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Endocrinology, doi:10.1210/en.2003-0481
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Endocrinology Vol. 144, No. 12 5615-5622
Copyright © 2003 by The Endocrine Society

Insulin Inhibition of Transcription Stimulated by the Forkhead Protein Foxo1 Is Not Solely due to Nuclear Exclusion

Wen-Chi Tsai, Nisan Bhattacharyya, Li-Ying Han, John A. Hanover and Matthew M. Rechler

Diabetes Branch (W.-C.T., N.B., L.-Y.H., M.M.R.) and Laboratory of Cell Biochemistry and Biology (J.A.H.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1758

Address all correspondence and requests for reprints to: Dr. M. M. Rechler, Building 10, Room 8D12, 9000 Rockville Pike, Bethesda, Maryland 20892-1758. E-mail: mrechler{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The FOXO family of forkhead transcription factors stimulates the transcription of target genes involved in many fundamental cell processes, including cell survival, cell cycle progression, DNA repair, and insulin sensitivity. The activity of FOXO proteins is principally regulated by activation of protein kinase B (PKB)/Akt by insulin and other cytokines. PKB/Akt phosphorylates three consensus sites in FOXO proteins, leading to their export from the nucleus and the inhibition of FOXO-stimulated transcription. It has been widely accepted that the decreased transcription results from reduced abundance of FOXO proteins in the nucleus. In the present study we mutated Leu375 to alanine in the nuclear export signal of Foxo1 (mouse FOXO1), so that it would remain in the nucleus of H4IIE rat hepatoma cells after insulin treatment, and determined whether insulin could still inhibit transcription stimulated by the Foxo1 mutant. Despite the retention of the Foxo1 mutant in the nucleus, insulin inhibited L375A-Foxo1-stimulated transcription to the same extent as transcription stimulated by wild-type Foxo1. Similar results were obtained using reporter plasmids containing the rat IGF-binding protein-1 promoter or a minimal promoter with three copies of the insulin response element to which FOXO proteins bind. We conclude that insulin can inhibit Foxo1-stimulated transcription even when nuclear export of Foxo1 is prevented, indicating that insulin inhibition can occur by direct mechanisms that do not depend on altering the subcellular distribution of the transcription factor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DECREASED INSULIN inhibition of the transcription of two key enzymes in gluconeogenesis, phosphoenolpyruvate carboxykinase and the catalytic subunit of glucose-6-phosphatase, contributes to the increased hepatic glucose production and hyperglycemia associated with type 2 diabetes (1, 2, 3). The promoters of both genes contain a consensus insulin response element (IRE) that confers on minimal promoters the ability to be negatively regulated by insulin (4, 5). A similar IRE is present in the promoter for IGF-binding protein-1 (IGFBP-1) (6, 7, 8), a protein that binds IGF-I and IGF-II and regulates their growth-promoting and insulin-like activities. IGFBP-1 transcription is increased in diabetic rat liver and is rapidly normalized by insulin (9, 10).

Insulin inhibition of IGFBP-1 gene expression is mediated by phosphatidylinositol 3-kinase (PI 3-kinase) (11, 12) and its downstream effector, serine/threonine-specific protein kinase B (PKB)/Akt (12). Recognition that a similar insulin signaling pathway in Caenorhabditis elegans inhibited the transcription factor Daf16 (13, 14), an ortholog of the FOXO subfamily of forkhead transcription factors (15), suggested that FOXO proteins might mediate insulin inhibition of transcription in mammalian cells. Three human FOXO proteins (FOXO1, FOXO3a, and FOXO4)1 and their mouse counterparts (Foxo1, Foxo3, and Foxo4) have been identified and extensively characterized (16, 17, 18, 19). They share a conserved central DNA-binding domain (the Fox box, residues 155–255 in the 652-amino acid Foxo1 used in the present study) and have a C-terminal trans-activation domain (20). The FOXO proteins bind to an IRE in the proximal promoter (21, 22, 23) of target genes involved in insulin sensitivity, cell survival, cell cycle progression, and DNA repair (24, 25, 26, 27) and stimulate their transcription.

After being activated by insulin or the closely related growth factor IGF-I, PKB/Akt phosphorylates FOXO proteins at three conserved sites (Thr24, Ser253, and Ser316 in Foxo1) (28, 29, 30). Phosphorylation of these sites leads to inhibition of FOXO-stimulated transcription (24, 31, 32, 33) and redistribution of the transcription factor from the nucleus to the cytoplasm (24, 34, 35). Brunet et al. (24) proposed that the export of FOXO from the nucleus after phosphorylation by PKB/Akt would prevent the transcription factor from activating its target genes. The concept that nuclear exclusion is the principal mechanism of FOXO regulation has been widely accepted and has been extrapolated to other growth factors and cytokines that activate PKB/Akt (36, 37, 38).

The present study examines whether insulin also can inhibit Foxo1-stimulated transcription by other mechanisms when redistribution of Foxo1 to the cytoplasm has been prevented. Our strategy was based on the demonstration by Biggs et al. (39), subsequently confirmed by other investigators (40, 41, 42), that Foxo1 was exported from the nucleus by the Crm1 export transporter that binds to a leucine-rich nuclear export signal (NES) in the presence of Ran-GTP (43, 44, 45, 46). Biggs et al. (39) reported that a Foxo1 mutant in which Leu375, a key residue in the NES (47), was mutated to alanine remained in the nucleus of CV1 monkey kidney cells after incubation with insulin (39). In the present study we demonstrate that L375A-Foxo1 is localized predominantly in the nucleus of H4IIE rat hepatoma cells after insulin treatment, and that despite nuclear localization of the mutant transcription factor, insulin inhibits transcription stimulated by L375A-Foxo1 and wild-type Foxo1 to the same extent. We conclude that insulin can inhibit Foxo1-stimulated transcription by additional mechanisms besides simple relocation to the cytoplasm.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The plasmid pCMV5-c-Myc-Foxo1 (henceforth, pCMV5-Foxo1) was provided by J. Nakae and D. Accili (29, 33). p925-GL3 Basic, a luciferase reporter plasmid containing nucleotides -925 to +79 of the rat IGFBP-1 promoter (7, 33), was previously described. pCMV5 empty vector was prepared by XbaI digestion of pCMV5-Foxo1 to delete the Foxo1 insert.

The plasmid pGL3-Promoter was obtained from Promega Corp. (Madison, WI); plasmid Bluescript II (KSII+) was purchased from Stratagene (La Jolla, CA); DMEM (low glucose) was obtained from Invitrogen (Carlsbad, CA); fetal bovine serum was purchased from HyClone Laboratories (Logan, UT); Humulin U-100 regular insulin was obtained from Eli Lilly & Co. (Indianapolis, IN); anti-c-Myc mouse monoclonal immunoglobulin G (9E-10) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey antimouse antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA); LY294002, an inhibitor of PI 3-kinase, was obtained from Calbiochem (San Diego, CA); restriction enzymes and QuikChange Site-Directed mutagenesis kit were purchased from Stratagene; Centri-Sep columns were obtained from Princeton Separations (Adelphia, NJ); plasmid purification kits were obtained from Qiagen (Valencia, CA); and DNA sequencing kits were purchased from PE Applied Biosystems (Foster City, CA).

Cell cultivation
H4IIE rat hepatoma cells (48) were grown as monolayer cultures in low glucose DMEM supplemented with 10% fetal bovine serum and were incubated in a humidified 95% air/5% CO2 atmosphere at 37 C. Cultures were passaged weekly (when they reached confluence) at a ratio of 1:20 using trypsin-EDTA. Fresh stocks were thawed after 8–10 passages.

Construction of L375A-Foxo1 expression vector
Full-length wild-type Foxo1 was excised from pCMV5-Foxo1 using XbaI and was ligated into XbaI-digested pBluescript vector to generate pBluescript-Foxo1. The L375A mutation was introduced into pBluescript-Foxo1 by overlapping PCR. The 5' fragment was generated by PCR using primers 490-GAC CTC ATC ACC AAG GCC ATC-510 (numbered with respect to ATG = 1) and 1142-GGG GAC GAG AGA AGG TTG GCA TTA TCC AGA AGG TTC-1106 [in which CT has been mutated to GC (underlined)] and contained an internal BglII site (545-AGA TCT 550). The 3' fragment was generated using primer 1106-GAA CCT TCT GGA TAA TGC CAA CCT TCT CTC GTC CCC-1142 and 1489-GGC CCA TCA TTA CAT TTT GGC CCA GGA C-1462 and contained an internal AgeI site (1425-ACC GGT-1430). The 5' and 3' fragments were amplified by PCR using the two nonoverlapping primers. The resulting fragment containing the L375A mutation was digested with BglII and AgeI and ligated into pBluescript-Foxo1 that had been digested with BglII and AgeI to generate pBluescript-L375A-Foxo1. L375A-Foxo1 was excised with XbaI and ligated into the dephosphorylated XbaI sites of pCMV5-c-Myc. The orientation and sequence of the insert containing the mutation were confirmed by DNA sequencing.

Construction of pGL3-IRE-3x and pGL3-GC-3x reporter genes
Three tandem copies of the wild-type IRE were inserted into the pGL3-Promoter plasmid (which contains a simian virus 40 promoter upstream from a luciferase reporter gene) to construct pGL3-IRE-3x. The sense strand of IRE-3x contained three copies of the IRE (GCA AAA CAA ACT TAT TTT GAA; the inverted palindrome is underlined). The antisense strand of IRE-3x contained three complementary copies of the IRE plus a BglII site (GATC) at the 5' end and a KpnI site (GTCA) at the 3' end. Plasmid pGL3-GC-3x containing three copies of an inactive G/C-C/A mutant IRE (49, 50) was constructed similarly using oligonucleotides in which G was substituted for C and C substituted for A (shown in bold) in both half-sites of the inverted palindrome (GCA AAA GAA ACT TCT TTT GAA). The G/C-C/A mutant IRE does not bind FOXO1 (21). Annealed and phosphorylated IRE-3x and GC oligonucleotides were ligated into the pGL3-promoter vector (digested with KpnI and BglII and dephosphorylated) using a ligation kit (Takara Biomedical, Inc., Berkeley, CA). The sequences of the IRE-3x and GC-3x inserts were confirmed.

Immunofluorescence
H4IIE cells were transiently transfected with wild-type or mutant pCMV5-c-myc-Foxo1 and incubated with or without insulin, and the subcellular distribution of transfected Foxo1 was studied by fluorescence microscopy as previously described (35). In brief, H4IIE cells were plated onto 60-mm dishes at 3–3.75 million cells/dish and incubated overnight in serum-supplemented medium. The cells then were transfected with the indicated plasmids using Lipofectamine and Plus Reagent (Invitrogen) according to the manufacturer’s protocol (1 µg DNA/4 µl Plus Reagent/5 µl Lipofectamine). After 24 h the cells were trypsinized, and approximately 20,000 cells/well were seeded into 8-well slide culture chambers (Nalge Nunc International, Naperville, IL). After overnight incubation, the medium was changed to serum-free medium (DMEM containing 0.1% BSA) for 24 h, after which insulin (1 µg/ml, final concentration) was added to half of the wells for 1 h. Cells were fixed using a solution of 2% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) and permeabilized in 0.5% Triton X-100. Myc-tagged Foxo1 was visualized using anti-Myc monoclonal antibody (2 µg/ml, final concentration) and FITC-conjugated AffiniPure donkey antimouse IgG (5 µg/ml, final concentration.). Slides were examined using Axiovert TV-100 and TV-200 fluorescence microscopes (Zeiss, New York, NY). Only transfected cells were fluorescent; FITC-stained cells were not observed when primary or secondary antibodies were omitted, and only a small percentage of the cells identified using Sytox nuclear stain were fluorescent.

Digital images of fluorescent cells were taken using the UltraView Confocal Imaging System in conjunction with a Zeiss Axiovert TV-100 microscope. Single cells were analyzed using Open Lab Image Analysis Software (Improvision, Inc., Lexington, MA). Cytoplasmic to nuclear ratios were calculated using the HIS colorspy feature of Open Lab. Pixel intensities were determined at 10 points in the nucleus, 10 points in the cytoplasm, and 10 background points for 5–7 cells in each condition, averaged, and analyzed statistically. In addition, z-axis sections (7- to 10-µm slices, ~10–15 slices/cell) of a representative cell for each condition also were analyzed. Cell and nuclear boundaries were estimated, and the mean intensities were determined in the nuclear and cytoplasmic areas of each section and summed. In addition, the nuclear/cytoplasmic ratio was determined by volume rendering using the Open Lab software.

Transient transfection and luciferase assay
H4IIE cells were transfected as described previously (51). The day before transfection, cells (3–3.75 million/dish) were plated onto 60-mm tissue culture dishes in 3 ml DMEM containing 10% serum and were typically 70–100% confluent at the time of transfection. Diethylaminoethyl dextran stock solution (2 mg/ml in 0.15 M NaCl; Amersham Pharmacia Biotech, Piscataway, NJ) was diluted with an equal volume of Tris-buffered saline [25 mM Tris (pH 7.5), 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM MgCl2, and 0.6 mM Na2HPO4]. Plasmid DNA (~5 µg diluted in 100 µl Tris-buffered saline) was mixed with 100 µl diethylaminoethyl dextran-Tris-buffered saline and incubated at room temperature. [Typically, 3 µg pCMV5 expression vector, 2 µg luciferase reporter, and 40 ng pRSV ß-galactosidase (33) were used.] After 15 min, 190 µl of the mixture were added to each dish. Fifteen minutes later, 3 ml DMEM containing 10% serum were added, and the incubation was continued overnight. After medium change and a second overnight incubation, the medium was replaced with serum-free DMEM containing 0.1% BSA with or without recombinant human insulin (0.25 µg/ml). After 24 h the cells were washed twice with ice-cold PBS and harvested for luciferase assay. After the addition of 360 µl lysis buffer [100 mM potassium phosphate (pH 7.8) and 0.2% Triton X-100], cells were scraped and centrifuged (4 C, 14,000 rpm, 5 min). Supernatant from each sample was transferred to a fresh tube. Luciferase activities of the supernatants were measured using a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) with a Dual Light chemiluminescent reporter gene assay kit (Tropix, PE Applied Biosystems, Bedford, MA) as specified by the manufacturer. Assays were performed in duplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
L375A-Foxo1 is retained in the nucleus after insulin treatment
The effect of insulin treatment on the subcellular localization of wild-type and L375A-Foxo1 transfected into H4IIE cells was examined by fluorescence microscopy. In the absence of insulin, wild-type and L375A-Foxo1 were localized predominantly in the nucleus in all fluorescent cells (Fig. 1Go). After incubation with insulin, wild-type Foxo1 redistributed to the cytoplasm, whereas L375A-Foxo1 remained predominantly in the nucleus. The cells depicted in the upper panel of Fig. 1Go are representative of more than 70 cells examined in each condition (tabulated in Fig. 1Go, lower panel) and show the same predominant localization of wild-type and mutant Foxo1 to the nucleus or cytoplasm.



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  		FIG. 1. Fluorescence microscopy of representative cells transfected with wild-type or L375A-Foxo1 and incubated with insulin. Top panel, Cells were transfected with wild-type (WT) or L375A-Foxo1, incubated with or without insulin for 1 h, fixed, and incubated with mouse monoclonal antibodies to the c-Myc epitope and fluorescein-labeled second antibody. Photographs of representative cells are shown. Bottom panel, The number of fluorescently labeled cells that were more intensely labeled in the nucleus or cytoplasm was determined. The ratio of the total number of cells with predominantly nuclear labeling to the total number of cells with predominantly cytoplasmic labeling from three experiments is shown.

 
The relative amounts of nuclear and cytoplasmic Foxo1 in individual cells transfected with wild-type or mutant Foxo1 and incubated with or without insulin were determined by quantifying the levels of nuclear and cytoplasmic fluorescence in whole cells (Fig. 2Go) and confirmed in sections of digital images of representative cells (Fig. 3Go). The ratios of average pixel intensities (cytoplasm/nucleus) in whole cells in the absence of insulin were 0.27 for wild-type Foxo1 and 0.31 for L375A-Foxo1 (Fig. 2Go). After incubation with insulin, wild-type Foxo1 was preferentially localized to the cytoplasm (cytoplasm/nucleus = 5.3), whereas L375A-Foxo1 remained predominantly in the nucleus (cytoplasm/nucleus = 0.19; Fig. 2Go).



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  		FIG. 2. The effect of insulin treatment on cytoplasmic to nuclear ratios of c-Myc-Foxo1 immunofluorescence in H4IIE rat hepatoma cells transfected with wild-type or L375A-Foxo1. Cells were transfected with wild-type Foxo1 (upper panels) or L375A-Foxo1 (lower panels), and treated (right panels) or not treated (left panels) with insulin as described in Materials and Methods. The mean ± SD of fluorescence intensity in five to seven cells examined for each condition is plotted.

 


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  		FIG. 3. Quantification of the effect of insulin on the subcellular distribution of wild-type (WT) and L375A-Foxo1 in sections of individual cells. Z-Sections of digital confocal images of representative fluorescent cells transfected with WT or L375A-Foxo1 were analyzed using the Open Lab program. The mean fluorescence intensity in the nucleus and cytoplasm was determined in each of the sections and summed. The percentage of total fluorescence in the nucleus is plotted in the absence of insulin ({blacksquare}) and after insulin treatment ({square}). Relative nuclear and cytoplasmic volumes (47%:53%) were estimated from the mean intensity values after incubation with insulin. The cytoplasmic signal in the absence of insulin was too low to allow accurate estimates of cytoplasmic volume.

 
To exclude the possibility that some of the observed nuclear fluorescence might have arisen from overlying or underlying cytoplasm, we also analyzed z-sections of digital images of representative cells (Fig. 3Go). Analysis of cell sections confirmed that the redistribution of wild-type Foxo1 to the cytoplasm after insulin treatment was much greater than that observed for L375A-Foxo1. In the absence of insulin, greater than 80% of total Foxo1 fluorescence was present in the nucleus with either the wild-type or mutant construct. After insulin treatment, nuclear fluorescence of wild-type Foxo1 decreased by 55%, whereas a much smaller decrease in nuclear fluorescence (14%) was observed with L375A-Foxo1. Although the extent of redistribution of wild-type Foxo1 from nucleus to cytoplasm determined by optical sectioning was less complete than that indicated by analysis of whole cells, nuclear export was decreased by 75% in the L375A-Foxo1 mutant compared with that in wild-type Foxo1. Together these results indicate that insulin treatment of H4IIE cells causes a major redistribution of wild-type Foxo1 from nucleus to cytoplasm that is not seen with the L375A-Foxo1 NES mutant, which is predominantly retained in the nucleus after insulin treatment.

Insulin inhibits transcription stimulated by L375A-Foxo1
The ability of insulin to inhibit transcription stimulated by wild-type or L375A-Foxo1 was examined by transient transfection of H4IIE rat hepatoma cells. A luciferase reporter (pGL3-IRE-3x) that contains three tandem copies of the IRE upstream from a minimal simian virus 40 promoter was used (Fig. 4Go). Reporter plasmids lacking an IRE insert (pGL3 empty vector) or containing a mutant IRE that could not bind Foxo1 (pGL3-GC-3x) (21, 49, 50)) were used as negative controls. Stimulation of transcription was dependent on both Foxo1 and the IRE (Fig. 4Go, left panel). In the absence of insulin, wild-type and L375A-Foxo1 stimulated pGL3-IRE-3x promoter activity approximately 25-fold relative to pCMV5 empty vector; no significant stimulation was seen with pGL3-GC-3x.



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  		FIG. 4. Stimulation of pGL3-IRE-3x transcription by wild-type and L375A-Foxo1 is IRE dependent and inhibited by insulin. Left panel, No insulin. Cells were cotransfected with pCMV5 empty vector, wild-type (WT) Foxo1 or L375A-Foxo1, and pGL3-IRE-3x, pGL3-GC-3x, or pGL3 empty vector luciferase reporter plasmids. Fold stimulation of luciferase activity normalized to pGL3 empty vector is plotted for pGL3-GC-3x ({square}) and pGL3-IRE-3x ({blacksquare}; mean ± SE; n = 4). Right panel, Effect of insulin. Luciferase activity also was determined in the same experiments after insulin treatment. The ratio of luciferase activity in the presence and absence of insulin [(+Insulin)/(-Insulin) x 100] using the pGL3-IRE-3x reporter for WT or L375A-Foxo1 is expressed relative to the pCMV5 empty vector in the same experiment. The mean ± SE (n = 6) are plotted. Identical results were obtained when the relative luciferase activity (+Insulin/-Insulin) using pGL3-IRE-3x was normalized to the activity observed using pGL3 empty vector or pGL3-GC-3x (results not shown). [With the pCMV5 empty vector, luciferase activity in the presence of insulin was 174 ± 20% (±SE; n = 6) of that in the absence of insulin. Similar stimulation was seen with pGL3 empty vector and pGL3-GC-3x, indicating that this is a property of the expression vector-reporter system and not of IRE- and Foxo1-dependent transcription.]

 
Insulin inhibited transcription stimulated by L375A-Foxo1 or wild-type Foxo1 to the same extent (Fig. 4Go, right panel). In cells transfected with L375A-Foxo1, luciferase activity in the presence of insulin was 31 ± 6% (mean ± SE; n = 6) of that in cells not treated with insulin compared with 32 ± 5% (mean ± SE; n = 5) for wild-type Foxo1. Thus, insulin inhibited L375A-Foxo1-stimulated transcription in pGL3-IRE-3x to the same extent as transcription stimulated by wild-type Foxo1, even though L375A-Foxo1 remained predominantly in the nucleus after insulin treatment.

Similar results were obtained using a luciferase reporter containing the native rat IGFBP-1 promoter, p925-GL3. In the absence of insulin, wild-type and L375A-Foxo1 stimulated transcription of the p925-GL3 luciferase reporter containing the rIGFBP-1 promoter by 5-fold (Fig. 5Go). Insulin treatment decreased transcription stimulated by wild-type and L375A-Foxo1 to about 35% of that in cells transfected with the same plasmids that did not receive insulin. Insulin also inhibited promoter activity in cells transfected with the pCMV5 empty vector. Inhibition of basal transcription has been observed previously and attributed to insulin inhibition of endogenous Foxo proteins (33, 52, 53, 54). Consistent with this interpretation, H4IIE cells synthesize Foxo3 (53, 55) and Foxo1 (33, 56), and insulin stimulates the phosphorylation of endogenous Foxo3 at two PKB/Akt sites, Thr32 and Ser253 (53).



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  		FIG. 5. Insulin inhibits IGFBP-1 promoter activity stimulated by wild-type and mutant Foxo1. H4IIE cells were cotransfected with pCMV5 empty vector, pCMV5-Foxo1 [wild-type (WT)] or pCMV5-L375A-Foxo1, and the p925-GL3 luciferase reporter. The cells were incubated with or without insulin, and luciferase activity was determined. Relative luciferase activity (mean ± SE from 8–10 experiments) in the absence ({square}) and presence ({blacksquare}) of insulin is plotted. The activity with pCMV5 empty vector in the absence of insulin is taken as 100.

 
PI 3-kinase mediates insulin inhibition of L375A-Foxo1-stimulated transcription
Insulin inhibition of Foxo1-stimulated transcription is mediated by PI 3-kinase (32). Inhibition of PI 3-kinase activity with LY294002 abolished insulin inhibition of IGFBP-1 transcription stimulated by either wild-type or L375A-Foxo1 (Fig. 6Go). With wild-type Foxo1, abrogation of insulin inhibition of transcription by LY294002 could be secondary to blocking insulin-induced nuclear exclusion of Foxo1 (24, 34, 35) or be a direct effect on transcription. Our results indicate that LY294002 also can block the direct effects of insulin on transcription, because it abolishes insulin inhibition of L375A-Foxo1-stimulated transcription when nuclear export does not occur.



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  		FIG. 6. Insulin inhibition of IGFBP-1 promoter activity stimulated by wild-type (WT) and mutant Foxo1 requires PI 3-kinase. H4IIE cells were cotransfected with pCMV5, pCMV5-Foxo1 (WT), or pCMV5-L375A-Foxo1 expression vectors and the p925-GL3 luciferase reporter. Cells were preincubated for 30 min with ({blacksquare}) or without ({square}) 50 µM LY294002, after which half of the cells in each group were treated with insulin. Relative luciferase activity [(+Insulin)/(-Insulin) x 100; mean ± SE] in the absence and presence of LY294002 in three to five experiments] is plotted. [In the absence of insulin, luciferase activity in the presence of LY294002 compared with its absence was 141 ± 31% (±SE; n = 3) for pCMV5 empty vector and 44 ± 8% (n = 8) for WT and L375A-Foxo1.]

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been widely accepted that phosphorylation of the three consensus PKB/Akt sites in Foxo1 and other FOXO proteins after incubation with insulin or other cytokines inhibits FOXO-stimulated transcription of target genes by inducing the export of FOXO proteins from the nucleus (24). In the present study we demonstrate that insulin can inhibit Foxo1-stimulated transcription even when export of Foxo1 from the nucleus is prevented by mutation, providing direct evidence that inhibition can occur in intact cells by mechanisms unrelated to subcellular redistribution of the transcription factor.

The C-terminal trans-activation domain of Foxo1 contains a leucine-rich NES (39, 47, 57).2 Previous studies had shown that leptomycin B, which inhibits binding of the nuclear export transporter Crm1 to the NES (43, 44, 45, 46), blocks the export of FOXO proteins from the nucleus (39, 40, 41). The effect of leptomycin B on the ability of insulin to inhibit FOXO-stimulated transcription was examined in only one of these studies, however, and the results were inconclusive. Leptomycin B decreased insulin inhibition of FOXO4-stimulated transcription in A14 mouse fibroblasts by only 60%, suggesting that both export-dependent and export-independent mechanisms contributed to inhibition (40).

Because of these uncertainties, we decided to evaluate whether insulin could inhibit IGFBP-1 transcription in H4IIE cells when Crm1-mediated nuclear export of Foxo1 was blocked by mutating Leu375 to alanine in the NES (Fig. 5Go). Biggs et al. (39) had shown that the Leu375Ala mutation abolished nuclear export of an N-terminal Foxo1 fragment (1–380) in CV1 cells. This truncated construct lacks the C-terminal trans-activation domain (20, 58, 59), however, so that its effect on transcription could not be studied. We introduced the Leu375Ala mutation into full-length Foxo1. In contrast to the dramatic redistribution of wild-type Foxo1 to the cytoplasm after incubation with insulin, L375A-Foxo1 remained predominantly in the nucleus of transfected H4IIE cells. This result was quantified by determining the relative intensities of wild-type and mutant Foxo1 in the nucleus and cytoplasm of digital images of representative cells and cell sections. As the nuclear and cytoplasmic volumes of H4IIE cells are comparable, the ratio of nuclear/cytoplasmic fluorescence intensities reflects the relative abundance of Foxo1 in the nucleus. Although the decrease in wild-type Foxo1 in the nucleus after insulin treatment would be sufficient to account for the approximately 65% decrease in Foxo1-stimulated transcription, the minimal decrease in nuclear L375A-Foxo1 after incubation with insulin is too low to account for the decreased transcription.

The ability of insulin to inhibit transcription stimulated by L375A-Foxo1 was examined in reporter constructs in which luciferase was fused to two different insulin-sensitive promoters: a 925-nucleotide rat IGFBP-1 promoter (7) and a minimal promoter containing three copies of the IRE. L375A-Foxo1 stimulated transcription from both promoters in the absence of insulin. With both reporters, insulin inhibited transcription stimulated by wild-type Foxo1 and L375A-Foxo1 to the same extent. These results directly demonstrate that insulin is able to inhibit Foxo1-stimulated transcription when nuclear export of the transcription factor is prevented and indicate that insulin can inhibit transcription by other mechanisms in addition to inducing the subcellular redistribution of Foxo1. We propose that direct regulation of transcription also might contribute to the regulation of FOXO-induced transcription by other growth factors [IGF-I (24), epidermal growth factor (36), nerve growth factor (60), and platelet-derived growth factor (61)] and cytokines [TGFß (62), erythropoietin (37, 63, 64), thrombopoietin (65), IL-3 (38), and IL-2 (66)] that have been reported to activate PKB/Akt and phosphorylate FOXO proteins. Insulin inhibition of L375A-Foxo1-stimulated transcription was abolished when PI 3-kinase was inhibited, indicating that direct transcription regulation as well as nuclear export are dependent on PI 3-kinase. Insulin-induced phosphorylation of the PKB/Akt phosphorylation sites, Thr24 and Ser253, in L375A-Foxo1 (results not shown) is consistent with the possibility that PKB/Akt mediates PI 3-kinase-dependent regulation of Foxo1-stimulated transcription.

Parallel studies also were performed with a second mutant, Thr24Ala-Foxo1, which had been shown in several studies to be localized to the nucleus (34, 35, 39), but the results were inconclusive. Although T24A-Foxo1 localized predominantly to the nucleus in most H4IIE cells after insulin treatment, nuclear retention was less complete than with L375A-Foxo1 in individual sectioned cells (results not shown). Scheimann et al. (67) also observed intermediate levels of nuclear retention in HepG2 cells, but the Thr24Ala mutation did not affect the export of green fluorescent protein (GFP)-FOXO1 to the cytoplasm in HEK293 cells (55). Insulin inhibited IGFBP-1 promoter activity stimulated by wild-type and T24A-Foxo1 to the same extent, but insulin inhibition of IRE-3x-promoter activity stimulated by T24A-Foxo1 was decreased by 50% (results not shown). Scheimann et al. (67) saw a similar intermediate reduction of insulin inhibition of T24A-FOXO1-stimulated transcription in HepG2 cells, but Guo et al. (32) observed no decrease in insulin inhibition. Thus, our findings with T24A-Foxo1 are consistent with those obtained with L375A-Foxo1, but are more difficult to interpret in light of the partial inhibition of redistribution observed with the Thr24Ala mutation.

During the preparation of our manuscript, Zhang et al. (42) reported results consistent with our conclusion that inhibition of FOXO-stimulated transcription by insulin does not depend solely on nuclear exclusion. They mutated Ser256, the PKB/Akt site at the C-terminal end of the Fox box in FOXO1, to aspartate. Both FOXO1 T24A/S319A and FOXO1 T24A/S256D/S319A localized to the nucleus of HEK-293 cells in serum-free medium, whereas wild-type FOXO1-GFP was predominantly cytoplasmic. Basal transcription in the absence of insulin was increased by FOXO1 T24A/S319A, but not by FOXO1 T24A/S256D/S319A, suggesting that introduction of the S256D mutation into FOXO1 T24A/S319A to mimic insulin-induced phosphorylation of the PKB/Akt site decreased basal FOXO1-stimulated transcription without affecting nuclear export.

The mechanism by which insulin inhibits FOXO-stimulated transcription in the absence of nuclear export could involve inhibition of DNA binding to the promoter or regulation of trans-activation. Two studies have reported inhibition of FOXO binding to the IRE under conditions mimicking insulin stimulation in in vitro gel shift assays. Cahill et al. (68) showed that dimers of the phosphoserine-phosphothreonine-specific binding protein 14-3-3 (69, 70) bound to Daf16 when the PKB/Akt sites equivalent to T24 and S316 of Foxo1 were phosphorylated and prevented the transcription factor from binding to the IRE. Zhang et al. (42) reported that substituting aspartate for Ser256 at the C-terminal end of the Fox box decreased binding of FOXO1 to the IRE by 50%. Insulin-stimulated phosphorylation of Thr24 and Ser253 in L375A-Foxo1 could decrease binding to the IRE by either of these mechanisms.

Insulin also can regulate Foxo1-stimulated trans-activation (33). This was demonstrated in constructs in which the C-terminal trans-activation domain of Foxo1 was fused to a heterologous Gal4 DNA-binding domain (71). Insulin inhibited Gal4 promoter activity stimulated by the fusion protein without significantly affecting the nucleocytoplasmic distribution of the fusion constructs (71). IRE-dependent FOXO-stimulated transcription also may be regulated by coactivators that bind to FOXO proteins: CBP/p300 (52), estrogen receptor {alpha} (59, 72), androgen receptor (72), Hoxa5 (73), and Hoxa10 (74). Whether any of these coactivator interactions are involved in the inhibition of FOXO-stimulated transcription by insulin is presently unknown.

Multiple mechanisms of regulating transcription such as we have observed for Foxo1 have been described for the yeast transcription factor, Pho4. Phosphorylation of multiple sites that determine nuclear export, nuclear import, and transcription activity contributes to the regulation of Pho4-stimulated transcription and allows adaptation to low and high phosphate environments (75, 76). Under low phosphate conditions, unphosphorylated Pho4 is imported into the nucleus, and its export from the nucleus is inhibited. Nuclear Pho4 binds to Pho2 and stimulates the transcription of genes that help overcome phosphate deprivation. Under high phosphate conditions, Pho4 becomes phosphorylated at two sites that promote nuclear export and a third site that prevents nuclear import. Although Pho4 was retained in the nucleus after mutation of these three sites, mutation of a fourth phosphorylation site was required before Pho4 could form complexes with Pho2 to activate transcription. Given the number of vital cell functions in which genes whose transcription is regulated by FOXO proteins are involved (reviewed in Ref.27), the ability to directly regulate transcription as well as the nuclear abundance of FOXO proteins makes it possible to achieve tighter control of FOXO-regulated gene expression.


   Acknowledgments
 
We thank Derek LeRoith and Peter Nissley for critical reading of the manuscript.


   Footnotes
 
This work was presented in part at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, June 19–23, 2002.

Abbreviations: FITC, Fluorescein isothiocyanate; FOXO, forkhead box transcription factor, subfamily O; Foxo1, mouse FOXO1; GFP, green fluorescence protein; IGFBP-1, IGF-binding protein-1; IRE, insulin response element; NES, nuclear export signal; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B.

1 The human (FOXO) and mouse (Foxo) members of the FOXO subfamily were previously known as FKHR (FOXO1, Foxo1), FKHRL1 (FOXO3a, Foxo3), and AFX (FOXO4, Foxo4). Back

2 FOXO4 contains a single NES (300-LELLDGLNL-308) (40 ) in a similar location to the Foxo1 NES (368-MENLLDNLNL-377) (39 ), whereas FOXO3a has two adjacent NES in this location (369-LTDMAGTMNL-378 and 386-LMDDLLDNITL-396 (41 ). Mutation of both NES sites abolished the export of FOXO3a from the nucleus, but the individual contributions of the two sites was not examined (41 ). Zhao et al. (Program of the 85th Annual Meeting of The Endocrine Society, Philadelphia, PA, June 19–22, Abstract P1–369) identified additional Crm1-independent and Crm1-dependent NES sites in N-terminal fragments of FOXO1 coupled to GFP, but the importance of these sites for export of full-length FOXO1 is unknown. Back

Received April 16, 2003.

Accepted for publication September 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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