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Published as doi: 10.1096/fj.07-9627com.
 (The FASEB Journal. 2008;22:1124-1134.)
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(The FASEB Journal. 2008;22:1124-1134.)
© 2008 FASEB

Inhibition of apoptosis by MAD1 is mediated by repression of the PTEN tumor suppressor gene

Sabine Rottmann1,2, Stefanie Speckgens1, Juliane Lüscher-Firzlaff and Bernhard Lüscher3

Abteilung Biochemie und Molekularbiologie, Institut für Biochemie, Universitätsklinikum, RWTH Aachen University, Aachen, Germany

3Correspondence: Institut für Biochemie, Universitätsklinikum, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail: luescher{at}rwth-aachen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The MYC/MAX/MAD network of transcriptional regulators controls distinct aspects of cell physiology, including cell proliferation and apoptosis. Within the network MAD proteins antagonize the functions of MYC oncoproteins, and the latter are deregulated in the majority of human cancers. While MYC sensitizes cells to proapoptotic signals, the transcriptional repressor MAD1 inhibits apoptosis in response to a broad range of stimuli, including oncoproteins. The molecular targets of MAD1 that mediate inhibition of apoptosis are not known. Here we describe the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumor suppressor gene as a target of MAD1. By binding to the proximal promoter region, MAD1 downregulated PTEN expression. PTEN functions as a lipid phosphatase that regulates the phosphatidylinositol 3-kinase/AKT pathway. Indeed MAD1-dependent repression of PTEN led to activation of AKT and subsequent stimulation of the antiapoptotic NF-{kappa}B pathway. Interfering with AKT function affected the control of Fas-induced apoptosis by MAD1. In addition, knockdown of PTEN using small interfering RNA (siRNA) or the lack of PTEN rendered cells insensitive to inhibition of apoptosis by MAD1. These findings identify the PTEN gene as a target of the MYC-antagonist MAD1 and provide a molecular framework critical for the ability of MAD1 to inhibit apoptosis.—Rottmann, S., Speckgens, S., Lüscher-Firzlaff, J., Lüscher, B. Inhibition of apoptosis by MAD1 is mediated by repression of the PTEN tumor suppressor gene.


Key Words: AKT • MYC • PI3K • transcription


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE TRANSCRIPTIONAL REGULATORS of the MYC/MAX/MAD network control multiple aspects of cell behavior, including proliferation, apoptosis, and transformation, through the regulation of a substantial set of target genes (1 , 2) . Originally, MAD proteins (i.e., MAD1, MXI1, MAD3, and MAD4) were identified as antagonists of MYC oncoproteins (3 4 5) . An additional MYC antagonist is MNT (6 , 7) . MAD and MNT proteins function as transcriptional repressors that inhibit transformation, proliferation, and apoptosis of cells (8) . Transcriptional repression depends on the ability of these proteins to interact with MAX through the basic region/helix-loop-helix/leucine zipper domain and to recruit a repressor complex through the mammalian ortholog of yeast Sin3 (mSin3) -interaction domain (8) . Repression of apoptosis has been documented for MAD1 (see below), for MAD3 (9) and MNT (10 , 11) . The first observation implicating MAD1 in the inhibition of apoptosis was made in mad1–/– mice. Hematopoietic cells isolated from these animals revealed increased sensitivity to apoptosis-inducing conditions (12) . Consistent with this finding, myeloid precursor cells of mad1 transgenic animals are less sensitive to limiting cytokine levels (13) . These studies were corroborated in tissue culture cells. Activation of MAD1 expression in U2OS cells stably transfected with tetracycline-regulatable Mad1 (U2OS-tet-MAD1 cells) results in a substantial reduction of apoptosis in response to Fas ligand, tumor necrosis factor (TNF) -related apoptosis-inducing ligand (TRAIL), and ultraviolet (UV) treatment (14) . Furthermore, apoptosis stimulated by the oncoproteins MYC and adenoviral E1a and by cytostatic drugs is inhibited by MAD1 in fibroblasts (14 , 15) . Inhibition of apoptosis depends on the recruitment of the repressor complex that contains histone deacetylase activity (14 , 16) . Together, these findings identify inhibition of apoptosis as a potent and biologically relevant function of MAD1.

Multiple pathways are implicated in mediating the effects of MYC on cell death and together provide a framework to explain the proapoptotic functions of this oncoprotein (17) . In contrast, little information is available about how MAD1 inhibits apoptosis. Recently, a set of MAD1-regulated genes has been identified, and the majority was involved in protein biosynthesis and metabolism (18) . No genes were detected that provide a direct link to apoptosis. Thus the relevant molecular targets that can explain the antiapoptotic effect of MAD1 remain to be determined.

We used the polymerase chain reaction (PCR)-select strategy to identify MAD1 target genes by using U2OS-tet-MAD1 cells (14) . The phosphatase and tensin homologue deleted on chromosome ten (PTEN) gene was identified as a repressed target. PTEN is a well-known tumor suppressor gene that is mutated or silenced in a broad range of human tumors (19 20 21) . PTEN functions as a lipid phosphatase with the key second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) as its main physiological substrate. PTEN dephosphorylates the D3 position on the inositol headgroup of PIP3. The reverse reaction is catalyzed by phosphatidylinositol 3-kinases (PI3K) that are activated in response to distinct signal transduction pathways and regulate various cellular processes, including apoptosis (20 , 22) . The generation of PIP3 results in recruitment and activation of the kinase AKT. Several substrates and downstream effectors of AKT, including the NF-{kappa}B family of transcriptional regulators, are involved in negatively regulating apoptosis (20 , 22) . By reducing the levels of PIP3, PTEN is an inhibitor of AKT activation and subsequent processes. In addition, PTEN functions as a protein phosphatase that appears to be involved in controlling cell motility (23 , 24) .

Despite the importance of PTEN in modulating cell physiology and in tumorigenesis, relatively little is known about the regulation of the expression of the PTEN gene. Only recently have several studies described and identified transcriptional regulators, including the tumor suppressor p53, Egr-1, peroxisome proliferator-activated receptor gamma (PPAR{gamma}), NF-{kappa}B, c-Jun (a component of the AP-1 family), and CBF-1, which control PTEN expression (25 26 27 28 29 30) . Together, these studies suggest that the expression of PTEN is under multiple levels of control and is targeted by several different signal transduction pathways.

Germline mutations in the PTEN gene have been identified in a large proportion of patients with Cowden and Bannayan-Riley-Ruvalcaba syndrome (21) . A substantial number of these mutations affect the coding region, and the majority result in the inactivation of the lipid phosphatase activity of PTEN. In addition to these mutations, in some families with the above syndromes the promoter of the PTEN gene is altered and thus potentially affects the regulation of PTEN expression (31) . Indeed a recent study suggests that germline deletion of an E-box DNA response element in the PTEN promoter results in repression of the PTEN gene (32) . E-box DNA elements are recognized by a number of transcriptional regulators, including upstream transcription factor (USF) and different dimeric complexes of members of the MYC/MAX/MAD network (33) . In vitro studies have shown that USF factors are involved in stimulating PTEN expression (32) . In parallel to this study, we have now identified 2 E-boxes in the promoter region of PTEN and provide evidence that MAD1 directly represses PTEN. This finding results in the activation of AKT and subsequently in inhibition of apoptosis. Interfering with this pathway is sufficient to block the MAD1 effect on apoptosis, demonstrating that PTEN is a critical target gene for the antiapoptotic function of MAD1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
U2OS, HEK293, SAOS, and HeLa cells and immortalized pten+/– and pten–/– mouse embryo fibroblasts (MEFs) (kindly provided by Tak Mak, University of Toronto, Toronto, ON, Canada) (27) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). HL60 cells were grown in RPMI 1640 and 10% FCS. For differentiation, HL60 cells were grown in flasks coated with 2% agarose M (Pharmacia Biotech Inc., Uppsala, Sweden) to prevent adherence. Exponentially growing HL60 cells (105 cells/ml) were treated with 1.6 x 10–8 M 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich, Taufkirchen, Germany). Selection and propagation of U2OS-tet-MAD1 clones was performed in the presence of 1 mg/ml tetracycline (14) . For induction of MAD1 expression, cells were washed extensively in medium containing FCS and then cultivated in the absence of tetracycline for the times indicated.

PCR-select
To identify MAD1-regulated genes, we used the Clontech PCR-Select (PR99466, Clontech, Mountain View, CA, USA) protocol according to the manufacturer’s specifications. The enriched sequences were either cloned into the pZERO vector, sequenced, and analyzed by database searches or labeled and used as probes to hybridize a Unigene filter (RZPD; Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany) with 36,000 human EST clones. We identified PTEN sequences with both methods.

RT-PCR
For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the endogenous PTEN gene, RNA of one 10-cm cell culture dish was harvested using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Potential DNA contaminations were removed by DNase treatment (DNA-free kit, Ambion, Austin, TX, USA). cDNA synthesis was performed with Oligo(dT)15 primer (Promega, Madison, WI, USA) and StrataScript RT-enzyme (Stratagene, Cedar Creek, CA, USA). cDNA reaction (5 µl of each) was used in preparing a PCR mastermix for 10 individual PCR assays using HotStar Taq Polymerase (Qiagen). The PCR conditions were as follows: 95°C for 15 min, 94°C for 30 s, and 50°C (β-GUS); or 53°C (PTEN) for 30 s and 72°C for 30 s. The primer sequences used were, for β-GUS: 5'-CTCATTTGGAATTTTGCCGATT and 5'-CCGAGTGAAGATCCCCTTTTTA (product size 120 bp), and for PTEN: 5'-GGACGAACTGGTGTAATGATATG and 5'-TCTACTGTTTTTGTGAAGTACAGC (do not recognize the pseudogene, product size 671 bp; ref. 34 ). Aliquots were removed from the PCR machine at the end of every 72°C cycle step, mixed with DNA gel loading buffer, and stored at 4°C. The products were separated on agarose gels and quantified using Quantity One 4.4.0 software (Bio-Rad, Hercules, CA, USA).

Chromatin immunoprecipitation (ChIP)
For ChIP assays, 1 x 107 U2OS-tet-MAD1 cells (clone M19) or 3 x 107 HL-60 cells were used per sample point. The cells were crosslinked, and the different protein-DNA complexes were immunoprecipitated as described previously (35 , 36) . The following antibodies were used: anti-MAD1 (C-19), anti-MAX (C-17), anti-MYC (C-19), anti-Myb (C-19), and anti-Cytochrome c (H-104) (Santa Cruz Biotechnology, Santa Cruz, CA USA). PCRs were performed with different numbers of cycles or with dilution series of input DNA to determine the linear range of the amplification; all results shown fall within this range. Primers: promoter E-box region 5'-CCGAGCAAAGGAAGAAGACGAC-3' and 5'-AGGAGGGTTCAAAAGGAGGTGG-3'; intron 3 E-box region 5'-CCGAGCAAAGGAAGAAGACGAC-3' and 5'-AGGAGGGTTCAAAAGGAGGTGG-3' (see Fig. 2 A for location, sequences derived from AH007803). The PCR-products were separated on agarose gels and documented using the Bio-Rad Quantity One 4.4.0 software.


Figure 1
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  		Figure 1. Repression of the PTEN gene by MAD1. A) The cDNAs obtained after the PCR-select protocol from RNA of U2OS-tet-MAD1 cells grown in the presence or absence of tetracyline for 12 h (i.e., without or with MAD1, respectively) were labeled and used as probes on Unigene filters. Differentially labeled spots were identified, and the corresponding DNA sequence information was retrieved. Portions of the filters are shown that contain PTEN sequences. B) MAD1 expression was induced in U2OS-tet-MAD1 cells (clone M19) by removing tetracycline for 8 h. The expression of the PTEN gene in these and in control cells was measured by RT-PCR using specific primers and the indicated number of PCR cycles (top panel). For control, β-GUS was amplified (bottom panel). The products were analyzed on agarose gels. C) RNA was prepared from exponentially growing HL60 cells (Exp.) or from cells treated with TPA (1.6x10–8 M) for 12 h. The analysis of PTEN and β-GUS was performed as described in B. D) HeLa cells were transiently transfected with control plasmids or plasmids expressing MAD1, MAD1{Delta}BR, or MAD1{Delta}N, as indicated. In general, 40–50% of the cells were transfected, as determined by EGFP staining. Whole cell lysates were analyzed for expression of PTEN and AKT by Western blotting (WB) (left panel). Parallel samples were stained for MAD1 using a polyclonal MAD1 serum (right panel). The arrowhead identifies a nonspecific band. E) HeLa cells were transiently transfected with plasmids expressing MYC, MAD1, and MNT, as indicated. The analysis was performed as described in D.


Figure 2
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  		Figure 2. MAD1 interacts with the PTEN promoter. A) A schematic representation of the PTEN gene (deduced from GeneBank accession number AF067844). Exons (E) are drawn to scale. Introns are indicated with their respective length in nucleotides. The numbering starts at the ATG that is marked in E1. Within 4 kb of the promoter region, two consensus MYC/MAX/MAD E-boxes were identified, as indicated. An additional E-box is indicated in the third intron. B) U2OS-tet-MAD1 cells (clone M19) were grown in the presence or absence of tetracyline for 8 h. The cells were then crosslinked, and protein-DNA complexes were immunoprecipitated with the antibodies indicated (anti-MYC: C-19; anti-Cyto c: H-104; anti-MAD1: C-19). The bound DNA was analyzed by PCR using primers that amplify the promoter region containing the E-boxes or the intron 3 E-box. For comparison, serial dilutions of input chromatin DNA were evaluated. C) HL60 cells were grown in the presence or absence of TPA as indicated. Chromatin immunoprecipitations were performed with antibodies recognizing the indicated proteins (c-MYC: C-19; MAX: C-17; MAD1: C-19; c-Myb: C-19). PCRs were done as described in B. D) HEK293 cells were cotransfected with pGL3-PTEN2484/961-luc (1 µg) and plasmids expressing p53 (0.5 µg), MAD1 (2 µg), and MAD1{Delta}BR (2 µg), as indicated. A typical experiment is shown.

Western blots and AKT kinase assays
Cells were lysed in F-buffer (10 mM Tris-HCl, pH 7.05; 50 mM NaCl; 30 mM Na4P2O7; 50 mM NaF; 5 µM ZnCl2; 100 µM Na3VO4; 1% Triton X-100; 1 mM PMSF; 5 U/ml {alpha}2-macroglobulin; 2.5 U/ml pepstatin A; 2.5 U/ml leupeptin; 0.15 mM benzamidin) and the lysates were prepared as described (37) . Aliquots of these lysates were separated on 12.5% SDS-PAGE gels and blotted for AKT ({alpha}-Akt1/2, H-136, Santa Cruz), phosphorylated AKT ({alpha}-P473-Akt, Cell Signaling, Danvers, MA, USA), or PTEN (mAb A2B1, Santa Cruz). Additional antibodies used were against caspase 3 (H-277, Santa Cruz), PARP1 ({alpha}-poly (ADP-ribose) polymerase1; Roche, Mannheim, Germany), actin (C-4, MP Biomedicals, Heidelberg, Germany), MAD1 (C19, Santa Cruz), and EGFP (FL, Santa Cruz).

AKT kinase assays were performed essentially as described (38) . In brief, transiently transfected 293HEK or HeLa cells were harvested in F-buffer, and AKT was immunoprecipitated using 0.5 µg {alpha}-Akt1/2 and Protein G-beads at 4°C for 90 min. The immunoprecipitates were washed in F-buffer and in AKT-buffer (20 mM HEPES, pH 7, 4; 10 mM MgCl2; 10 mM MnCl2). Kinase reactions were performed in 30 µl AKT-buffer containing 200 µM ATP, 5 µCi {gamma}-32P-ATP (Hartmann Analytik, Braunschweig, Germany), 5 mM β-glycerophosphate, 2 mM DTT, and 3 µg histone H2B at 30°C for 20 min. The reactions were stopped by adding 4x sample buffer, and the proteins were analyzed by SDS-PAGE, Coomassie blue staining, and autoradiography. Wortmannin (Sigma, 200 nM) was added to cell cultures 30 min prior to harvesting the cells.

Reportergene and apoptosis assays
Transient transfections were performed using the calcium phosphate precipitation method as described previously (39) or ExGen500 (Fermentas, St. Leon-Rot, Germany) as recommended by the manufacturer. The following plasmids were used: pCMV-hu-MAD1, pCMV-hu-MAD1{Delta}BR, pCMV-hu-MAD1{Delta}N (40) , pEQ176-β-Gal (to standardize for transfection efficiency), p(Gal4)4-mintk-luc (39) , pGL3–5xNF-{kappa}B-luc reporter, pCMV-Gal4-p65RelA (from Michael Kracht, Justus Liebig University, Giessen, Germany), pCMV-I{kappa}B, pCMV-p50NF-{kappa}B, pCMV-p65RelA (from Lienhard Schmitz, Justus Liebig University, Giessen, Germany), pCMV5-PDK1 (from Andreas Barthel, Ruhr University, Bochum, Germany), pcDNA4-TO-MycHis-PTEN (from Mark M. Moasser, University of California, San Francisco, CA, USA), pCR3-Fas receptor (from Jürg Tschopp, University of Lausanne, Epalinges, Switzerland), pEGFP-C1 (Clontech), pCMV-p53 (from Martin Scheffner, University of Konstanz, Konstanz, Germany), pGL3-PTEN2526/427-luc (from Tak Mak, University of Toronto, Toronto, ON, Canada), pBABE-puro (from Gerhard Evan, University of California, San Francisco, CA, USA), and pSUPER (from René Bernards, The Netherlands Cancer Institute, Amsterdam, Netherlands). pGL3-PTEN2484/961-luc was cloned using restriction sites from pGL3-PTEN2526/427-luc. The following PTEN-specific sequences were used: 5'-GTCAGAGGCGCTATGTGTA in pSUPER-siPTEN#1 and 5'-AGACAAAGCCAACCGATAC in pSUPER-siPTEN#2. pSUPER-control contained an unrelated sequence with no identity to any human gene.

For apoptosis assays, 2 x 104 HeLa cells, 12 x 104 HEK293 cells, or 8 x 104 Saos cells were seeded per well of a 12-well plate and transfected the next day. For these assays, the different effector plasmids were coexpressed with pEGFP-C1 and pCR3-Fas to identify transfected cells and to sensitize the cells to agonistic Fas-specific antibodies, respectively. At 24 h after transfection, the cells were treated with 50 ng/ml anti-Fas (clone CH11; Upstate, Charlottesville, VA, USA) for 3 h. Thereafter, the cells were fixed in 4% paraformaldehyde at 4°C for 23 min, washed once with PBS, and then analyzed by fluorescence microscopy using the Analysis software (Olympus, Tokyo, Japan). The transfected EGFP-expressing cells were counted, and the apoptotic subset was determined. Terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-stainings were performed according to the instructions of the manufacturer (Roche Diagnostics). Wortmannin (200 nM) was added to the cultures 2 h prior to inducing apoptosis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PTEN is a MAD1 target gene
U2OS-tet-MAD1 cells are derived from the osteosarcoma line U2OS and express MAD1 under the control of a promoter regulated by tetracycline (tet-off system) (14) . U2OS cells express very low amounts of endogenous MAD1. On induction of MAD1 (i.e., removal of tetracycline), these cells show a slow growth phenotype, are contact inhibited at low cell density, and are less sensitive to apoptotic stimuli (14 , 41) . We performed a PCR-select screen using U2OS-tet-MAD1 cells to identify MAD1 target genes. Only a few differentially expressed clones were detected reproducibly in several independent experiments (42) . One gene observed to be downregulated consistently was PTEN (Fig. 1 A). This was verified in semiquantitative RT-PCR experiments on RNA isolated from U2OS-tet-MAD1 cell clones M19 and M37 grown in the presence or absence of MAD1 (Fig. 1B and data not shown). Induction of MAD1 considerably reduced the signal obtained with PTEN specific primers, while the expression of β-glucuronidase (β-GUS), a gene that served as control, was unchanged (Fig. 1B ). These findings were complemented by the analysis of HL60 cells, a human promyelocytic line that differentiates toward macrophages in response to the tumor promoter TPA. During this process MAD1 expression is activated (36 , 43) . Within 12 h of TPA treatment, PTEN expression was substantially reduced as determined by RT-PCR analysis (Fig. 1C ). Furthermore, PTEN expression, both by analyzing mRNA (data not shown) and protein (Fig. 1D ), was reduced by MAD1 in transiently transfected HeLa cells. No effect was seen with MAD1 mutants that are defective for DNA binding (MAD1{Delta}BR) or that cannot recruit the mSin3-HDAC repressor complex (MAD1{Delta}N) (Fig. 1D ). Similar results were obtained in HEK293 cells (data not shown). The repression of PTEN in response to MAD1 was in the range of 2- to 4-fold in the different experiments. In addition to MAD1, we also tested whether MYC, a MAD1 antagonist, and MNT, which also functions as a repressor, affect the expression of PTEN. MYC did not enhance PTEN expression in HeLa cells, while MNT had a small effect in some and no effect in other experiments (Fig. 1E ). This finding suggested that not all members of the MYC/MAX/MAD network regulate the PTEN gene under these experimental conditions. Thus, in four cell systems we observed a correlation between MAD1 and PTEN expression, which suggests that MAD1 directly or indirectly represses the PTEN gene.

Inspection of the PTEN promoter region revealed two 5'-CACGTG consensus E-boxes as well as several noncanonical potential binding sites in the promoter distal region (Fig. 2 A and data not shown) (27 , 44) . To address whether MAD1 binds to the region that contains the consensus E-boxes, we performed chromatin immunoprecipitation (ChIP) experiments. In M19 cells, MAD1 binding to the corresponding promoter region was observed on removal of tetracycline (Fig. 2B ). No signal above background was seen in the absence of MAD1 expression. Instead MYC was bound, an interaction that was lost on MAD1 induction that suggests MAD1 replaced MYC at the PTEN promoter (Fig. 2B ). Control ChIP with an irrelevant antibody (anti-Cytochrome c) resulted in background signals. Furthermore, neither MYC nor MAD1 were detected at an E-box located in intron 3 (Fig. 2A, B ). These findings were corroborated in HL60 cells. MAD1 binding to the promoter was induced during TPA-stimulated differentiation, whereas MYC binding was seen only in exponentially growing cells (Fig. 2C ). MAX binding was constant throughout the course of the experiment, an expected finding since MAX is the common dimerization partner of both MAD1 and MYC. No binding was seen to the intron 3 E-box, and also no binding was seen with a c-MYB-specific antibody used for control (Fig. 2C and data not shown). Together with the observed downregulation of PTEN by MAD1 (Fig. 1) , these findings suggest direct repression of PTEN by MAD1. To further evaluate these findings, we addressed whether MAD1 could interfere with the expression of a PTEN reporter gene construct activated by the tumor suppressor p53 (27) . We observed repression by MAD1 but not by MAD1{Delta}BR (Fig. 2D ), whereas the basal activity of this reporter gene construct was not significantly inhibited by MAD1 (data not shown). The effect was at least partially dependent on the two consensus E-Boxes, but a contribution of noncanonical binding sites cannot be excluded (unpublished observation). We find that MAD1 occupies the PTEN promoter and represses a PTEN reporter gene, strongly suggests that the repression of PTEN by MAD1 is direct.

MAD1 activates the PI3K-AKT pathway
To evaluate the downstream effects of PTEN repression, we analyzed the phosphorylation and activity of AKT. Phosphorylation at Thr308 and at Ser473 by PDK1 and the predicted PDK2, respectively, is critical for AKT activation (20) . Increased AKT phosphorylation at Ser473 was observed in response to MAD1 in HeLa (Fig. 3 A, B) and in 293 cells (data not shown). This phosphorylation was absent when cells were treated with Wortmannin, an inhibitor of PI3K, while AKT expression remained unchanged (Fig. 3A ). MAD1-induced phosphorylation was accompanied with increased AKT kinase activity toward histone H2B as substrate while MAD1{Delta}BR had no effect (Fig. 3C, D ). Kinase activity was blocked by Wortmannin or by coexpressing PTEN. Identical results were obtained in HEK293 and HeLa cells. These findings are consistent with an increase in PIP3 levels due to the inhibition of PTEN by MAD1 and a subsequent increase in AKT kinase activity.


Figure 3
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  		Figure 3. MAD1 stimulates AKT phosphorylation and kinase activity. A) HeLa cells were transiently transfected with control plasmids or plasmids expressing MAD1. Wortmannin (W) was added to a final concentration of 200 nM 30 min prior to harvesting. AKT (H-136) and phosphorylated AKT ({alpha}-P473-Akt) were detected by Western blotting (WB). B) Quantification of AKT phosphorylation. Mean values and SD of three independent experiments are shown. The signal obtained from control cells was arbitrarily set at 100. C) Cells were treated as described in B. AKT was immunoprecipitated ({alpha}-AKT), and kinase assays were performed with histone H2B as substrate. In the first lane, antibodies were omitted (c). An autoradiography (32P) and the corresponding Coomassie blue (CB) -stained gel are displayed. All the lanes shown are from one exposure of one gel. D) Quantification of AKT kinase activity. Mean values of two independent experiments are shown. The background phosphorylation of the control samples (lane c in C) was subtracted.

The PI3K-AKT kinase pathway is a potent negative regulator of apoptosis (20 , 22) . This occurs at least in part through phosphorylation and activation of the inhibitor of NK-{kappa}B (I{kappa}B) kinase, which leads to the inactivation of I{kappa}B and subsequently to the stimulation of the NF-{kappa}B signal transduction pathway. Therefore, we tested whether MAD1 expression affected NF-{kappa}B transactivation in HeLa cells. MAD1 stimulated a NF-{kappa}B reporter gene construct, an effect that was sensitive to the coexpression of I{kappa}B (Fig. 4 A). In addition, the activity of a Gal4-NF-{kappa}B fusion protein was significantly enhanced by coexpressing MAD1 but not by MAD1{Delta}BR (Fig. 4B ). Together these findings identify, in both HeLa and HEK293 cells (data not shown), MAD1-dependent regulation of AKT and the downstream effector NF-{kappa}B, which is involved in the control of apoptosis.


Figure 4
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  		Figure 4. MAD1 affects downstream targets of the PI3K-AKT pathway. A) HeLa cells were transfected with pGL3–5xNF-{kappa}B-luc reporter (200 ng), pEQ176-β-Gal (1 µg), pCMV-p50NF-{kappa}B (5 ng), pCMV-p65RelA (20 ng), pCMV-hu-MAD1 (500 ng), and pCMV-I{kappa}B (500 ng), as indicated. The expression of luciferase was standardized with β-galactosidase. The mean values and SD of 4 independent experiments performed in duplicates are shown. B) HeLa cells were transfected with p(Gal4)4-mintk-luc (200 ng), pEQ176-β-Gal (1 µg), pCMV-Gal4-p65RelA (50 ng), pCMV-hu-MAD1 (500 ng), and pCMV-hu-MAD1{Delta}BR (500 ng), as indicated. The mean values and SD of 3 independent experiments performed in duplicates are shown.

MAD1 inhibits apoptosis through stimulating the PI3K-AKT pathway
The results described above suggest that the PI3K-AKT pathway mediates MAD1-dependent inhibition of apoptosis. Therefore, we measured apoptosis in response to MAD1 and/or to modulators of this pathway. MAD1 expression inhibited, as shown previously (14) , apoptosis in HeLa cells induced by Fas (Fig. 5 ). For routine analysis apoptotic cells were identified morphologically (Fig. 5A, B ), findings that were corroborated using the TUNEL assay (data not shown). In addition, MAD1 reduced the activation of caspase-3 and the cleavage of the caspase substrate PARP1 (Fig. 5C ). The MAD1 effect was counteracted by Wortmannin and by PTEN (Fig. 5A, B ), indicating that activation of the PI3K-AKT pathway is important for MAD1-dependent inhibition of apoptosis. The treatments of cells with either Wortmannin or by expressing PTEN in the absence of an apoptotic signal was not sufficient to induce apoptosis under our experimental conditions (data not shown). Furthermore expression of PDK1 reduced substantially Fas-induced apoptosis. PDK1 has been demonstrated to be sufficient to activate AKT and thus allows assessing the role of endogenous AKT and downstream effectors (45) . PDK1 did not further enhance the MAD1 effect, which suggests that the PI3K-AKT pathway is maximally activated by MAD1 (Fig. 5B ). Comparable findings were made in HEK293 and SAOS cells (data not shown). Although PDK1 phosphorylates additional substrates in cells whose role in regulating apoptosis cannot be excluded, our findings suggest that activation of the PI3K-AKT pathway by MAD1 is critical for its antiapoptotic function.


Figure 5
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  		Figure 5. MAD1-dependent activation of the PI3K-AKT pathway mediates inhibition of apoptosis. A) HeLa cells were transiently transfected with pEGFP-C1, pCR3-Fas, and plasmids expressing the indicated proteins. The cells were treated with an agonistic Fas-specific antibody (CH11) to induce apoptosis 24 h after transfection. After 4 h, GFP-positive cells were analyzed and assessed for apoptotic morphology. Arrows identify some of the apoptotic cells. Wortmannin was added 30 min prior to the addition of the Fas-specific antibody. B) Quantification of the apoptosis assays. Mean values and SD of 4 independent experiments are displayed. For the PDK1-expressing samples mean values of two experiments are shown. In each experiment several fields were analyzed, and the percentage of apoptotic cells was determined. The significance of the experimental results was determined by the Student’s t test (2-sided); *P < 0.05; **P < 0.01; ***P < 0.001; {blacksquare}not significant. C) HeLa cells were transiently transfected with a MAD1 expressing construct, with pEGFP-C1, and with pCR3-Fas. Transfection efficiency was ~50% as evaluated by EGFP staining. The cells were then stimulated with an agonistic Fas-specific antibody (CH11) (+ lanes) or left unstimulated (– lanes), and cell extracts were prepared 4 h later. The indicated proteins were analyzed by Western blotting.

To corroborate a role of PTEN in MAD1-dependent inhibition of apoptosis, PTEN expression was depleted by transfecting several pSUPER constructs that express small interfering RNAs (siRNAs) specific for PTEN. Two constructs were selected that repressed PTEN expression when coexpressed with a plasmid-encoding PTEN (Fig. 6 A). This repression was sufficient to induce AKT phosphorylation (Fig. 6A ). In addition, both siRNA-expressing constructs efficiently downregulated endogenous PTEN (Fig. 6B ). Next, we analyzed the effect of PTEN knockdown on apoptosis and found that repression of PTEN was comparable in its antiapoptotic activity to the consequence of MAD1 expression (Fig. 6C ). No additive effect of PTEN-specific siRNA and MAD1 was measurable (Fig. 6C ), demonstrating that PTEN regulation is critical for the control of apoptosis by MAD1. Finally, we addressed whether immortalized pten–/– MEFs would be resistant to MAD1. We observed that pten–/– and the corresponding pten+/– MEFs were equally sensitive to Fas-induced apoptosis (Fig. 6D ). This finding was somewhat unexpected, but the analysis of AKT phosphorylation revealed no difference between these two immortalized lines (data not shown). However, and in support of the findings described above, MAD1 inhibited apoptosis in pten+/– MEFs but had no effect in pten–/– MEFs (Fig. 6D ). This condition was dependent on the ability of MAD1 to bind to DNA (Fig. 6D ) and suggests that cells are sensitive to MAD1 when the antiapoptotic PI3K-AKT pathway can be activated in response to PTEN repression. In contrast, MAD1 is inert in cells that do not possess this regulatory potential. Together our findings demonstrate that PTEN is essential to mediate inhibition of apoptosis by MAD1.


Figure 6
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  		Figure 6. Loss of PTEN abolishes MAD1-dependent repression of apoptosis. A) Whole cell lysates of HEK293 cells, transfected with a PTEN expression plasmid and with the indicated pSUPER plasmids, were analyzed for expression of exogenous PTEN (mAb A2B1) and phosphorylated AKT ({alpha}-P473-Akt) by Western blotting (WB). The control siRNA lane is from the same gel and equal exposure as the other lanes. B) HeLa cells were transfected with the indicated pSUPER plasmids pEGFP-C1 and pBABE-puro, selected in puromycin, and analyzed for the expression of endogenous PTEN, coexpressed GFP, and endogenous CK2β. C) HeLa cells were transiently transfected with pEGFP-C1, pCR3-Fas, pCMV-hu-MAD1, pSUPER-siPTEN#1, and pSUPER-siPTEN#2 using ExGen500 as indicated. In the samples without pSUPER-siPTEN, a pSUPER-control construct was cotransfected. The cells were treated with an agonistic Fas-specific antibody (CH11) to induce apoptosis 24 h after transfection. After 4 h, GFP-positive cells were analyzed and assessed for apoptotic morphology. Mean values and SD of multiple slides of 3 independent experiments are presented. D) Immortalized pten+/– and pten–/– MEFs were transfected with pEGFP-C1, pCR3-Fas, pCMV-hu-MAD1, and pCMV-hu-MAD1{Delta}BR as indicated. Apoptosis was induced and the number of apoptotic cells was determined as described in C. Mean values and SD of multiple slides of two independent experiments are presented.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings demonstrate that MAD1 activates the PI3K-AKT pathway by repressing PTEN expression and that this pathway is an important downstream effector of MAD1 to inhibit apoptosis. Thus PTEN is the first target gene that links MAD1 to the control of apoptosis. Interestingly, the PI3K regulatory subunit p85{alpha} was identified as a MAD1-activated gene (18) . Although, this effect is probably indirect, it suggests that MAD1 regulates the PI3K-AKT pathway at least at two distinct levels. The PI3K-AKT pathway not only interferes with apoptosis but also stimulates proliferation (20 , 22) . How then can the antiproliferative and antiapoptotic activities of MAD1 be reconciled? We suggest that the consequence of MAD1 function on the expression of the PTEN gene dominates the effect on apoptosis (Figs. 5 and 6) , while the potential proliferative impact of activating the PI3K-AKT pathway is antagonized by other MAD1 target genes. Indeed several genes have been identified that connect MAD1 to the control of cell proliferation (8) . Some repressed genes, including cyclin D2, provide a link to the ability of MAD1 to repress the cell cycle (18 , 36 , 46) . In addition, a number of genes involved in protein and DNA synthesis and in energy metabolism—activities that are required for efficient cell growth—are also downregulated by MAD1 (18) . Together, it is well possible that inhibition of the cell cycle and downregulation of metabolic activities dominate over the proliferation-stimulating activity of the PI3K-AKT pathway and thus provides an explanation for the ability of MAD1 to inhibit both proliferation and apoptosis.

The identification of MAD proteins as MYC antagonists led to the suggestion that MAD proteins might function as tumor suppressors. However, little evidence to support this hypothesis has been obtained (8 , 47) . Since the four MAD proteins are expressed in overlapping patterns in different tissues, and since MNT, an additional MYC antagonist, is expressed rather ubiquitously, loss of MAD or MNT function may be difficult to achieve in tumor cells. Furthermore, we suggest that the loss of the antiapoptotic activities of MAD and MNT proteins may be unfavorable for tumor cells. Indeed, not only loss of MAD1 but also of MAD3 and MNT results in enhanced apoptosis (9 10 11 12) . In the case of MAD proteins these effects appear to be restricted to certain cell types. This finding may reflect the observation that several MAD/MNT family members are expressed in all cell types analyzed. Thus, stimulation of apoptosis by repressing MAD/MNT protein activity may be rather difficult to achieve. In agreement with these data our preliminary findings using mad1/mxi1/mad3 triple knockout MEFs indicate that these cells do not display significantly enhanced apoptosis, probably due to the high MNT levels in these cells (unpublished observation). The findings summarized above indicate that MAD and MNT proteins have overlapping antiapoptotic functions. Together, these functions might be sufficiently important for tumor cells that the corresponding genes are infrequent targets of inactivation during malignant progression of cells.

What is the interplay between MYC and PTEN? Our ChIP data demonstrate that MYC can also bind to the PTEN promoter (Fig. 2) . In agreement PTEN was identified as a MYC target in a microarray screen, although it remains to be determined whether this is the result of a direct effect (48) . In addition the PTEN promoter was found in a search for MYC chromatin binding sites by ChIP (49) . However, unlike the findings with MAD1, exogenous expression of MYC was not sufficient to affect (i.e., induce or repress) PTEN transcription under the conditions used here (Fig. 1E and data not shown). This condition may be due to already high endogenous MYC levels in the tumor cells used, and thus it remains open whether PTEN is a direct MYC target gene. It is worth noting that MYC is bound to a surprisingly large number of promoters in different cell types, but the functional consequences may be distinct on different promoters (49 , 50) . Furthermore, MYC may control PTEN expression by indirect means. One is by inducing p19ARF, a protein that enhances p53 activity (51) . The tumor suppressor p53 in turn stimulates PTEN transcription (27) . Functionally the PI3K-AKT pathway and its antiapoptotic activities have been linked previously to MYC. MYC-induced apoptosis is inhibited by this pathway (52 , 53) , which is consistent with our previous observations that MAD1, but not MAD1{Delta}N, blocks efficiently MYC-induced apoptosis (14) . Thus, these findings define an intimate relationship between the MYC/MAX/MAD network and the PI3K-AKT pathway.

Our findings described here together with published work on the role of other transcriptional regulators, including p53, Egr-1, and NF-{kappa}B, in controlling PTEN expression demonstrate that this gene is regulated by both pro- and antiapoptotic signals (25 26 27 28 29 30) . Of particular interest is p53, which integrates many stress signals and translates these into cellular responses including apoptosis (54) . p53 activates PTEN expression by binding near the core promoter of PTEN, an aspect important for the ability of p53 to stimulate apoptosis (27) . Similarly, Egr-1 stimulates PTEN expression in response to UV light and results in enhanced apoptosis (29) . NF-{kappa}B transcriptional regulators mediate part of the antiapoptotic response triggered by the PI3K-AKT pathway (55) . A recent study defines NF-{kappa}B as a negative regulator of PTEN expression, providing evidence for a positive feedback loop in the inhibition of apoptosis by the PI3K-AKT pathway (28) . Finally, c-Jun, which has both pro- and antiapoptotic activities, promotes cellular survival by inhibiting PTEN transcription (25) . The association of MAD1 function and expression with stress signals is less well defined. Nevertheless, MAD1 expression is controlled by different cytokines that are also involved in the regulation of apoptosis (8) . Thus several different signal transduction pathways and associated transcriptional regulators that are associated with regulation of apoptosis control PTEN transcription.

These findings are consistent with the important function PTEN plays in regulating the PI3K-AKT signal transduction pathway and thus apoptosis as well as other aspects of cell behavior (20 , 22) . These studies suggest that different signaling pathways converge, at least in part, through the factors mentioned above, on the PTEN promoter. A first indication in support of this suggestion is provided by our observation that MAD1 antagonized the stimulation of the PTEN promoter by p53 (Fig. 2) . Future work will have to address the interaction of p53 and MAD1 in more detail. In addition, MAD1 and NF-{kappa}B factors may also interact to regulate PTEN expression. Since MAD1 stimulates the activity of NF-{kappa}B (Fig. 4) , this factor may provide positive enhancement of MAD1-dependent repression of PTEN. Also, recent findings suggest a role of NF-{kappa}B transcription factors in tumorigenesis (56) . Inhibition of PTEN might be one important function for this activity. Understanding the regulation of the expression of the PTEN tumor suppressor gene will shed more light on the role of the encoded protein in the control of cell behavior.


   ACKNOWLEDGMENTS
 
We thank A. Barthel, R. Bernards, G. Evan, M. Kracht, T. Mak, M. Moasser, M. Scheffner, L. Schmitz, and J. Tschopp for plasmids and cells. We are indebted to M. Wymann for helpful discussions and to J. Bernhagen, R. Lilischkis, and D. Litchfield for comments on the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB542 TP B8) to B.L.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Present address: Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, CA 92121, USA.

Received for publication August 14, 2007. Accepted for publication October 11, 2007.


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