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Originally published In Press as doi:10.1074/jbc.M508510200 on November 9, 2005

J. Biol. Chem., Vol. 281, Issue 2, 1091-1098, January 13, 2006
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The Forkhead Transcription Factor Foxo1 Bridges the JNK Pathway and the Transcription Factor PDX-1 through Its Intracellular Translocation*

Dan Kawamori, Hideaki Kaneto1, Yoshihisa Nakatani, Taka-aki Matsuoka, Munehide Matsuhisa, Masatsugu Hori, and Yoshimitsu Yamasaki

From the Department of Internal Medicine and Therapeutics (A8), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan

Received for publication, August 3, 2005 , and in revised form, September 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been shown that oxidative stress and activation of the c-Jun N-terminal kinase (JNK) pathway induce the nucleocytoplasmic translocation of the pancreatic transcription factor PDX-1, which leads to pancreatic beta-cell dysfunction. In this study, we have shown that the forkhead transcription factor Foxo1/FKHR plays a role as a mediator between the JNK pathway and PDX-1. Under oxidative stress conditions, Foxo1 changed its intracellular localization from the cytoplasm to the nucleus in the pancreatic beta-cell line HIT-T15. The overexpression of JNK also induced the nuclear localization of Foxo1, but in contrast, suppression of JNK reduced the oxidative stress-induced nuclear localization of Foxo1, suggesting the involvement of the JNK pathway in Foxo1 translocation. In addition, oxidative stress or activation of the JNK pathway decreased the activity of Akt in HIT cells, leading to the decreased phosphorylation of Foxo1 following nuclear localization. Furthermore, adenovirus-mediated Foxo1 overexpression reduced the nuclear expression of PDX-1, whereas repression of Foxo1 by Foxo1-specific small interfering RNA retained the nuclear expression of PDX-1 under oxidative stress conditions. Taken together, Foxo1 is involved in the nucleocytoplasmic translocation of PDX-1 by oxidative stress and the JNK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Diabetes mellitus is the most prevalent metabolic disorder all over the world. Impaired insulin secretion and whole body insulin resistance play major roles in the development and progression of diabetes. In particular, pancreatic beta-cell function, such as insulin biosynthesis and secretion, is often impaired under the chronic hyperglycemic conditions found in diabetes (1).

Several lines of evidence have implicated oxidative stress in the progression of beta-cell dysfunction in type 2 diabetes (1-8). Under diabetic conditions, reactive oxygen species are increased in many tissues and organs through activation of the mitochondrial electron transport chain (9) or acceleration of glycation reactions (1, 3) and cause various forms of tissue damage in patients with diabetes (10). Extracellular hyperglycemia readily causes intracellular hyperglycemia in beta-cells, leading to the induction of reactive oxygen species in pancreatic islets of diabetic animals as best represented by the enhanced expression of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2,3-nonenal (7, 8). In addition, because pancreatic islet cells express a relatively low amount of anti-oxidative enzymes, such as glutathione peroxidase and catalase (11), beta-cells are likely to be sensitive to oxidative stress. Indeed, we and other groups have recently demonstrated that in vivo antioxidant treatment can exert beta-cell protective anti-diabetic effects in animal models for type 2 diabetes (4-6).

It has been reported that the activity of the c-Jun N-terminal kinase (JNK)2 pathway, which is known to be activated by various stress signals, such as cytokines or oxidative stress (12, 13), is abnormally elevated in various tissues under diabetic conditions (14). As an underlying molecular mechanism of oxidative stress-mediated beta-cell deterioration, we have reported (15, 16) that JNK activation is involved in the reduction of insulin gene expression by oxidative stress and that suppression of the JNK pathway can protect beta-cells from glucose toxicity. In addition, we have shown (17, 18) that the JNK pathway is also important in insulin resistance by interfering with the insulin signaling pathway, suggesting that the JNK pathway plays a central role in the pathophysiology of diabetes.

Regarding the molecular mechanism of beta-cell deterioration, we have reported that JNK activation induces the nucleocytoplasmic translocation of the pancreatic transcription factor PDX-1 and thereby reduces PDX-1 DNA binding activity (16). PDX-1, also known as IPF-1, IDX-1, and STF-1, is a homeodomain-containing transcription factor that plays a pivotal role in pancreatic development, beta-cell differentiation (19-22), and in maintaining mature beta-cell function (19, 21, 22). As support for the implication of PDX-1 in beta-cell dysfunction, the reduction of insulin gene expression in beta-cells chronically exposed to high glucose conditions either in vivo or in vitro is often accompanied by a decrease of PDX-1 expression in nuclei (4, 5, 23-25).

Regulation of the intracellular localization of transcription factors is a crucial requirement for their action, and stimulus-dependent nuclear import can serve as a mechanism to regulate gene expression (26, 27). PDX-1 is reported to have a functional nuclear localization signal (28, 29) and undergoes nuclear translocation in response to several functional stimuli, such as high glucose or glucagon-like peptide-1 (30-32). Conversely, we have shown that PDX-1 also possesses a nuclear export signal and showed its nuclear export in a JNK-dependent manner (16). However, the precise mechanism as to how the JNK pathway induces the nuclear export of PDX-1 is still unknown.

The forkhead transcription factor Foxo1 (previously known as FKHR) is a mammalian homologue of DAF-16 and is known as one of the important fundamental transcription factors playing a key role in apoptosis, cellular proliferation, differentiation and glucose metabolism through regulating the transcription of various target genes (33, 34). It is reported that Foxo1 regulates hepatic gluconeogenesis and thus contributes to insulin resistance (35). Insulin inhibits the function of Foxo1 through Akt/PKB-mediated phosphorylation and nuclear exclusion (36) and thereby suppresses hepatic gluconeogenesis. Foxo1 was recently reported to inhibit PDX-1 gene transcription in pancreatic beta-cells (35, 37), suggesting that it is involved in the deterioration of beta-cell function. Moreover, because Foxo1 exhibits a counterlocalization to PDX-1 in beta-cells (37), Foxo1 translocation might modulate the nucleocytoplasmic translocation of PDX-1 by oxidative stress.

Here, we have investigated the missing link between the JNK pathway and PDX-1 function in beta-cells, and found that oxidative stress induces the nuclear translocation of Foxo1 through activation of the JNK pathway, which leads to the nucleocytoplasmic translocation of PDX-1. Our findings suggest the involvement of the JNK-Akt-Foxo1-PDX-1 axis in the impairment of PDX-1 function, and this pathway may explain part of the molecular mechanisms of beta-cell dysfunction found in diabetes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction enzymes, DNA polymerases, antibodies, and other modification enzymes were purchased from commercial suppliers (New England Biolabs, Beverly, MA; Toyobo, Tokyo, Japan; Sigma; Cell Signaling Technology, Inc., Beverly, MA; Santa Cruz Biotechnology, Santa Cruz, CA; and Zymed Laboratories, San Francisco, CA). PCR primers and oligonucleotides were purchased from Invitrogen Japan (Tokyo, Japan). Tissue culture media were purchased from Sigma and fetal calf serum from Invitrogen Japan.

Cell Culture—HIT-T15 cells (American Type Culture Collection number 1777) were maintained in RPMI 1640 medium containing 11.1 mM glucose, 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin at 5% CO2 and 37 °C. The HIT-T15 cells used in this study were between passages 80 and 100.

Immunocytochemistry—For immunostaining of cultured cells, the cells were seeded on a Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY) and cultured for 2 days in appropriate medium. After rinsing with phosphate-buffered saline three times, the cells were fixed with 4% paraformaldehyde for 20 min. For detection of Foxo1 and PDX-1, the cells were microwaved in citrate buffer for antigen retrieval before being incubated with blocking serum. Each slide was treated with a H2O2 solution to inactivate endogenous peroxidase. Detection of Foxo1 and PDX-1 was performed by the avidin-biotin complex method using the Vectastain Elite avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). After blocking, the cells were incubated for 60 min with an anti-Foxo1 rabbit IgG antibody diluted at a ratio of 1:100 (Cell Signaling Technology) or an anti-PDX-1 antibody diluted at 1:1000 (38) in phosphate-buffered saline containing 1% bovine serum albumin. The cells were subsequently incubated for 30 min with biotinylated anti-rabbit IgG (Vector Laboratories) at a dilution of 1:200. The sections were then incubated with avidin-biotin complex reagent for 30 min, and positive reactions were visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride substrate (Zymed Laboratories).

Isolation of Nuclear, Cytoplasmic, and Whole Cell Extracts and Western Blot Analyses—HIT-T15 cells were cultured in 60-mm diameter culture dishes until ~80% confluency. For isolation of nuclear extracts, the cells were then collected into microtubes, centrifuged for 20 s in a microcentrifuge, and resuspended in 200 µl of 10.0 mM Hepes, pH 7.9, containing 10.0 mM KCl, 1.5 mM MgCl2, and 0.5 mM dithiothreitol. After incubation at 4 °C for 15 min, the cells were lysed by passing 10 times through a 22-gauge needle. Next, the cells were centrifuged for 20 s in a microcentrifuge, and the supernatant (cytoplasmic fraction) was removed and frozen in small aliquots. The pellet, which contained the nuclei, was resuspended in 150 µl of 20 mM Hepes, pH 7.9, containing 20% v/v glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride and then stirred at 4 °C for 30 min. The nuclear extracts were then centrifuged for 20 min at 4 °C in a microcentrifuge. The supernatant was collected, aliquoted into small volumes, and stored at -80 °C.

For isolation of whole cell extracts, the cells were collected into microtubes, centrifuged for 1 min in a microcentrifuge, and resuspended in 150 µl of 20 mM Hepes, pH 7.9, containing 20% v/v glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride, and then stirred at 4 °C for 30 min. After centrifugation for 20 min at 4 °C in a microcentrifuge, the supernatant was collected as whole cell extracts, aliquoted into small volumes, and stored at -80 °C.

Western blot analyses were performed following standard procedures as described below. Nuclear, cytoplasmic, or whole cell proteins derived from each cell sample were fractionated by SDS-PAGE, blotted onto a nitrocellulose membrane (Optitran BA-S85, Schleicher & Schuell), and incubated overnight at 4 °C in phosphate-buffered saline containing a 1:1000 dilution of either rabbit anti-Foxo1, anti-Ser256-phosphospecific Foxo1, anti-Thr24-phosphospecific Foxo1, anti-Akt, anti-Ser473-phosphospecific Akt, anti-JNK antibody (Cell Signaling Technology), or anti-USF-1 antibody (Santa Cruz Biotechnology), a 1:5000 dilution of rabbit anti-PDX-1 antibody (38), or a 1:1000 dilution of goat anti-beta-actin antibody (Santa Cruz Biotechnology). The membrane was then incubated at room temperature for 60 min in phosphate-buffered saline with a 1:2000 dilution of anti-rabbit IgG or anti-goat IgG horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories), and then for 2 min with chemiluminescence reagent (PerkinElmer Life Sciences). The antigen-antibody complex was detected by exposure to a light-sensitive film for the appropriate exposure time. Relative protein amounts were estimated by densitometry after scanning using a Fluorchem IS-8000 image analyzer system (Alpha Innotech Corporation, San Leandro, CA).

Preparation of Recombinant Adenoviruses—Recombinant adenoviruses expressing the wild type (WT) and dominant negative (DN) form of JNK, the WT and DN form of Foxo1, and Foxo1-specific siRNA were prepared using the AdEasy system (kindly provided by Dr. Bert Vogelstein, the Johns Hopkins Oncology Center) (39). The generation of JNK WT- and DN-expressing adenoviruses was described previously (15). Expression plasmids for the WT and DN forms ({Delta}256) of mouse Foxo1 were kind gifts from Dr. J. Nakae (Kobe University Graduate School of Medicine, Kobe, Japan) (40). In brief, the coding region of WT and Foxo1 DN ({Delta}256) was cloned into the SalI-EcoRV site of the shuttle vector pAdTrack-CMV, and annealed double strand-synthesized hair-pin siRNA template oligonucleotides, including 21-bp sequences for Foxo1-specific siRNA (5'-AAAAGTCCTTCAGATTGTCTG-3') (41) was also cloned into the MluI-HindIII site of the shuttle vector pRNAT-H1.1/Adeno (GenScript, Piscataway, NJ). To allow for homologous recombination, the linearized plasmid containing Foxo1 WT or Foxo1 DN or Foxo1-specific siRNA and the adenoviral backbone plasmid pAdEasy-1 were introduced into electrocompetent Escherichia coli BJ5183. The linearized plasmids were transfected into the adenovirus packaging cell line 293 using Lipofectamine (Invitrogen), and the adenovirus titers were increased up to 1 x 108 plaque-forming units/ml in the 293 cells. Control adenovirus expressing green fluorescent protein (Ad-GFP) was prepared in the same manner. Adenovirus titers were further increased up to 1 x 1010 plaque-forming units/ml using the Virakit for Adenovirus5 recombinant adenovirus purification kit (Virapur LLC, San Diego, CA). The HIT-T15 cells were infected with various adenoviruses at 3 x 108 plaque-forming units/ml. Subsequent experiments were conducted 48 h after the initial addition of the virus. The efficiencies of the adenovirus-mediated gene transfers were between 80 and 95%.


Figure 1
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  		FIGURE 1.
Effects of oxidative stress on the intracellular localization of Foxo1. Immunocytochemical analyses of Foxo1 and PDX-1 were performed with HIT-T15 cells, which were left untreated or treated with H2O2 (50 µM) for 48 h (A and C). HIT-T15 cells were treated with or without H2O2 (50 µM) for 48 h, and nuclear and cytoplasmic proteins were extracted from the cells and subjected to Western blot analyses. Nuclear and cytoplasmic expression of Foxo1 and PDX-1 were evaluated by Western blot analyses using an anti-Foxo1 antibody (B) and an anti-PDX-1 antibody (C). Nuclear expression of USF-1 and cytoplasmic expression of beta-actin were evaluated by Western blot analyses using an anti-USF-1 antibody and an anti-beta-actin antibody, respectively (D). Nuclear expression of phosphorylated Foxo1 was evaluated by Western blot analyses using an anti-Ser256-phospho-specific Foxo1 antibody (E). 50 µg each of nuclear protein extracts and 100 µg each of cytoplasmic protein extracts were used. The relative ratio of Foxo1 was calculated by densitometry (B). The bar graph depicts the averages of the data obtained from five individual experiments, and data are expressed as means ± S.D. The same results were obtained in independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oxidative Stress Induces Nuclear Accumulation of Foxo1—To investigate the effect of oxidative stress on the expression and intracellular localization of Foxo1, we first examined the intracellular distribution of Foxo1 by immunostaining. In the pancreatic beta-cell-derived cell line HIT-T15, Foxo1 changed its intracellular localization from the cytoplasm to the nucleus under oxidative stress conditions (Fig. 1A). Next, to quantify the change in intracellular distribution of Foxo1 by oxidative stress, we performed Western blot analyses using nuclear and cytoplasmic protein extracts isolated from the HIT cells. As shown in Fig. 1B, the amount of nuclear Foxo1 was increased by treatment with 50 µM H2O2, whereas the amount of cytoplasmic Foxo1 was decreased (Fig. 1B). In contrast to Foxo1, the nuclear expression of PDX-1 was decreased and its cytoplasmic distribution was increased by oxidative stress (Fig. 1C). Also in immunostaining, PDX-1 showed nucleocytoplasmic translocation under oxidative stress conditions (Fig. 1C). The equivalence of nuclear and cytoplasmic components was verified by the expression of USF-1 (42) and beta-actin, respectively (Fig. 1D).

It is known that the intracellular distribution of Foxo1 is mainly determined by its phosphorylation status (36). Foxo1 is retained in the cytoplasm upon phosphorylation by Akt at multiple sites (36). Conversely, a decrease in phosphorylation of Foxo1 allows Foxo1 to remain in the nucleus. The phosphorylation of Foxo1 at Ser256, which was reported to be important in determining the intracellular localization of Foxo1 (36), was shown to be reduced under oxidative stress conditions (Fig. 1E). This is consistent with the observations of the nuclear accumulation of Foxo1 under oxidative stress conditions (Fig. 1B). These results thus indicate that oxidative stress induces the nuclear accumulation of Foxo1 via reducing its phosphorylation status of Foxo1.

Oxidative Stress Reduces Insulin-induced Phosphorylation of Akt—Next, to explore the upstream mechanism of oxidative stress-induced nuclear accumulation of Foxo1, we investigated the insulin-induced phosphorylation of Akt. Because Akt is primarily activated as a result of its phosphorylation at the Thr308 and Ser473 residues (43), we examined the effect of oxidative stress on the insulin-induced phosphorylation of Akt at Ser473 in HIT cells. Treatment with 17 nM insulin induced phosphorylation of Akt at Ser473 in a time-dependent manner in vehicle-treated control HIT cells. However, insulin-induced up-regulation of Akt phosphorylation at Ser473 was reduced by treatment with 50 µM H2O2 (Fig. 2A, upper panel). Following 15 min of insulin treatment, the phosphorylation of Akt at Ser473 was increased to 432 ± 45.0% of basal conditions (0 min). On the other hand, under oxidative stress conditions, the increase of Akt phosphorylation at Ser473 was reduced to 323 ± 32.1% of basal conditions. To confirm that equal amounts of protein were loaded among the samples, whole cell protein extracts were immunoblotted with an anti-Akt antibody (Fig. 2A, middle panel) and an anti-beta-actin antibody (Fig. 2A, lower panel). The graphs depict the averages of data obtained from three individual experiments, and data are expressed as means ± S.D. (Fig. 2B). Thus, these results indicate that oxidative stress reduces the insulin-induced phosphorylation of Akt, which leads to an alteration in the phosphorylation status of Foxo1 and its nuclear accumulation.


Figure 2
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  		FIGURE 2.
Effects of oxidative stress on insulin-induced phosphorylation of Akt. HIT-T15 cells were treated with or without H2O2 (50 µM) for 48 h. The cells were serum-starved for 16 h and treated with 17 nM insulin for the periods indicated. Phosphorylation of Akt was evaluated using whole cell protein extracts by Western blot analyses with an anti-Ser473-phosphospecific antibody (A, upper panel), an anti-Akt antibody (A, middle panel), and an anti-beta-actin antibody (A, lower panel). In the upper panel, 200 µg each of proteins, in the middle panel, 100 µg each of proteins, and in the lower panel, 50 µg each of proteins were used, respectively. The amount of Akt phosphorylated at Ser473 corrected for the amount of whole Akt was quantified by densitometry. The graph shows the averages of the data obtained from three individual experiments, and the data are expressed as means ± S.D. (B).

 
The JNK Pathway Is Involved in the Nuclear Translocation of Foxo1—Two major mitogen-activated protein kinase pathways, the JNK and p38 MAP kinase pathways, can be activated by a variety of extracellular stress signals, such as UV light, heat, and oxidative stress, and induce various cellular responses (12, 13). It has been suggested that activation of the JNK pathway is involved in beta-cell dysfunction by oxidative stress (15). In addition, we have recently reported that the nucleocytoplasmic translocation of PDX-1 induced by oxidative stress is mediated by activation of the JNK pathway (16). Hence, we next investigated the possible implication of the JNK pathway in this oxidative stress-induced nuclear accumulation of Foxo1.

When a dominant negative form of JNK (JNK DN) was expressed by the infection of a JNK DN-expressing adenovirus (Fig. 3A), the oxidative stress-induced nuclear accumulation of Foxo1 was greatly inhibited (Fig. 3B, upper panel). In contrast, no such effects were observed when a GFP-expressing adenovirus was infected (Fig. 3B, upper panel). Moreover, when a JNK WT was overexpressed by infection of a JNK WT-expressing adenovirus (Fig. 3A), Foxo1 showed nuclear accumulation even without oxidative stress (Fig. 3B, upper panel). As shown in Fig. 3B, lower panel, the cytoplasmic expression of Foxo1 also changed in contrast to the nuclear expression of Foxo1. A decrease in cytoplasmic Foxo1 was also induced by oxidative stress and inhibited by JNK DN (Fig. 3B, lower panel). These observations indicate that the oxidative stress-induced nuclear accumulation of Foxo1 is because of its translocation from the cytoplasm to the nucleus. These results also suggest that the JNK pathway partially mediates the oxidative stress-induced nuclear translocation of Foxo1. Consistent with our previous report (16), nuclear expression of PDX-1 was reduced by oxidative stress and overexpression of JNK and preserved by the expression of JNK DN, whereas the amount of whole cell PDX-1 was not affected (Fig. 3C). Thus, PDX-1 showed a counter localization to Foxo1.


Figure 3
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  		FIGURE 3.
Effects of the overexpression of wild type JNK and the dominant negative form of JNK on the intracellular localization of Foxo1 and PDX-1, and on the phosphorylation status of Akt. HIT-T15 cells were infected with adenoviruses expressing GFP, wild type JNK, or a dominant negative form JNK (JNK DN) 48 h prior to protein extraction. From 24 h after the infection, HIT-T15 cells were treated with or without H2O2 (50 µM) for 24 h, and nuclear, cytoplasmic, or whole cell proteins were extracted from the cells and subjected to Western blot analyses. The adenovirus-mediated overexpression of JNK and JNK DN was evaluated using whole cell protein extracts by Western blot analysis with an anti-JNK antibody (A). Intracellular localization of Foxo1 and nuclear and whole cell expression of PDX-1 were evaluated by Western blot analyses using an anti-Foxo1 antibody (B) and an anti-PDX-1 antibody (C), respectively. The phosphorylation of Akt was evaluated using whole cell protein extracts by Western blot analyses with an anti-Ser473-phosphospecific antibody (D, upper panel) and an anti-Akt antibody (D, lower panel). Nuclear expression of USF-1 and cytoplasmic expression of beta-actin were evaluated by Western blot analyses using an anti-USF-1 antibody and an anti-beta-actin antibody, respectively (E). In A, 100 µg of whole cell protein extracts were used. In B and C, 50 µg each of nuclear and 100 µg of cytoplasmic or whole cell protein extracts were used. In the upper panel of D, 200 µg each of whole cell protein extracts and in the lower panel of D, 100 µg each of whole cell protein extracts were used. In E, 50 µg each of nuclear and whole cell protein extracts were used. The same results were obtained in independent experiments.

 
The JNK Pathway Affects the Phosphorylation Status of Akt—Because the JNK pathway was shown to be involved in the intracellular translocation of Foxo1 induced by oxidative stress, we next investigated the involvement of the JNK pathway in Akt activity, which is the main regulatory component of Foxo1 intracellular localization (36).

As shown in Fig. 3D, upper panel, when cells were subjected to oxidative stress or JNK WT was overexpressed, the phosphorylation of Akt at Ser473 was reduced compared with basal conditions in which only GFP-expressing adenoviruses were infected. On the other hand, expression of a JNK DN by infection of a JNK DN-expressing adenovirus preserved the Ser473 phosphorylation of Akt even in the presence of oxidative stress. To confirm that equal amounts of protein were loaded among the samples, whole cell protein extracts were immunoblotted with an anti-Akt antibody (Fig. 3D). These observations suggested that the oxidative stress-induced impairment of Akt phosphorylation was partially mediated by the JNK pathway in pancreatic beta-cells. JNK is known to induce serine phosphorylation of insulin receptor substrate-1 and thereby reduces the activity of insulin receptor substrate-1 and also its downstream target Akt (17, 44). Taking these findings together as a molecular mechanism underlying this oxidative stress-induced nuclear translocation of Foxo1, the modification of the insulin receptor substrate-1-Akt pathway by JNK activated under oxidative stress conditions was suggested, and this reduced activity of Akt was also suggested to be an upstream mechanism of the decreased phosphorylation and nuclear localization of Foxo1. The confirmation of equal protein amounts loaded among the samples was obtained by the equal expression of nuclear USF-1 and whole cell beta-actin by Western blot analysis (Fig. 3E).

Foxo1 Directly Affects the Intracellular Localization of PDX-1—Our results indicated the JNK-mediated nuclear localization of Foxo1 under oxidative stress conditions. On the other hand, PDX-1 was shown to change its localization from the nucleus to the cytoplasm in a JNK-dependent manner under oxidative stress conditions (16). Furthermore, Foxo1 was reported to repress PDX-1 promoter activity via binding to the Foxa2 binding site in the PDX-1 promoter and inhibiting transcription (37) and thereby reducing the expression of PDX-1. Because Foxo1 shows a counterlocalization to PDX-1 in pancreatic beta-cells (37), this translocation might be an upstream mechanism of PDX-1 translocation from the nucleus to the cytoplasm.


Figure 4
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  		FIGURE 4.
Effects of the overexpression of wild type Foxo1 and the dominant negative form of Foxo1 and Foxo1 knockdown by siRNA on nuclear expression of PDX-1. HIT-T15 cells were infected with adenoviruses expressing GFP, wild type Foxo1, Foxo1-{Delta}256 mutant (a dominant negative form of Foxo1, Foxo1 DN), or Foxo1-specific siRNA (siFoxo1) 48 h prior to nuclear protein extraction. From 24 h after the infections, HIT-T15 cells were treated with or without H2O2 (50 µM) for 24 h, and nuclear and whole cell proteins were extracted from the cells and subjected to Western blot analyses. Adenovirus-mediated overexpression was evaluated by Western blot analyses using an anti-Foxo1 antibody for wild type Foxo1 (A) and an anti-Thr24-phosphospecific Foxo1 antibody for the Foxo1-{Delta}256 mutant (B). Knockdown of Foxo1 by an adenovirus expressing a specific siRNA was also evaluated by Western blot analyses using an anti-Foxo1 antibody (C). Nuclear expression of USF-1 was evaluated by Western blot analyses using an anti-USF-1 (D). Nuclear and whole cell expression of PDX-1 was evaluated by Western blot analyses using an anti-PDX-1 antibody (E and F). Whole cell expression of beta-actin was evaluated by Western blot analyses using an anti-beta-actin antibody (E and F). In A and B, 20 µg each of nuclear proteins were used. In C, D, the upper panel of E, and the upper panel of F, 50 µg each of nuclear proteins were used. In the middle panels of E and F, 100 µg each of whole cell protein extracts and in the lower panel of E and F, 50 µg each of whole cell protein extracts were used. Same results were obtained in independent experiments.

 
To clarify the relationship between Foxo1 and PDX-1 and the possible involvement of Foxo1 in the intracellular localization of PDX-1, we investigated the direct effect of the overexpression or reduction of Foxo1 using an adenovirus expressing wild type Foxo1 (Foxo1 WT) and a dominant negative form of Foxo1 (Foxo1 DN) and a Foxo1-specific siRNA expressing adenovirus vectors. At first, adenovirus-mediated overexpression of Foxo1 WT and Foxo1 DN and a knockdown effect of Foxo1-specific siRNA were confirmed by Western blot analysis using nuclear protein extracts from adenovirus-infected or siRNA-treated 293 cells (data not shown). Thereafter, the adenoviruses were introduced to the experiments using the HIT cells. Foxo1 expression was actually induced in the HIT cells infected with Foxo1 WT-expressing adenovirus compared with the cells infected with GFP-expressing adenovirus (Fig. 4A). The expression of Foxo1 DN was confirmed by Western blot analysis using an anti-Thr24-phosphospecific Foxo1 antibody, because this mutant Foxo1 is a truncated fragment containing only 256 aa of the N terminus (40). In nuclear protein extracts from Foxo1 DN-infected HIT cells, truncated Foxo1 was observed at ~27 kDa; conversely no blotted band was observed within nuclear proteins from the control GFP-infected HIT cells (Fig. 4B). The knockdown effect of Foxo1-specific siRNA was also confirmed by Western blot analysis using nuclear protein extracts from adenoviral siRNA-transfected HIT cells. The expression of Foxo1 was significantly reduced by infection of a Foxo1-specific siRNA expressing adenovirus (Fig. 4C). In contrast to the modification of Foxo1 expression by adenoviruses, the nuclear expression of USF-1 did not change in the HIT cells infected with these adenoviruses (Fig. 4D).

As shown in Fig. 4E, when Foxo1 was expressed in HIT cells by infection of a wild type Foxo1 expression adenovirus, the amount of nuclear PDX-1 was reduced even without oxidative stress or JNK overexpression. The expression of Foxo1 DN did not significantly affect the nuclear expression of PDX-1, both with and without oxidative stress. In contrast to the change in nuclear PDX-1 expression, the whole cell expression of PDX-1 was not affected by the status of Foxo1 expression. As shown in Fig. 4F, the reduction in Foxo1 by a specific siRNA restored nuclear PDX-1 even in the presence of oxidative stress. These results indicate that Foxo1 directly affects the nuclear expression of PDX-1 by altering the intracellular localization of PDX-1 and also show the possible involvement of Foxo1 in oxidative stress-induced, JNK pathway-mediated nucleocytoplasmic translocation of PDX-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we showed that oxidative stress induces the nuclear translocation of Foxo1 through activation of the JNK pathway and that Foxo1 bridges the JNK pathway and PDX-1 through its intracellular translocation. Our findings provide new insights into the molecular background of the JNK-induced deterioration of beta-cell function.

Although this study showed that Foxo1 directly induces PDX-1 translocation under oxidative stress conditions, the precise mechanism underlying this phenomenon is yet to be clarified. However, from our findings, this effect of Foxo1 seems to be dependent on the quantity of Foxo1 protein expressed. The overexpression of Foxo1 by adenoviral gene transfer reduced the nuclear expression of PDX-1; in contrast, the reduction of Foxo1 by expression of a Foxo1-specific siRNA that reduced Foxo1 protein restored the nuclear expression of PDX-1 under oxidative stress conditions. On the other hand, the expression of a dominant negative form of Foxo1 that can bind to the Foxo1 binding sites of its target genes but cannot transactivate its targets because of the lack of a transactivation domain could not rescue the reduction of nuclear PDX-1 resulting from oxidative stress. These results suggest that the function of Foxo1 in regulating the intracellular localization of PDX-1 might not be due to a transcriptional event, although Foxo1 is reported to reduce PDX-1 promoter activity by competing with Foxa2 for DNA binding to the PDX-1 promoter (37). Indeed, in our finding, the whole cell amount of PDX-1 was not altered by the modification of Foxo1 expression by adenovirus. In our experiments, we investigated the effect of adenoviral Foxo1 modification 48 h after its infection. Thus, at present we cannot exclude the possibility that a longer period of Foxo1 modification alters the whole cell expression of PDX-1. Taken together, the protein interaction between Foxo1 and PDX-1 is suggested as the underlying mechanism of PDX-1 translocation. However, what kind of molecular event occurs between the Foxo1 protein and the PDX-1 protein remains unclear. Some molecular actions, such as a modification of acetylation status between these two transcription factors, competition for binding to the transcriptional co-activator p300/CBP, or spatial inhibition of DNA binding might be possible. Both Foxo1 and PDX-1 are also known to interact with p300, which has the ability to modify the acetylation status of its partners (45, 46). Moreover, the acetylation status of Foxo1 is reported to be involved in the regulation of its functions (47). Further studies need to be performed to clarify the detailed mechanism underlying the Foxo1-induced nucleocytoplasmic translocation of PDX-1.

The insulin signaling pathway is widely known as one of the most important signal transduction pathways, especially in insulin target organs, such as the liver, skeletal muscle, and adipose tissue (48, 49). In addition, in beta-cells, insulin signaling pathways are reported to play an important role in maintaining cellular function (50-54). Particularly, Akt/PKB, one of the pivotal components in the insulin signaling pathway, is reported to play various critical roles in beta-cells, such as cell growth, differentiation, and cellular survival by the prevention of apoptosis (55-57). In our findings, the phosphorylation of Akt was reduced under oxidative stress conditions, suggesting that Akt is involved in the oxidative stress-mediated nuclear translocation of Foxo1. Moreover, because nuclear translocation of Foxo1 induced the nucleocytoplasmic translocation of PDX-1, we assume that the reduced activity of Akt is involved in PDX-1 translocation. The pancreatic beta-cell-specific insulin receptor-disrupted mice showed a characteristic phenotype resembling the pathophysiological condition seen in type 2 diabetic patients, such as impairment of insulin biosynthesis and glucose-stimulated insulin secretion (52). These reports and our findings together confirm the importance of insulin signaling pathway in beta-cells.

Insulin has been shown to induce the nuclear translocation of PDX-1 (32), and Foxo1 may be involved in this phenomenon. Insulin activates Akt phosphorylation and thereby induces the cytoplasmic translocation of Foxo1. Reduced nuclear expression of Foxo1 might restore nuclear expression of PDX-1, because suppression of Foxo1 nuclear expression by an siRNA for Foxo1 restored nuclear PDX-1 (Fig. 4F).

Apart from under pathological conditions, Foxo1 should not have the ability to affect the intracellular localization of PDX-1, unless it has a certain physiological role. Although highly speculative, PDX-1 activity may need to be suppressed under certain circumstances. One possibility is that Foxo1 acts to protect cells from various stimuli, such as oxidative stress. DAF-16, the Foxo1 orthologue of Caenorhabditis elegans, has the ability to suppress cellular growth and proliferation and thus extends life span (58). By contrast, PDX-1 functions as an accelerator of beta-cell functions, such as insulin transcription, growth, and proliferation (59). Just as DAF-16 in C. elegans, Foxo1 might protect beta-cells against cellular damage by suppressing PDX-1 function and thereby arresting cellular functions and growth. In fact, in hepatocytes, Foxo1 undergoes nuclear translocation in response to oxidative stress (47) and thereby is suggested to prevent cellular damages.

In conclusion, oxidative stress induces the nuclear translocation of Foxo1 through activation of the JNK pathway, which leads to the nucleocytoplasmic translocation of PDX-1 and thereby explains, at least in part, the molecular mechanism for beta-cell dysfunction found in diabetes.


   FOOTNOTES
 
* This study was supported in part by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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

1 To whom correspondence should be addressed: Dept. of Internal Medicine and Therapeutics (A8), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan. Tel.: 81-6-6879-3633; Fax: 81-6-6879-3639; E-mail: kaneto{at}medone.med.osaka-u.ac.jp.

2 The abbreviations used are: JNK, c-Jun N-terminal kinase; WT, wild type; DN, dominant negative type; GFP, green fluorescent protein; USF-1, upstream stimulating factor-1; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jun Nakae (21st Century Center of Excellence Program, Kobe University Graduate School of Medicine, Kobe, Japan) for providing precious plasmids and Dr. Christopher J. Rhodes (Pacific Northwest Research Institute, Seattle, WA) for valuable suggestions. We also thank Chikayo Yokogawa for superb secretarial work and Yuko Sasaki for excellent technical assistance, and Dr. Helena Akiko Popiel for valuable comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Poitout, V., and Robertson, R. P. (2002) Endocrinology 143, 339-342[Abstract/Free Full Text]
  2. Robertson, R. P. (2004) J. Biol. Chem. 279, 42351-42354[Free Full Text]
  3. Matsuoka, T., Kajimoto, Y., Watada, H., Kaneto, H., Kishimoto, M., Umayahara, Y., Fujitani, Y., Kamada, T., Kawamori, R., and Yamasaki, Y. (1997) J. Clin. Investig. 99, 144-150[Medline] [Order article via Infotrieve]
  4. Tanaka, Y., Gleason, C. E., Tran, P. O., Harmon, J. S., and Robertson, R. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10857-10862[Abstract/Free Full Text]
  5. Kaneto, H., Kajimoto, Y., Miyagawa, J., Matsuoka, T., Fujitani, Y., Umayahara, Y., Hanafusa, T., Matsuzawa, Y., Yamasaki, Y., and Hori, M. (1999) Diabetes 48, 2398-2406[Abstract]
  6. Ihara, Y., Yamada, Y., Toyokuni, S., Miyawaki, K., Ban, N., Adachi, T., Kuroe, A., Iwakura, T., Kubota, A., Hiai, H., and Seino, Y. (2000) FEBS Lett. 473, 24-26[CrossRef][Medline] [Order article via Infotrieve]
  7. Ihara, Y., Toyokuni, S., Uchida, K., Odaka, H., Tanaka, T., Ikeda, H., Hiai, H., Seino, Y., and Yamada, Y. (1999) Diabetes 48, 927-932[Abstract]
  8. Gorogawa, S., Kajimoto, Y., Umayahara, Y., Kaneto, H., Watada, H., Kuroda, A., Kawamori, D., Yasuda, T., Matsuhisa, M., Yamasaki, Y., and Hori, M. (2002) Diabetes Res. Clin. Pract. 57, 1-10[Medline] [Order article via Infotrieve]
  9. Nishikawa, T., Edelstein, D., Du, X. L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, M. A., Beebe, D., Oates, P. J., Hammes, H. P., Giardino, I., and Brownlee, M. (2000) Nature 404, 787-790[CrossRef][Medline] [Order article via Infotrieve]
  10. Brownlee, M. (2001) Nature 414, 813-820[CrossRef][Medline] [Order article via Infotrieve]
  11. Tiedge, M., Lortz, S., Drinkgern, J., and Lenzen, S. (1997) Diabetes 46, 1733-1742[Abstract]
  12. Davis, R. J. (2000) Cell 103, 239-252[CrossRef][Medline] [Order article via Infotrieve]
  13. Minden, A., and Karin, M. (1997) Biochim. Biophys. Acta 1333, F85-F104[Medline] [Order article via Infotrieve]
  14. Hirosumi, J., Tuncman, G., Chang, L., Gorgun, C. Z., Uysal, K. T., Maeda, K., Karin, M., and Hotamisligil, G. S. (2002) Nature 420, 333-336[CrossRef][Medline] [Order article via Infotrieve]
  15. Kaneto, H., Xu, G., Fujii, N., Kim, S., Bonner-Weir, S., and Weir, G. C. (2002) J. Biol. Chem. 277, 30010-30018[Abstract/Free Full Text]
  16. Kawamori, D., Kajimoto, Y., Kaneto, H., Umayahara, Y., Fujitani, Y., Miyatsuka, T., Watada, H., Leibiger, I. B., Yamasaki, Y., and Hori, M. (2003) Diabetes 52, 2896-2904[Abstract/Free Full Text]
  17. Nakatani, Y., Kaneto, H., Kawamori, D., Hatazaki, M., Miyatsuka, T., Matsuoka, T. A., Kajimoto, Y., Matsuhisa, M., Yamasaki, Y., and Hori, M. (2004) J. Biol. Chem. 279, 45803-45809[Abstract/Free Full Text]
  18. Kaneto, H., Nakatani, Y., Miyatsuka, T., Kawamori, D., Matsuoka, T. A., Matsuhisa, M., Kajimoto, Y., Ichijo, H., Yamasaki, Y., and Hori, M. (2004) Nat. Med. 10, 1128-1132[CrossRef][Medline] [Order article via Infotrieve]
  19. Edlund, H. (1998) Diabetes 47, 1817-1823[Abstract]
  20. Offield, M. F., Jetton, T. L., Labosky, P. A., Ray, M., Stein, R. W., Magnuson, M. A., Hogan, B. L., and Wright, C. V. (1996) Development (Camb.) 122, 983-995[Abstract]
  21. Sander, M., and German, M. S. (1997) J. Mol. Med. 75, 327-340[CrossRef][Medline] [Order article via Infotrieve]
  22. Habener, J. F., and Stoffers, D. A. (1998) Proc. Assoc. Am. Physicians 110, 12-21[Medline] [Order article via Infotrieve]
  23. Zangen, D. H., Bonner-Weir, S., Lee, C. H., Latimer, J. B., Miller, C. P., Habener, J. F., and Weir, G. C. (1997) Diabetes 46, 258-264[Abstract]
  24. Olson, L. K., Sharma, A., Peshavaria, M., Wright, C. V., Towle, H. C., Robertson, R. P., and Stein, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9127-9131[Abstract/Free Full Text]
  25. Olson, L. K., Qian, J., and Poitout, V. (1998) Mol. Endocrinol. 12, 207-219[Abstract/Free Full Text]
  26. Yoneda, Y. (2000) Genes Cells 5, 777-787[Abstract]
  27. Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve]
  28. Moede, T., Leibiger, B., Pour, H. G., Berggren, P., and Leibiger, I. B. (1999) FEBS Lett. 461, 229-234[CrossRef][Medline] [Order article via Infotrieve]
  29. Hessabi, B., Ziegler, P., Schmidt, I., Hessabi, C., and Walther, R. (1999) Eur. J. Biochem. 263, 170-177[Medline] [Order article via Infotrieve]
  30. Rafiq, I., Kennedy, H. J., and Rutter, G. A. (1998) J. Biol. Chem. 273, 23241-23247[Abstract/Free Full Text]
  31. Rafiq, I., da Silva Xavier, G., Hooper, S., and Rutter, G. A. (2000) J. Biol. Chem. 275, 15977-15984[Abstract/Free Full Text]
  32. Elrick, L. J., and Docherty, K. (2001) Diabetes 50, 2244-2252[Abstract/Free Full Text]
  33. Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A., and Ruvkun, G. (1997) Nature 389, 994-999[CrossRef][Medline] [Order article via Infotrieve]
  34. Accili, D., and Arden, K. C. (2004) Cell 117, 421-426[CrossRef][Medline] [Order article via Infotrieve]
  35. Nakae, J., Biggs, W. H., III, Kitamura, T., Cavenee, W. K., Wright, C. V., Arden, K. C., and Accili, D. (2002) Nat. Genet. 32, 245-253[CrossRef][Medline] [Order article via Infotrieve]
  36. Biggs, W. H., III, Meisenhelder, J., Hunter, T., Cavenee, W. K., and Arden, K. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7421-7426[Abstract/Free Full Text]
  37. Kitamura, T., Nakae, J., Kitamura, Y., Kido, Y., Biggs, W. H., III, Wright, C. V., White, M. F., Arden, K. C., and Accili, D. (2002) J. Clin. Investig. 110, 1839-1847[CrossRef][Medline] [Order article via Infotrieve]
  38. Watada, H., Kajimoto, Y., Umayahara, Y., Matsuoka, T., Kaneto, H., Fujitani, Y., Kamada, T., Kawamori, R., and Yamasaki, Y. (1996) Diabetes 45, 1478-1488[Abstract]
  39. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2509-2514[Abstract/Free Full Text]
  40. Nakae, J., Barr, V., and Accili, D. (2000) EMBO J. 19, 989-996[CrossRef][Medline] [Order article via Infotrieve]
  41. Hribal, M. L., Nakae, J., Kitamura, T., Shutter, J. R., and Accili, D. (2003) J. Cell Biol. 162, 535-541[Abstract/Free Full Text]
  42. Read, M. L., Clark, A. R., and Docherty, K. (1993) Biochem. J. 295, 233-237[Medline] [Order article via Infotrieve]
  43. Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J., Frech, M., Cron, P., Cohen, P., Lucocq, J. M., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 31515-31524[Abstract/Free Full Text]
  44. Aguirre, V., Uchida, T., Yenush, L., Davis, R., and White, M. F. (2000) J. Biol. Chem. 275, 9047-9054[Abstract/Free Full Text]
  45. Qiu, Y., Guo, M., Huang, S., and Stein, R. (2002) Mol. Cell. Biol. 22, 412-420[Abstract/Free Full Text]
  46. Perrot, V., and Rechler, M. M. (2005) Mol. Endocrinol. 19, 2283-2298[Abstract/Free Full Text]
  47. Frescas, D., Valenti, L., and Accili, D. (2005) J. Biol. Chem. 280, 20589-20595[Abstract/Free Full Text]
  48. Kahn, C. R. (2003) Exp. Diabesity Res. 4, 169-182[Medline] [Order article via Infotrieve]
  49. Okamoto, H., and Accili, D. (2003) J. Biol. Chem. 278, 28359-28362[Abstract/Free Full Text]
  50. Rhodes, C. J. (2005) Science 307, 380-384[Abstract/Free Full Text]
  51. Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938[CrossRef][Medline] [Order article via Infotrieve]
  52. Kulkarni, R. N., Bruning, J. C., Winnay, J. N., Postic, C., Magnuson, M. A., and Kahn, C. R. (1999) Cell 96, 329-339[CrossRef][Medline] [Order article via Infotrieve]
  53. Kulkarni, R. N., Winnay, J. N., Daniels, M., Bruning, J. C., Flier, S. N., Hanahan, D., and Kahn, C. R. (1999) J. Clin. Investig. 104, R69-R75[Medline] [Order article via Infotrieve]
  54. Leibiger, B., Leibiger, I. B., Moede, T., Kemper, S., Kulkarni, R. N., Kahn, C. R., de Vargas, L. M., and Berggren, P. O. (2001) Mol. Cell 7, 559-570[CrossRef][Medline] [Order article via Infotrieve]
  55. Tuttle, R. L., Gill, N. S., Pugh, W., Lee, J. P., Koeberlein, B., Furth, E. E., Polonsky, K. S., Naji, A., and Birnbaum, M. J. (2001) Nat. Med. 7, 1133-1137[CrossRef][Medline] [Order article via Infotrieve]
  56. Wrede, C. E., Dickson, L. M., Lingohr, M. K., Briaud, I., and Rhodes, C. J. (2002) J. Biol. Chem. 277, 49676-49684[Abstract/Free Full Text]
  57. Dickson, L. M., and Rhodes, C. J. (2004) Am. J. Physiol. 287, E192-E198
  58. Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993) Nature 366, 461-464[CrossRef][Medline] [Order article via Infotrieve]
  59. Kulkarni, R. N., Jhala, U. S., Winnay, J. N., Krajewski, S., Montminy, M., and Kahn, C. R. (2004) J. Clin. Investig. 114, 828-836[CrossRef][Medline] [Order article via Infotrieve]

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