract and the purified protein. Approximately 0.65 µg nuclear protein was loaded on lane 1. The amount of protein loaded on lane 2 corresponded to that purified from 6.5 mg starting nuclear protein. B, Western immunoblot analysis of the partially purified protein using antibodies to USF-1 and USF-2. The electrophoresed protein (same amount as shown in lane 2 of A) was transferred to a nitrocellulose membrane and blotted with respective antibodies. Both USF-1 (43 kDa) and USF-2 (44 kDa) were detected. C, EMSA analyses using the purified protein and the IGFBP-1 AARU probe. The results of competition assay (left) and supershift assay (right) are shown. The arrows indicate the position of complex I, and the arrowhead on the right shows the position of the supershifted band. NE, Nuclear extract; USF, USF consensus competitor; M1, AARU-M1 competitor; M2, AARU-M2 competitor (see Fig. 1).

 
Partial purification of complex I protein
The nuclear extract obtained as described above (209 ml, 1348 mg protein) was diluted to 0.1 M NaCl with HMEG buffer [20 mM HEPES-KOH (pH 7.8), 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF, 14 µg/ml aprotinin, 1 µg/µl leupeptin, 1 µg/µl pepstatin, 1 mM benzamidine, 1 mM DTT, 2 mM Na2VO4, and 2 mM NaF]. The sample was loaded onto a column of SP-Sepharose FF (150-ml bed volume; Amersham Pharmacia Biotech) preequilibrated with HMEG buffer containing 0.1 M KCl. This column was washed with 4 vol HMEG buffer containing 0.1 M KCl and eluted with a linear gradient of KCl from 0.1–0.5 M. The activity of each fraction was determined by EMSA as described above. The fractions containing the activity to form the retarded band were pooled, diluted to 0.05 M KCl with HMEG buffer, and loaded onto a column of DEAE-Sepharose FF (30-ml bed volume; Amersham Pharmacia Biotech). The column was washed and eluted with a linear gradient of KCl from 0.05–0.4 M. The active fractions were pooled, diluted to 0.1 M KCl with HMEG buffer, and further purified by affinity chromatography using heparin-Sepharose CL-6B (bed volume, 5 ml; Amersham Pharmacia Biotech). The column was washed with 4 vol HMEG buffer containing 0.2 M KCl and eluted with a linear gradient of KCl from 0.2–0.6 M. Then the pooled active fractions were subjected to DNA affinity chromatography according to the method of Kadonaga et al. (34). The AARU oligonucleotides (Fig. 1Go) that had been annealed and phosphorylated were ligated to yield polymers. The DNA affinity resin was prepared by coupling the ligated polymers to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech). The sample was diluted to 0.1 M KCl with buffer [20 mM HEPES-KOH (pH 7.8), 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.1% Nonidet P-40, 0.5 mM PMSF, 14 µg/ml aprotinin, 1 mM benzamidine, 1 mM DTT, 2 mM Na2VO4, and 2 mM NaF], mixed with poly(dI-dC)/poly(dI-dC) to a final concentration of 10 µg/ml, and allowed to stand for 15 min. The column containing 2 ml resin was equilibrated with buffer [20 mM HEPES-KOH (pH 7.8), 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.1% Nonidet P-40, 0.5 mM PMSF, 14 µg/ml aprotinin, 1 mM benzamidine, 1 mM DTT, 2 mM Na2VO4, and 2 mM NaF] containing 0.1 M KCl, loaded with the sample, and eluted with a stepwise increase in the KCl concentration (0.1, 0.2, 0.3, 0.4, and 0.5 M). The active fractions were collected and subjected to a second cycle of DNA affinity chromatography.



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  		Figure 1. Structure of an AARU of the rat IGFBP-1 gene and the sequences of the probes and competitors used in EMSA. A schematic representation of the structure of the proximal region of the rat IGFBP-1 gene with the sequence of AARU is shown at the top. IRE and GRE in AARU are indicated by the lines above the sequence, and the E box sequence and the putative binding sites for HNF-3 are underlined. The sequences of the sense strand of the probes and competitors used in EMSA are also shown; their respective names are indicated.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our previous study showed that the region between -77 and -112 bp upstream of the rat IGFBP-1 gene promoter was important for this gene to respond to amino acid deprivation. This region, tentatively termed an amino acid response unit (AARU), contains binding sites for several transcription factors. Fig. 1Go shows the sequence of AARU, putative binding sites of transcription factors, and the sequences of the probes and competitors used for electrophoresis mobility shift assays (EMSA) in the present study. We attempted to identify the transcription factor(s) that mediate the up-regulation of IGFBP-1 in response to protein malnutrition in vivo.

The nuclear extracts of the liver of rats fed C (12% casein) and PF, respectively, were subjected to EMSA using an AARU probe (-77 to –114). As shown in Fig. 2Go, among the several shifted bands observed, the intensity of one band (complex I) was consistently stronger in PF-fed rats than in C-fed rats. The protein composing the complex I appeared to be the factor that responds to protein malnutrition by increasing its binding capability to the AARU.



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  		Figure 2. Nuclear extract of rat liver contains a factor that undergoes increased binding to IGFBP-1 AARU under the condition of protein deprivation. Liver extracts were prepared from rats fed C and PF, respectively, and subjected to EMSA using a 32P-labeled IGFBP-1 AARU probe as described in Materials and Methods. The positions of the free probe and a protein-probe complex termed complex I are designated by arrows. Each lane represents the result using extract from an individual rat (n = 5).

 
Given that the AARU region contains two IREs and a GRE, we next examined whether the retarded band consisted of IRE- or GRE-binding protein bound to the AARU probe (Fig. 3Go). Competition assay was performed using unlabeled AARU itself, and IGFBP-1-IRE DNA and IGFBP-1-GRE DNA as competitors (Fig. 1Go). A 50-fold molar excess of unlabeled AARU competitor eradicated the band of complex I. The complex I was also eradicated by the IGFBP-1-GRE competitor, but was not affected by the IGFBP-1-IRE competitor. Thus, the factor may be the GR itself, or another factor that binds to GRE or to a different sequence within the IGFBP-1-GRE DNA. Figure 4Go shows the result of a competition assay using a competitor DNA that included the consensus GRE sequence. This competitor did not affect the formation of the retarded band, indicating that the factor involved is less likely to be the GR. We cannot, however, exclude the possibility that GR is one of the components of this complex, given that the affinity of activated GR for the IGFBP-1-GRE relative to that for the consensus GRE is not known.



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  		Figure 3. The factor that forms complex I binds to the position containing GRE, but not to that containing IRE in IGFBP-1 AARU. Liver nuclear extracts of rats fed C and PF, respectively, were used for EMSA as described in Materials and Methods and Fig. 2Go. Assays were performed in the absence and presence, respectively, of a 50-fold molar excess of competitor DNAs (Fig. 1Go). AARU, Unlabeled AARU oligo DNA (-77 to -112); IRE, oligo DNA containing IGFBP-1 IRE (-88 to -116); GRE, oligo DNA containing IGFBP-1 GRE (-77 to -95).

 


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  		Figure 4. The complex I protein is not the GR. Liver nuclear extracts of rats fed C and PF, respectively, were used for EMSA as described in Materials and Methods and Fig. 2Go. Assays were performed in the absence and presence, respectively, of different amounts of a competitor DNA that contained a GRE consensus sequence (Fig. 1Go).

 
The GRE sequence in the IGFBP-1 AARU contains an E box-like sequence (CACGGG, Fig. 1Go). The E box, the consensus sequence of which is CANNTG, is known as the binding site for USF. We next examined the possibility that the PF-responsive factor was a USF. Nuclear extracts were preincubated with anti-USF-1 or anti-USF-2 antibody before being subjected to EMSA. As shown in Fig. 5Go, both USF-1 and USF-2 antibodies caused a disappearance of complex I. A faint band, which probably resulted from the supershift, appeared after treatment with each of the antibodies (arrowhead). The specificity of the binding was verified using the USF consensus sequence and mutant oligonucleotides as competitors (Fig. 6Go). An excess amount of USF consensus DNA, which contained the CACGTG sequence but was unrelated to IGFBP-1 AARU, strongly blocked the formation of complex I. Mutation of the CACGGG sequence in the IGFBP-1 AARU (AARU-M1 and AARU-M2) resulted in complete loss of ability to compete with the AARU probe.



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  		Figure 5. The complex I protein is a mixture of USF-1 and USF-2. Liver nuclear extracts of rats fed C and PF, respectively, were incubated with antibodies to USF-1 and USF-2, then used for EMSA as described in Materials and Methods and Fig. 2Go. The arrowhead on the right designates the position of the band derived from a supershift induced by the antibodies.

 


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  		Figure 6. The complex I protein binds to the E box sequence in the rat IGFBP-1 AARU. Liver nuclear extracts of rats fed C and PF, respectively, were used for EMSA as described in Materials and Methods and Fig. 2Go. Assays were performed in the absence and presence, respectively, of a 50-fold molar excess of competitor DNAs (Fig. 1Go). USF consensus, Oligo DNA containing a consensus USF-binding sequence but unrelated to IGFBP-1 AARU; AARU-M1 and AARU-M2, oligo DNAs similar to IGFBP-1 AARU with mutations in the E box sequence.

 
To further characterize the identity of complex I, the proteins bound to the probe were partially purified using gel chromatography of SP-Sepharose, diethylaminoethyl-Sepharose, heparin-Sepharose, and DNA affinity Sepharose. Figure 7AGo is a Coomassie brilliant blue-stained gel showing the purified proteins. We could not obtain a preparation pure enough to determine its amino acid sequence. The partially purified protein preparation was subjected to Western immunoblotting using anti-USF-1 and anti-USF-2 antibodies. The preparation was found to contain both USF-1 and USF-2 (Fig. 7BGo). EMSA using this preparation produced an essentially single retarded band (Fig. 7CGo, left). The addition of a 50-fold excess of USF consensus DNA reduced the intensity of the band. AARU-M1 and AARU-M2 had no effect. A supershift assay was performed using the antibodies to USFs (Fig. 7CGo, right). Both anti-USF-1 and USF-2 antibodies caused a reduction in the intensity of the band corresponding to complex I and the appearance of a new band (arrowhead on the right). The effect of the USF-1 antibody was more potent than that of the USF-2 antibody, which might be explained by the relative abundance of each protein. In addition, the position of the remaining band differed slightly between USF-1 and USF-2 antibodies. The band that remained at the position of complex I after USF-1 antibody treatment migrated more slowly than did the band that remained after USF-2 treatment (arrows on the right). The bands are probably composed of USF-2 homodimer and USF-1 homodimer, respectively, bound to the probe. The band of complex I totally disappeared in response to the combination of these antibodies.

We next investigated whether the augmentation of complex I by PF could be accounted for by a change in either USF-1 or USF-2. The amounts of USF-1 and USF-2 in the nuclear proteins of the liver of rats fed C or PF were estimated by Western immunoblotting (Fig. 8Go). Feeding PF caused 1.7- and 2.5-fold increases, respectively, in USF-1 and USF-2 levels. In addition, cytosolic protein was immunoprecipitated by antibodies to USF-1 and USF-2 and immunoblotted using the respective antibodies. The results showed that the amount of USF protein is much lower in the cytosol than in the nucleus (data not shown), suggesting that USFs are primarily localized in the latter. These results indicate that the increased AARU-binding activity of USFs in the liver of PF-fed rats is attributable to increases in USF-1 and USF-2.



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  		Figure 8. Protein deprivation increases USF-1 and USF-2 in rat liver. The amounts of USF-1 and USF-2 in the nuclear extracts of the liver of rats fed C or PF were analyzed by Western immunoblotting. A and C, Autoradiogram of USF-1 (A) and USF-2 (B) immunoblots. Each lane represents the result of an individual rat. B and D, Quantitative analyses of the results of A and C, respectively. Values are the mean ± SEM relative to the mean of C. * and **, P < 0.05 and P < 0.01, respectively, vs. C, by unpaired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A great body of information has accumulated on the mechanism by which hormonal and nutritional factors regulate the expression of responsive genes. In many genes that are regulated, for example, by glucose, fatty acids, sterols, and vitamins, the precise pathways leading to changes in gene expression by these factors have been well characterized (35). Much less is known, however, about gene regulation by protein nutrition or amino acid availability. Although many genes have been identified as being under the control of protein nutrition, there is presently no confirmed consensus sequence for the amino acid responsiveness common to these genes. Only limited information is available on transcription factors that play important roles in amino acid regulation of gene expression. Further, the signaling pathway by which information on the amount or balance of available amino acids is transmitted to the transcriptional machinery has not yet been revealed for any of these factors (36). Recently, activating transcription factor 2 (ATF-2) has been identified as essential for amino acid responsiveness of the CCAAT enhancer binding protein (C/EBP)-related gene (37), although the mechanism of regulation of ATF-2 is not known. Harding et al. (38) recently delineated a pathway by which amino acid availability regulates ATF-4, a transcription factor related to ATF-2.

The IGFBP-1 gene has garnered recent attention because IGFBP-1 is not only metabolically important, but is also highly responsive to nutritional and hormonal factors. We have been using the rat IGFBP-1 gene promoter as a suitable model for studying the mechanism of gene regulation by protein and/or amino acid nutrition.

We previously showed that the effect of a protein-free diet on the hepatic gene expression of IGFBP-1 can be mimicked in hepatoma cell cultures by incubating the cells in an amino acid-free medium. Further analyses using DNA constructs of the IGFBP-1 promoter and the luciferase reporter enabled us to identify within the IGFBP-1 promoter a short region that contributes to the regulation by amino acids (30). This region, tentatively termed an AARU, is located at -112 to -77 bp upstream from the transcription initiation site of the IGFBP-1 gene. The next logical step is to identify the trans-acting factors acting on AARU to mediate the effect of protein nutrition. Binding sites for several transcription factors exist within AARU and include the IRE, the GRE, and an HNF-3-binding site. Among the factors bound to these elements, IRE-binding protein and GR do not seem to mediate the effect of protein malnutrition, based on the finding that the plasma concentration of insulin is not much affected by a protein-free diet (39) and that the GRE in IGFBP-1 AARU has been reported not to be functional in vivo (40). In the present study using EMSA, we showed that nuclear extract of rat liver contained some components whose binding activity to AARU increased when the rats were fed a protein-free diet. Experiments incorporating competitor oligonucleotides and mutant oligonucleotides revealed that the factor was not a GR or an IRE binder. By supershift assays using the respective antibodies, the factor was determined to be a mixture of USF-1 and USF-2. This result is in line with the observation by Crissey et al. (41) that the binding of USF-1 to a CTCGGG site in mouse IGFBP-1 promoter is rapidly increased in partial hepatectomy-induced expression of the IGFBP-1 gene.

USF was originally characterized as a factor that activates the adenovirus major late promoter (42). USF belongs to the basic/helix-loop-helix/leucine zipper family of transcription factors, the members of which include c-Myc (43), Max (44), transcription factor EB (45), transcription factor E3 (46), and sterol regulatory element-binding protein (47). Purification of USFs from HeLa cells revealed that USF activity involves two polypeptides with apparent molecular masses of 43 and 44 kDa, which are referred to as USF-1 and USF-2, respectively (48, 49). cDNAs for USF-1 and USF-2 have been reported in humans, mice, and other species (50, 51). The USF proteins form hetero- and homodimers and bind to the E box motif (CACGTG) (52).

Despite the fact that molecular aspects of USF have been well characterized, its biological role remains poorly understood. USF proteins are suggested to be involved in cell cycle regulation, one of the known mechanisms of which is an antagonistic action against the function of c-Myc (53). A wide number of genes have emerged as being under the control of USFs. These include the genes for {alpha}-crystalline (54), myosin light chain (55), C/EBP{alpha} (56), liver-type pyruvate kinase (57, 58), {alpha}1(I) collagen (59), and fatty acid synthetase (60). Wang and Sul (60) showed that regulation of the fatty acid synthetase gene by fasting and refeeding is mediated by changes in the content of USF-1 proteins; such changes, notably, differed from the changes observed herein. They found that the liver nuclear extract of fasted rats contained a much higher amount of a 17-kDa protein that was recognized by anti-USF-1 antibody, whereas that of refed rats contained mainly the 45-kDa form. Taking their results and ours together, it seems apparent that the mechanism of regulation of USF-1 activity by energy intake (and insulin) and that by protein malnutrition are different. Other factors may play a role in determining whether 45-kDa USF-1 induces or represses the IGFBP-1 gene.

In the previous study we showed that gene expression of the three subtypes of HNF-3, a member of the forkhead transcription factors, is differentially regulated by protein nutrition, insulin, and glucocorticoid (61). Protein malnutrition increases HNF-3{gamma} mRNA, whereas insulin deficiency up-regulates HNF-3ß and -3{gamma}. In addition, dexamethasone treatment increases the levels of HNF-3{alpha} and -3ß mRNA. Interestingly, the AARU of the IGFBP-1 promoter contains an HNF-3-binding site, which is involved in up-regulation of IGFBP-1 gene expression (62, 63). To further complicate the situation, IREs existing in this region overlap the HNF-3 site. The mechanism of regulation of the IGFBP-1 gene by insulin via these IREs has been well characterized. Insulin-induced phosphorylation of serine and threonine residues of FKHR transcription factor, another member of the forkhead family of proteins, causes exclusion of this protein from the nucleus, resulting in a reduced transcription rate of the IGFBP-1 gene (26, 27, 28, 29). However, it should be noted that the roles of FKHRs and HNF-3 in regulation of the IGFBP-1 gene by insulin are still controversial (64, 65). Figure 9Go shows our working hypothesis of the mechanism by which hormonal and nutritional factors control the expression of the IGFBP-1 gene through the AARU region. The effects of insulin, glucocorticoid, and protein nutrition on the activity of the IGFBP-1 promoter are mediated by many transcription factors, i.e. FKHR, GR, and members of HNF-3 and USFs. The complicated interaction of many transcription factors may make it possible to fine-tune the activity of this promoter.



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  		Figure 9. A hypothetical model for the mechanism of regulation of the IGFBP-1 gene by protein malnutrition and hormonal factors through multiple transcription factors. Protein malnutrition increases USF-1 and -2 by a presently unknown mechanism, which leads to stimulation of IGFBP-1 gene transcription through the E box. In addition, protein malnutrition stimulates the gene expression of HNF-3{gamma} (61 ), which, in turn, binds to the HNF-3 site. Thus, USFs and HNF-3{gamma} may work cooperatively to stimulate IGFBP-1 gene transcription under protein malnutrition. The GR may not work through the GRE of this region in vivo (40 ).

 
The increased binding of USF in PF-fed rats may not be attributed solely to an increased USF protein level. Involvement of other mechanisms, such as facilitated translocation to the nucleus, alteration in phosphorylation status, facilitation of dimerization, and/or increased affinity to E box, cannot be excluded at present, and should be further investigated. Among these other mechanisms, regulation by translocation, which is the major regulatory mechanism in the case of the action of insulin on FKHR and AFX (29, 66), would not contribute much; USFs are localized mainly in the nucleus of cells in the liver, even in C-fed rats (Matsukawa, T., Y. Inoue, H. Kato, and T. Noguchi, unpublished observation).

At present we do not have direct evidence that USF actually stimulates IGFBP-1 gene expression in vivo. We recently observed in a cell culture system that cotransfection of USF proteins strongly stimulated IGFBP-1 promoter activity, whereas only a small amount (0.1 µg) of the expression vector of USF-1 or USF-2 resulted in a 10-fold increase in the activity. In addition, mutation of the E box sequence in AARU attenuated the response of this promoter, although considerable response (approximately half that of the wild-type promoter) still remained (Kitamura, Y., Y. Inoue, T. Matsukawa, Z. W. Fu, T. Noguchi, and H. Kato, unpublished results). These findings suggest that the E box in AARU is important in the response to USFs but an additional mechanism, probably through other E box-like sequences in this promoter, has an auxiliary role. It should be noted that the partial hepatectomy-induced USF-1 binding mentioned above (41) is to the same sequence (CACGGG) but at a different position (-170 to -165) in the IGFBP-1 promoter of mouse. Intensive study using many mutant constructs in a cell culture system is underway. It is difficult, however, to prove the role of USFs in an in vivo system; the transgenic approach, given that USFs are inhibitory to growth and cell proliferation, presents problems (67). However, accumulated evidence from cell culture systems and the results of the present study suggest an active role of USFs in IGFBP-1 regulation in vivo.

Our observation that USFs are regulated by protein nutritional status could imply that they play roles more important than that of the regulator of the IGFBP-1 gene. Based on the fact that both USF-1 and USF-2 are ubiquitously expressed among many tissues, and that the E box motif is very common to a number of genes, it is reasonable to postulate that USFs take part in the regulation by protein nutrition of a wide range of genes. It bears mentioning that USFs play a known role not only as E box binders, but also as initiator sequence-binding proteins (68, 69, 70).

The mechanism by which protein deprivation up-regulates USF-1 and USF-2 is currently being investigated. Regulation at the level of transcription, mRNA stabilization, translation, protein degradation, or some combination thereof probably contribute to the regulation process. Our preliminary studies have to date shown that changes in mRNA levels do not seem to be involved. In addition, we observed in several cell lines that USFs tend to increase by amino acid limitation without changes in their mRNA levels (Kitamura, Y., Y. Inoue, T. Matsukawa, Z. W. Fu, T. Noguchi, and H. Kato, unpublished results). As described above, Harding et al. (38) have recently shown that up-regulation of ATF-4 plays a pivotal role in C/EBP-related gene expression by amino acids. They demonstrated that amino acid limitation activates mGCN2, which, in turn, increases translation of ATF-4. As yet, it is not known to what degree this mechanism is related to the regulation of IGFBP-1 gene or the regulation of USF activity. In any case, elucidation of the mechanism of response to protein deprivation would constitute a great advance in our knowledge of the specific mechanisms mediating the effects of amino acids and protein nutrition on gene expression.


   Acknowledgments
 


   Footnotes
 
This work was supported by Grant-in-Aid for Scientific Research 10460056 (to T.N.) and 12460054 (to H.K.) from the Japan Society for the Promotion of Science.

1 T.M. and Y.I. contributed equally to this work. Back

Abbreviations: AARU, Amino acid response unit; ATF, activating transcription factor; C, 12% casein diet; C/EBP, CCAAT enhancer binding protein; DTT, dithiothreitol; GRE, glucocorticoid response element; HMEG buffer, 20 mM HEPES-KOH (pH 7.8), 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF, 14 µg/ml aprotinin, 1 µg/µl leupeptin, 1 µg/µl pepstatin, 1 mM benzamidine, 1 mM DTT, 2 mM Na2VO4, and 2 mM NaF; HNF, hepatocyte nuclear factor; IGFBP, IGF-binding protein; IRE, insulin response element; PF, protein-free diet; PMSF, phenylmethylsulfonylfluoride; TBS-T, 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1 mM EDTA, and 0.1% Tween 20; USF, upstream stimulatory factor.

Received April 30, 2001.

Accepted for publication August 1, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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