HIV-1 Accessory Inhibits the Effect of Insulin on the Foxo Subfamily of Forkhead Transcription Factors by Interfering With Their Binding to 14-3-3 Potential Clinical Implications Regarding the Insulin Resistance of HIV-1–Infected Patients Tomoshige Kino,1 Massimo U. De Martino,1 Evangelia Charmandari,1 Takamasa Ichijo,1 Taoufik Outas,2 and George P. Chrousos1

HIV-1 accessory protein Vpr arrests host cells at the interferes with the suppressive effects of insulin on G2/M phase of the cell cycle by interacting with mem- FOXO-mediated transcription of target via 14-3-3. bers of the 14-3-3, which regulate the Vpr thus may contribute to the tissue-selective insulin activities of “partner” molecules by binding to their resistance often observed in HIV-1–infected individuals. phosphorylated serine or threonine residues and chang- Diabetes 54:23–31, 2005 ing their intracellular localization and/or stability. Vpr does this by facilitating the association of 14-3-3 to its partner protein Cdc25C, independent of the latter’s phosphorylation status. Here we report that the same he HIV-1 protein Vpr, a 96–amino acid virion- interfered with and altered the activity of associated accessory protein, is important for another 14-3-3 partner molecule, Foxo3a, a subtype of virus propagation in vivo and has multiple other the forkhead transcription factors, by inhibiting its association with 14-3-3. Foxo3a’s transcriptional activ- Tfunctions (1–6). Vpr is packaged in significant ity is normally suppressed by insulin-induced transloca- quantities into viral particles (7,8) and is imported into the tion of this protein from the nucleus into the cytoplasm. nucleus early after infection (9). Vpr participates in the Vpr inhibited the ability of insulin or its downstream nuclear translocation of the HIV-1 preintegration complex protein kinase Akt to change the intracellular localiza- (10,11) and also functions as a coactivator of several tion of Foxo3a preferentially to the cytoplasm. This steroid hormone receptors (12,13). Vpr is detected in sera HIV-1 protein also interfered with insulin-induced co- of HIV-1–infected patients, and when it is administered precipitation of 14-3-3 and Foxo3a in vivo and antago- extracellularly, it penetrates cell membranes, enters into nized the negative effect of insulin on Foxo3a-induced the cytoplasm and nucleus of cells, and exerts its actions transactivation of a FOXO-responsive promoter. More- over, Vpr antagonized insulin-induced suppression of in both cytoplasmic and nuclear compartments. Thus, the the mRNA expression of the glucose 6-phosphatase, biologic effects of Vpr may be exerted in neighboring or manganese superoxide dismutase, and sterol carrier distant tissues that are not directly infected with the HIV-1 protein 2 genes, which are known targets of insulin and virus (14–16). FOXO, in HepG2 cells. These findings indicate that Vpr In addition to the above-indicated activities, Vpr effi- ciently arrests the host cell cell cycle at the G2/M bound- ary (17–19). To discover one or more molecules that From the 1Pediatric and Reproductive Endocrinology Branch, National Insti- support Vpr’s cell cycle–arresting activity, we recently tute of Child Health and Human Development, National Institutes of Health, performed yeast two-hybrid screening assays using wild- Bethesda, Maryland; and the 2Women’s Cancer Section, Pathologic Branch, National Cancer Institute, Bethesda, Maryland. type and mutant Vpr molecules as baits (20). We found Address correspondence and reprint requests to Tomoshige Kino, MD, PhD, that Vpr physically interacted with 14-3-3 proteins (20), Pediatric and Reproductive Endocrinology Branch, National Institute of Child which regulate numerous cellular activities by changing Health and Human Development, National Institutes of Health, 10 Center Dr. MSC 1109, Building 10, Clinical Research Center, Room 1-3140, Bethesda, MD the intracellular location and/or stability of “partner” mol- 20892-1109. E-mail: [email protected]. ecules after binding to phosphorylated serine/threonine Received for publication 29 March 2004 and accepted in revised form residues in special sequences of these molecules (21,22). 27 September 2004. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3- Vpr bound to 14-3-3 at the COOH-terminal domain of the phosphate dehydrogenase; G6Pase, glucose 6-phosphatase; IRS, insulin-re- latter, outside the binding sites for the phosphorylated sponsive sequence; MnSOD, manganese superoxide dismutase; SCP2, sterol carrier protein 2. amino acids of partner proteins. In a recent study from our © 2005 by the American Diabetes Association. laboratory, Vpr supported the complex formation of 14-3-3 The costs of publication of this article were defrayed in part by the payment of page and one of its partner proteins, Cdc25C, independent of charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the phosphorylation state of the latter, and prevented

DIABETES, VOL. 54, JANUARY 2005 23 Vpr INHIBITS THE EFFECT OF INSULIN ON Foxo3a

Cdc25C translocation into the nucleus (20). Thus, it seems and 329 (40,42). The phosphorylated NH2-terminal threo- that Vpr arrests the host cells at the G2/M phase of the cell nine and middle region serine residues act as high-affinity cycle by retaining 14-3-3 and Cdc25C in the cytoplasm. binding sites for 14-3-3 dimers (43). The FOXO-bound Infection with HIV-1 results in AIDS, which is charac- 14-3-3 dimer then masks the forkhead domain of the terized by profound defects in the immune system that former, as well as its closely located nuclear localization lead to opportunistic infections and neoplastic processes signal; thus, binding of 14-3-3 inhibits access of FOXO to (23). In addition, these patients frequently develop malnu- DNA and stimulates its translocation from the nucleus to trition/body weight loss and wasting, as well as growth the cytoplasm (42,43). Two nuclear export signals located retardation in children, states associated with paradoxi- in the NH2- and COOH-termini of Foxo1, respectively, cally increased levels of serum glucose, triglycerides, influence the latter process (42). These pieces of evidence cholesterol, and insulin (24–26). These pieces of evidence therefore indicate that FOXO proteins function as tran- indicate that tissues of HIV-1–infected patients are char- scription factors suppressed by insulin, and their binding acterized by reduced sensitivity to insulin, i.e., insulin to 14-3-3 is a crucial step in insulin’s ability to exert its resistance. inhibitory actions. In parallel, insulin and/or Akt may also Insulin regulates diverse physiologic functions of cells regulate FOXO-induced transcriptional activity indepen- and tissues, such as carbohydrate and lipid metabolism, dent of their effect through 14-3-3, for example, by directly protein synthesis, DNA replication, cell growth and differ- affecting the nuclear export/import and DNA-binding of entiation, and inhibition of apoptosis (27). Binding of FOXOs or via other as yet unknown mechanisms (42,43). insulin to its receptor stimulates many signaling cascades Because Vpr binds 14-3-3 and changes its binding spec- via phosphorylation-mediated reactions and activates sev- ificity to its partner protein Cdc25C, we hypothesized that Vpr might also modulate the binding activity of 14-3-3 to eral transcription factors that, finally, regulate expression other partner molecules, the FOXO family proteins. We of target molecules (28). Insulin-responsive genes have at show that Vpr inhibits the association of 14-3-3 and least eight distinct consensus insulin-responsive se- Foxo3a (FKHR-L1) and antagonizes insulin’s effect on quences (IRSs) in their promoter regions that positively or Foxo3a. Our results indicate that Vpr may be one of the negatively respond to insulin stimuli (29). Consensus viral factors that participate in the mechanism of insulin sequences, such as those of activator protein 1, Ets, E-box, resistance seen in some HIV-1-infected patients. and thyroid transcription factor 2, mediate positive tran- scriptional effects of insulin, whereas an element with the consensus sequence T(G/A)TTT(T/G)-(G/T), also referred RESEARCH DESIGN AND METHODS Plasmids. pCDNA3-Vpr and -VprR80A, which express wild-type and R80A to as the PEPCK-like motif, mediates the inhibitory effect mutant Vpr, respectively, were described previously (12). pCMV-FLAG-Vpr of insulin on several insulin-responsive genes (29). The and -VprR80A, which respectively express FLAG epitope-tagged wild-type Vpr forkhead in human rhabdomyosarcoma (FKHR or Foxo1), and R80A mutant Vpr, were also described previously (12). HA-FKHR-L1 one of the members of the FOXO subfamily of the fork- (HA-Foxo3a), HA-FKHR-L1-T32, S253, S315A, and HA-AKT-⌬PH, which ex- press the hemagglutinin epitope-tagged wild-type Foxo3a (FKHR-L1) and head proteins that share the forkhead DNA-binding do- Foxo3a (FKHR-L1) mutant that harbors mutations replacing a threonine and main and play diverse roles in developmental and serines located at amino acids 32, 253, and 312 to alanines, and constitutively metabolic functions, has recently been shown to bind this active Akt were gifts from Dr. M. Greenberg (Harvard Medical School, Boston, PEPCK-like motif (30). MA) (40). pEGFP-C1-Foxo3a, which expresses the enhanced green fluores- cent protein (EGFP)-fused Foxo3a, was constructed by subcloning the full Insulin regulates the activity of several FOXO proteins, coding sequence of Foxo3a (FKHR-L1) from HA-FKHR-L1 into pEGFP-C1 such as Foxo3a (FKHR-L1) and Foxo4 (AFX), in addition (Clontech, Palo Alto, CA). 3xIRS-Luc, which has the luciferase under the to Foxo1 (FKHR) (31–33). In the absence of insulin, they control of three FOXO-responsive elements, was a gift from Dr. K.L. Guan are located in the nucleus, bind to their responsive pro- (University of Michigan Medical School, Ann Arbor, MI) (44). pGEXT-4T3- Foxo3a and -Cdc25C were constructed by subcloning Foxo3a and Cdc25C moters, and activate the transcription rate of their target cDNAs into pGEX-4T3 in an in-flame manner, respectively. pHook-1, pSV40- genes, including the key gluconeogenesis PEPCK, ␤-Gal, and Bluescript SK (ϩ) were purchased from Invitrogen (Carlsbad, CA), the IGF-binding protein 1, and the glucose 6-phosphatase Promega (Madison, WI), and Stratagene (La Jolla, CA), respectively. A probe (G6Pase) enzyme, which dephosphorylates glucose to for the Northern blot analysis of the G6Pase gene was a gift from Dr. J. Chou facilitate its excretion from the liver (29,31,34,35). FOXO (National Institutes of Health, Bethesda, MD). Cell culture, transfection, reporter assays, and detection of EGFP- proteins also regulate mRNA expression of the manganese fused Foxo3a. Human cervical carcinoma HeLa cells and human hepatoma superoxide dismutase and sterol carrier protein 2 genes, HepG2 cells were purchased from American Type Culture Collection (Rock- which are also responsive to insulin (36,37). The former ville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium supple- plays an important role in the protection of tissues from mented with 10% of fetal bovine serum, 100 units/ml penicillin, 1 ␮g/ml oxidative stress (38), and the latter is involved in the streptomycin sulfate, and 25 mmol/l HEPES. Cells were transfected with either the Lipofectin or CaPO4 method, as described previously (12,13). For intracellular transport of cholesterol and phospholipids the experiments to detect EGFP, HeLa cells were seeded in 20-mm-diameter and in the activation of involved in fatty acyl CoA dishes 1 day before transfection and 0.3 ␮g per well of pEGFP-C1-Foxo3a and transacylation (39). Vpr-expressing plasmids were used for the transfection. Cells were treated Once insulin induces the phosphorylation of specific with 100 nmol/l insulin after 24 h of transfection or were transfected with 0.2 ␮g per well of HA-AKT-⌬PH to phosphorylate expressed Foxo3a. The EGFP- serine and threonine residues of these FOXO proteins via fused Foxo3a was detected under a Leica DM IRB inverted microscope (Leica activation of Akt or protein kinase B, these phosphory- Microsystems, Wetzlar, Germany). Images were captured with a charge- lated amino acids create binding sites for 14-3-3; then, coupled device camera (Shimazu, Kyoto, Japan) and analyzed with the Open binding of 14-3-3 to FOXOs induces translocation of the Lab software (Improvision, Coventry, U.K.). For reporter assays, HeLa cells were transfected with 1 ␮g per well of HA-FKHR-L1 (HA-Foxo3a) or HA- complexes into the cytoplasm and hence abolition of their FKHR-L1-T32, S253, or S312A, and 1 ␮g per well of Vpr-expressing plasmids, transcriptional effects (40,41). For example, insulin phos- together with 1.5 ␮g per well of 3xIRS-Luc and 0.5 ␮g per well of pSV40-␤-Gal. phorylates human Foxo1 at threonine 24 and serines 256 For keeping the same amounts of DNA, empty vectors or Bluescript SK (ϩ)

24 DIABETES, VOL. 54, JANUARY 2005 T. KINO AND ASSOCIATES

FIG. 1. Vpr inhibits the ability of insulin and Akt to induce translocation of EGFP-fused Foxo3a from the nucleus to the cytoplasm. A: Vpr inhibits the ability of insulin to induce nucleus-to-cytoplasm translocation of EGFP-Foxo3a in HeLa cells. HeLa cells were transfected with pEGFP-C1- Foxo3a and/or pCDNA3-Vpr and stimulated with 100 nmol/l insulin. B: Vpr inhibits the ability of the constitutively active Akt to induce nucleus-to-cytoplasm translocation of EGFP-Foxo3a in HeLa cells. HeLa cells were transfected with pEGFP-C1-Foxo3a, HA-AKT-⌬PH, and/or pCDNA3-Vpr. C: Wild-type Vpr but not Vpr R80A blocks insulin’s ability to induce nucleus-to-cytoplasm translocation of EGFP-Foxo3a in HeLa cells. HeLa cells were transfected with pEGFP-C1-Foxo3a and/or pCDNA3-Vpr or -VprR80A and stimulated with 100 nmol/l insulin. Cells exhibited different intracellular distribution patterns of EGFP-Foxo3a and were categorized into five types, depending on this distribution. Cells of each distribution pattern were counted and shown in the x-axis as percentages to the total cell number counted. N, nuclear localization; C, cytoplasmic localization; WT, wild type. were used. Twenty-four hours after the transfection, cells were treated with Cruz Biotechnology) in Western blots. Phosphorylated Foxo1, Foxo3a, and 100 nmol/l insulin, and cell lysates were collected after an additional 24-h Akt were also detected in Western blot by using anti-Foxo1, -Foxo3a, and -Akt, incubation. Luciferase and ␤-galactosidase activities were determined as which specifically recognize these molecules that are phosphorylated at described previously (12). Luciferase activity was divided by ␤-galactosidase specific amino acids (Santa Cruz Biotechnology). The membranes were activity to account for transfection efficiency. treated with secondary antibodies, and blotted proteins were visualized by the Coimmunoprecipitation assay. HeLa cells were grown in 175-cm2 flasks ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech, and transfected with 15 ␮g of each indicated plasmid. Cells were treated with Piscataway, NJ) and exposed to high-performance chemiluminescence films 100 nmol/l insulin 24 h after transfection. After an additional 24 h, cells were (Hyperfilm ECL; Amersham Pharmacia Biotech). lysed with buffer that contained 50 mmol/l Tris-HCl (pH 8.0), 120 mmol/l NaCl, In vitro binding assay. 35S-labeled Vpr was generated by in vitro translation 0.5% NP-40, and 1 Tab/50 ml Complete tablets (Roche Molecular Biochemi- and tested for interaction with bacterially produced and purified GST-Foxo3a cals, Indianapolis, IN), and extracts were centrifuged at 400g at 4°C for 10 min. or -Cdc25C immobilized on glutathione-sepharose beads in buffer that con- Supernatants were collected to obtain the whole homogenates (20). They tained 50 mmol/l Tris-HCl (pH 8.0), 50 mmol/l NaCl, 1 mmol/l EDTA, 0.1% were incubated with anti–14-3-3␤ (K-19) antibody (Santa Cruz Biotechnology, NP-40, 10% glycerol, and 0.1 mg/ml BSA at 4°C for 1.5 h. After vigorous Santa Cruz, CA), which cross-reacts with multiple 14-3-3 proteins, at 4°C for washing with the buffer, proteins were eluted and separated on a 14% 3 h. Protein-antibody complexes were subsequently harvested by adding SDS-PAGE gel. Gels were fixed and exposed on film. Expression and protein A/G agarose PLUS (Santa Cruz Biotechnology) and were washed three purification of GST, and GST-Foxo3a and -Cdc25C were confirmed by running times with the buffer that contained 20 mmol/l Tris-HCl (pH 8.0), 100 mmol/l these proteins on a 4–20% SDS-PAGE gel and by visualizing with SimplyBlue NaCl, 0.5% NP-40, and 1 Tab/50 ml Complete tablets. Samples were separated SafeStain (Invitrogen). on 12 or 16% SDS-PAGE gels and blotted to a nitrocellulose membrane, which Northern blot analyses. HepG2 cells were plated on 150-mm-diameter was subsequently treated with anti-hemagglutinin antibody (Santa Cruz dishes 1 day before the experiments. They were transfected with pCDNA3-Vpr Biotechnology). Expressed Vpr was detected with anti-FLAG (M2) antibody and pHook-1. Cells were subsequently treated with 100 nmol/l insulin or (Sigma, St. Louis, MO) in whole homogenates. Inputs of hemagglutinin vehicle for 24 h. Transfection-positive cells were then collected through the (HA)-Foxo3a and 14-3-3 were detected in 10% of whole homogenates. Foxo1 epitope expressed from pHook-1 following the company’s instruction (13), was detected in 10% whole homogenates by using anti-Foxo1 antibody (Santa and total RNA was isolated with TRIZOL (Invitrogen). Purified total RNA was

DIABETES, VOL. 54, JANUARY 2005 25 Vpr INHIBITS THE EFFECT OF INSULIN ON Foxo3a run on a formaldehyde-denaturing gel and was blotted to a nylon membrane, which was subsequently treated with 32P-labeled probe directed to G6Pase or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was washed in SSPE buffers and was exposed to an X-ray film. Band density of G6Pase or GAPDH was measured by using the NIH Image software (National Institutes of Health, Bethesda, MD). The expression of G6Pase was corrected for the expression of GAPDH. Results represent the mean and SE of three independent experiments. Akt inhibitor 1L-6-hydroxymethyl-chio-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate was purchased from Calbiochem- Novabiochem (La Jolla, CA). Quantitative real-time PCR for the evaluation of G6Pase, manganese superoxide dismutase, and sterol carrier protein 2 (SCP2) mRNA levels. HepG2 cells (1 ϫ 106) were cultured in serum-free medium for 48 h, plated on a 100-mm dish, and transfected with pCDNA3-Vpr wild-type or R80A, and pHook-1. The cells were subsequently treated with 100 nmol/l insulin or vehicle for 24 h. Transfection-positive cells were then collected through the epitope expressed from pHook-1 as per company instructions (13), and total RNA was isolated with TRIZOL (Invitrogen). The reverse transcription reaction was carried out using random hexamers with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), as reported previously (45). For detecting mRNA levels of G6Pase, manganese superoxide dismutase (MnSOD) and SCP2 and control human acidic ribo- somal phosphoprotein P0 (RPLP0), primer pairs (G6Pase: forward primer 5Ј-CTACTCTTTCCATCTTTCAG-3Ј, reverse primer 5Ј-GAGTTAGGAAGCACT GATC-3Ј; MnSOD: forward primer 5Ј-CAGATAGCTCTTCAGCCTG-3Ј, reverse primer 5Ј-CACGCTGCGCTTGAAGAAG-3Ј; SCP2: forward primer 5Ј-GAA GCTGTTCCAACCAG-3Ј, reverse primer 5Ј-GAGTTAGGAAGCACTGATC-3Ј; RPLP0: forward primer 5Ј-CGCGACCTGGAAGTCCAACT-3Ј; reverse primer 5Ј-CCATCAGCACCACAGCCTTC-3Ј) were used. The real-time PCR, consisting of the heat activation of the Taq polymerase (at 95°C for 10 min) and the subsequent 62 PCR cycles (denaturing at 95°C for 15 s, annealing/extension at 62°C for 1 min) was performed in triplicate using the SYBR Green PCR Master Mix (Applied Biosystems) in an ABI PRIZM 7700 SDS lightcycler (Applied Biosystems). Obtained threshold cycle values of G6Pase, MnSOD, and SCP2 were normalized for those of RPLP0, and their relative mRNA expressions were shown as fold induction over the baseline. The dissociation curves of used primer pairs showed a single peak, and samples after PCRs had a single expected DNA band in an agarose gel analysis (data not shown).

RESULTS Vpr inhibits insulin- and Akt-induced cytoplasmic translocation of EGFP-Foxo3a. To test our hypothesis, we first examined the effects of Vpr on the nucleus to cytoplasm translocation of EGFP-Foxo3a induced by in- sulin in HeLa cells (Fig. 1A). In the absence of insulin, the majority of cells had EGFP-Foxo3a in the nucleus with minimal distribution in the cytoplasm (Fig. 1A, top left). Coexpression of Vpr did not affect the subcellular local- ization of EGFP-Foxo3a (Fig. 1A, bottom left). Addition of 100 nmol/l insulin, however, strongly shifted the intracel- lular localization of EGFP-Foxo3a from the nucleus to the cytoplasm (Fig. 1A, top right), whereas addition of Vpr moderately retained EGFP-Foxo3a in the nucleus (Fig. 1A, bottom right). These results indicate that Vpr antagonized FIG. 2. A: Vpr inhibits the ability of insulin to induce 14-3-3 and Foxo3a association in HeLa cells. HeLa cells were transfected with HA-FKHR- insulin’s effect on the nucleus to cytoplasm translocation L1 (HA-Foxo3a) and/or pCMV-FLAG-Vpr and pHook-1. HA-Foxo3a/14- of Foxo3a. 3-3 complexes were coprecipitated with anti-hemagglutinin antibody and blotted with anti–14-3-3␤ antibody. Expression of FLAG-Vpr, We next used a constitutively active form of an Akt HA-Foxo3a, and 14-3-3 was tested in 10% of whole homogenates, which mutant instead of insulin, which directly phosphorylates were used for the coprecipitation reactions. Phosphorylated forms of Foxo3a, bypassing several intermediate phosphorylation- Foxo3a (Thr 32), Akt (Thr 308), and Akt (Ser 473) were also detected in 10% whole homogenates in Western blots. B: Vpr R80A did not dependent reactions induced by insulin (Fig. 1B). Expres- interfere with association of 14-3-3 and Foxo3a in HeLa cells. HeLa sion of this mutant Akt induced cytoplasmic redistribution cells were transfected with HA-FKHR-L1 (HA-Foxo3a) and/or pCMV- of EGFP-Foxo3a in a manner similar to insulin (Fig. 1B, FLAG-Vpr WT or -VprR80A and pHook-1. HA-Foxo3a/14-3-3 or Vpr/14- 3-3 complexes were coprecipitated with anti-hemagglutinin antibody top right). Vpr also antagonized this effect of Akt on EGFP- or anti-FLAG antibody, respectively, and blotted with anti–14-3-3␤ Foxo3a localization, partially retaining it in the nucleus antibody. Expression of FLAG-Vpr, HA-Foxo3a, and 14-3-3 was tested in 10% of whole homogenates, which were used for the coprecipitation (Fig. 1B, bottom right). reactions. C and D: Vpr does not bind to Foxo3a in vitro. In vitro– We examined Ͼ100 cells that were cotransfected with translated and 35S-labeled Vpr was mixed with bacterially produced EGFP-Foxo3a- and Vpr-expressing plasmids and treated and purified GST-Foxo3a or -Cdc25C immobilized on glutathione- sepharose beads, and their interaction was tested (C). Purified GST, with 100 nmol/l insulin and categorized them into five GST-Cdc25C, and -Foxo3a were also run on a 4–20% SDS-PAGE and groups that have different distribution patterns from com- visualized with SimplyBlue SafeStain (D).

26 DIABETES, VOL. 54, JANUARY 2005 T. KINO AND ASSOCIATES

FIG. 3. Vpr inhibits the suppressive effect of insulin on Foxo3a-induced target gene transactivation in HeLa cells. HeLa cells were transfected with HA-FKHR-L1 (HA-Foxo3a) (WT) or HA-FKHR-L1-T32, S253, S315A (Mut), and/or pCDNA3-Vpr or -VprR80A, together with 3xIRS-Luc and pSV40-␤-Gal. Ⅺ, mean ؎ SE values in the absence of 100 nmol/l insulin; f, mean ؎ SE values in the presence of 100 nmol/l insulin. *P < 0.01. plete nuclear localization to complete cytoplasmic local- Vpr antagonizes the negative effect of insulin on the ization. We demonstrated cell numbers in each group as transcriptional activity of Foxo3a. We next examined percentage of the total numbers of examined cells (Fig. the effects of Vpr on Foxo3a-induced transactivation of a 1C). In this analysis, the spectrum of cells that demon- responsive promoter to address the functional significance strated different EGFP-Foxo3a distribution was from the of the observed inhibitory effect of Vpr on the insulin- cells that showed complete nucleus versus cytoplasm induced association of Foxo3a and 14-3-3 (Fig. 3). We used localization in the absence of insulin, consistent with the 3xIRS-Luc, which contains three FOXO-responsive ele- previous experiment. Addition of 100 nmol/l insulin strong- ments that drive the luciferase reporter gene, as a ly shifted EGFP-Foxo3a from the nucleus into the cyto- reporter plasmid. Overexpression of Foxo3a stimulated plasm. Expression of wild-type Vpr weakly shifted local- this promoter activity by fourfold, and addition of 100 ization of EGFP-Foxo3a in the nucleus in the absence of nmol/l insulin suppressed Foxo3a-stimulated transacti- insulin. In the presence of insulin, however, Vpr expres- vation by 60%. Wild-type Vpr reduced insulin-induced sion partially retained EGFP-Foxo3a in the nucleus, sug- inhibition of the Foxo3a-stimulated transcription, gesting that Vpr moderately antagonized insulin’s effect on whereas it did not have a discernible effect on the basal Foxo3a subcellular distribution. In contrast to wild-type activity of the promoter. VprR80A, however, failed to Vpr, VprR80A mutant, which is defective in binding to antagonize insulin-induced suppression of the Foxo3a 14-3-3 (20), did not affect the nucleus-to-cytoplasm trans- activity. A Foxo3a mutant (Mut), which has amino acid location of EGFP-Foxo3a induced by insulin. substitutions at threonine 32 and serines 253 and 312 to Vpr inhibits insulin-induced association of 14-3-3 and alanines and, thus, is unable to bind 14-3-3, stimulated the Foxo3a in vivo. We previously demonstrated that Vpr promoter activity in an insulin-independent manner. Vpr through its COOH-terminal portion binds 14-3-3 outside did not affect the transcriptional activity of this mutant the phosphopeptide-binding pocket of the latter located Foxo3a. These results are consistent with the observation COOH-terminal (20). Because Vpr inhibited insulin- obtained in the subcellular localization study and the induced nucleus-to-cytoplasm translocation of EGFP- coimmunoprecipitation experiments, in which Vpr respec- Foxo3a, we hypothesized that binding of Vpr to 14-3-3 tively suppressed cytoplasmic redistribution of EGFP- might interfere with association of 14-3-3 and FOXO Foxo3a and inhibited the association of Foxo3a and 14-3-3 proteins induced by insulin. We thus examined this possi- induced by insulin. However, Vpr might also affect FOXO- bility in a coimmunoprecipitation assay in HeLa cells (Fig. induced transcriptional activity by a mechanism(s) inde- 2A). In the absence of insulin, Foxo3a was coprecipitated pendent of 14-3-3. Indeed, insulin and Akt may regulate with 14-3-3 (Fig. 2A, top gel, lane 1). Addition of 100 nmol/l FOXOs in multifaceted ways not all fully understood insulin increased the association of 14-3-3 and Foxo3a (42,43). (Fig. 2A, top gel, lane 2). However, once Vpr was coex- Vpr antagonizes insulin’s effect on the expression of pressed, this viral protein inhibited the coprecipitation of the G6Pase mRNA and other two insulin/Foxo-re- 14-3-3 and Foxo3a that was induced by insulin (Fig. 2A, top sponsive genes in HepG2 cells. We examined the effect gel, lane 4). In control studies, insulin stimuli phosphory- of Vpr on the expression of endogenous G6Pase mRNA, a lated Foxo3a (Fig. 2A, third top gel) and upstream Akt gene whose transcription is stimulated by FOXO proteins (Fig. 2A, bottom three gels), indicating that Vpr did not and, thus, is negatively regulated by insulin (29). We inhibit the Foxo3a/14-3-3 association by suppressing phos- transfected HepG2 cells with the Vpr-expressing plasmids, phorylation reactions on these molecules induced by in- harvested transfection-positive cells, and examined G6Pase sulin. In addition, VprR80A, which cannot bind 14-3-3, did mRNA expression in Northern blots. Expression of Vpr did not affect the association of these two molecules in con- not have an obvious effect on the G6Pase mRNA abun- trast to the wild-type Vpr (Fig. 2B). Finally, Vpr did not bind dance, whereas 100 nmol/l insulin suppressed it by 65%. Foxo3a in vitro (Fig. 2C). These results further strengthen Addition of Vpr expression with the same concentration of the possibility that Vpr interfered with the association of insulin almost reversed the suppressive effect of insulin on 14-3-3 and Foxo3a via direct binding to 14-3-3. the G6Pase mRNA expression (Fig. 4A, top two gels). In

DIABETES, VOL. 54, JANUARY 2005 27 Vpr INHIBITS THE EFFECT OF INSULIN ON Foxo3a

FIG. 4. Vpr antagonized insulin-induced suppression of mRNA expression of the insulin/Foxo-responsive G6Pase, MnSOD, and SCP2 genes in HepG2 cells. A and B: Vpr reduces the suppressive effect of insulin on the expression of endogenous G6Pase mRNA in HepG2 cells. HepG2 cells were transfected with Vpr-expression plasmids and pHook- 1 and treated with 100 nmol/l insulin or vehicle. The mRNA abundance of G6Pase and GAPDH was detected by North- ern blots (A, top two gels). Endogenous Foxo1 and its phosphorylated form (Thr 24) were detected in samples obtained in the same experiments in Western blots (B, -bottom two gels). Mean ؎ SE values of the G6Pase expres sion corrected for the expression of GAPDH in three independent experiments are shown in B.*P < 0.01. C: Insulin suppresses G6Pase mRNA expression via activation of Akt in HepG2 cells. HepG2 cells were treated with 100 nmol/l insulin in the presence or absence of 10 ␮mol/l of the Akt inhibitor 1L-6-hydroxymethyl-chio-inositol 2-(R)-2-O- methyl-3-O-octadecylcarbonate. The mRNA abundance of G6Pase and GAPDH was detected by Northern blots (top two gels), and expressed Foxo1 (third gel) and its phos- phorylated form at threonine 24 (bottom gel) were identi- fied in Western blots. D: Wild-type Vpr antagonized insulin- induced suppression of G6Pase, MnSOD, and SCP2 mRNA expression, whereas the 14-3-3 binding–defective Vpr R80A mutant lost this effect. HepG2 cells were transfected with wild-type Vpr- or R80A mutant–expressing plasmids and pHook-1 and were treated with 100 nmol/l insulin or vehi- cle. The mRNA expression of G6Pase, MnSOD, SCP2, and RPLP0 was evaluated by real-time PCR, using specific primer pairs. Obtained threshold cycle values of G6Pase, MnSOD, and SCP2 were normalized for those of RPLP0, ؎ and their relative mRNA expressions are shown as mean SE values of fold induction over baseline. *P < 0.01. the same experiment, Foxo1, which is a major FOXO MnSOD, or SCP2, whereas addition of insulin significantly protein in HepG2 cells, was phosphorylated at threonine suppressed it. Wild-type Vpr strongly attenuated the neg- 24 in Western blots (Fig. 4A, bottom two gels). Mean Ϯ SE ative effect of insulin on their mRNA expression of these values of the G6Pase expression corrected for the expres- genes, whereas the 14-3-3 binding–defective Vpr R80A mu- sion of GAPDH from three independent experiments are tant exerted no such effects. Taken together, our results shown in Fig. 4B. In this cell line, an Akt kinase inhibitor suggest that Vpr suppresses insulin’s effect on FOXO- 1L-6-hydroxymethyl-chio-inositol 2-(R)-2-O-methyl-3-O- induced expression of endogenous genes, such as those of octadecylcarbonate reversed insulin-induced suppres- G6Pase, MnSOD, and SCP2, possibly by inhibiting insulin- sion of G6Pase mRNA expression in Northern blots and induced association of 14-3-3 and FOXO proteins in HepG2 suppressed phosphorylation of Foxo1 in Western blots cells. (Fig. 4C), indicating that insulin, at least in part, impaired G6Pase mRNA expression by inhibiting Foxo activity in this cell line. Taken together, these findings indicate that DISCUSSION Vpr disrupts insulin-induced regulation of G6Pase gene In the present study, we investigated the effect of HIV-1 expression by altering the interaction of Foxo with 14-3-3. Vpr on the insulin-induced modulation of Foxo3a activity. However, it is possible that Vpr inhibits some of insulin’s We demonstrated that Vpr antagonized both the insulin- effect on G6P via non–14-3-3–mediated and Akt-induced translocation of Foxo3a from the nucleus mechanisms. to the cytoplasm. It also inhibited the association of Because there are several conflicting reports indicating Foxo3a with 14-3-3 induced by insulin in a coimmunopre- limited effect of FOXO proteins on G6Pase expression cipitation assay. Furthermore, Vpr antagonized the nega- (31,46), we also tested Vpr on two other Foxo-responsive tive effect of insulin on Foxo3a-induced transactivation of genes, MnSOD and SCP2, using real-time PCR in the same a responsive promoter and also inhibited the suppressive HepG2 cells (Fig. 4D). Expression of wild-type Vpr or the effect of insulin on the mRNA expression of the endoge- R80A mutant did not affect mRNA expression of G6Pase, nous G6Pase, MnSOD, and SCP2 genes in HepG2 cells.

28 DIABETES, VOL. 54, JANUARY 2005 T. KINO AND ASSOCIATES

Fig. 4. Continued.

Because the VprR80A mutant, which is defective in bind- known as AIDS-associated insulin resistance and lipodys- ing to 14-3-3, lost all of these activities and because trophy syndrome, especially when given long-term therapy wild-type Vpr inhibited insulin-induced coprecipitation of with anti-HIV drugs, such as protease inhibitors and nu- 14-3-3 and Foxo3a, our results strongly suggest that Vpr cleotide/nonnucleotide inhibitors inhibits binding of 14-3-3 to phosphorylated FOXO pro- (26). AIDS patients who have this syndrome have carbo- teins and, thus, antagonizes insulin’s negative regulation of hydrate intolerance and dyslipidemia and may also de- their activity. It is still possible, however, that Vpr regu- velop overt diabetes, along with their cardiovascular lates insulin/FOXO-responsive genes by 14-3-3–bypassing sequelae, particularly atherosclerotic coronary artery dis- mechanisms (42,43,46). ease (24,25,48–50). The cause of this syndrome is not clear The above effects of Vpr may be relevant to several but seems to be multifactorial. The HIV-1 infection itself biologic changes observed in HIV-1–infected lymphocytes seems to contribute to the development of these patho- and monocytes or nonimmune tissues, such as adipose logic changes or increases the vulnerability of patients to tissue, that are primary targets of HIV-1 (47). However, the develop the syndrome upon uptake of anti–HIV-1 drugs; majority of insulin-responsive tissues, such as muscle and some AIDS patients develop the characteristic features liver, have not been reported to be infected directly with and metabolic disturbances of the syndrome before treat- HIV-1. The latter may not be necessary for the metabolic ment (26,51,52). Our results suggest that Vpr might con- actions of Vpr, which is expressed and secreted by host tribute to the insulin resistance of AIDS patients through cells infected by HIV-1 and is detected in extracellular both its FOXO-related insulin action blockade and its fluids, such as plasma and cerebrospinal fluid, of HIV-1– previously described glucocorticoid receptor coactivator infected patients (14). This is corroborated by extracellular activity (12,13). A recent report indicated that FOXO plays administration of synthetic Vpr, which readily penetrates an important role in the adipocyte differentiation (53); the cell membrane and causes cell cycle arrest or exerts thus, it is also possible that Vpr influences development of other effects in HIV-1–uninfected cells (15,16), indicating lipodystrophic phenotype seen in this syndrome by mod- that Vpr may also act as a paracrine or endocrine “hor- ulating FOXO activity. mone,” acting on neighboring or distant cells that are not Vpr inhibited insulin-induced association of 14-3-3 and infected with HIV-1. Foxo3a in a coimmunoprecipitation assay. This indicates AIDS patients frequently develop a pathologic state that binding of Vpr to 14-3-3 interferes with the interac-

DIABETES, VOL. 54, JANUARY 2005 29 Vpr INHIBITS THE EFFECT OF INSULIN ON Foxo3a tion of phosphorylated serine and threonine residues of deficiency virus type 1 (HIV-1) Vpr functions as an immediate-early protein Foxo3a to a phosphopeptide-binding pocket located in during HIV-1 infection. J Virol 73:4101–4109, 1999 10. Vodicka MA, Koepp DM, Silver PA, Emerman M: HIV-1 Vpr interacts with 14-3-3. Vpr binds 14-3-3 at the COOH-terminal portion of the nuclear transport pathway to promote macrophage infection. Genes the latter, outside the phosphopeptide-, includ- Dev 12:175–185, 1998 ing the eighth ␣-helix of 14-3-3 (20). Because this portion 11. Heinzinger NK, Bukinsky MI, Haggerty SA, Ragland AM, Kewalramani V, of 14-3-3 also plays a role in the creation of the phos- Lee MA, Gendelman HE, Ratner L, Stevenson M, Emerman M: The Vpr phopeptide-binding cleft, possibly by stabilizing the struc- protein of human immunodeficiency virus type 1 influences nuclear ture of 14-3-3 (54,55), it seems that Vpr reduces the binding localization of viral nucleic acids in nondividing host cells. Proc Natl Acad SciUSA91:7311–7315, 1994 activity of 14-3-3 to the phosphorylated serine and threo- 12. Kino T, Gragerov A, Kopp JB, Stauber RH, Pavlakis GN, Chrousos GP: The nine residues of FOXO proteins. In contrast, we previously HIV-1 virion-associated protein vpr is a coactivator of the human glucocor- reported that Vpr facilitated binding of 14-3-3 and Cdc25C ticoid receptor. J Exp Med 189:51–62, 1999 independent of the phosphorylation state of Cdc25C (20). 13. Kino T, Gragerov A, Slobodskaya O, Tsopanomichalou M, Chrousos GP, These results indicate that Vpr influences the binding ac- Pavlakis GN: Human immunodeficiency virus type-1 (HIV-1) accessory protein Vpr induces transcription of the HIV-1 and glucocorticoid-respon- tivity of 14-3-3 to its partners in different directions, de- sive promoters by binding directly to p300/CBP coactivators. J Virol 76: pending on the partner molecule with which it associates. 9724–9734, 2002 In addition to the forkhead transcription factors, 14-3-3 14. Levy DN, Refaeli Y, MacGregor RR, Weiner DB: Serum Vpr regulates proteins influence the function of numerous proteins productive infection and latency of human immunodeficiency virus type 1. involved in growth factor, hormone, and cytokine signal- Proc Natl Acad SciUSA91:10873–10877, 1994 15. Henklein P, Bruns K, Sherman MP, Tessmer U, Licha K, Kopp J, de ing, as well as apoptosis, and cell cycle regulation, such as Noronha CM, Greene WC, Wray V, Schubert U: Functional and structural IRS-1, Raf-1, MEKK1, Bcr, calmodulin/calmodulin kinase, characterization of synthetic HIV-1 Vpr that transduces cells, localizes to protein kinase C, c-Cbl, BAD, and NF-AT, through direct the nucleus, and induces G2 cell cycle arrest. J Biol Chem 275:32016– binding to their phosphorylated serine/threonine residues 32026, 2000 (21,56). Modulation of the activities of these proteins by 16. Mirani M, Elenkov I, Volpi S, Hiroi N, Chrousos GP, Kino T: HIV-1 protein Vpr suppresses IL-12 production from human monocytes by enhancing Vpr might contribute to the development of other patho- glucocorticoid action: potential implications of Vpr coactivator activity for logic manifestations observed in HIV-1–infected patients. the innate and cellular immunity deficits observed in HIV-1 infection. We conclude that the HIV-1 accessory protein Vpr coun- J Immunol 169:6361–6368, 2002 teracts the negative regulation of insulin on the FOXO 17. He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR: Human subfamily of the forkhead proteins possibly by inhibiting immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69:6705–6711, association between these proteins and 14-3-3. Our results 1995 may be relevant to pathogenetic mechanisms that account 18. Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, Chen IS: The human for the development of insulin resistance and related met- immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 abolic disturbances observed in HIV-1–infected individuals. ϩ M phase of the cell cycle. J Virol 69:6304–6313, 1995 19. Re F, Braaten D, Franke EK, Luban J: Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2- ACKNOWLEDGMENTS cyclin B. J Virol 69:6859–6864, 1995 20. Kino T, Gragerov A, Valentin A, Tsopanomihalou M, Ilyina-Gragerova G, We thank Drs. G.N. Pavlakis, A. Gragerov, J. Nakae, and D. Erwin-Cohen R, Chrousos GP, Pavlakis GN: Vpr protein of human immu- Accili for insightful comments; Drs. M. Greenberg, K.L. nodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex Guan, and J. Chou for kindly providing plasmids; and K. formation with Cdc25C: implications for cell cycle arrest. J Virol. In press 21. Aitken A: 14-3-3 and its possible role in co-ordinating multiple signaling Zachman and L. Chheng for excellent technical assistance. pathways. Trends Cell Biol 6:341–347, 1996 22. 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