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Molecular and Cellular Biology, January 2008, p. 529-538, Vol. 28, No. 2
0270-7306/08/$08.00+0     doi:10.1128/MCB.00533-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Thalidomide Induces Limb Anomalies by PTEN Stabilization, Akt Suppression, and Stimulation of Caspase-Dependent Cell Death{triangledown}

Jürgen Knobloch,1* Ingo Schmitz,2 Katrin Götz,1 Klaus Schulze-Osthoff,2 and Ulrich Rüther1

Institute for Animal Developmental and Molecular Biology,1 Institute of Molecular Medicine, Heinrich Heine University, D-40225 Düsseldorf, Germany2

Received 29 March 2007/ Returned for modification 19 June 2007/ Accepted 30 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thalidomide, a drug used for the treatment of multiple myeloma and inflammatory diseases, is also a teratogen that causes birth defects, such as limb truncations and microphthalmia, in humans. Thalidomide-induced limb truncations result from increased cell death during embryonic limb development and consequential disturbance of limb outgrowth. Here we demonstrate in primary human embryonic cells and in the chicken embryo that thalidomide-induced signaling through bone morphogenetic proteins (Bmps) protects active PTEN from proteasomal degradation, resulting in suppression of Akt signaling. As a consequence, caspase-dependent cell death is stimulated by the intrinsic and Fas death receptor apoptotic pathway. Most importantly, thalidomide-induced limb deformities and microphthalmia in chicken embryos could be rescued by a pharmacological PTEN inhibitor as well as by insulin, a stimulant of Akt signaling. We therefore conclude that perturbation of PTEN/Akt signaling and stimulation of caspase activity is central to the teratogenic effects of thalidomide.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The application of the sedative drug thalidomide to pregnant women resulted in congenital defects in thousands of human fetuses. The evaluation of numerous case reports revealed that many organs, like the eye and heart, could be affected by thalidomide, but variable limb truncations were reported to be the most frequent defect (36). Remarkably, the teratogenic effect of thalidomide is strictly species specific. While mice and rats are thalidomide resistant, certain nonhuman primates, rabbits, and chickens show embryopathy, including a high frequency of limb truncations and microphthalmia (small eyes) upon thalidomide exposure (12, 26, 32).

Recently, we have established primary limb bud cells (PLBCs) isolated from chicken embryos, primary human embryonic fibroblasts (HEFs), and the chicken embryo as suitable model systems to study the molecular basis of thalidomide teratogenicity (20). By using these systems, we found that thalidomide-induced oxidative stress enhances signaling through bone morphogenetic proteins (Bmps). This causes hyperexpression of the Bmp target gene and secreted Wnt antagonist Dickkopf1 (Dkk1) with subsequent down-regulation of Wnt-mediated β-catenin activity, resulting in increased apoptosis. In vivo, thalidomide-induced apoptosis causes tissue degradation during early embryonic development, resulting in limb truncations and microphthalmia (20).

Apoptosis is triggered by at least two major signaling routes, namely, the extrinsic death receptor and the intrinsic mitochondrial pathway. Both pathways result in the activation of intracellular cysteine proteases, called caspases. In the extrinsic pathway, ligation of death receptors, such as Fas (CD95, APO-1), tumor necrosis factor (TNF) receptor 1 (TNF-R1), or TNF-related apoptosis-inducing ligand (TRAIL) receptors, triggers the recruitment of the initiator caspase-8 or -10 into a death-inducing signaling complex (31). In contrast, the mitochondrial death pathway is initiated by the mitochondrial release of cytochrome c into the cytosol (46). Once released, cytochrome c binds to the adapter protein Apaf-1, which enables the subsequent binding and activation of caspase-9. The mitochondrial apoptosis pathway is inhibited by antiapoptotic members of the Bcl-2 family. Both the death receptor and the mitochondrial pathway are interconnected by the proapoptotic Bcl-2 protein Bid. Upon cleavage by caspase-8, the truncated Bid protein triggers the release of cytochrome c and caspase-9 activation (7). Both pathways then activate effector caspase-3, -6, or -7, which lead to cell death via the cleavage of several cellular substrates.

The protein kinase Akt (protein kinase B) protects cells from caspase-mediated cell death and is both necessary and sufficient for cell survival. Accordingly, Akt inhibits several proapoptotic proteins, such as glycogen synthase kinase-3β (Gsk3β), Bad, caspase-9, and Forkhead transcription factors (5, 8, 19). The major upstream regulator of Akt is the phosphatidylinositide 3-OH kinase (PI3K), which is activated by a variety of transmembrane receptors. In the case of insulin, activation of its receptor causes the tyrosine phosphorylation of insulin receptor substrate (IRS) proteins that serve as docking sites for a number of downstream effector molecules, including the regulatory p85 subunit of PI3K (11). Upon stimulation, PI3K catalyzes the generation of phosphatidylinositide-3,4,5-triphosphate (PIP3), thereby recruiting phosphoinositide-dependent protein kinase (PDK) and Akt to the plasma membrane. At the membrane, PDK phosphorylates Ser473 and Thr308 residues of Akt, which is required for maximal activation of Akt (19).

The activation of Akt is antagonized by the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates PIP3 and thereby inhibits PI3K/Akt-mediated survival signaling (24). PTEN itself is regulated by phosphorylation on a cluster of serine and threonine residues at its C-terminal tail. Dephosphorylation promotes both, its activation through enhanced recruitment to the plasma membrane as well as its susceptibility to proteasomal degradation (24).

There is increasing evidence that oxidative stress interferes with signaling through Bmps, Wnts, and Akt and that Wnt and Akt pathways converge on Gsk3β (18, 38, 41, 43, 45). We have previously shown that thalidomide-induced oxidative stress results in induction of Bmp expression, which in turn leads to down-regulation of Wnt-mediated β-catenin and a concomitant increase of Gsk3β activity (20). Our previous results, however, also indicated that besides inhibition of Wnt signaling, additional pathways might contribute to thalidomide-induced cell death and teratogenicity. In the present study we found in HEFs and in the chicken embryo that stabilization of PTEN and the subsequent down-regulation of Akt activity are important events, which are involved not only in the proapoptotic effects but also in the developmental defects induced by thalidomide.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chicken embryos and drug treatment. Fertilized eggs were purchased from Deindl GmbH (Rietberg-Varensell, Germany). Embryos were incubated, staged according to the criteria of Hamburger and Hamilton (HH), and exposed to racemic thalidomide (Sigma, St. Louis, MO) at 0.75 mg/kg of egg weight at HH stages 4/5 as described before (20). Ten microliters of dipotassium bisperoxo (pyridine-2-carboxyl) oxovanadate [bpV(pic)] (10 or 100 mM; Alexis, Grünberg, Germany) or bovine insulin (250 nM or 2.5 µM, Sigma) supplemented with 0.1% Fast Green were applied per embryo at HH stages 17 to 19 by injection through the extraembryonic membranes. Embryos were analyzed for anomalies at day 6 or 7 of embryonic development as described before (20).

Primary cells and drug treatment. Isolation of PLBCs and HEFs and preincubation before drug treatment was done as described previously (20). Treatment with thalidomide (38.7 µM), human BMP4 (300 ng/ml; R&D Systems, Minneapolis, MN), mouse Noggin (300 ng/ml; R&D Systems), anti-human DKK1 antibody (350 ng/ml; R&D Systems), bpV(pic), insulin, cycloheximide (CHX) (10 µg/ml; Sigma), MG132 (10 µM; Calbiochem, Darmstadt, Germany), wortmannin (10 nM; Alexis), PD98059 (10 µM; Calbiochem), caspase inhibitors [quinoline-Val-Asp (OCH3)-CH2-O-Ph (QVD-oPh), N-acetyl-Val-Asp (OMe)-Val-Ala-Asp (OMe)-fluoromethyl ketone (zVDVAD-fmk), N-acetyl-Ile-Glu (OMe)-Thr-Asp (OMe)-fluoromethyl ketone (zIETD-fmk), and N-acetyl-Leu-Glu (OMe)-His-Asp (OMe)-fluoromethyl ketone (zLEHD-fmk); MP Biomedicals, Heidelberg, Germany; all used at 20 µM], anti-TNF-R1 (10 µg/ml) (42), anti-FasL (10 µg/ml) (34), and anti-TRAIL antibodies (10 µg/ml) (clone 2E5) (ALX-804-296; Alexis) was performed in OptiMEM1 (Invitrogen, Carlsbad, CA) plus 1% fetal calf serum. Unless otherwise noted, all treatments were done for 6 hours. Inhibitors were applied once at the beginning of incubation. Pretreatment with phenyl N-t-butylnitrone (PBN) (2 mM) (Sigma) was done for 1 hour.

Transfections and RNA interference. HEFs were transfected with expression constructs by the use of Lipofectamine (Invitrogen) 24 h prior to thalidomide treatment. Plasmids encoding constitutively active Akt (membrane-targeted Akt [mtAkt]), dominant-negative kinase-dead (K179/A) Akt (2), c-FLIPshort (22), Bcl-2 (48), IRS-1F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989) (14), or IRS-1{Delta}C were used. The latter was cloned by the method of Tanaka and Wands (39). Using pBS-human-IRS-1 (Addgene plasmid 11359) as a template, a cDNA encoding amino acids 1 to 516 was cloned into pEF6/V5-His A (Invitrogen). The following primers were used for amplification: forward, 5' GGA TCC AGC ATG GCG AGC CCT CCG GAG AG 3'; reverse, 5' GAA TTC ATC TGC AGC ACT GGC TGC TTC 3'. Inhibition of PTEN expression was done by using the SignalSilence PTEN small interfering RNA (siRNA) kit (human specific) (catalog no. 6250; Cell Signaling, Danvers, MA) according to the instructions of the manufacturer.

TOPflash assay. Plasmids with lymphoid enhancer-binding factor (LEF)/T-cell factor (TCF) binding sites (TOPflash) (21) upstream of a luciferase reporter gene were transiently transfected into HEFs using Lipofectamine. If applicable, HEFs were cotransfected with PTEN-specific siRNA (see above). Following transfection, cells were exposed to Wnt3a-conditioned medium (a kind gift from Ya-Wei Qiang, NCI) or to control medium (20) with or without thalidomide and anti-Dkk1 antibody (350 ng/ml) for 24 h prior to the luciferase assay. Values were normalized for transfection efficiency by cotransfection with a pSV-β-galactosidase vector and for the number of living cells. Luciferase activity was measured using the Dual-Light system (Applied Biosystems, Foster City, CA).

Detection of transcriptional activity and cell death. RNA isolation and semiquantitative reverse transcription-PCR (sq-RT-PCR) were done according to standard protocols. Primer sequences and PCR conditions are available upon request. Cell death in HEFs was measured by trypan blue staining as described previously (20).

Western blotting. Protein extraction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were performed according to standard protocols. Antibodies for Akt (catalog no. 9272), active Akt (phosphorylated on Ser473; catalog no. 9271), PTEN (catalog no. 9552), Ser380-phosphorylated PTEN (catalog no. 9551S, all from Cell Signaling) and antiactin antibody (catalog no. A2066; Sigma) were used according to the manufacturers' instructions. Following incubation with appropriate horseradish peroxidase-coupled secondary antibodies, signals were detected by the ECL PLUS detection system (GE Healthcare, Freiburg, Germany).

ELISA. For quantification of PTEN protein, PTEN-specific enzyme-linked immunosorbent assays (ELISAs) were performed as described previously (29). Briefly, HEFs were lysed in luciferase lysis buffer (Dual-Light system; Applied Biosystems), and the lysates were concentrated with Microcon centrifugal filters (30-kDa cutoff; Millipore Corporation, Billerica, MA). Then, 5 µg protein extract was diluted in 200 µl carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and coated onto MaxiSorp ELISA plates (Nunc, Roskilde, Denmark). After blocking, PTEN was detected by a polyclonal PTEN antibody (catalog no. 9552; Cell Signaling), a biotinylated secondary antibody (R&D Systems), streptavidin-horseradish peroxidase (R&D Systems), and 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (Sigma). The enzyme reaction was stopped by the addition of sulfuric acid, and substrate conversion was measured at 450 nm. PTEN activity in cell lysates of HEFs, PLBCs, or limb buds was determined by measuring the conversion of PIP3 to PIP2 using a commercial kit (ELISA for detection and quantification of PTEN phosphatase activity, catalog no. K-2300; Echelon, Salt Lake City, UT).

Caspase glo assay. Proteins were extracted from HEFs or limb buds (for one sample, limb buds were pooled from 15 embryos) as described before (34). Thirty micrograms (HEFs) or 5 µg (limb buds) protein was used in the caspase glo assay to measure caspase-3 and -7 activity according to the instructions of the manufacturer (Promega, Heidelberg, Germany). Caspase activity was measured every 10 min for 3 h after addition of the substrate, and the values obtained after 90 min were included in the figures.

Statistical analyses. Two-tailed Student's t tests were done using Microsoft Excel.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Thalidomide induces activation of effector caspases in HEFs and limb buds. We have recently reported that thalidomide induces cell death in HEFs and chicken embryos (20). To investigate whether this effect is mediated by caspases, we measured the activity of effector caspase-3 and -7 in thalidomide-treated HEFs. Within the first 5 hours of incubation, thalidomide-treated HEFs did not reveal increased caspase activity compared to the control cells (Fig. 1A). However, after 6 hours of treatment, caspase activity was induced threefold above the activity of the controls (Fig. 1A). Longer incubation times did not further enhance this effect (Fig. 1A). Importantly, caspase activation corresponded to the kinetics of cell death in HEFs, which was also not observed earlier than after 6 hours of thalidomide treatment (20).


Figure 1
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  		FIG. 1. Thalidomide induces caspase activation in HEFs and limb buds. HEFs (A) and chicken embryos (B) were treated with thalidomide (thal) or a solvent control. After the indicated times, proteins were extracted from HEFs or pooled wing/limb buds of 15 embryos. The activity of effector caspases was measured using a luminometric caspase assay. Values were normalized to the values for negative controls (untreated HEFs and limb buds of solvent-treated embryos). The data represent the means ± standard deviations (error bars) from three (A) and four (B) individual experiments, respectively. nv, normalized values.

 
Thalidomide induces most limb and eye defects in chicken embryos when applied at HH stages 4/5 at 750 µg/kg of egg weight (20). Using these conditions, we investigated thalidomide-induced molecular pathology in vivo. Thalidomide-induced limb anomalies are the consequence of increased cell death in limb buds at HH stages 23/24 (20). Thus, we determined caspase activity in cell lysates extracted from pooled limb buds of thalidomide-exposed and nonexposed control embryos at HH stages 23/24. In response to thalidomide, caspase activity was induced about 7.5-fold than that of controls (Fig. 1B).

Thalidomide induces apoptosis via the intrinsic apoptotic pathway and Fas signaling. In order to elucidate through which apoptotic pathway thalidomide stimulates effector caspases, we performed inhibitor studies in HEFs. After 6 hours of treatment, thalidomide increased cell death 5.5- to 6.6-fold above that of the controls (Fig. 2A and B). The general caspase inhibitor QVD-oPh (6) completely counteracted thalidomide-induced cell death (Fig. 2A), demonstrating that thalidomide causes caspase-dependent apoptosis. We next tested inhibitors specific for several initiator caspases. Both the caspase-9 inhibitor zLEHD-fmk (25) and the caspase-8 inhibitor zIETD-fmk (25) counteracted thalidomide-induced apoptosis to ~50% and ~40%, respectively (Fig. 2A). In contrast, the caspase-2 inhibitor zVDVAD-fmk (6) did not influence thalidomide-induced apoptosis (Fig. 2A).


Figure 2
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  		FIG. 2. Thalidomide-induced cell death is caspase dependent and mediated by Fas death receptor and mitochondrial signaling. (A) HEFs were incubated with the indicated caspase inhibitors or specific antibodies blocking TNF, Fas, or TRAIL death receptor/ligand signaling and treated with thalidomide (thal) (+) or a solvent control (–). (B) HEFs were transiently transfected with plasmids encoding the caspase-8 inhibitor c-FLIPshort or Bcl-2 or with the empty vector control. After 6 h of incubation in the presence (+) or absence (–) of thalidomide (thal), the ratios of dead to live cells were determined and normalized to the values for negative controls (solvent-treated cells). The data represent the means ± standard deviations (error bars) from eight individual experiments. Values that are significantly different from those of thalidomide-treated control cells are shown as follows: *, P < 10–4; **, P < 10–8. Cell death is shown in normalized values (nv). A value of 1 corresponds to 1 to 2% cell death. {alpha}FasL, anti-FasL.

 
Caspase-9 activation requires cytochrome c release from mitochondria, a process that is antagonized by Bcl-2 (46). Transient expression of Bcl-2 in thalidomide-treated HEFs significantly counteracted drug-induced apoptosis (Fig. 2B), confirming that thalidomide partially induces apoptosis via the intrinsic pathway. The short form of the antiapoptotic FLICE-inhibitory protein (c-FLIPshort) competes with caspase-8 for death receptor binding, thereby specifically antagonizing caspase-8-induced activation of effector caspases (22). Transient expression of c-FLIPshort in thalidomide-treated HEFs significantly counteracted thalidomide-induced apoptosis (Fig. 2B), indicating an involvement of death receptors. The use of blocking antibodies against TNF-R1 or TRAIL did not influence thalidomide-induced apoptosis, indicating that TNF or TRAIL receptors are not activated by thalidomide in embryonic fibroblasts. However, blocking FasL with specific neutralizing antibodies inhibited thalidomide-induced apoptosis to ~40% (Fig. 2A). Thus, thalidomide induces apoptosis through both the Fas death receptor and mitochondrial pathway.

Thalidomide down-regulates Akt activity in embryonic cells and in chicken limb buds. Akt signaling antagonizes the intrinsic and extrinsic apoptotic pathways (4, 5, 8, 19). To elucidate whether thalidomide modulates Akt activity, we treated PLBCs, HEFs, and chicken embryos with thalidomide. Akt activity was determined by Western blot analyses using an activation-specific antibody detecting phosphorylated Ser473 of Akt. In comparison to solvent controls, thalidomide strongly decreased the level of phosphorylated and hence active Akt in PLBCs, HEFs, and limb buds of chicken embryos at HH stages 23/24 (Fig. 3A to C). In contrast, as determined by phosphorylation-independent antibody, the total amount of Akt was not influenced by thalidomide treatment.


Figure 3
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  		FIG. 3. Thalidomide suppresses Akt signaling via up-regulation of active PTEN. Western blot analyses (A to E) or PTEN activity ELISAs (F) were performed with protein extracts isolated from PLBCs, HEFs, or wing/limb buds pooled from 50 embryos to analyze the effect of thalidomide (thal) on expression and activity of Akt and PTEN. Cells and embryos were treated as indicated in the figure. Each Western blot represents one representative example of at least two independent experiments. The data of the PTEN activity ELISAs represent the means ± standard deviations (error bars) from three individual experiments. pAkt, Akt phosphorylated on Ser473; pPTEN, PTEN phosphorylated on Ser380; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate.

 
Thalidomide-induced Bmp signaling increases the amount of active PTEN. A major antagonist of Akt is PTEN (24). Thus, we asked whether thalidomide-induced down-regulation of Akt activity could be mediated by PTEN. Western blot analyses revealed that thalidomide enhanced the level of PTEN protein in PLBCs, HEFs, and HH stage 23/24 chicken limb buds (Fig. 3A to C). We did not find differences in the amount of phosphorylated/inactive PTEN between thalidomide-treated HEFs and controls by the use of a phospho-Ser380-PTEN-specific antibody (Fig. 3B). Thalidomide induces Bmp expression in chicken limb buds and in HEFs (20). Treatment of HEFs with recombinant Bmp4 for 6 hours resulted in a dramatic increase of PTEN protein levels (Fig. 3D). Notably, the simultaneous application of the Bmp inhibitor Noggin (49) neutralized this effect (Fig. 3D). Noggin also counteracted thalidomide-induced up-regulation of PTEN protein amounts and promoted PTEN phosphorylation on Ser380 in HEFs (Fig. 3B).

Thalidomide-induced up-regulation of Bmp expression depends on the generation of reactive oxygen species (ROS), and PBN, a reagent that neutralizes oxidative stress by trapping free radicals, counteracts thalidomide-induced apoptosis completely (20). To investigate the hierarchy of events in thalidomide-induced signaling, we treated HEFs with either thalidomide or with a combination of thalidomide and PBN. PBN counteracted thalidomide-induced up-regulation of PTEN (Fig. 3E), confirming the generation of ROS as a prerequisite for the impact of thalidomide on Bmp and PTEN signaling.

As oxidative stress often inhibits rather than increases the catalytic activity of phosphatases (23), we further determined PTEN lipid phosphatase activity by measuring the conversion from PIP3 to PIP2. Thalidomide enhanced PTEN activity 1.7- to 1.9-fold above solvent controls in PLBCs, HEFs, and limb buds of chicken embryos at HH stages 23/24 (Fig. 3F). Consistent with promoting PTEN phosphorylation (Fig. 3B), Noggin caused a reduction of PTEN activity in solvent-treated cells (Fig. 3F). More importantly, Noggin almost completely neutralized the induction of PTEN activity in thalidomide-treated HEFs and PLBCs (Fig. 3F).

Thalidomide-induced Bmp signaling protects active PTEN from proteasomal degradation. We next investigated the mechanism of the Bmp-dependent increase of PTEN levels in response to thalidomide treatment. Semiquantitative RT-PCR analyses did not reveal any influence of thalidomide on PTEN transcription in HEFs (Fig. 4A). To analyze whether thalidomide affects PTEN translation, we treated HEFs with thalidomide or a solvent control and added the translational inhibitor CHX after 2.5 h. At this time point, thalidomide-treatment already resulted in increased Bmp4 levels (data not shown) but did not yet up-regulate PTEN (Fig. 4B). In solvent-treated cells, CHX caused a continuous reduction of PTEN (Fig. 4B), indicating a rapid protein turnover under normal conditions. Thalidomide increased the PTEN protein level slightly after 4 hours and about threefold after 6 and 8 hours. The addition of CHX, however, did not influence thalidomide-induced PTEN up-regulation after 4 and 6 hours and only slightly reduced PTEN levels after 8 hours (Fig. 4B). Thus, these data demonstrate that the elevated PTEN levels are primarily not mediated by a thalidomide-induced increase of PTEN translation. The slight reduction after 8 hours might be explained by limitations in translation of Bmps and/or other, hitherto unknown signaling molecules acting upstream of PTEN in thalidomide-induced molecular pathology.


Figure 4
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  		FIG. 4. Thalidomide protects PTEN from proteasomal degradation. (A) To investigate the effect of thalidomide (thal) on PTEN transcription, sq-RT-PCRs were performed with RNA of thalidomide-treated (+) or untreated (–) HEFs. Amplification of hypoxanthine phosphoribosyltransferase (HPRT) mRNA was used as a control. (B and C) HEFs were treated with dimethyl sulfoxide (DMSO) or thalidomide. After 2.5 h, cycloheximide (CHX) (10 µg/ml) or MG132 (10 µM) was added, as indicated. At various time points, PTEN protein levels were measured in cell extracts by ELISA and normalized to the values for the solvent control. The data represent the means ± standard deviations (error bars) from three individual experiments. nv, normalized values.

 
A translation-independent effect of thalidomide was further supported by experiments using MG132, a proteasome inhibitor (3), which increased PTEN protein levels in solvent-treated cells (Fig. 4C). Notably, the effect of MG132 was very similar to that of thalidomide at all measured time points, and the combination of MG132 with thalidomide did not significantly up-regulate PTEN further compared to treatment with the single agents alone (Fig. 4C). Thus, these data suggest that thalidomide up-regulates active PTEN by inhibiting its proteasomal degradation, thereby resulting in the down-regulation of Akt activity.

Stimulation of Akt signaling counteracts thalidomide-induced apoptosis. To investigate whether down-regulation of Akt activity is responsible for thalidomide-induced cell death, we transfected HEFs with a construct encoding constitutively active (membrane-targeted) Akt (mtAkt). Compared to nontransfected HEFs, the transient expression of mtAkt counteracted thalidomide-induced cell death (Fig. 5A). For a control, we transiently expressed dominant-negative Akt that slightly induced cell death in HEFs independently of thalidomide treatment (Fig. 5A). Interestingly, insulin, a stimulator of PI3K/Akt signaling, partially prevented thalidomide-induced apoptosis in HEFs in a concentration-dependent manner (Fig. 5B).


Figure 5
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  		FIG. 5. Stimulation of the insulin/IRS/PI3K/Akt pathway inhibits thalidomide-induced apoptosis. (A and C) HEFs were transfected with plasmids encoding constitutively active membrane-targeted Akt (mtAkt), dominant-negative Akt (dnAkt), or dominant-negative IRS-1 (IRS-1F6 and IRS-1{Delta}C) mutants or with the empty vector (control). Twenty-four hours posttransfection, cells were treated with thalidomide (thal) (+) or solvent (–) (B and D). HEFs were incubated with the indicated concentrations of insulin, with 10 nM wortmannin, or with 10 µM PD098059. After 6 h of incubation in the presence (+) or absence (–) of thalidomide (thal), the ratios of dead to live cells were determined and normalized to the values for the solvent control. The data represent the means ± standard deviations (error bars) from nine (A) and six (B to D) individual experiments. Values that are significantly different (P < 10–3) from the values for thalidomide-treated (nontransfected) cells (A and B) or thalidomide- and insulin-treated cells (C and D) are indicated by an asterisk. nv, normalized values.

 
Canonical insulin signaling involves the stimulation of IRS-1 and -2 with subsequent activation of the PI3K/Akt and/or the Grb2/Erk (extracellular signal-regulated kinase) pathway (11). To elucidate whether the rescue by insulin involves downstream signaling through IRS proteins, we transiently expressed dominant-negative IRS-1 mutants that compete with endogenous IRS-1 and -2 (40). These mutants are incapable of interacting with PI3K because they lack either essential C-terminal tyrosine phosphorylation sites (IRS-1F6) or the entire C terminus (IRS-1{Delta}C). Noteworthy, the protecting effect of insulin on thalidomide-induced apoptosis was counteracted by the two dominant-negative IRS-1 mutants (Fig. 5C) and, furthermore, by the PI3K inhibitor wortmannin (44) but not by PD098059, an inhibitor of the Erk pathway (27) (Fig. 5D). Thus, thalidomide-induced apoptosis is at least partially caused by down-regulation of Akt signaling and can be counteracted by stimulation of insulin/IRS/PI3K signaling.

Thalidomide-induced PTEN activity causes apoptosis independently of Dkk1. To elucidate the relevance of thalidomide-induced PTEN activity for increased apoptosis, we treated HEFs with a combination of thalidomide and bpV(pic). In the nanomolar range, bpV(pic) is a specific and efficient inhibitor of PTEN activity with a 50% inhibitory concentration of 31 nM in vitro (33). At concentrations of up to 100 nM, bpV(pic) reduced thalidomide-induced apoptosis to ~50% (Fig. 6A). As reported before, thalidomide-induced apoptosis is partially caused by a Bmp-mediated up-regulation of Dkk1 expression (20). Accordingly, a Dkk1-blocking antibody reduced thalidomide-induced apoptosis to approximately 50% in HEFs (Fig. 6A). This neutralizing effect was enhanced up to 85% in HEFs treated with a combination of thalidomide, bpV(pic), and anti-Dkk1 antibody (Fig. 6A).


Figure 6
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  		FIG. 6. Inhibition of PTEN counteracts thalidomide-induced apoptosis. (A) Effect of the PTEN inhibitor bpV(pic). HEFs were treated with the indicated concentrations of bpV(pic) and with a solvent control or with thalidomide (thal) in the presence and absence of neutralizing DKK1 antibodies (anti-Dkk1 antibody [{alpha}Dkk1]). (B) Effect of siRNA-mediated down-regulation of PTEN. HEFs were transfected with a nontargeted control or a PTEN-specific siRNA and treated with thalidomide or solvent 24 h posttransfection. The efficiency of siRNA-mediated silencing of PTEN expression was verified by Western blot analyses. Cell death in panels A and B was determined 6 h after thalidomide treatment and is given as the means ± standard deviations (error bars) from eight individual experiments. Values that were significantly different from the values for thalidomide-treated cells (P < 10–5 [ast]) are indicated. Values that were significantly different (P < 0.01) from the values for the corresponding thalidomide- and bpV(pic)-treated cells (A) or thalidomide-treated cells transfected with PTEN siRNA (B) are indicated (#). (C) Effect on LEF/TCF-dependent reporter gene activity. HEFs were transfected with TOPflash and with the PTEN-specific siRNA as indicated. Incubation was performed in Wnt3a-conditioned medium or control medium in the presence (+) or absence (–) of Dkk1-blocking antibodies. Values were normalized to the values for TOPflash-transfected cells incubated with control medium and to the number of living cells. The data represent means ± standard deviations (error bars) from four experiments. Significant differences (P < 10–4) between two values are indicated by an asterisk. (D) Effect on FasL expression. HEFs, incubated with the PTEN-specific siRNA in the presence and absence of Dkk1-blocking antibodies as described above, were treated with thalidomide or a solvent control. After 6 h, mRNA was harvested and subjected to RT-PCR using FasL- or hypoxanthine phosphoribosyltransferase (HPRT)-specific primers. One representative of two individual experiments is shown. nv, normalized values.

 
The involvement of PTEN in thalidomide-induced apoptosis was confirmed by the use of PTEN-specific siRNA. Transfection of HEFs with PTEN-specific siRNA reduced the PTEN protein level of thalidomide-treated HEFs to that of untreated (and nontransfected) HEFs (Fig. 6B). In contrast to a nontargeted negative-control siRNA, transfection with PTEN-specific siRNA resulted in a clear inhibition of thalidomide-induced apoptosis. This effect was significantly enhanced through the simultaneous application of the anti-Dkk1 antibody (Fig. 6B). sq-RT-PCR demonstrated that siRNA-mediated silencing of PTEN expression did not influence thalidomide-induced Dkk1 transcription (data not shown), and Western blot analyzes revealed that treatment with the anti-Dkk1 antibody did not influence drug-induced PTEN protein levels in HEFs (data not shown). Thus, thalidomide-induced Bmp signaling affects Dkk1 and PTEN independently of each other.

Thalidomide-induced Bmp signaling causes a Dkk1-mediated inhibition of the Wnt/β-catenin pathway (20). Dkk1 prevents binding of the Wnt ligand to the low-density lipoprotein receptor-related protein 5/6 coreceptor. As a consequence, Gsk3β becomes derepressed and phosphorylates β-catenin, resulting in its degradation (28). Besides Wnt signaling, phosphorylation by Akt also represses Gsk3β activity, thereby stabilizing β-catenin (4). To elucidate whether thalidomide-induced down-regulation of Wnt/β-catenin and Akt signaling may converge at Gsk3β/β-catenin, we performed a TOPflash reporter gene assay as a readout for β-catenin activity. In line with our recent data (20), thalidomide counteracted Wnt3a-induced TOPflash activity to ~70% in HEFs (Fig. 6C). As expected, blocking of Dkk1 with an anti-Dkk1 antibody significantly restored thalidomide-induced down-regulation of β-catenin activity (Fig. 6C). Interestingly, inhibition of PTEN expression by the use of a PTEN-specific siRNA also antagonized thalidomide-induced down-regulation of β-catenin activity (Fig. 6C). Although the combined use of PTEN-specific siRNA and anti-Dkk1 antibody significantly enhanced this neutralizing effect, it did not completely restore the negative impact of thalidomide on β-catenin activity (Fig. 6C). These data indicate that thalidomide-induced down-regulation of Wnt and Akt signaling partially converge at Gsk3β/β-catenin to induce apoptosis.

To investigate whether down-regulation of Wnt or Akt signaling is involved in thalidomide-induced Fas death receptor signaling, we demonstrated up-regulation of FasL expression in response to thalidomide by sq-RT-PCR (Fig. 6D). Interestingly, this up-regulation was partially abolished by siRNA-mediated knockdown of PTEN expression, but not by the anti-Dkk1 antibody (Fig. 6D). These results indicate that PTEN/Akt but not Dkk1/Wnt signaling is involved in the activation of the Fas pathway upon thalidomide treatment.

A PTEN/IRS-dependent negative-feedback loop counterregulates thalidomide-induced apoptosis. Induction of apoptosis in HEFs and PLBCs is maximal after 6 hours of thalidomide treatment and thereafter declines, reaching less than 50% of the peak level after 16 h of incubation (20). Interestingly, by sq-RT-PCR, we found that after 8 and 12 h, thalidomide induced a slight but reproducible increase of IRS-2 but not IRS-1 transcripts in HEFs and PLBCs that was, however, not detectable at earlier or later time points of thalidomide exposure (Fig. 7A). The increase of IRS-2 transcription after 12 h was completely neutralized by Noggin and bpV(pic) but not by the Dkk1 blocking antibody (Fig. 7B and C). We did not find up-regulation of IRS-1 or -2 in the limb buds of thalidomide-treated chicken embryos at HH stages 23/24, when thalidomide strongly induces Bmp expression, PTEN activity, and apoptosis (Fig. 1B and 3C and F) (20). However, IRS-2 transcription was enhanced at later stages (HH stage 25/26; Fig. 7D). Transient expression of IRS-1F6 and IRS-1{Delta}C, which compete with endogenous IRS-1 and -2 (40), did not influence thalidomide-induced apoptosis after up to 8 hours of treatment in HEFs. However, both mutants partially prevented the decrease of the level of apoptosis at longer incubation times (Fig. 7E). Consistent with the up-regulation of IRS expression and activity being PTEN dependent, bpV(pic)—in contrast to its neutralizing effect at short incubation times—maintained thalidomide-induced apoptosis almost at the peak level after 12 and 16 h of treatment (Fig. 7E). In summary, these data provide evidence for a PTEN/IRS-dependent feedback loop that counterregulates thalidomide-induced apoptosis.


Figure 7
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  		FIG. 7. A PTEN/IRS feedback mechanism counterregulates thalidomide-induced apoptosis. (A to D) HEFs, PLBCs, or chicken embryos were treated with thalidomide (thal) (+) or a solvent control and/or Noggin, bp(V)pic or anti-Dkk1 antibody ({alpha}Dkk1). After the indicated time points, the RNA was isolated from cultured cells or wing/limb buds pooled from 60 embryos, and IRS-1 and -2 expression was investigated by sq-RT-PCR. Amplification of elongation factor 1{alpha} (EF1{alpha}) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used for control. The results of one experiment representative of two individual experiments are shown. (E) HEFs were treated with thalidomide, a solvent control, and/or bpV(pic) and transfected with IRS-1F6 or with IRS-1{Delta}C as indicated. At the indicated time points, the ratios of dead to live cells were determined and normalized to the values for the solvent control. The data represent the means ± standard deviations (error bars) from six individual experiments. RT0, control RT-PCR without reverse transcriptase.

 
Stimulation of the PI3K/Akt pathway rescues thalidomide embryopathy. In order to verify these data for thalidomide-induced embryopathy, we tested whether the activation of PI3K/Akt signaling in the embryo might rescue thalidomide-caused developmental defects. In agreement with our previous study (20), thalidomide induced limb truncations and microphthalmia independently from each other in 22 and 23% of the embryos, respectively (Fig. 8A to F). Limb and eye defects occurred uni- and bilaterally with different severities. Limb truncations were observed on wing and/or hind limbs. In all cases, both proximal structures (upper and/or lower long bones) and distal structures (autopods) were affected. In extreme cases, embryos displayed amelia (the complete absence of a limb). Since the frequency and distribution of limb and eye abnormalities were very similar to those in our pervious study, refer to Knobloch et al. (20) for a detailed description of the phenotypes.


Figure 8
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  		FIG. 8. Insulin and bpV(pic) prevent thalidomide-induced limb truncations and microphthalmia in chicken embryos. (A and B) Thalidomide-treated 6-day-old embryos with truncated hind limbs are shown. In panel A, the left hind limb (LH) was affected, and in panel B, the right hind limb (RH) was affected. (C and D) Heads of 7-day-old control (C) and thalidomide-treated embryos displaying microphthalmia of the left eye (D) are shown. (E and F) Embryos were treated with insulin (250 nM or 2.5 µM) or bpV(pic) (0.1 or 1 pmol) in the presence (+) or absence (–) of thalidomide (thal). The percentage of embryos with limb truncations (E) or microphthalmia (F) is shown. For a single experiment, between 45 and 60 embryos were analyzed for each kind of treatment. The data in panels E and F represent mean values ± standard deviations (error bars) from four independent experiments. Values that are significantly different (P < 0.01 by the t test) from the values for thalidomide-treated embryos are indicated by an asterisk.

 
The application of insulin at HH stages 17 to 19 to thalidomide-treated embryos caused a significant reduction in the number of embryos showing limb or eye defects in a dosage-dependent manner (Fig. 8E and F). In order to rescue thalidomide-induced phenotypes via PTEN inhibition, we applied bpV(pic) at low dosages to thalidomide-treated embryos to assure that the drug specifically blocks PTEN. We found that application of 1 pmol bpV(pic) per egg significantly reduced thalidomide-induced limb truncations and microphthalmia (Fig. 8E and F). Thus, these results clearly indicate that PTEN-mediated inhibition of Akt activity contributes to thalidomide-induced embryopathy.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, we and others have shown that thalidomide causes oxidative stress-induced apoptosis during embryonic development to provoke birth defects, such as limb truncations and microphthalmia (20, 30). Thalidomide-generated ROS enhance expression of Bmps, resulting in hyperexpression of the Bmp target gene and Wnt antagonist Dkk1. The subsequent suppression of Wnt/β-catenin signaling contributes to apoptosis. However, since Dkk1 inhibition only partially neutralizes thalidomide effects in isolated cells and in vivo, thalidomide-induced Bmp signaling has been assumed to cause apoptosis and embryopathy also independently of Dkk1 (20).

Here we show that such a Dkk1-independent signal is provided by the thalidomide-induced activation of the phosphatase PTEN. The Bmp-specific inhibitor Noggin abolished thalidomide-induced up-regulation of PTEN protein levels and activity. Thalidomide did not influence PTEN transcription or translation but led to stabilization of the phosphatase, like proteasome inhibitor MG132. Moreover, the combined addition of thalidomide and MG132 did not significantly further up-regulate the cellular PTEN protein level from that of treatment with each agent alone. We conclude that thalidomide-induced Bmp signaling protects active, dephosphorylated PTEN from degradation by the proteasome. Since PTEN is an antagonist of the PI3K/Akt pathway and acts upstream of Akt, this hypothesis is supported by our finding that thalidomide suppresses Akt activity. Consistent with our data, PTEN has two canonical PEST motifs, which characterize short-lived proteins degraded by the ubiquitin pathway, and Bmps have been demonstrated to stabilize active PTEN in a breast cancer cell line by preventing its degradation by the proteasome (45, 47).

Insulin has been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying mesoderm (progress zone [PZ]) required for proximo-distal limb outgrowth during early embryonic development (9). The limb buds of amelic wingless (wl) and limbless (ll) mutant chick embryos form at the proper time in development but fail to undergo further outgrowth and subsequently degenerate, because they lack a thickened AER. Insulin induces proliferation in the thin AER of explanted wl or ll limb buds, leading to the formation of structures that resemble normal AERs. Moreover, the application of exogenous insulin causes directed outgrowth of these mutant limb buds in vitro (10). Interestingly, the application of insulin during early embryonic development rescued thalidomide-induced limb truncations that include amelia. Since inhibition of PTEN caused similar rescue effects, we conclude that insulin governs limb outgrowth and neutralizes thalidomide effects via stimulation of the PI3K/Akt pathway. We have previously shown that limb truncations in thalidomide embryopathy are the consequence of increased apoptosis in the AER and in the PZ (20). Stimulation of Akt signaling in embryonic cells by insulin treatment, expression of constitutively active Akt, or inhibition of PTEN partially neutralized thalidomide-induced apoptosis. Our results therefore indicate that besides down-regulation of Wnt/β-catenin signaling (20), suppression of Akt signaling is an important event in thalidomide-induced apoptosis in the AER and PZ.

In the canonical pathway, Wnt ligands activate Disheveled (Dsh), resulting in its interaction with the Axin-Gsk3β complex. This leads to the inhibition of Gsk3β, thereby preventing β-catenin degradation (17). Gsk3β inhibition neutralizes thalidomide-induced apoptosis and rescues embryopathy (20). Since thalidomide induces expression of the Wnt antagonist Dkk1, we had previously concluded that Gsk3β is hyperactivated by thalidomide merely as a consequence of suppression of Wnt activity. In agreement with this hypothesis, we found β-catenin activity to be down-regulated in response to thalidomide (20; this study). Besides the Wnt pathway, Akt also inhibits Gsk3β through phosphorylation of Gsk3β in the Axin complex. Nevertheless, Akt requires activated Dsh to stimulate β-catenin activity (15). Factors, such as insulin, that stimulate Akt through the PI3K pathway but do not activate Dsh fail to cause an increase of active β-catenin (13). Furthermore, constitutively active membrane-targeted forms of Akt also do not enhance β-catenin activity (1), most likely because membrane-anchored Akt is unable to interact with the Axin-Gsk3β complex in the cytosol (15). In contrast to these constitutively active forms, wild-type Akt is activated at the plasma membrane and then translocates to the cytosol (37). Since down-regulation of PTEN expression in thalidomide-treated embryonic cells enhances β-catenin activity, we believe that suppression of Wnt and Akt signaling converges at Gsk3β in thalidomide-induced molecular pathology. However, both insulin and expression of constitutively membrane-anchored Akt partially neutralized thalidomide-induced apoptosis. This indicates that thalidomide-induced Akt suppression also contributes to increased apoptosis independently of Gsk3β.

Our findings that thalidomide induces caspase-dependent apoptosis by two different apoptotic pathways, the intrinsic pathway and the Fas death receptor pathway, exactly fit this hypothesis. Gsk3β paradoxically exerts pro- and antiapoptotic functions, since it promotes cell death caused by the intrinsic pathway but inhibits death receptor pathways (4). On the other hand, Akt signaling is known to counteract both the intrinsic pathway and the Fas death receptor pathway. For example, Akt inactivates Bad, a proapoptotic member of the Bcl-2 family, which activates the intrinsic pathway, and Akt phosphorylation of Forkhead transcription factors blocks expression of FasL (5, 8). We therefore believe that suppression of Wnt and Akt signaling in response to thalidomide hyperactivates Gsk3β with subsequent stimulation of the intrinsic apoptotic pathway. In a Gsk3β-independent manner, Akt suppression might additionally cause the activation of Fas signaling, thereby surpassing the blocking effect of Gsk3β on this death receptor pathway. In agreement with this hypothesis, inhibition of PTEN but not of Dkk1 neutralized thalidomide-induced FasL expression.

PTEN blocks the insulin/IRS/PI3K/Akt pathway by converting PIP3 to PIP2. However, PTEN overexpression in breast cancer cell lines has been shown to induce expression of IRS-2 that subsequently is activated by tyrosine phosphorylation and binds to the p85 subunit of PI3K (35). The authors suggested a feedback mechanism that restores antiapoptotic IRS-2/PI3K signaling via the increase of PIP3 levels. We have shown that thalidomide induces IRS-2 transcription in HEFs in a PTEN-dependent manner at a time point when apoptosis already declines. This decline of cell death is counteracted by the expression of dominant-negative IRS mutant proteins that compete with endogenous IRS-1 and -2 for binding to upstream activators. Consistently, thalidomide did not up-regulate IRS-2 in limb buds at HH stages 23/24, when Bmp expression and apoptosis are most strongly induced, but did so only at HH stages 25/26, when thalidomide-induced Bmp expression and apoptosis can hardly be detected (J. Knobloch, unpublished observation). By taking the study mentioned above in consideration, we postulate a PTEN/IRS-2-dependent negative-feedback mechanism that counteracts thalidomide-induced apoptosis with delay. However, from our data, we cannot exclude a role for IRS-1 in this scenario.

Thalidomide is an effective treatment in various inflammatory diseases, such as Crohn's disease and rheumatoid arthritis, due to its anti-inflammatory and immunosuppressive effects. Although the exact mechanisms are still unknown at this point, down-regulation of TNF alpha (TNF-{alpha}) is central for the inhibitory action of thalidomide. Thalidomide stimulates the intrinsic apoptotic pathway via Akt suppression but not Fas signaling in human monocytes, which are a major source of proinflammatory mediators, such as TNF-{alpha} (16). This indicates that the molecular mechanisms responsible for the teratogenic and immunosuppressive effects of thalidomide share similarities but also exhibit differences.

In summary, we suggest the following model for thalidomide-induced molecular pathology in embryonic development (Fig. 9). The generation of ROS by thalidomide increases signaling through Bmps. In turn, Dkk1 and PTEN are hyperactivated, resulting in the suppression of Wnt/β-catenin and Akt signaling with subsequent activation of the intrinsic apoptotic pathway and Fas signaling. Increased apoptosis in the PZ and AER during early embryonic limb development finally causes limb truncations. A PTEN/IRS-dependent negative-feedback loop that counterregulates apoptosis is activated with a delay. Thalidomide-induced limb truncations and microphthalmia can be rescued by the same reagents, such as activators of Wnt or Akt signaling, indicating that thalidomide-induced molecular pathologies for limb and eye defects are essentially identical.


Figure 9
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  		FIG. 9. Model to explain thalidomide-induced molecular pathology responsible for limb truncations and microphthalmia. For details, see text. IR, insulin receptor.

 


   ACKNOWLEDGMENTS
 
We thank Wioletta Hörschken, Peter Sikorski, Carina Meyer, and Daniel Scholtyssik for excellent technical assistance and Dario R. Alessi, Ya-Wei Qiang, Michael Quon, and Harald Wajant for providing reagents.

This work was supported in part by the SFB 590 (U.R.).


   FOOTNOTES
 
* Corresponding author. Present address: University of Cologne, Medical Clinic III, Department of Pneumology, Kerpener Strasse 62, D-50924 Cologne, Germany. Phone: 49-221-4784191. Fax: 49-221-47887031. E-mail: juergen.knobloch{at}uk-koeln.de Back

{triangledown} Published ahead of print on 26 November 2007. Back


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 DISCUSSION
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Molecular and Cellular Biology, January 2008, p. 529-538, Vol. 28, No. 2
0270-7306/08/$08.00+0     doi:10.1128/MCB.00533-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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