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Originally published In Press as doi:10.1074/jbc.M404349200 on June 11, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36650-36659, August 27, 2004
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Insulin-response Element-binding Protein 1

A NOVEL Akt SUBSTRATE INVOLVED IN TRANSCRIPTIONAL ACTION OF INSULIN*

Betty C. Villafuerte{ddagger}§, Lawrence S. Phillips¶, Madhavi J. Rane||, and Weidong Zhao¶**

From the {ddagger}Division of Endocrinology and Metabolism, Department of Medicine, University of Louisville, Louisville, Kentucky 40202, the Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, and the ||Division of Nephrology, Department of Medicine, University of Louisville, Louisville, Kentucky 40202

Received for publication, April 20, 2004 , and in revised form, June 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the cis-acting elements that mediate the actions of insulin on gene transcription have been defined for a significant number of genes, the transcription factors responsible for the transactivation of these target sequences remain unknown. In this report, we identified a novel transcription factor that binds and transactivates the insulin-response elements of the insulin-like growth factor-binding protein-3 and other insulin responsive genes. This factor is a target of insulin signal transduction downstream of the phosphatidylinositol 3'-kinase/protein kinase B (Akt) pathway. Akt phosphorylates this factor in vivo and in vitro. Changes in expression level, phosphorylation, and nuclear translocation modulate the transactivation effects of the factor, and its expression is decreased in conditions of diabetes and insulin deficiency. Identification of a novel target of Akt that appears to mediate signals specific for insulin action should provide further insight into the mechanism of insulin action at the genomic level.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin regulates metabolism by altering transcription of many genes, and defects in basal and insulin-regulated gene expression may be important in the etiology of "non-insulin dependent" type 2 diabetes mellitus (1). Although no single consensus insulin response element (IRE)1 has been identified, some genes whose transcription is inhibited by insulin appear to share a common IRE core motif. But because other well defined IREs appear to be different from this sequence and from each other (2), it has been postulated that no common trans-acting factor will be associated with all IREs. Insulin mediates its actions through several distinct signal transduction pathways, and multiple pathways of insulin action may be involved in the regulation of gene transcription by insulin. Although insulin-initiated signaling cascades induce changes in nuclear protein phosphorylation, few transcription factors that are phosphorylated in response to insulin have been identified, and none has been unequivocally proven to mediate the effect of insulin on gene transcription. Furthermore, the mechanisms by which these factors transactivate IREs are largely unexplored.

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier protein of the mitogenic peptide IGF-1 in the circulation, and insulin is an important regulator of IGFBP-3 in vivo (3). We reported previously that insulin enhances IGFBP-3 gene transcription through a cis-regulatory IRE localized to the –1150 to –1124-bp region (4). Binding of insulin-responsive nuclear factors to the IGFBP-3 IRE is regulated in vivo; diabetes reduces DNA-protein binding activity of the IRE in rat liver nuclear extracts. Southwestern blotting with an IGFBP-3 IRE probe pointed to an alteration in the quantity and/or activity of a 70- and 90-kDa nuclear protein that binds to the IRE as a potential mechanism for the control of IGFBP-3 transcription by insulin (4). In search of the trans-acting factors that bind to the IRE of the IGFBP-3 gene, we screened a rat liver cDNA library to identify candidate genes involved in IRE-mediated regulation of IGFBP-3 gene transcription. We report here the identification of a novel transcription factor that binds to the IRE, and transactivates the IGFBP-3 gene. The factor has similar actions on other insulin-responsive genes, and is a target of insulin signal transduction downstream of both the phosphatidylinositol 3'-kinase/Akt and the mitogen-activated protein kinase pathways. Structurally, this factor contains motifs that are normally associated with extracellular matrix protein but not with other identified transcription factors, suggesting that it belongs to a different class of DNA-binding factor. Insulin treatment alters the phosphorylation state and transactivation potential of the factor, and insulin deficiency in a diabetes model is associated with decreased hepatic mRNA and protein expression of the factor. To recognize its activity in promoting insulin-dependent gene transcription, we named this factor insulin response element-binding protein 1 (IRE-BP1). Because IRE-BP1 is likely a mediator of insulin action on multiple target genes, future studies of the activation and actions of IRE-BP1 may provide important insights into the pathogenesis and treatment of diabetes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast One-hybrid cDNA Library Screening—Using the yeast onehybrid system to screen a rat liver cDNA library (Clontech, Palo Alto, CA), three tandem repeats of the IGFBP-3 IRE (–1150 to –1117 bp) was inserted upstream of a His3 reporter gene under the control of GAL4-responsive promoter, and the resulting plasmid was transformed into YM4271 yeast. Yeast containing the target element were co-transformed with an activation domain (AD) library that contains fusions between target-independent AD (GAL4 AD) and cDNA from normal rat liver. Colonies were selected on His–/Leu– plates with 15 mM 3-amino-1,2,4-triazole, and we picked 79 yeast clones. After DNA sequencing and confirmation of the ability of the cDNAs to transactivate a GAL4 promoter linked to a LacZ reporter gene, the cDNA was subcloned into a prokaryotic expression vector for further studies.

{lambda} Phage cDNA Library Screening—A 935-bp IRE-BP1 cDNA was used as a probe to screen 108 plaques of a {lambda} bacteriophage rat brain cDNA library (Uni-Zap XR, Stratagene, La Jolla, CA) as per the manufacturer's protocol. After isolation of the plaques that hybridized with the cDNA probe, the pBluescript phagemid was rescued with the VCSM 12 helper phage, and the cDNA was sent for automated sequencing.

Rapid Amplification of cDNA 5'-Ends—Using random hexamers, poly(A)+ mRNA from rat cerebellum (Clontech, Palo Alto, CA) was used as a template for reverse transcription and first strand cDNA synthesis. This was followed by PCR amplification using a forward primer consisting of the anchor primer from the SMART II kit (Clontech), and a reverse primer corresponding to nucleotides 337–312 of the 3.4-kb IRE-BP1 cDNA: 5'-TTGGTGACCTCGAAGTCTTCAATAG-3'. This was repeated several times until no additional 5' cDNA was obtained.

Bacterial Expression of IRE-BP1—A 1503-bp (+1641/+3144) translated cDNA encoding the sequences of IRE-BP1 was subcloned in-frame into the prokaryotic expression vector (pET-32a from Novagen Inc., Madison, WI), transformed into the AD494(DE3) strain of Escherichia coli, and grown at 37 °C until it reached an A600 of 0.6. Isopropyl-{beta}-D-thiogalactoside was added to a final concentration of 1 mM 3 h before harvest. The fusion protein was purified by affinity chromatography on immobilized His-bind metal chelation resin (Qiagen Inc., Valencia, CA), and used for gel shift and Western analysis.

Production of Antibody—The C-segment antibody is an epitope of the rat IRE-BP1 between amino acids 786 and 800 and has the following sequence: acetylated Cys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Ty-Iso-Pro-Asn-Gly-Lys-Leu. To develop the N-segment antibody, we used the peptide fragment between amino acids 233 and 247, with the following sequence: acetylated Cys-Arg-As-Gly-Gly-Thr-Tyr-Lys-Glu-Thr-Gly-Asp-Glu-Tyr-Arg. Production of the anti-rabbit polyclonal antibodies and its affinity purification were accomplished by BIOSOURCE Inc., Hopkinton, MA.

Gel Mobility Shift Analysis—Double-stranded oligonucleotides corresponding to the published sequences of the IREs identified from various genes were used for competition (4–9). These include the IREs identified from IGFBP-3 (5'-AATTCAAGGGTATCCAGGAAAGTCTCCTTCAAG-3'), glyceraldehyde-6-phosphate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase (5'-AAGTTCCCCAACTTTCCCGCCTCTCAGCCTTTGAAAG-3'), IGFBP-1 (5'-GCCTCATTATTCCTGCCCACCAAT-3'), IGF-1 (5'-GCCTCATTATTCCTGCCCACCAAT-3'), amylase (5'-TATTTTGCGTGAGAGTTTCTAAAAGTCCAT-3'), phosphoenolpyruvate carboxykinase or PEPCK (5'-TGGTGTTTTGACAAC-3'), tyrosine aminotransferase or TAT (5'-GACTAGAACAAACAAGTCCTGCGTA-3'), prolactin (5'-ATCTATTTCCGTCATTAAGATA-3'), and the consensus sequence for NF{kappa}B binding (5'-GGGACTTTCCGGGACTTTCC-3') (10).

Farwestern Blotting—Double-stranded oligonucleotides corresponding to the IREs of IGFBP-3, IGF-1, and IGFBP-1 genes were end-labeled with [{gamma}-32P]ATP, then incubated with 20 µg of rat hepatic nuclear extract, and subjected to electrophoresis on a 5% polyacrylamide gel. The gel-shift bands were transferred to nitrocellulose membrane, and denatured with decreasing concentrations of guanidine HCl, initially at 6 M concentration for 15 min, then at 3, 1.5, 0.75, 0.375, and 0.18 M, then washed with phosphate-buffered saline. The blot was blocked with 5% milk in phosphate-buffered saline, then incubated with antibodies to IRE-BP1, Sp1, and peroxisome proliferator-activated receptor {gamma}, as indicated. The protein was visualized with chemiluminescent luminol reagent.

Immunoprecipitation and Western Blotting—Total cell lysates from HepG2 cells were incubated with rabbit IgG and protein G-agarose at 4 °C for 30 min, then centrifuged. The pre-cleared lysates were transferred to a fresh microcentrifuge tube, incubated with IRE-BP1 cAb or nAb overnight at 4 °C, then with protein G-agarose for 1 h, and centrifuged. The agarose pellet was washed with RIPA buffer 4 times, then run on Western blot. The blotted protein was probed with the IRE-BP1 antibodies as indicated.

Two-dimensional Gel Electrophoresis—For two-dimensional electrophoresis, cell lysates were dissolved in 2 M thiourea, 7 M urea, 65 mM CHAPS, 58 mM dithiothreitol, and 4.5% ampholytes (pH 4–6). Immobiline dry strips were rehydrated with a 155-µl sample at 25 °C overnight. IEF was performed for a total of 71 750 V-h, with the voltage ramped linearly from 500 to 3500 V during the first 5 h and maintained for 15.5 h at 3500 V. Prior to the second dimension, strips were equilibrated 2 times for 5 min each in equilibration buffer 1 (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris, pH 8.6, 2% dithiothreitol) and another 5 min in equilibration buffer 2 (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris, pH 6.8, 2.5% iodacetamide, a trace of bromphenol blue). Vertical second dimension gels were prepared and run (11).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mechanism of Stimulation of IGFBP-3 Gene Transcription by Insulin—Hepatic IGFBP-3 synthesis occurs in nonparenchymal cells, particularly Kupffer and sinusoidal endothelial cells, and we have shown previously that insulin stimulates IGFBP-3 gene transcription in these cells (12, 13). To begin to characterize the mechanism of stimulation of IGFBP-3 transcription by insulin, we used transcription elongation "nuclear run-on" assays (Fig. 1a), and found that the relative transcription rate of IGFBP-3/{beta}-actin was increased 3.6-fold in hepatic nonparenchymal cells after adding 10 nM insulin for 6 h. Stimulation by insulin was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting that the effect of insulin on IGFBP-3 gene transcription is dependent on the induction of a secondary factor(s). Insulin thus appeared to induce the synthesis and/or binding of a protein to the IRE, and the candidate insulin response factor appeared to be –70 and 90 kDa in size (4).



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  		FIG. 1.
Identification of the IRE-binding protein. a, nuclear run-on assays were performed with nuclei isolated from hepatic nonparenchymal cells exposed to insulin or vehicle, with or without cycloheximide. Transcription rates were determined by densitometric scanning of autoradiographs. b, binding of rat hepatic nuclear extracts and recombinant NF{kappa}B p50 to the IGFBP-3 IRE was done using gel shift assay, and the shifted bands were incubated with antibodies against NF{kappa}B p50 and NF{kappa}B p65. c, hepatic expression of HBP1 was detected by Northern blotting, using liver RNAs extracted from normal and streptozotocin-diabetic rats. d, the protein structure of rat IRE-BP1, as predicted by SMART (Entrez). The GenBank accession number for the nucleotide sequence of rat IRE-BP1 is AF439916. e, the fusion protein expressed from the 1.5-kb IRE-BP1 cDNA with a Trx-His tag was detected by antibody against the His peptide tag (left panel) and antipeptide serum developed against a 15-amino acid residue of IRE-BP1 or COOH-terminal antibody (right panel). f, Western blot of hepatic nuclear extracts pooled from 3 normal (NL) and 3 streptozotocin-diabetic (DM) rats, probed with anti-IRE-BP1 antibody. Two pooled extracts are shown. The blot was reprobed with anti-Sp1 antibody to show that equal amounts of nuclear protein was loaded.

 
Cloning of the IRE-binding Protein—Using a 34-bp DNA probe based on the IRE in the rat IGFBP-3 gene (4), we screened a rat liver cDNA library using the yeast one-hybrid system, and obtained 79 clones. After sequencing of the cDNA and binding studies to the IGFBP-3 IRE using gel shift assay, we found that 76 of the 79 clones were false positives, whereas 3 clones produced proteins that bound to the IGFBP-3 IRE. Two of the proteins are known transcription factors, NF{kappa}B p65 and HBP1 (high mobility group box containing protein 1) (14). The third factor was a novel gene that was not previously reported to the GenBankTM. As shown in Fig. 1b, E. coli-expressed NF{kappa}B p50 (the binding subunit of NF{kappa}B, which forms a heterodimer with the p65 subunit) binds to the IGFBP-3 IRE (lane 5), and NF{kappa}B p50 antibody produced a supershift of the band (lane 6). However, antibodies against NF{kappa}B p65 or NF{kappa}B p50 did not alter binding of rat liver nuclear extract to the IGFBP-3 IRE (lanes 3 and 4 versus 2). Furthermore, the level of expression of NF{kappa}B p65 in diabetic rat liver was unchanged compared with normal liver, and co-transfection of expression plasmids of either NF{kappa}B p50 or p65 with target constructs containing the IGFBP-3 IRE linked to a luciferase reporter showed neither activation nor repression of the reporter gene (not shown). Whereas HBP1 may be involved in the regulation of gene transcription by insulin, the tissue distribution of HBP1 is ubiquitous (14), and not restricted to insulin-sensitive tissues, and HBP1 mRNA is not stimulated by insulin in vivo (Fig. 1c). Therefore, neither NF{kappa}B p65 nor HBP1 appears to be the factor in rat liver that transactivated IGFBP-3, so we examined the role of the novel gene in mediating insulin-regulated gene transcription.

The original IRE-BP1 cDNA was 936 bp in size, and was used as a probe to screen a rat brain cDNA library, leading to the identification of 8 overlapping clones that were aligned to produce a 3404-bp contiguous sequence. Use of 5' rapid amplification of cDNA ends provided an additional 1.6-kb DNA of contiguous 5' sequence. The present IRE-BP1 cDNA is 5043 bp in length, and includes an open reading frame with 3144 bp of translated sequence followed by 1899 bp of 3'-untranslated sequence (GenBank accession number AF439916). The DNA is predicted to encode a protein of 1047 amino acids with a molecular mass of ~107 kDa.

Predicted Protein Structure of IRE-BP1—IRE-BP1 contains multiple cysteine-rich motifs, and the NH2 domain is predicted to form 13 epidermal growth factor (EGF)-like repeats (Fig. 1d). Within the EGF-like repeats is a sushi domain (15). The carboxyl-half of IRE-BP1 is organized into three fibronectin type III (FN3) domains, a motif containing ~100 amino acids that can bind to DNA and interacting proteins (16). A solitary calcium-binding EGF-like domain is also present near the COOH terminus.

We generated an antibody to an oligopeptide corresponding to the carboxyl-terminal segment (amino acids 786–800) of rat IRE-BP1 (cAb). In addition, a 1503-bp (+1641/+3144) translated sequence of the cDNA that encodes the 50-kDa carboxyl portion of the protein was expressed in E. coli as a His6-tagged thioredoxin (Trx) fusion protein, and were purified with Ni2+-nitriloacetate. Fig. 1e shows a Western blot of the induction control (Trx) and the fusion protein (Trx-IRE-BP1) in E. coli lysate. Both the IRE-BP1 antibody and histidine antibody recognized a 65–70-kDa protein, consistent with the predicted size of the fusion protein containing 20 kDa Trx. In Western blotting experiments using hepatic nuclear extracts from normal and streptozotocin-diabetic rats, the IRE-BP1 cAb recognized a 90-kDa protein (Fig. 1f), consistent with the size of the insulin-responsive protein recognized in Southwestern blots using an IGFBP-3 IRE probe (4). The size of the nuclear factor recognized by cAb appears to be smaller than the predicted size of the protein, however, suggesting that cleavage of the protein may occur. IRE-BP1 expression was reduced in the livers of streptozotocin-diabetic rats compared with normal rats, implying regulation by insulin.

IRE-BP1 Binds to Multiple IREs—To study the regulation of IRE-BP1 binding, we used the Trx-IRE-BP1 fusion protein for gel mobility shift analysis. We found that the fusion protein produced a gel shift band with an IGFBP-3 IRE probe, and this band was competed by unlabeled IGFBP-3 IRE oligonucleotide but not by an unrelated NF{kappa}B oligonucleotide (Fig. 2a). We then used this system to determine whether IRE-BP1 is also recognized by the IREs of other insulin-responsive genes (also shown in Fig. 2a). Gel mobility shift assays showed that the IREs from the glyceraldehyde-3-phosphate dehydrogenase, IGFBP-1, IGF-I, and amylase genes competed fully for IRE-BP1 binding to the IGFBP-3 IRE, competition was weaker with the IREs from the PEPCK and TAT genes, and there was little competition by the prolactin IRE. Except for the amylase IRE, competition was similar for binding of rat liver nuclear extracts to the IGFBP-3 IRE, presumably reflecting interactions with native IRE-BP1 (Fig. 2b). To further confirm that IRE-BP1 is an IRE-binding protein, we assessed the immunoreactivity of the endogenous protein that binds to the IRE. A Farwestern blot showed that the shifted bands formed between the nuclear extracts and the IREs of IGFBP-3, IGF-I, and IGFBP-1 reacted strongly with the antibodies against IRE-BP1 (Fig. 2c). The shifted bands immunoreacted with both IRE-BP1 cAb and a second antibody developed against an oligopeptide corresponding to the amino segment (amino acids 233–247) of rat IRE-BP1 (nAb). The nAb reacted as two bands under the nondenaturing conditions of the gel shift assay, suggesting that IRE-BP1 may bind as homodimer to the IREs. Specificity of the reaction was shown with a Sp1 antibody, which recognized only Sp1 reacting with the IGF-I IRE as reported previously (17), and a peroxisome proliferator-activated receptor {gamma} antibody used as a negative control. Interestingly, in these studies the Farwestern technique was more informative than supershift analysis; because denaturation with the use of the Farwestern technique separates individual proteins, it is possible that IRE-BP1 binds to cofactors that interfere with antibody epitope binding, or that the binding of IRE-BP1 to the IRE limits reaction with the antibody. In combination, these findings indicate that the endogenous protein that binds to the IREs studied appears to contain immunoreactive IRE-BP1. Whereas the data show specificity of IRE-BP1 recognition, interactions of IRE-BP1 with multiple genes suggest that IRE-BP1 may be involved in coordinating a variety of responses to insulin. Forkhead box transcription factor 01 (Foxo1) is a transcriptional activator that was previously reported to act through the IRE sequence of the IGFBP-1 gene (18), however, a rabbit polyclonal antibody directed against an epitope mapping to amino acids 471–598 of Foxo1 did not react with the IGFBP-1 IRE by this far Western technique, although we were able to detect the presence of Foxo1 in the same preparation of nuclear protein using Western blotting (data not shown).



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  		FIG. 2.
DNA binding specificity of IRE-BP1. a, gel mobility shift assay was conducted with Trx-IRE-BP1 fusion protein and 32P-labeled IGFBP-3 IRE probe. Competition was done with a 50-fold molar excess of unlabeled double-stranded oligonucleotides corresponding to the IGFBP-3 IRE, NF{kappa}B consensus binding site, and double-stranded oligonucleotides corresponding to the identified IREs of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IGFBP-1, PEPCK, IGF-1, prolactin, TAT, and amylase (left panel). The arrow indicates the position of the IRE-BP1-IRE band. b, binding of normal rat hepatic nuclear extracts to the IGFBP-3 IRE was competed in the same manner as Trx-IRE-BP1. c, gel shift bands formed between nuclear extracts and 32P-labeled IGFBP-3, IGF-I, and IGFBP-1 IREs were transferred to a nitrocellulose membrane (upper panel), the proteins were denatured by guanidine HCl, and the blot was probed successively with anti-IRE-BP1, anti-Sp1, and anti-peroxisome proliferator-activated receptor {gamma} antibodies. This experiment was repeated 2 times.

 
IRE-BP1 Transactivates the Insulin-response Sequence—To test the transactivation potential of IRE-BP1, we subcloned the cDNA into a mammalian expression vector (PCR 3.1), and transiently cotransfected the vector into COS-7 cells with target constructs containing the IGFBP-3 IRE linked to a luciferase reporter (pGL3 promoter). The results showed that a 3.4-kb IRE-BP1 cDNA (+1641/+5043 bp), which includes 1503 bp of translated carboxyl sequence and 1899 bp of 3'-untranslated sequence, increased IRE-linked reporter activity 14-fold but had only a 2-fold effect on the control reporter vector (Fig. 3a). Addition of 10–8 M insulin had little effect on the expression with the control vector but produced 3-fold stimulation of the IRE reporter in the presence of IRE-BP1. Thus, there are two components to the stimulation of the IGFBP-3 IRE by IRE-BP1. First, IRE-BP1 appears to bind to the insulin response element and confers basal promoter activity to the reporter gene. Second, modulation of the action of IRE-BP1 by insulin enhanced reporter transcription further, probably through mechanisms that involve phosphorylation or other post-translational effects. Similar findings were obtained with an IGFBP-3 IRE reporter gene transfected into primary cultures of hepatic nonparenchymal cells, where the endogenous factors that bind to the promoter region of IGFBP-3 are presumably localized (shown in Fig. 3b). Compared with an empty expression vector, the IRE region increased reporter activity by 17-fold even in the absence of insulin, consistent with induction of basal promoter activity by the transcription factors that bind to the IRE. Addition of insulin at 10–6 M increased reporter activity by a further 2.3-fold, suggesting post-translational modulation of the bound factors by insulin. Therefore, transfected IRE-BP1 transactivates IGFBP-3 IRE by a mechanism likely to be similar to that of the endogenous factor in hepatic cells, in which insulin increased gene transcription above the activity conferred by the factors that bind to the IRE in the basal state.



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  		FIG. 3.
Transactivation of the insulin-responsive sequences by IRE-BP1. a, cotransfection of COS-7 cells with a 3.4-kb IRE-BP1 expression vector and an IGFBP-3 IRE-luciferase reporter (pGL3 IRE). Control included pGL3 promoter vector without the IRE. b, hepatic nonparenchymal cells were transfected with pGL3 vector or with IGFBP-3 IRE reporter construct (pGL3 IRE). Insulin was added in increasing doses, and luciferase activity was measured. c, cotransfection of COS-7 cells with the 3.4-, 4.8-, and 5.043-kb IRE-BP1 cDNA expression constructs and IGFBP-3 IRE luciferase reporter as in a. d, schematic representation of cDNA constructs used for co-tranfection studies in a and c, and for the DNA binding study shown in Fig. 2a. e, coupled in vitro transcription and in vitro translation was conducted using rabbit reticulocyte lysate in the presence of [35S]methionine. The 3.4-kb IRE-BP1 cDNA expression constructs were translated from 3 reading frames to verify the correct translation frame of the protein. f, cotransfection of the 3.4-kb IRE-BP1 expression vector with IGFBP-1, IGF-1, and prolactin IREs linked to luciferase reporter gene into COS-7 cells. Control represents vector without IRE-BP1 cDNA. Luciferase assay was done 48 h after cotransfection.

 
Unexpectedly, cDNA constructs containing additional 5' sequence (4.8–5.04 kb) increased IRE reporter activity to a much lesser extent than the 3.4-kb cDNA construct (Fig. 3c), consistent with a negative regulatory element in the region 5' to the 3.4-kb sequence. Structural analysis revealed that the 3.4-kb cDNA (transcriptionally active cDNA) contains three FN3-like domains and the apparent COOH terminus, including the DNA-binding domain (scheme shown in Fig. 3d). Because the added sequence contains most of the EGF-like repeats, these elements may have a silencing effect. The truncated carboxyl-half of the protein (~50 kDa) thus appears to be sufficient for transcriptional stimulation. To confirm that this cDNA was expressed in the proper context for transfection, coupled in vitro transcription and in vitro translation was conducted with a reticulocyte lysate system to confirm that the transcriptionally active expression vector encodes the predicted 50-kDa size protein, as shown in Fig. 3e.

To determine whether IRE-BP1 regulates transcription of other insulin responsive genes, we cotransfected an expression vector that encodes the transcriptionally active cDNA, together with target constructs that contain insulin-responsive sequences identified previously from the IGFBP-1, IGF-1, and prolactin genes. As shown in Fig. 3f, compared with vector alone, IRE-BP1 did not significantly alter the basal transcription rate of the IGFBP-1 IRE reporter gene. However, with addition of insulin, IRE-BP1 decreased IGFBP-1 IRE reporter transcription by 63.2 ± 0.7%, compared with 24.7 ± 5% with vector alone (p < 0.05). Thus, IRE-BP1 enhanced the negative effect of insulin on IGFBP-1 IRE transcription. By contrast, IRE-BP1 increased basal IGF-1 IRE transcription by 4.7 ± 0.1-fold, and by 3.5 ± 0.3-fold in the presence of insulin (both p < 0.05 versus vector only); and IRE-BP1 had no significant effect on the transcription rate of the prolactin IRE. Thus, the ability of IRE-BP1 to activate IRE reporter transcription correlates with its ability to bind to the specific IRE sequence of the gene.

Proteolysis and Subcellular Distribution of IRE-BP1—IRE-BP1 cDNA is predicted to encode a protein of ~107 kDa. Co-transfection studies showed, however, that the truncated carboxyl-half of the cDNA, which encodes a 50-kDa protein, is transcriptionally active. Furthermore, addition of 5' sequence to the cDNA attenuates the transcriptional activity of the expression vector (shown in Fig. 3c). To determine whether truncation of the protein is required for its biological activation, we separated the cytoplasmic and the nuclear proteins from HepG2 cells (19), and studied the fractionated extracts by Western blotting using both cAb and nAb. The results showed that the nAb recognized the –120-kDa band in the cytoplasmic extracts, but reacted poorly with the nuclear extracts (Fig. 4a). The increased size from the predicted 107 kDa may be secondary to post-translational modifications of the protein. In contrast, the cAb recognized both the 120-kDa band in the cytoplasmic extracts and a 50-kDa protein in the nuclear extracts. Furthermore, exposure of cells to insulin (10–7 M) for 16 h appeared to decrease cytoplasmic IRE-BP1, and increase the 50-kDa carboxyl portion of IRE-BP1 in the nucleus.



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  		FIG. 4.
Proteolysis, subcellular and tissue distribution of IRE-BP1. a, HepG2 cells were subfractionated into cytosolic and nuclear fractions by detergent disruption of cell membrane and high salt extraction of crude nuclei. Samples were subjected to immunoprecipitation with nAb or cAb, followed by Western blotting with the same antibodies. b, confocal microscope image of HepG2 cells grown in the absence or presence of insulin. Cells were immunostained with IRE-BP1 nAb or cAb, and optical sections in the center of the nuclei were obtained with a Zeiss confocal microscope. Magnifications, x630. c, antisense RNA probes corresponding to the 170-bp Kpn/XhoI (+2270 to +2440) fragment of IRE-BP1 and the 250-nucleotide mouse {beta}-actin transcript were used for ribonuclease protection assay of rat tissues from various organs.

 
We also analyzed the subcellular distribution of the protein by confocal laser scanning microscopy. HepG2 cells were incubated in the presence or absence of insulin for 72 h, and immunostained with the cAb or nAb (Fig. 4b). Optical sections through the nucleus showed that the nAb immunoreactivity was localized predominantly to the cytoplasm, and tended to aggregate in the perinuclear area. In contrast, cAb immunoreactivity was localized predominantly to the nucleus, even in the absence of stimulation by insulin, whereas insulin increased cAb staining in both the cytoplasm and nucleus. Together with the findings of our cotransfection assays, these data demonstrate both proteolysis and potential functional relevance. The high molecular mass protein (120-kDa band) appeared to be restricted mostly to the cytoplasm, but the truncated portion containing the carboxyl end (50-kDa band) was localized predominantly to the nucleus. Thus, the 50-kDa protein may represent the mature form that translocates to the nucleus and activates IRE-BP1-dependent transcription.

Tissue Distribution of IRE-BP1—Use of a 250-bp {beta}-actin and a 170-bp IRE-BP1 probe in a ribonuclease protection assay demonstrated expression of IRE-BP1 in multiple organs (Fig. 4c). These studies also confirmed decreased hepatic expression of IRE-BP1 in streptozotocin-induced diabetic rats as compared with normal rats. Densitometric analysis of IRE-BP1 expression normalized to {beta}-actin expression showed that IRE-BP1 expression was highest in the brain, followed by liver, small intestine, kidney, subcutaneous fat, and spleen. IRE-BP1 is thus distributed in target tissues that are critical for the peripheral and central actions of insulin, and hepatic IRE-BP1 expression is responsive to insulin/diabetes status.

IRE-BP1 Is Regulated through Insulin Signaling Pathways—Because insulin stimulates gene transcription through both the mitogen-activated protein extracellular signal-regulated kinase (ERK) and the PI 3-kinase/Akt pathways, we tested the ability of Akt and ERK to phosphorylate IRE-BP1 in vitro (Fig. 5a). Akt and ERK kinases were immunoprecipitated from insulin-treated COS-7 cells, and kinase reactions were performed with Trx fusion proteins described above. Akt and ERK phosphorylated Trx-IRE-BP1 within 20 min, but failed to phosphorylate the negative controls (Trx alone and the p50 subunit of NF-{kappa}B). IRE-BP1, therefore, may be a physiological substrate for both Akt and ERK.



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  		FIG. 5.
Regulation of IRE-BP1 by insulin signal transduction. a, Trx and Trx-IRE-BP1 fusion protein were expressed in E. coli, incubated with Akt or ERK in the presence of [{gamma}-32P]ATP, and analyzed by SDS-PAGE. E. coli-expressed NF{kappa}B p50 was used as negative control. b, COS-7 cells were transfected with either IRE-BP1 (pCR IRE-BP1, black bars) or control vector (pCR vector, white bars), plus Akt 1 myr or Akt K179M or the control vector (pUSE amp) as indicated, with IGFBP-3 IRE-luciferase reporter constructs. Luciferase activity was normalized to total protein. The data represent results of three independent experiments. c, cells were cotransfected with IGFBP-3 IRE-luc and with either wild-type Akt1 and/or ERK2, treated with (black bars) or without insulin (white bars) overnight. The average of six independent experiments are shown. d, protein extracts (250 µg/gel) from HepG2 cells were subjected to two-dimensional gel electrophoresis and immunoblotted for IRE-BP1 using cAb. The relative positions of the spots were directly compared by overlaying co-electrophoresed markers on the gels. The leftward shift in pI toward the acidic end exhibited by the lower gels is indicated by horizontal arrows.

 
To determine whether Akt could modulate IRE-BP1 action in vivo, we examined the effects of Akt1 on basal and IRE-BP1-induced IGFBP-3 IRE reporter activity in COS-7 cells (Fig. 5b). Overexpression of IRE-BP1 increased transcription of the IRE reporter 6-fold as compared with the control vector (lane 5 versus lane 1); insulin treatment at 10 nM for 24 h further increased IRE-BP1-activated transcription by 107.7 ± 2% (lane 6 versus 5). The effect of Akt myr (constitutively activated enzyme) on IRE-BP1-increased IRE activity was similar to that observed for insulin (lane 7 versus 6), whereas kinase-inactive Akt K179M did not change IRE-BP1-activated transcription above that induced by IRE-BP1 alone (lane 8 versus 5). Control studies lacking the IRE-BP1 expression construct revealed that both insulin and Akt myr slightly increased the reporter gene, but Akt K179M did not (lanes 2–4 versus lane 1). Activated Akt1 therefore mimics the ability of insulin to enhance IRE-BP1-induced transcription, suggesting that insulin stimulation of IRE-BP1 may be mediated in part through phosphorylation by Akt.

We also investigated the difference between signaling from Akt and ERK on IRE-BP1 activation (Fig. 5c). As in the previous experiment, IRE-BP1 increased IRE reporter activity 6.8-fold (lane 3 versus lane 1). Similar to Akt myr, wild-type Akt1 expression stimulated IRE-BP1-induced IRE activity to the same extent as 10 nM insulin (lane 7 versus lane 4), and insulin treatment had no additive effect on Akt-stimulated transcription (lane 8 versus lane 7). By contrast, ERK2 decreased IRE-BP1-induced IRE transcription by 45 ± 4% (lane 5 versus lane 3), but did not completely abolish the effect of IRE-BP1. This inhibitory effect of ERK on IRE-BP1 activation was partially reversed by adding insulin (lane 6 versus lane 5). When ERK2 and Akt1 were expressed together, the inhibitory effect of ERK2 appeared to predominate over the stimulatory effect of Akt (lane 9 versus 7). These data therefore suggest that ERK2-mediated phosphorylation of IRE-BP1 inhibits its function, whereas Akt-mediated phosphorylation of IRE-BP1 enhances its function.

Finally, to demonstrate insulin- and Akt-mediated in vivo phosphorylation of IRE-BP1, we used the two-dimensional proteomic approach. In this experiment, HepG2 cells were incubated with vehicle (control) or 10 nM insulin for 20 min, in the presence or absence of the phosphatidylinositol 3'-kinase inhibitor LY294002 (10 µM), or the cells were transfected with a constitutively active Akt expression vector (Akt myr) without the addition of insulin. Then the cells were lysed, total extracts were separated by two-dimensional electrophoresis, and IRE-BP1 was detected by immunoblotting with an antibody that recognized the COOH-terminal epitope. As shown in Fig. 5d, the main immunoreactive band that migrated to the predicted pI of IRE-BP1 (indicated by down arrows), contains at least four spots of similar molecular mass (–50 kDa) but differing pI values (ranging between pI 7.1 and 7.8). Stimulation of HepG2 cells with insulin resulted in a shift of the spots to a more acidic pI. Because phosphorylation will increase the negative charge of the protein, a shift to a more acidic isoelectric point is suggestive of a phosphorylation event. To confirm that the electrophoretic shift resulted from the post-translational effect of Akt, we showed that cells that were transfected with Akt exhibited a pI shift similar to that of insulin-treated cells. Furthermore, inhibition of PI 3'-kinase prior to stimulation by insulin prevented the pI shift, confirming that the PI 3'-kinase-Akt cascade may participate in insulin-induced IRE-BP1 regulation of HepG2 cells. Other spots on the left side of the gels represent either nonspecific binding or other post-translational modifications of IRE-BP1, including tyrosine phosphorylation and glycosylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we report the identification of a novel transcription factor that shares structural domains with the notch, insulin, and IGF-1 receptors, but not with other DNA binding factors. EGF-like modules contain about 45 amino acids, including six cysteine residues that characteristically paired to form disulfide bonds (20, 21), and IRE-BP1 contains 13 EGF-like repeats in the NH2 terminus. Although EGF-like motifs are found in many proteins with diverse functions, the EGF-like segment of IRE-BP1 has strong similarity to the notch-related proteins (22, 23); the nucleotide 567 to 1031 sequence of rat IRE-BP1 exhibits 62% homology with the extracellular domain of human notch receptor 4 (24). The carboxyl-half of IRE-BP1, however, contains three FN3 repeats, instead of the six to seven ankyrin repeats usually seen in notch receptors (25). Similar to the EGF-like domain, FN3 domains have been identified in different proteins of diverse function, such motifs have been identified in cytokines and protein-tyrosine kinase receptors, including the insulin and IGF-1 receptors (16). It is interesting that IRE-BP1 has three consecutive FN3 domains in the COOH terminus, a structure in common with the extracellular juxtamembrane region of the IGF-1 and insulin receptors, suggesting the possibility of a shared function with these receptors (26).

IRE-BP1 appears to fulfill several key criteria as an insulin responsive DNA-binding factor. First, IRE-BP1 binds to the IREs of multiple insulin-responsive genes, and activates transcription through such elements. In this study, we demonstrated that IRE-BP1 is an endogenous hepatic nuclear protein that binds to the IREs previously identified for the IGF-1, IGFBP-1, and IGFBP-3 genes (4, 6, 27). IRE-BP1 transactivates IRE transcription in a direction similar to the positive or negative effects of insulin on different genes. It acts to stimulate the IRE of the IGFBP-3 and IGF-1 genes that are positively regulated by insulin, and it acts to inhibit the IRE of the IGFBP-1 gene, which is negatively regulated by insulin. Second, IRE-BP1 is regulated by insulin at the mRNA and protein levels. We found that insulin deficiency in streptozotocin-diabetic rats was associated with reduced hepatic expression of the 90-kDa IRE-BP1 protein. This finding is consistent with our previous Southwestern blotting study with the IGFBP-3 IRE probe in which a 90-kDa insulin-responsive DNA-binding protein was shown to be decreased in diabetes (4). The hepatic nuclear extracts from diabetic rats also exhibited reduced DNA-protein complex formation with an IGFBP-3 IRE probe, presumably underlying the decreased hepatic transcription of the IGFBP-3 gene observed in diabetes. Induction of IRE-BP1 by insulin is also consistent with the results of our nuclear run-on assay in which inhibition by cycloheximide implied that induction of a secondary protein is necessary for insulin activation of IGFBP-3 gene transcription. Third, insulin appears to stimulate phosphorylation of IRE-BP1 through the PI 3-kinase-Akt pathway. The active form of Akt enhances IRE-BP1 transactivation of the IRE. Whereas IRE-BP1 may be directly phosphorylated by Akt, the primary sequence of IRE-BP1 does not contain the consensus sequence predicted for an Akt substrate (RXRXX(S/T)) (28). However, IRE-BP1 encodes a peptide sequence that is consistent with a pattern of the p70 S6 kinase substrate (Lys-Glu-Arg-Cys-Gln-Ser1036-Thr-Ser-Leu), a signaling kinase downstream of Akt, and others have reported that Akt phosphorylates sequences that are different from the archetypal motif (29). Therefore, the possibility that IRE-BP1 also contains a non-typical Akt motif cannot be excluded at present. In addition, IRE-BP1 is predicted to express the PX(S/T)P domain (Gly-Ala-P-Glu-Thr883-Pro-Thr-Gln-Pro and Ser-Gln-Pro-Thr-Thr920-Pro-Val-Pro-Leu), a typical motif for proline-directed kinase such as ERK, and Thr883 is followed by a FXXP peptide, a sequence that closely resembles an ERK docking site (FXFP) (30). Determination of the residues of IRE-BP1 that are phosphorylated after insulin treatment is ongoing in our laboratory.

Our investigation is limited by the use of the IGFBP-3 IRE reporter gene, which is transcriptionally active in COS-7 and hepatic nonparenchymal cells but not in hepatocytes (12). However, our studies suggest that IRE-BP1 could function as a general mediator of the transcriptional action of insulin in hepatic tissues because IRE-BP1 also binds to the IREs identified for IGF-1 and IGFBP-1 genes, two genes known to be expressed predominantly in hepatocytes (3). Although IRE-BP1 does not bind directly to the IREs previously identified for the hepatic PEPCK and TAT genes, the IREs from both genes coincide with elements required for induction of transcription by glucocorticoids, suggesting that insulin might function by interfering with either binding and/or transactivation of glucocorticoid-induced factors (1). Thus, IRE-BP1 could potentially act to modulate IRE function of these genes without binding directly to the IREs. Furthermore, the differential effects of IRE-BP1 on the promoter regions of the different genes may also be determined by the presence of different groups of proteins that interact with IRE-BP1. For example, we detected Sp1 as a protein that may interact with IRE-BP1 in the IGF-1 promoter region (shown in Fig. 2c), but not in the IRE region of the IGFBP-1 and IGFBP-3 genes. At present, we have not eliminated the possibility that the promoter effects of IRE-BP1 may be induced by competing away a repressor, such as hepatic nuclear factor 3, which has been shown to support glucocorticoid-induced gene transcription of the IGFBP-1 and PEPCK genes through their insulin response sequences (31).

The finding that the carboxyl domain of IRE-BP1 is able to elicit transcriptional activation of the IRE, whereas inclusion of the amino domain attenuates the transactivation of the IRE, suggests the presence of a negative regulatory region in the 5' end, and/or the possibility that the protein must be truncated to be transcriptionally active. Our studies imply that IRE-BP1 undergoes proteolysis, with the cleaved cytosolic half of the protein being transported to the nucleus. In signal transduction involving the notch receptor, the intracellular domain of the receptor is released by proteolysis, and translocates to the nucleus to act as a transcriptional co-activator, whereas the NH2-terminal fragment containing EGF-like repeats remains localized to the plasma membrane (20, 25, 32, 33). In the regulation of ErbB4, the receptor undergoes proteolysis, with the cleaved cytosolic half of the protein being transported to the nucleus (34, 35). However, the nuclear-transported cytosolic half of ErbB4 is inactive in transcription assays while the carboxyl-terminal tail drives transcription from a GAL4 reporter gene, indicating that the protein undergoes further processing in the nucleus (36). Our studies indicate similarities between the processing of IRE-BP1 and the ErbB4 and notch receptors. IRE-BP1 cDNA encodes a 120-kDa protein but we detected a 90-kDa immunoreactive band in hepatic nuclei. Only the carboxyl-terminal 50-kDa region appears to activate transcription of a luciferase reporter gene driven by the insulin response DNA binding element, suggesting that sequential proteolysis of IRE-BP1 may be necessary for its transduction effect. Because only a small amount of processed protein fragments are needed to confer full signal transduction activity, the present model of IRE-BP1 proteolysis is based largely on the functional assays of transcriptional activity of the truncated protein, and requires further study.

IRE-BP1 appears to be regulated by insulin in a manner similar to other factors that affect IRE transcription. The Foxo1 is a target of Akt action, and can mediate the negative regulatory effects of insulin on the IGFBP-1 and the Fas ligand gene promoters (37, 38). Phosphorylation by Akt results in the redistribution of Foxo1 to the cytoplasm, inhibiting forkhead-induced transactivation of IGFBP-1 and reducing IGFBP-1 gene transcription, and gain-of-function mutations of Foxo1 result in the development of diabetes (39). Our investigation suggests that instead of sequestering IRE-BP1 in the cytosol, insulin may increase nuclear entry of the carboxyl portion of IRE-BP1 to activate insulin responsive genes. Furthermore, IRE-BP1 contains the peptide sequences LSVLS (at amino acids 374–378) and DRSR (at amino acids 603–606), which have been identified as optimal substrates for cleavage of sterol regulatory element-binding protein-1 (40). Such cleavage is required for the release from the endoplasmic reticulum and subsequent transit into the nucleus, where truncated sterol regulatory element-binding protein modulates the transcription of genes involved in fatty acid and cholesterol synthesis. Whether the nuclear translocation or proteolysis of IRE-BP1 is related to phosphorylation by Akt or whether IRE-BP1 is subject to a similar mechanism of proteolysis as sterol regulatory element-binding protein-1, are appropriate subjects for future investigations.

In summary, IRE-BP1 is regulated by insulin at the mRNA, protein, and post-translational levels. Post-translational regulation appears to involve both phosphorylation and proteolysis. Therefore, the mechanisms by which insulin induces responses through IRE-BP1 could involve de novo synthesis of protein that leads to increased DNA binding to the IRE. These events may account for the delayed effect of insulin on the transcription of some genes. Phosphorylation of prebound IRE-BP1 through the PI 3-kinase-Akt pathway may alternatively mediate a rapid effect on transcription of target genes. Based on our data, we propose that IRE-BP1 is a transcription factor that activates multiple insulin-responsive genes. Identification of this molecular target of insulin action should increase our understanding of the regulation of gene transcription by insulin, and may help us determine how this function is linked to the pathogenesis of type 2 diabetes.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant DK52965, grants from EmTech Biotechnology Development Inc., Georgia Institute of Technology FRCP, and the Walter F. and Avis Jacobs Foundation (to B. C. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Postdoctoral fellow in the Division of Endocrinology. Back

§ To whom correspondence should be addressed. Tel.: 502-852-4048; Fax: 502-852-2492; E-mail: bcvill01{at}louisville.edu.

1 The abbreviations used are: IRE, insulin response element; IGFBP-3, insulin-like growth factor binding protein-3; IGF-1, insulin-like growth factor-1; AD, activation domain; IRE-BP1, insulin response element-binding protein 1; PEPCK, phosphoenolpyruvate carboxykinase; cAB, antibody against carboxyl-terminal segment (amino acids 786–800) of rat IRE-BP1; nAb, antibody against amino segment (amino acids 233–247) of rat IRE-BP1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HBP1, high mobility group box containing protein 1; EGF, epidermal growth factor; FN3, fibronectin III; ERK, extracellular signal-regulated kinase; Foxo1, forkhead box transcription factor 01; Trx, thioredoxin; PI 3-kinase, phosphatidylinositol 3-kinase. Back


   ACKNOWLEDGMENTS
 
We are grateful for the technical assistance of Saroja Devi, for the thoughtful comments of Drs. Stephen B. Winters and Haian Fu.



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 DISCUSSION
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