University of South Florida Scholar Commons

Integrative Biology Faculty and Staff ubP lications Integrative Biology

2005 Suppression of FOXO1 Activity by FHL2 through SIRT1 Mediated deacetylation Yonghua Yang University of South Florida

Huayan Hou University of South Florida

Edward M. Haller University of South Florida, [email protected]

Santo V. Nicosia University of South Florida

Wenlong Bai University of South Florida

Follow this and additional works at: https://scholarcommons.usf.edu/bin_facpub

Scholar Commons Citation Yang, Yonghua; Hou, Huayan; Haller, Edward M.; Nicosia, Santo V.; and Bai, Wenlong, "Suppression of FOXO1 Activity by FHL2 through SIRT1 Mediated deacetylation" (2005). Integrative Biology Faculty and Staff Publications. 265. https://scholarcommons.usf.edu/bin_facpub/265

This Article is brought to you for free and open access by the Integrative Biology at Scholar Commons. It has been accepted for inclusion in Integrative Biology Faculty and Staff ubP lications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The EMBO Journal (2005) 24, 1021–1032 | & 2005 European Molecular Biology Organization | All Rights Reserved 0261-4189/05 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL Suppression of FOXO1 activity by FHL2 through SIRT1-mediated deacetylation

Yonghua Yang, Huayan Hou, FOXO4 (AFX, acute-lymphocytic-leukemia-1) (Parry et al, Edward M Haller, Santo V Nicosia 1994), and the recently cloned FOXO6 (Jacobs et al, 2003). and Wenlong Bai* As demonstrated by genetic and biochemical analysis of lower organisms and mammalian systems, the FOXO factors Departments of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine and Programs of Molecular Oncology, are critical regulators of stress responses, insulin sensitivity, Drug Discovery and Experimental Therapeutics, H Lee Moffitt Cancer longevity, and oncogenesis. For example, in Caenorhabditis Center, Tampa, FL, USA elegans, the FOXO-related protein Daf-16 increases longevity and signals formation of the dauer larva, which is a stage of Forkhead box class O (FOXO) proteins are transcription developmental arrest and reduced metabolic activity that factors that function downstream of the PTEN tumor enhances survival during periods of environmental stress suppressor and directly control the expression of genes (Kenyon et al, 1993). Mutation of the insulin/IGF1 receptor involved in apoptosis, cell cycle progression, and stress homolog Daf-2 or of Age-1, the catalytic subunit of phosphoi- responses. In the present study, we show that FOXO1 nositide 3-kinase (PI3K), mimics the effects of Daf-16, thus interacts with four and a half LIM 2 (FHL2) in prostate suggesting that the insulin/IGF receptor–PI3K pathway nega- cancer cells. This interaction occurred in the nucleus and tively regulates Daf-16 function. Consistent with this premise, was enhanced by lysophosphatic acid. FHL2 decreased the Daf-16 and its human homologs contain multiple conserved transcriptional activity of FOXO1 and the expression of phosphorylation sites for members of the protein kinase B known FOXO target genes and inhibited FOXO1-induced family (AKT-1, AKT-2, AKT3, and SGK-1), all of which func- apoptosis. Interestingly, SIRT1, a mammalian homolog of tion downstream of PI3K (Brunet et al, 1999). yeast Sir2, bound to and deacetylated FOXO1 and inhibited Phosphorylation of the FOXO factors by PKB results in their its transcriptional activity. FHL2 enhanced the interaction nuclear export, their interaction with the 14-3-3 proteins in of FOXO1 and SIRT1 and the deacetylation of FOXO1 by the cytosol, and consequently in the abrogation of their Sirtuin-1 (SIRT1). Overall, our data show that FHL2 in- transcriptional activity. Recent studies suggest that kinases hibits FOXO1 activity in prostate cancer cells by promoting in addition to PKB inhibit FOXO activity (Brunet et al,2001; the deacetylation of FOXO1 by SIRT1. Woods et al, 2001; Hu et al, 2004), as do nuclear hormone The EMBO Journal (2005) 24, 1021–1032. doi:10.1038/ receptors (Schuur et al, 2001; Zhao et al, 2001; Dowell et al, sj.emboj.7600570; Published online 3 February 2005 2003; Li et al, 2003) and other forkhead factors (Seoane et al, Subject Categories: chromatin & transcription; molecular 2004). biology of disease The PI3K–PKB pathway is active in and enhances the Keywords: acetylation; FKHR; forkhead; prostate cancer; growth and survival of many types of human cancer cells. SIR2 Conversely, the FOXO factors, which function downstream of the PI3K antagonist PTEN in cancer cells, inhibit cell cycle progression and promote cell death. These factors accomplish these actions by modulating the expression of genes encoding apoptosis and growth regulatory proteins by two methods: Introduction (1) direct binding to the insulin response sequence (IRS) in The forkhead transcription factors are winged helical proteins gene promoters and (2) ‘tethering’ to other transcription first known for their role in development. Each of the more factors (Ramaswamy et al, 2002). Examples of genes regu- than 90 members of the forkhead family contains a conserved lated by direct binding of the FOXO factors to DNA include DNA binding domain with a butterfly-shaped structure of those encoding apoptotic proteins such as Bim and Fas three a-helices and two characteristic large loops or ‘wings’ ligand, and examples of genes regulated by ‘tethering’ in- (Kaufmann and Kno¨chel, 1996). clude those encoding the cell cycle regulators such as cyclin Members of the forkhead family, as classified by phyloge- G2 and cyclin D1. Both wild-type FOXO and FOXO mutants netic analysis of the amino-acid sequence of the DNA binding that cannot bind DNA modulate the expression of cyclin D1 domain, include the forkhead box class O (FOXO) factors. and cyclin G2 (Ramaswamy et al, 2002). There are four FOXO factors: FOXO1 (FKHR, Forkhead in FHL2 (four and a half LIM 2) was first identified as a rhabdomyosarcoma) (Galili et al, 1993; Kaestner et al, 2000), protein differentially expressed in normal human myoblasts FOXO3a (FKHRL1, FKHR-like 1) (Anderson et al, 1998), and their malignant counterparts, rhabdomyosarcoma cells; thus, it is also named DRAL (Downregulated in *Corresponding author. Department of Pathology, University of South Rhabdomyosarcoma LIM Protein) (Genini et al, 1997). Florida, College of Medicine, 12901 Bruce B Downs Blvd, MDC 11, FHL2 is a LIM-only protein with five LIM domains whereby Tampa, FL 33612-4799, USA. Tel.: þ 1 813 974 0563; the LIM domain in the amino terminus consists only of the Fax: þ 1 813 974 5536; E-mail: [email protected] second half of the consensus motif. Although not required for Received: 6 September 2004; accepted: 7 January 2005; published normal heart development (Chu et al, 2000), FHL2 may online: 3 February 2005 control the hypertrophic response to stress in cardiomyocytes,

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where it is enriched (Kong et al, 2001, Purcell et al, 2004). human prostate cancer. b-Galactosidase (b-gal)-based screen- The present study shows that FHL2 interacts with FOXO1 ing identified several positive clones, and sequencing of both in vitro and in prostate cancer cells. Binding of FHL2 to cDNA identified one of the positive clones as full-length FOXO1 inhibits the transcriptional activity of FOXO1 by FHL2 fused in-frame to the VP16 activation domain. facilitating its deacetylation through Sirtuin-1 (SIRT1), the To determine whether FHL2 and FOXO1 interact in mam- mammalian homolog of the yeast Sir2 deacetylase. Our data malian cells, we ectopically expressed Myc-FHL2 and Flag- raise the interesting possibility that interaction of FHL2 with FOXO1 in DU145 cells, which are human prostate tumor cells. FOXO1 and SIRT1 may promote prostate tumorigenesis in Cell extracts were immunoprecipitated and immunoblotted response to increased stress during aging. with antibodies to the tags. In cells expressing both proteins, antibody to Myc co-precipitated FOXO1, and antibody to Flag Results co-precipitated Myc-FHL2 (Figure 1A). Co-precipitation did not occur in cells expressing Myc-FHL2 or Flag-FOXO1 alone, FHL2 interacts with the amino terminus of FOXO1 and thus is not the result of antibody crossreactivity. and inhibits the transcriptional activity of FOXO1 To substantiate the interaction data, the subcellular dis- The functions of the amino terminus of FOXO1, in contrast to tribution of FHL2 and FOXO1 in DU145 cells was determined that of the carboxyl-terminal activation domain and of the by immunofluorescence microscopy. As shown in Figure 1B, central DNA binding domain, are unclear. The amino termi- a significant amount of ectopic FOXO1 was nuclear (panel nus interacts with transcriptional coactivators (Puigserver B2). This finding is consistent with the presence of functional et al, 2003) and thus may contain an activation function. To PTEN, and thus of relatively low amounts of PKB activity, in better define its function and to identify additional binding DU145 cells (Ramaswamy et al, 1999). Some cytoplasmic partners, we used the amino terminus (amino acids 1–150) of staining was detected, presumably because the cells were in FOXO1 as bait in a two-hybrid screen of a cDNA library from medium containing 5% serum, which may activate PKB.

AC− ++− Flag-FOXO1 FOXO1(1−150) −−++Myc-FHL2 IP: Anti-Flag Interaction with FOXO1 IB: Ant-Myc Input

IP: Anti-Myc GST-FHL2 GST 1/2 LIM 1 LIM 2 LIM 3 LIM 4 + IB: Anti-Flag GST-LIM (3&1/2) GST 1/2 LIM 1 LIM 2 LIM 3 + IP: Anti-Flag GST-LIM (2&1/2) GST 1/2 LIM 1 LIM 2 + IB: M2 GST-LIM (1&1/2) GST 1/2 LIM 1 − IP: Anti-Myc IB: Anti-FHL2 GST-LIM (1/2) GST 1/2 −

GST GST −

BDB1. DAPI B2. FOXO1 B3. FHL2 B4. 2-Color B5: 3-Color 120

100

80

60

40 B6: 2-Color confocal 20 deconvolution Luciferase activity (RLU) image 0 Gal4-FOXO1(1−150) 0 0.05 0.05 0.05 0.05 0 (µg) FHL2 0 0 0.2 0.4 1 0.2 (µg)

Figure 1 FHL2 interacts with FOXO1 and inhibits the transcriptional activity of the amino terminus of FOXO1. (A) DU145 cells were transfected with Myc-FHL2 (1 mg) and Flag-FOXO1 (1 mg). Cell extracts were immunoprecipitated with rabbit anti-Flag or anti-Myc antibody. Immunoprecipitates were immunoblotted with anti-Myc, rabbit anti-Flag, M2 anti-Flag, or goat anti-FHL2 antibody as indicated. (B) DU145 cells in medium containing 5% sFBS were transfected with Myc-FHL2 (1 mg) and Flag-FOXO1 (1 mg). Cells were stained with DAPI and incubated with anti-FHL2 and M2 antibodies. Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red, detects FHL2) or FITC (green, detects FOXO1). Colocalization was visualized by high-resolution imaging with deconvolution microscopy (panel B6). (C) DU145 cells were transfected with 1 mg of His-FOXO1(1–150), and cell extracts were incubated with GSTor with GST-FHL2 fusion proteins prebound to glutathione beads. Proteins bound to the beads and the input (50% of extract protein used for precipitations) were immunoblotted with goat anti-FOXO1 antibody. (D) DU145 cells were transfected with Gal4 reporter 3 Â17merLuc (firefly, 0.3 mg) and pRL-null (Renilla, 0.01 mg) and the indicated amounts of Gal4-FOXO1(1–150) and Myc-FHL2. Activity of the firefly luciferase was normalized to that of the Renilla luciferase and expressed as relative luciferase unit (RLU).

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FHL2 exhibited a similar pattern of distribution as did FOXO1 tional activity of FOXO1, we transfected DU145 cells with a (panel B3) and apparently colocalized with FOXO1 in the luciferase reporter construct containing three copies of the nucleus, as indicated by the yellow color in the merged image IRS (3 Â IRSLuc) (Guo et al, 1999; Tang et al, 1999) and with (panel B4). The colocalization is more obvious in the high- plasmids encoding FHL2 and full-length FOXO1. Ectopic resolution deconvolution image (panel B6). FHL2 and FOXO1 expression of FOXO1 increased luciferase activity (and thus did not colocalize in the cytosol, thus suggesting that FHL2 the expression of luciferase), and coexpression of FHL2 interacts preferentially with nuclear FOXO1. substantially reduced its capacity to do so (Figure 2B). To define the region of FHL2 that interacts with FOXO1, FHL2 did not affect FOXO1 abundance (Figure 2B, inset) cell extracts prepared from DU145 cells expressing His-tagged and thus apparently decreases the specific activity of FOXO1. FOXO1 (amino acids 1–150) were incubated with recombi- Another gene activated by FOXO1 by direct DNA binding is nant FHL2 proteins of various lengths fused to glutathione the gene encoding Fas ligand. To determine whether the S-transferase (GST). As determined by ‘pull-down’ assays inhibitory effects of FHL2 on FOXO1 activity translate into with glutathione beads, full-length FHL2 interacted with changes in the abundance of endogenous proteins encoded FOXO1, as did FHL2 lacking the Lim3 and Lim4 domains by FOXO1-responsive genes, we monitored amounts of Fas (Figure 1C). Deletion of the Lim2 domain, however, ablated ligand in DU145 cells ectopically expressing FOXO1 or FOXO1 the interaction of FHL2 with FOXO1. Thus, binding of FHL2 and FHL2 by immunoblotting. Expression of FOXO1 in- to FOXO1 requires Lim2 and perhaps also the one and a half creased the abundance of Fas ligand, and coexpression of Lim domain in the amino terminus of FHL2. FHL2 reduced the extent of this increase (Figure 2C). These When fused to the DNA binding domain of Gal4, the data support the results of the reporter assay in Figure 2B and amino terminus of FOXO1 (amino acids 1–150) activated importantly show that the inhibition of the mode of FOXO1 the Gal4 reporter gene (Figure 1D). This finding indicates action through ‘direct DNA binding’ by FHL2 is not limited to that this region contains an intrinsic activation function. reporter genes. As estimated using green fluorescence protein Cotransfection of increasing amounts of FHL2 progressively (GFP) vectors, the transfection efficiency of DU145 cells in inhibited Gal4 activation by FOXO1, thus suggesting that the current conditions is about 40% (data not shown). FHL2 is a FOXO1 suppressor. Therefore, the data shown underestimate the effects of FOXO1 on Fas ligand expression. FHL2 inhibits gene expression induced by direct binding Our earlier studies show that FOXO1 induces the apoptosis of FOXO1 to DNA and the apoptosis of cells by FOXO1 of DU145 cells (Li et al, 2003). As shown here, FHL2 FOXO1 activates the transcription of the IGFBP1 gene by suppresses the transcriptional activity of FOXO1 in reporter directly binding to the IRS in the IGFBP1 promoter assays and prevents the accumulation of Fas ligand (and (Figure 2A). To determine whether FHL2 affects the transcrip- presumably of other apoptotic proteins encoded by FOXO1- responsive genes). Thus, we predict that FHL2 will inhibit FOXO1-induced apoptosis. To test this prediction, DU145 cells were transfected with CD20 and either FOXO1 or FOXO1 and A Direct DNA binding C − + + FOXO1 −−+ FHL2 FHL2. After FACS-based sorting, the apoptotic index of CD20- FOXO1 FasL positive cells was determined by the Annexin V method. 3 × IRS LuciferaseLuciferase FOXO1 increased the apoptotic index about two-fold in four IGFBP1 promoter β-Actin independent experiments, and FHL2 reduced the extent of this increase (Figure 3A). We also found that caspase-3 was B 30 active in cells expressing FOXO1 but was not appreciably FOXO1 active in cells expressing both FOXO1 and FHL2 (Figure 3B). ) 4 25 These findings show that FHL2 reverses the apoptotic actions − 0 0.1 0.3 0.6 FHL2

10 of FOXO1 in DU145 cells. Such actions may or may not × 20 involve upregulation of the gene encoding Fas ligand. As shown below (Figure 8D), FOXO1 also increases the expres- 15 sion of the apoptotic protein Bim, and FHL2 antagonizes this action of FOXO1. 10 Lysophosphatidic acid promotes the nuclear 5

FOXO1 activity (RLU accumulation of FHL2 and the formation of FHL2-FOXO1 complexes and inhibits FOXO1 activity 0 in prostate cancer cells FOXO1 0 0.1 0.3 0.6 0.6 0.6 0.6 000(µg) FHL2 0 00 00.1 0.3 0.6 0.1 0.3 0.6 (µg) Prostate cancer cells express FHL2, and nuclear accumulation of FHL2 correlates with the progression of prostate cancers Figure 2 FHL2 suppresses FOXO1 action through the mode of to higher Gleason scores (Muller et al, 2002). Thus, factors direct DNA binding. (A) Diagram depicting FOXO1 action by direct DNA binding. (B) DU145 cells were transfected with 3 Â IRSLuc that promote the nuclear translocation of FHL2 may en- (0.3 mg), pCMVbGal (0.3 mg), and the indicated amounts of FOXO1 hance prostatic tumorigenesis by inhibiting the expression and FHL2 vectors. FOXO1 activity is expressed as relative luciferase of FOXO-responsive genes. Such factors include the serum units (RLU) normalized to b-gal activity. (C) DU145 cells were factor lysophosphatidic acid (LPA), and the effects of LPA on transfected with Flag-FOXO1 (4 mg) and Myc-FHL2 (4 mg). Cell extracts were immunoblotted with anti-Fas ligand antibody 24 h the subcellular location of FHL2 and FOXO1 and on the post-transfection. A b-actin immunoblot is included as a loading activity of FOXO1 and its interaction with FHL2 were deter- control. mined.

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A Vector FOXO1 FOXO1+FHL2 monitored FOXO1 activity in DU145 cells expressing 104 104 104 CD20+ cells CD20+ cells CD20+ cells 3 Â IRSLuc and either vector alone or vector encoding 3 3 3 10 10 10 siRNA to FHL2 or to GFP (negative control). As shown in 102 102 102 Figure 5A, expression of FHL2 siRNA substantially reduced 101 101 101 the abundance of endogenous FHL2, whereas expression of FL3-H 7-AAD FL3-H 7-AAD FL3-H 7-AAD 100 100 100 GFP siRNA had little effect. LPA reduced FOXO activity in 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 cells expressing empty vector or GFP siRNA but did not FL2-H Annexin-PE FL2-H Annexin-PE FL2-H Annexin-PE reduce FOXO activity in cells expressing FHL2 siRNA 1 2 3 4 Exp. no. (Figure 5B). Thus, depletion of FHL2 attenuates the inhibi- 11.03 26.225 26.31 18.39 Vector tory effects of LPA on FOXO1 activity. These experiments 32.64 42.42 52.68 39.69 FOXO1 were repeated in JCA1 cells (Figure 5C) and 267B cells 12.51 27.145 39.71 29.27 FOXO+FHL2 (Figure 5D), and similar results were obtained. Overall, the results of the studies shown in Figures 4 and 5 2.5 B suggest that LPA may promote prostate tumorigenesis by ) sending FHL2 to the nucleus where it interacts with and 405 2.0 inhibits the antitumor activity of FOXO1.

1.5 Relief of FHL2-mediated suppression of FOXO1 activity by the inhibition or depletion of SIRT1 1.0 The inhibitory affects of FHL2 on FOXO1 activity suggest that FHL2 functions as a corepressor of FOXO1 activity. Because 0.5 corepressors often recruit histone deacetylases (HDACs) to

Caspase-3 activity (OD transcriptional complexes, we asked whether inhibition of 0.0 − + + FOXO1 FOXO1 activity by FHL2 requires HDAC activity. In these −−+ FHL2 experiments, DU145 cells were transfected with 3 Â IRSLuc and with plasmids encoding FHL2, FOXO1, or both; cells Figure 3 FHL2 suppresses FOXO1-induced apoptosis. (A) DU145 cells were transfected with pCMV-CD20 (3 mg), Flag-FOXO1 (2.5 mg), were subsequently treated with and without nicotinamide, an and Myc-FHL2 (2.5 mg) as indicated. At 16 h post-transfection, inhibitor of the SIRT family of HDACs. At 5 mM, nicotinamide CD20-positive cells were separated from CD20-negative cells by partially blocked the inhibition of FOXO1 activity by FHL2; at FACS-based sorting. The apoptotic index of CD-20 positive cells was 20 mM, nicotinamide completely blocked this process determined as described in Materials and Methods. Representative profiles are shown in the top graphs. Apoptotic indexes from four (Figure 6A). Nicotinamide also increased FOXO1 activity independent experiments are shown in the table below the graphs. in cells ectopically expressing FOXO1 but not FHL2. These (B) DU145 cells were transfected with FOXO1 (4 mg) and FHL2 findings show that FHL2 (both exogenous and endogenous) (4 mg) as indicated. The activity of capase-3 was determined as inhibits FOXO1 activity in a manner dependent on SIRT described in Materials and methods. activity. Among the members of the SIRT family, SIRT1 is the closest mammalian homolog of yeast Sir2. To assess the Serum-starved DU145 cells were treated with and without need for SIRT1 in the FHL2 inhibition of FOXO1 activity, we LPA for 10 h, and nuclear and cytosolic extracts were immu- depleted DU145 cells of SIRT1 by RNA interference. Western noblotted with antibodies to FOXO1 and FHL2. FOXO1 was blots showed substantial reductions in SIRT1 abundance in predominantly nuclear in both serum-starved and LPA-trea- cells expressing SIRT1 siRNA but not in cells expressing GFP ted cells (Figure 4A). In contrast, FHL2 resided in both the siRNA (Figure 6B). Depletion of SIRT1 had no effect on cytosol and nucleus of serum-starved cells and translocated FOXO1 abundance. As monitored by reporter assays with to the nucleus in response to LPA, as has been reported 3 Â IRSLuc, FHL2 inhibited FOXO1 activity in cells expressing previously (Muller et al, 2002). Similar to ectopically ex- GFP siRNA, but did not inhibit FOXO1 activity in cells pressed proteins, endogenous FOXO1 interacted with FHL2, expressing SIRT1 siRNA (Figure 6C). Thus, depletion of as demonstrated by co-precipitation of FHL2 with anti-FOXO1 SIRT1 abrogates the inhibitory effects of FHL2 on FOXO1 antibody (Figure 4B). There were more FOXO1–FHL2 com- activity. plexes in LPA-treated cells than in serum-starved cells; this presumably reflects the increased abundance of FHL2 in the SIRT1 interacts with and deacetylates FOXO1 nucleus and not an increase in binding affinity of the two and inhibits FOXO1 activity proteins for each other. To determine the mechanism by which SIRT1 represses Co-precipitation experiments were also performed on con- FOXO1 activity, we first asked whether SIRT1 and FOXO1 trol and LPA-treated JCA1 cells (Figure 4C) and 267B1 cells interact. DU145 cells were transfected with plasmids encod- (Figure 4D), and results similar to those in Figure 4B were ing Flag-SIRT1, HA-FOXO1, or both, and co-precipitation obtained. JCA1 cells are prostate cancer cells with an intact experiments were performed. Antibody to anti-Flag co-pre- PTEN, as are DU145 cells, and 267B cells are immortalized cipitated HA-FOXO1 and antibody to HA co-precipitated Flag- but nontumorigenic prostate epithelial cells. These results SIRT1 from cells expressing both proteins but not either show that enhancement of FOXO1–FHL2 interaction by LPA protein alone (Figure 7A). Thus, SIRT1 associates with is not limited to DU145 cells. FOXO1 in vivo. To assess the effects of the LPA-induced nuclear localiza- In addition to histones, SIRT1 also deacetylates non-his- tion of FHL2 on the transcriptional activity of FOXO1, we tone proteins such as p53 (Luo et al, 2001; Vaziri et al, 2001).

1024 The EMBO Journal VOL 24 | NO 5 | 2005 &2005 European Molecular Biology Organization SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

A − + LPA C Flag FOXO1 IP antibody NC NC + − + LPA (20 µM)

FHL2 IP: Anti-FOXO1 IB: Anti-FHL2 FOXO1

PARP IP: Anti-FOXO1 IB: Anti-FOXO1

Hsp60

IB: Anti-PARP

B Flag FOXO1 IP antibody D Flag FOXO1 IP antibody + − + LPA (20 µM) + − + LPA (20 µM)

IP: Anti-FOXO1 IP: Anti-FOXO1 IB: Anti-FHL2 IB: Anti-FHL2

IP: Anti-FOXO1 IB: Anti-FOXO1 IP: Anti-FOXO1 IB: Anti-FOXO1

IB: Anti-PARP

IB: Anti-PARP

Figure 4 Interaction of endogenous FHL2 and FOXO1 in the nucleus of PTEN-intact prostate cancer cells in the presence or absence of LPA. (A) DU145 cells were starved in serum-free medium for 36 h and treated with or without 20 mM LPA for 10 h. Nuclear and cytosolic extracts were immunoblotted with anti-FHL2, anti-FOXO1, anti-Hsp60, and anti-PARP antibodies as indicated. Hsp60 and PARP are cytosolic and nuclear proteins, respectively, and are used as controls for subfractionation. (B–D) DU145 (B), JCA1 (C), and 267B1 (D) cells were starved and treated with 20 mM LPA as in (A). Nuclear extracts were immunoprecipitated with rabbit anti-FOXO1 or rabbit anti-Flag (as a control) antibody. The immunoprecipitates were immunoblotted with anti-FHL2 or goat anti-FOXO1 antibody as indicated. Immunoblotting with anti-PARP antibody without prior immunoprecipitation was included as a control.

Therefore, in our next set of experiments, we asked whether not in the presence of nicotinamide. These data are in SIRT1 deacetylates FOXO1. Flag-FOXO1 was expressed in agreement with recent reports by Motta et al (2004) and DU145 cells with either empty vector or vector encoding Brunet et al (2004), which show that SIRT1 deacetylates and SIRT1 or a SIRT1 mutant that lacks deacetylase activity inhibits the activity of FOXO factors. (SIRT1HY) (Vaziri et al, 2001). Cell extracts were immuno- precipitated with antibody to Flag and immunoblotted with Enhancement of the interaction of FOXO1 and SIRT1 antibody to acetyl-lysine. Cells expressing SIRT1 contained by FHL2 less acetylated Flag-FOXO1 than did cells expressing empty Our data show that FHL2 does not inhibit FOXO1 activity in vector or SIRTHY (Figure 7B). SIRT1 did not appreciably the absence of SIRT1 activity (Figure 6). Thus, FHL2 may reduce amounts of acetylated Flag-FOXO1 in cells receiving recruit SIRT1 to FOXO1, thus allowing SIRT1 to interact with 5 mM nicotinamide. These data show that SIRT1 reduces the and deacetylate FOXO1 and inhibit FOXO1 activity (Figure 7). abundance of acetylated FOXO1 in a manner dependent on its As demonstrated by co-precipitation assays of ectopically deacetylase activity. Consistent with its capacity to increase expressed proteins, Myc-FHL2 interacts with Flag-SIRT1 FOXO1 activity (see Figure 6A), nicotinamide also increased (Figure 8A). Co-precipitation was observed in cells expres- the abundance of acetylated FOXO1 in cells expressing sing both tagged proteins but not either protein alone. FOXO1 in the absence of ectopic SIRT1. To determine whether FHL2 modulates the interaction A third set of experiments examined the effects of SIRT1 on of FOXO1 with SIRT1, we cotransfected DU145 cells with the transcriptional activity of FOXO1 using the 3 Â IRSLuc plasmids encoding Flag-FOXO1 and increasing amounts of Myc- reporter construct. As shown in Figure 7C and D, ectopic FHL2, and co-precipitation assays were performed. Expres- expression of SIRT1 reduced FOXO activity in the absence but sion of Myc-FHL2 substantially increased the abundance

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A Vector FHL2 GFP siRNA A Vector 12 FOXO1 FHL2 10 FOXO1 + FHL2 8 FHL2 β-Actin 6

4 Fold activation B 4 − LPA 2 + LPA 3 0 Nicotinamide 0 5 20 (mM) 2 B +++ +Flag-FOXO1 +++− Myc-FHL2 1 −−SIRT1 GFP siRNA FOXO1 activity (RLU) 0 SIRT1 siRNA Vector FHL2 GFP β-Actin

C 2 − LPA + LPA Flag-FOXO1 1.5

1 C 1.0 0.5 0.8

FOXO1 activity (RLU) 0.6 0 siRNA Vector FHL2 GFP 0.4

0.2 D 2.5 −

LPA FOXO1 activity (RLU) + LPA 0.0 2 Flag-FOXO1 + + + + − +++ 1.5 Myc-FHL2 siRNA −−SIRT1 GFP 1 Figure 6 Inhibition of FOXO1 activity by FHL2 requires SIRT1 activity. (A) DU145 cells were transfected with 3 Â IRSLuc (0.3 mg) 0.5 and pCMVbGal (0.3 mg). As indicated, cells were cotransfected with FOXO1 activity (RLU) Flag-FOXO1 (0.3 mg) and Myc-FHL2 (0.3 mg). Transfected cells re- 0 ceived 5 or 20 mM nicotinamide for 24 h. Luciferase activity was siRNA Vector FHL2 GFP determined and normalized to b-gal activity. Fold activation was Figure 5 Inhibition of endogenous FOXO1 activity by LPA requires calculated by dividing the normalized activity of cells expressing FHL2. (A) DU145 cells were transfected with 4 mg of pcDNA3 FOXO1 and FHL2 by that of the corresponding vector controls. (B) (vector) or the indicated siRNA vectors. Cell extracts were immu- DU145 cells were transfected with Flag-FOXO1 (3 mg), Myc-FHL2 noblotted with anti-FHL2 or anti-b-actin antibody. (B–D) DU145 (3 mg), and 6 mg of SIRT1 or GFP siRNA. Cell extracts were immu- (B), JCA1 (C), and 267B1 (D) cells were transfected with 3 Â IRSLuc noblotted with anti-SIRT1, anti-b-actin, or M2 antibody. (C) DU145 (0.3 mg), pRL-null (0.01 mg), and 1 mg of the indicated siRNA vectors. cells were transfected with 3 Â IRSLuc (0.3 mg), pRL-null (0.01 mg), Luciferase activity was assayed and normalized as in Figure 1D. Flag-FOXO1 (0.3 mg), and 0.6 mg of the indicated siRNA vectors. Luciferase activity was assayed and normalized as in Figure 1D.

of FOXO1-associated SIRT1 without affecting the total abun- siRNA did not. These studies show that FHL2 suppresses dance of Flag-FOXO1 or endogenous SIRT1. Increased FOXO1 activity by SIRT1-mediated deacetylation of FOXO1. binding of SIRT1 to FOXO1 was paralleled by reductions in amounts of acetylated Flag-FOXO1 (Figure 8B). In other experiments, Flag-FOXO1, Myc-FHL2, and SIRT1 FHL2 inhibits gene expression regulated by the were expressed in DU145 cells. Myc-FHL2 substantially re- ‘tethering’ of FOXO1 to other transcription factors duced the abundance of acetylated Flag-FOXO1 in the ab- As mentioned in Introduction, the FOXO factors repress the sence but not in presence of SIRT1 siRNA (Figure 8C). More transcription of the cyclin D1 gene by a mechanism that does importantly, depletion of SIRT1 ablated the capacity of FHL2 not require the direct interaction of FOXO1 with the IRS to inhibit endogenous gene expression by FOXO1. As shown (Ramaswamy et al, 2002; Schmidt et al, 2002). This finding in Figure 8D, expression of FOXO1 increased the abundance suggests that FOXO1 represses cyclin D1 gene transcription of Fas ligand and Bim. Coexpression of FHL2 reduced the by ‘tethering’ to other sequence-specific transcription factors extent of increase, whereas coexpression of FHL2 and SIRT1 (Figure 9A). To determine whether FHL2 affects the capacity

1026 The EMBO Journal VOL 24 | NO 5 | 2005 &2005 European Molecular Biology Organization SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

A − ++ − HA-FOXO1 of FOXO1 to modulate gene expression by ‘tethering’, we −−++Flag-SIRT1 transfected LNCaP cells with a luciferase reporter driven by IP: Anti-Flag the cyclin D1 promoter and with plasmids encoding FHL2 IB: Anti-HA and FOXO1. As shown in Figure 9B, expression of FOXO1 IP: Anti-HA decreased the activity of the cyclin D1 promoter, and coex- IB: Anti-Flag pression of FHL2 ablated its capacity to do so. We also monitored amounts of cyclin D1 protein in cells IP: Anti-HA IB: Anti-HA expressing Flag-FOXO1 and Myc-FHL2. Consistent with the results of the reporter assay, FOXO1 reduced the abundance IP: Anti-Flag of cyclin D1 in the absence but not in the presence of ectopic IB: M2 FHL2. Together, the data in Figures 2 and 9 show that FHL2 B ++++ +Flag-FOXO1 inhibits the transcriptional activity of FOXO1 regardless of + − + −−Nicotinamide whether FOXO1 directly binds DNA or associates with other −−++ − SIRT1 transcription factors. −−−− + SIRT1HY IP: M2 IB: Anti-acetyl-Lys Discussion Despite the need for FOXO1 for multiple biological processes, IP: M2 little is known about the cofactors that potentially regulate its IB: Anti-Flag transcriptional activity. Our study shows that FHL2 interacts with the amino terminus of FOXO1, a putative transactivation C 2.5 region (Puigserver et al, 2003). Several findings indicate that FHL2 negates the transcriptional activity of FOXO1 by pro- 2 moting its deacetylation by SIRT1 (Figure 10). First, FHL2 1.5 colocalizes and interacts with FOXO1 in prostate cancer cells. The interaction of endogenous FHL2 and FOXO1 occurs in the 1 nucleus and is enhanced by the serum growth factor LPA. Second, FHL2 inhibits the activation of an IRS-driven reporter 0.5 gene by FOXO1 in the presence but not in the absence of FOXO1 activity (RLU) 0 SIRT1 activity. This was demonstrated by use of SIRT1 siRNA FOXO1 0 0.3 0.3 0.3 0.3 0 0 0 (µg) and the SIRT inhibitor nicotinamide. FHL2 also attenuates the (µ SIRT1 0 0 0.05 0.2 0.80.05 0.2 0.8 g) expression of endogenous FOXO1-targeted genes by FOXO1 and inhibits FOXO1-induced apoptosis. Third, SIRT1 binds D 140 − SIRT1 FOXO1, decreases its acetylation, and inhibits its transcrip- 120 + SIRT1 tional activity. Fourth, FHL2 interacts with SIRT1 and pro- 100 motes its interaction with FOXO1. Finally, FHL2 decreases 80 FOXO1 acetylation in a manner dependent on the deacetylase 60 activity of SIRT1. 40 (% of control)

FOXO1 activity Our data in Figure 1D showed that the amino terminus of 20 FOXO1 contains an intrinsic activation function that is sup- 0 Nicotinamide 0 20 mM pressed by FHL2 binding. However, FHL2 also suppressed cyclin D1 repression by FOXO1. It is thus likely that the amino Figure 7 SIRT1 binds to FOXO1 and inhibits FOXO1 by deacetylat- ing FOXO1. (A) DU145 cells were transfected with HA-FOXO1 (1 mg) terminus is a region essential for both transcriptional activa- and Flag-SIRT1 (1 mg). Cell extracts were immunoprecipitated with tion and repression. Because FHL2 antagonizes both the rabbit anti-Flag or mouse anti-HA antibody, and immunoprecipi- activation and the repression functions of FOXO1, it differs tates were immunoblotted with rabbit anti-HA or M2 anti-Flag from classical transcriptional corepressors. It functions more antibody as indicated. (B) DU145 cells were transfected with 4 mg of either Flag-FOXO1, pcDNA-SIRT1, pcDNA-SIRT1HY, or pcDNA3 like an adaptor that couples SIRT1 to FOXO1 and thus allows and treated with or without 5 mM nicotinamide as indicated. Cell SIRT1 to deacetylate FOXO1. In addition to SIRT1 recruit- extracts were immunoprecipitated with M2 anti-Flag antibody. The ment, the decreases in the acetylation and activity of FOXO1 immunoprecipitates were immunoblotted with an anti-acetyl lysine by FHL2 may also involve interference with the interaction antibody (upper panel) or rabbit anti-Flag antibody (lower panel). (C) DU145 cells were transfected with 3 Â IRSLuc (0.3 mg), pRL-null between FOXO1 and histone acetyl transferases associated (0.01 mg), and the indicated amounts of Flag-FOXO1 and pcDNA3 with transcriptional coactivators such as PGC-1, which inter- SIRT1. Luciferase activity was assayed and normalized as in acts with the amino terminus of FOXO1 (Puigserver et al, Figure 1D. (D) DU145 cells were transfected with 3 Â IRSLuc 2003). (0.3 mg), pRL-null (0.01 mg), Flag-FOXO1 (0.3 mg), and 0.3 mgof either pcDNA3 or pcDNA-SIRT1. Transfected cells were treated It has been well established that acetylation of histones with or without nicotinamide for 24 h as indicated. Luciferase generates novel adhesive surfaces required for the recruit- activity was assayed and normalized as in Figure 1D. FOXO1 ment of transcription complexes (Agalioti et al, 2002). activity is expressed as % of control and was calculated by normal- Acetylation is also known to regulate the DNA binding izing the activity in cells transfected with SIRT1 to that of the vector controls (as 100%). activity of transcription factors such as p53 (Gu and Roeder, 1997) and E2F1 (Martinez-Balbas et al, 2000). FOXO1 con- tains multiple lysine residues, the acetylation of which might regulate its direct transcriptional activity and ‘tethering’

&2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 5 | 2005 1027 SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

A − ++− Myc-FHL2 C ++ + + Flag-FOXO1 −− ++Flag-SIRT1 + − + + Myc-FHL2 IP: Anti-Myc −−SIRT1 GFP siRNA IB: Anti-Flag IP: M2 IB: Anti-acetyl-Lys IP: Anti-Flag IB: Anti-Myc IP: M2 IB: Anti-Flag IP: Anti-Myc IB: Anti-FHL2

IP: Anti-Flag IB: M2

− B Myc-FHL2 D ++++Flag-FOXO1 −−+++Myc-FHL2 0 0.1 0.3 0.6 µg −−−SIRT1 GFP siRNA + + + + Flag-FOXO1

IB: Anti-Myc FasL

IB: Anti-SIRT1 Bim IP: Anti-Flag IB: Anti-SIRT1 IP: Anti-Flag Flag-FOXO1 IB: Anti-acetyl-Lys IP: Anti-Flag β-Actin IB: Anti-Flag

Figure 8 FHL2 decreases FOXO1 acetylation by promoting the interaction of FOXO1 with SIRT1. (A) DU145 cells were transfected with Myc- FHL2 (1 mg) and Flag-SIRT1 (1 mg) as indicated. Cell extracts were immunoprecipitated with anti-Myc or rabbit anti-Flag antibody, and immune complexes were immunoblotted with rabbit anti-Flag, anti-Myc, M2 anti-Flag, or anti-FHL2 antibody. (B) DU145 cells were transfected with Flag-FOXO1 (0.3 mg) and the indicated amounts of Myc-FHL2. Cell extracts were immunoprecipitated and immunoblotted with the indicated antibodies. (C) DU145 cells were transfected with Flag-FOXO1 (3 mg), Myc-FHL2 (3 mg), and 6 mg of siRNA vectors. Cell extracts were immunoprecipitated with the indicated antibodies. (D) DU145 cells were transfected with Flag-FOXO1 (3 mg), Myc-FHL2 (3 mg), and 6 mg of the siRNA vectors. Cells were harvested 24 h after transfection, and amounts of Fas ligand, Bim, and b-actin (loading control) were determined by immunoblotting.

(Figure 10). FHL2–SIRT1 complex may deacetylate FOXO1 at regulates the ‘tethering’ through its ability to interact with two separate lysine residues, one of which is critical for DNA both FOXO1 and b-catenin. binding whereas the other for ‘tethering’. Alternatively, the Although the current study focuses on FOXO1, it is possi- complex may deacetylate FOXO1 at a single lysine residue ble that FHL2 also modulates the activities of other FOXO that, at acetylated status, provides a ‘docking’ site for inter- factors. The FOXO factors share a similar domain structure action with a ‘ying-yang’ transcriptional cofactor that is and mode of activation/inactivation by phosphorylation. involved in FOXO1 action through either direct transcrip- Indeed, recent studies by Motta et al (2004) and Brunet et al tional activation or ‘tethering’, depending on the promoter (2004) show that SIRT1 deacetylates and inhibits the activity context. While we have presented multiple evidences for the of FOXO3a, FOXO4, and FOXO1. On the other hand, in the involvement of SIRT1-mediated deacetylation in the suppres- studies by Fukuoka et al (2003) and Van der Horst et al sion of direct transcriptional activity of FOXO1 by FHL2, it (2004), acetylation of FOXO4 inhibited its activity. Thus, remains to be determined whether deacetylation is involved whether our findings on FOXO1 extend to other FOXO factors in the relief of FOXO1-mediated suppression of cyclin D1 requires further study. expression by FHL2. For example, FHL2 has been shown to Previous studies (Nakamura et al, 2000; Li et al, 2001, be a coactivator for b-catenin (Wei et al, 2003) that upregu- 2003) have shown that FOXO1 acts downstream of PTEN to lates the expression of cyclin D1 (Tetsu and McCormick, induce the apoptosis of prostate cancer cells. Our current 1999). It is possible that the effect of FHL2 on cyclin D1 work suggests that FHL2 may negate the antitumor activity of involves its interaction with b-catenin. Although the data in PTEN by inactivating FOXO1 in these cells. The enhanced Figure 9 show that the effect of FHL2 on cyclin D1 depends on interaction of FHL2 with FOXO1 in LPA-treated cells supports FOXO1, it does not exclude the possibility that FOXO1 is the existence of an additional pathway in prostate cancer cells ‘tethered’ to b-catenin on cyclin D1 promoter and that FHL2 that inhibits FOXO activity independently of PKB. Androgens

1028 The EMBO Journal VOL 24 | NO 5 | 2005 &2005 European Molecular Biology Organization SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

A C − ++− FOXO1 et al, 2000). Second, FHL2 may cooperate with androgens to FOXO1 −−++FHL2 inhibit FOXO1 activity. We (Li et al, 2003) have shown that androgens inhibit FOXO1 activity by promoting the interac- Cyclin D1 'Tethering' tion of FOXO1 with the androgen receptor. Consistent with β-Actin the idea that FHL2 promotes tumorigenesis, others have ? shown that (1) stimulation of the Rho signaling pathway Luciferase induces the translocation of FHL2 to the nucleus and the Cyclin D1 promoter subsequent activation of androgen receptor-dependent genes B 16 (Muller et al, 2002), (2) FHL2 is a serum-inducible transcrip- tional coactivator of AP-1 (Morlon and Sassone-Corsi, 2003); 14 and (3) FHL2 is a coactivator of b-catenin (Wei et al, 2003). 12 These proteins have been implicated in prostate tumorigen- 10 esis. In yeast, the abundance of Sir2 increases in response to 8 caloric restriction and sublethal levels of stress. Because 6 extension of the lifespan of C. elegans by Sir2 requires the 4 FOXO-related protein Daf-16 (Tissenbaum and Guarente, 2001), it may appear paradoxical that SIRT1 inhibited 2 FOXO1 activity. Recently, SIRT1 was shown to suppress the

Cyclin D1 promoter activity (RLU) 0 induction of proapoptotic genes by FOXO3a but to enhance FOXO1 0 0.1 0.3 0.6 0.6 0.6 0.6 0 (µg) the induction of genes involved in growth inhibition (e.g. FHL2 00000.1 0.3 0.6 0.6 (µg) p27Kip1) and stress responses (e.g. GADD45) (Brunet et al, Figure 9 FHL2 suppresses the ‘tethering’ mode of FOXO1 action. 2004). Moreover, deacetylation of FOXO1 by SIRT1 potenti- (A) Diagram depicting FOXO1 action through ‘tethering’. (B) LNCaP ates the FOXO1-dependent expression of p27Kip1 and manga- cells were transfected with the pGL2-CD1 reporter plasmid (0.3 mg), nese superoxide dismutase (Daitoku et al, 2004). Similarly, pRL-null (0.01 mg), and the indicated amounts of Flag-FOXO1 and deacetylation of FOXO4 by SIRT1 relieves the inhibition of Myc-FHL2. Luciferase activity was assayed and normalized as in Figure 1D. (C) DU145 cells were transfected with Flag-FOXO1 (4 mg) FOXO4 activity by CREB-mediated acetylation and enhances and 4 mg of either Myc-FHL2 or pcDNA3. Amounts of endogenous the expression of p27Kip1 (Van der Horst et al, 2004). These cyclin D1 protein were determined by immunoblotting with anti- studies suggest that Sir2 and its mammalian homologs may cyclin D1 antibody. A b-actin immunoblot is included as a loading increase organismal longevity by shifting the FOXO-depen- control. dent response away from apoptosis and toward stress resis- tance and growth inhibition, which would explain the need for Daf-16 in the increase of longevity by members of Sir2 family. However, SIRT1 has recently been shown to augment Ac Ac SIRT1 Ac Acetylase FHL2 TNFa-induced apoptosis by inhibiting the transcriptional FOXO1 FOXO1 FOXO1 activity of NF-kB (Yeung et al, 2004), suggesting a positive Inactive Active Low activity role of SIRT1 in the apoptosis of mammalian cells. It remains Ac Ac to be determined whether the effect of FHL2 on the activity of DNA binding and 'Tethering' and FOXO1 is sensitive to stress conditions or depends on cell transcriptional transcriptional type, FOXO subtype, or the promoter context of specific FOXO activation repression target genes. (e.g. Bim) (e.g. cyclin D1) Because the members of Sir2 family and the FOXO factors increase longevity, our finding that FHL2 inhibits FOXO1 Cell death Cell cycle progression through SIRT1 reveals a potential novel function for this LIM protein in the control of longevity. Cancer is considered an aging disease. This is particularly true for prostate cancers, Prostate tumorigenesis whose incidence increases with age more rapidly than does that of other types of cancer. The inhibition of apoptosis by Figure 10 A model of how FHL2 regulates FOXO1 activity by SIRT1-mediated deacetylation. Active FOXO1 suppresses prostate interaction of FHL2, FOXO1, and SIRT1 in prostate cancer tumorigenesis by inhibiting cell cycle progression and by inducing cells may represent a novel molecular pathway the interrup- apoptosis. FOXO1 activation in prostate cancer cells requires acet- tion of which could lead to the development of strategies that ylation at yet to be defined sites. These sites are essential for its prolong lifespan without increasing the risk of prostate modulation of gene expression by both direct DNA binding and ‘tethering’. FHL2 promotes prostate tumorigenesis by serving as an cancer. adaptor that couples SIRT1 and FOXO1 and thus inhibits FOXO1 Although the current data were generated in prostate action in prostate cells. cancer cells, the functional interaction between FHL2, SIRT1, and FOXO1 may also occur in other human cancers, even though the nature of the interaction may vary. In this regard, it is interesting that FHL2 was initially identified as a promote prostate tumorigenesis, and FHL2 may enhance protein that is downregulated in rhabdomyosarcoma (Genini their tumor-promoting activity in two ways. First, FHL2 is a et al, 1997), whereas FOXO1 was identified as a gene in- coactivator for the androgen receptor and colocalizes with volved in chromosomal translocation in the same disease the receptor in the nucleus of prostate epithelial cells (Muller (Galili et al, 1993).

&2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 5 | 2005 1029 SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

Materials and methods Immunological assays. For immunoprecipitations, cells in 100- or 60-mm dishes were transfected as described for reporter assays and Constructs were treated as indicated in the figures. Whole-cell extracts or m 1 Flag-FKHR (Tang et al, 1999), 3 Â IRSLuc (Guo et al, 1999; Tang nuclear extracts were incubated with 4 g of antibody for 4 h at 4 C et al, 1999), and pGL2-CD1 (Tetsu and McCormick 1999) have been and subsequently with protein G-agarose beads for an additional described in previous studies. To generate the mammalian and 4 h. Beads were washed four times, and the immunoprecipitates were heated in SDS–PAGE sample buffer. After brief centrifugation, bacterial expression vectors for FHL2, full-length FHL2 cDNA was the supernatants were resolved on an 8% SDS gel for subsequent amplified by polymerase chain reaction (PCR) using the positive clone identified from yeast two-hybrid screening as a template and immunoblotting analysis. Commercially available antibodies in- was inserted into the BamH1 and XhoI sites of pCMV-tag-3B cluding M2 anti-Flag (Sigma), rabbit anti-Flag (Sigma), anti-Myc (Stratagene) and pGEX-4T-1 (Pharmacia), respectively. A mamma- (Santa Cruz, 9E10), rabbit anti-FOXO1 (Santa Cruz, H-128), and lian vector expressing the amino terminus of FOXO1 (1–150) was mouse anti-HA (Berkeley Antibody Laboratories, HA11) antibodies were used for immunoprecipitations. constructed by inserting the BamH1/PstI fragment of pFA-CMV- For immunoblotting, cell extracts, nuclear extracts, or immuno- FKHR (Li et al, 2003) into pcDNA4-V5-His(A) (Invitrogen). The HA- FOXO1 vector was constructed by inserting the BamH1/XbaI precipitates were separated on SDS–polyacrylamide gels and fragment from Flag-FKHR into pcDNA3-HA (Invitrogen). To transferred to nitrocellulose membranes. Membranes were incu- generate SIRT1 expression vectors, wild-type SIRT1 and mutant bated with antibody, and proteins detected by the antibody were SIRT1 were excised from retroviral vectors (Vaziri et al, 2001) and visualized with ECL. Besides the antibodies described above for inserted into the BamH1 and BstX1 sites of pcDNA3.1 or pcDNA3- immunoprecipitations, additional antibodies used for immunoblot- ting analyses include goat anti-FHL2 (Santa Cruz, C16), anti-Fas Flag. SIRT1 and FHL2 siRNAs were subcloned into pSilencer-Neo ligand (Santa Cruz, N-20), anti-b-actin (Sigma), anti-Hsp60 (Santa (Ambion). The corresponding oligonucleotides for generating the SIRT1 siRNA are 50-GAT CCG CTT CAG TGG CTG GAA CAG TTT Cruz, H-1), anti-PARP (Santa Cruz, H-250), goat anti-FOXO1, anti- CAA GAG AAC TGT TCC AGC CAC TGA AGT TTT TTG GAA A-30 SIRT1 (Upstate Biotechnologies), rabbit anti-HA (Cell Signaling), (forward) and 50-AGC TTT TCC AAA AAA CTT CAG TGG CTG GAA anti-acetyl lysine (Upstate Biotechnologies), anti-Bim (BD Pharmin- CAG TTC TCT TGA AAC TGT TCC AGC CAC TGA AGC G-30 gen), and anti-cyclin D1 (Santa Cruz, HD11) antibodies. Nuclear and cytosolic fractions were prepared according to a standard (reverse). The corresponding oligonucleotides for generating the protocol (Dignam et al, 1983). FHL2 siRNA are 50-GAT CCG TGA CTG AGC GCT TTG ACT GTT CAA GAG ACA GTC AAA GCG CTC AGT CAT TTT TTG GAA A-30 For immunofluorescence and deconvolution imaging, DU145 (forward) and 50-AGC TTT TCC AAA AAA TGA CTG AGC GCT TTG cells transfected with Myc-FHL2 and Flag-FOXO1 (wild type) were ACT GTC TCT TGA ACA GTC AAA GCG CTC AGT CAC G-30 cultured in 5% FBS and stained with DAPI and goat anti-FHL2 or (reverse). The oligonucleotides for GFP siRNA were provided by with M2 anti-Flag antibodies. FHL2 and FOXO1 were detected by Ambion. chicken anti-goat IgG conjugated with Alexa Fluor 594 (red)- and fluorescein isothiocyanate (FITC; green)-conjugated anti-mouse IgG (Molecular Probes), respectively. Fluorescent microscopic images were obtained with a Nikon Diaphot microscope using a Photo- Yeast two-hybrid screening metrix PXL cooled CCD camera. The microscope was equipped with The Matchmaker Two-Hybrid System 3 (Clontech) was the appropriate filters for three-color imaging of cells and with a used to screen the human prostate MATCHMAKER cDNA motorized stage for obtaining z-series images. Digital image files library (Clontech). The bait plasmid pGBKT-FOXO1 (1–150) were processed and deconvolved using Oncor Image software Bam Pst was constructed by inserting the H1– I fragment of (Oncor Inc.). High-resolution images of the deconvolved and 3-d pFA-CMV-FKHR (Li et al, 2003) into the NdeI–PstI site of GBKT7. reconstructed image z-series stacks were processed for presentation BamH1 and NdeI were blunt ended with Klenow I before with Adobe Photoshop. ligation. The screening was performed in AH109 yeast cells according to the protocol provided by the Clontech Matchmaker Two-Hybrid System. Caspase-3 assays. Caspase-3 activity of cell extracts normalized for protein abundance was determined by the Caspase-3/CPP32 Colorimetric Assay Kit (BD Biosciences, Pharmingen) following a Cell culture, transfections, and reporter assays. For reporter protocol from the vendor. Absorption at 405 nm was measured in an analysis, DU145 cells were plated in 12-well plates in Dulbecco’s MRX microplate reader (DYNEX Technologies, Chantilly, VA). modified Eagle’s medium containing 10% fetal bovine serum at Caspase-3 activity is expressed as OD per mg protein. 1.5 Â105 cells/well. LNCaP, JCA1, and 267B1 cells were plated in 405 RPMI 1640 at 2 Â105 cells/well. At 1 day after plating, cells were transfected by Lipofectamine Plus or Lipofectamine 2000 following Cell sorting and apoptotic assays. The apoptotic index of trans- the protocol provided by Gibco/BRL. Transfected cells were fected cells was determined with the Annexin V PE Apoptosis cultured in medium containing 5% charcoal-stripped FBS (sFBS) Detection Kit (BD Pharmingen). Cells grown in 100 mm dishes were for 24 h. Then, cell lysates were prepared and luciferase activity was cotransfected with CD20 plasmid that expresses a cell surface determined using the luciferase assay systems or the dual luciferase antigen. At 16 h post-transfection, the cells were collected in PBS assay system from Promega Corporation (Madison, WI). b-Gal containing 2.5 mM EDTA, washed twice with PBS, and incubated at 41C for 60 min with FITC-conjugated mouse anti-CD20 antibody activity was determined as previously described (Lee et al 2000; Lee 6 and Bai, 2002). Luciferase activity was normalized to Renilla (Becton-Dickinson, 20 ml/10 cells). After staining with anti-CD20 luciferase activity or to b-gal activity. antibody and washing, the cells were incubated with Annexin V-PE and 7-amino-actinomycin D (7-AAD) for 30 min at 251C. CD20- positive cells were separated from the CD20-negative cells by FACS- GST pull-down assays. Plasmids expressing GST fused to full-length based cell sorting. The apoptotic index of transfected cells was or truncated FHL2 were transformed into the BL21 (DE3) strain. determined by flow cytometry. Cell sorting and flow cytometry Transformed bacteria were cultured at 371C until OD600 had reached analysis were performed on a Becton-Dickinson FACScan (Moun- 0.8. IPTG (0.5 mM) was added and the culturing continued tain View, CA). overnight at 301C. Bacterial lysates containing GST fusion proteins were prepared by sonication in buffer containing 50 mM Tris, 10 mM NaCl, 1mM EDTA, 6 mM MgCl2, 1 mM DTT, and 1 mM PMSF and Acknowledgements were incubated with extracts of DU145 cells transfected with His- FOXO1(1–150). FOXO–FHL2 complexes were precipitated with We thank Dr FG Barr for the permission to use FKHR in the studies, Glutathione Sepharose 4B Beads (Amersham Pharmacia, Piscat- Drs KL Guan and Terry G Unterman for FKHR plasmids, Dr Robert away, NJ). The beads were then washed with the binding buffer Weinberg for the SIRT1 retroviral plasmids, and Drs Nancy Olashaw containing 0.5% NP-40 and heated for 5 min in SDS–PAGE sample and Paul Byvoet for reading the manuscript. FACS analyses were buffer. After brief centrifugation, the supernatant was resolved on performed in the Flow Cytometry core facility at H Lee Moffitt an 8% SDS gel. The presence of FOXO1(1–150) in the precipitates Cancer Center and Research Institute. This work was supported by was detected by immunoblotting with goat anti-FOXO1 antibody the Public Health Service grant CA79530 (WB) and a DOD Prostate (Santa Cruz, N18). Cancer grant DAMD17-02-1-0140 (WB).

1030 The EMBO Journal VOL 24 | NO 5 | 2005 &2005 European Molecular Biology Organization SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al

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