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Published as doi: 10.1096/fj.06-7603com.
 (The FASEB Journal. 2007;21:1410-1421.)
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Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway

Jürgen Knobloch*, John D. Shaughnessy, Jr.{dagger} and Ulrich Rüther*,1

* Institut für Entwicklungs-und Molekularbiologie der Tiere, Heinrich-Heine-Universität, Düsseldorf, Germany; and

{dagger} Donna D. and Donald M. Lambert Laboratory for Myeloma Genetics, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

1Correspondence: Institut für Entwicklungs-und Molekularbiologie der Tiere, Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany. E-mail: ruether{at}uni-duesseldorf de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thalidomide, a sedative originally used to treat morning sickness and now used to treat leprosy and multiple myeloma, is also a teratogen that induces birth defects in humans such as limb truncations and microphthalmia. However, the teratogenic mechanism of action of this drug remains obscure. Thalidomide induces limb and eye defects in the chicken embryo at an EC50 of 50 µg/kg egg wt and apoptosis in primary human embryonic fibroblasts (HEFs) at an EC50 of 8.9 µM. Using these model systems, we demonstrate by semiquantitative reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization that thalidomide-induced oxidative stress enhances signaling through bone morphogenetic proteins (Bmps). This leads to up-regulation of the Bmp target gene and Wnt antagonist Dickkopf1 (Dkk1) with subsequent inhibition of canonical Wnt/ß-catenin signaling and increased cell death as shown by trypan blue and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Thalidomide-induced cell death was dramatically reduced in HEFs and in embryonic limb buds by the use of inhibitors against Bmps, Dkk1, and Gsk3ß, a ß-catenin antagonist acting downstream of Dkk1 in the Wnt pathway. Most interestingly, blocking of Dkk1 or Gsk3ß dramatically counteracts thalidomide-induced limb truncations and microphthalmia. From this, we conclude that perturbing of Bmp/Dkk1/Wnt signaling is central to the teratogenic effects of thalidomide.—Knobloch, J., Shaughnessy, Jr., J. D., Rüther, U. Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway


Key Words: limb truncations • microphthalmia • teratogenic mechanism • programmed cell death


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE TERATOGENICITY OF THALIDOMIDE, a sedative used in early pregnancy, resulted in congenital defects in thousands of human fetuses. Numerous studies have revealed that many tissues and organs like eyes and heart, for example, are affected by thalidomide during embryonic development. However, variable limb truncations such as amelia (absence of limbs) and phocomelia (proximal limb truncations) are most frequent (1 2 3) . Current hypotheses presume that thalidomide interferes with limb outgrowth by inducing oxidative stress in the limb mesenchyme and/or by inhibiting angiogenesis (4 , 5) . However, the molecular pathways essential for mediating thalidomide teratogenicity remain unknown.

Studies of the soluble Wnt inhibitor Dickkopf1 (Dkk1) revealed that canonical Wnt/ß-catenin signaling is involved in limb morphogenesis (6 , 7) . Dkk1 is known to promote programmed cell death (PCD) in the developing limb (6) , and Dkk1 expression is induced by Bmp4 and Bmp5 (6 , 8) . However, since all Bmps with important functions during limb development (Bmp2, -4, -5, and -7) signal through the same Bmp type I receptor (BmpR-IA) in the limb mesenchyme (9 10 11) , it is likely that also Bmp2 and Bmp7 might regulate Dkk1 expression. Given that thalidomide can activate DKK1 expression in multiple myeloma plasma cells (12) and that over-expression of Dkk1 in chicken wing buds causes wing truncations (6) , we hypothesized that DKK1 might be the principle mediator of thalidomide-induced embryopathy.

Remarkably, the teratogenic effect of thalidomide is strictly species specific. Thalidomide exposure to certain nonhuman primates in utero induces limb malformations identical to those seen in humans. Whereas standard model organisms like mice and rats are thalidomide-resistant, New Zealand White rabbits and chickens show embryopathies, including a high frequency of limb truncations on exposure to thalidomide (13 14 15 16) . In humans and rabbits, the result of thalidomide treatment is primarily phocomelia, whereas in chicken limbs both proximal and distal structures are affected. In extreme cases, all three organisms display amelia.

Several reports indicate that differences in the potential to counteract thalidomide-induced oxidative stress might be the explanation for the species specificity of the teratogenic properties of the drug. Thalidomide induces oxidative stress through the formation of free radical-initiated reactive oxygen species (ROS) in limb bud cells and embryos of thalidomide-sensitive rabbits but not in those of thalidomide-resistant mice or rats (4 , 17) . The cellular response to ROS production primarily consists of removal of free radicals through the reduced-glutathione (GSH)-dependent detoxification pathways. GSH is oxidized to glutathione disulfide in the detoxification processes, leading to a shift in the intracellular redox potential, thus resulting in a more oxidative environment. Dramatic oxidative intracellular redox potentials can modulate signaling and gene expression thereby inducing apoptosis (4) . The overall embryonic redox potential and especially that of limb buds are much more oxidative in thalidomide-sensitive rabbits than in thalidomide-resistant rats. Furthermore, rat limb buds possess higher GSH stores to buffer redox potentials altered by ROS than do rabbit limb buds (4) . Thus, it has been suggested that thalidomide-resistant species are less susceptible to thalidomide-induced ROS formation and redox misregulation than thalidomide-sensitive species.

In this study, we show that thalidomide-induced limb defects and microphthalmia (small eyes) are caused by an oxidative stress mediated up-regulation of Bmp signaling. As a consequence, Dkk1 expression is induced, leading to an inhibition of canonical Wnt signaling and increased PCD. Both thalidomide-induced PCD and birth defects can be inhibited by blocking Dkk1 or by activating canonical Wnt signaling downstream of the ligand-receptor interaction. Furthermore, we demonstrate the relevance of the data generated in the chicken model system by showing that thalidomide induces identical molecular changes in primary human embryonic cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chicken embryos and drug treatment
Fertilized eggs were purchased from Deindl (Rietberg-Varensell, Germany). They were incubated at 38°C in a humidified incubator, windowed after one day and staged according to Hamburger and Hamilton (HH stage). Racemic thalidomide (Celgene, Summit, NJ), thalidomide enantiomers, or phthalimide (Sigma, St. Louis, MO, USA) was solved in DMSO at 5 mg/ml and further diluted in PBS. Embryos were exposed to drugs by dropping 15 µg to 3 mg thalidomide or phthalimide per kg egg wt (~1–200 µg per embryo) in 50 µl DMSO/PBS as a single dose at HH stages 0–13 onto the embryo. Fifty micrograms of thalidomide or phthalimide in 10 µl DMSO were injected into the extraembryonic blood vessels of HH 19 stage embryos. LiCl (99% pure, Sigma) was solved in sterile PBS; 0.1, 0.6, or 3.2 mg LiCl were applied at HH stages 17–19 by dropping 500 µl of a 5, 28, or 151 mM LiCl solution onto the embryo. Recombinant Noggin, Dkk1 specific goat IgG (R&D Systems, Minneapolis, MN, USA), or serum of nonimmunized goats (Sigma) was solved in 0.1% BSA/0.01% Fast Green/PBS; 10 or 100 ng Noggin, 5 or 50 ng Dkk1 specific goat IgG, or 50 ng serum of nonimmunized goats were applied to a single embryo at HH stages 17–19 by injecting a 10 µl solution through the extraembryonic membranes.

Primary cells and drug treatment
Primary limb bud cells (PLBCs) were isolated from 60 pooled wing and hind limb buds of HH stages 23/24 embryos. Fibroblasts were gained from HH stage 34 chicken embryos or from 13.5 d.p.c. mouse embryos. Human embryonic fibroblasts were a kindly gift from Matthias Maass (18) . Before the reagents were added, all cell types were cultured for 16 h in DMEM (Life Technologies, Carlsbad, CA, USA) plus 10% fetal calf serum (FCS) and 2% chicken serum. Treatment with thalidomide, phthalimide, recombinant human (rh) BMP4 (300 ng/ml; R&D Systems), juglone (5 µM; Sigma), recombinant mouse Noggin (30, 300, and 1000 ng/ml or 300 ng/ml; see Results), anti-human-DKK1 antibody (35, 350, and 1000 ng/ml or 350 ng/ml; see Results), LiCl (3, 30, 60 mM or 30 mM; see Results) and/or (2Z,3E)-6-bromoindirubin-3-oxime (BIO, Gsk3-inhibitor IX; 50, 500, and 1000 nM or 500 nM or 50 and 500 nM; see Results; Calbiochem, San Diego, CA, USA) were performed in Optimem1 (Invitrogen, Carlsbad, CA, USA) plus 1% FCS. Unless otherwise noted, all treatments with thalidomide and rh-BMP4 and associated controls were done for 6 h. Inhibitors were applied once at the beginning of incubation with the exception of LiCl that was added 150 min before harvesting the cells for further analyses. Pretreatment with phenyl N-t-butylnitrone (PBN; 2 mM; Sigma) was done for 1 h. Incubations with juglone were performed for 2 h, if applicable, after a pretreatment with PBN, Noggin, anti-Dkk1 antibody, LiCl, or Gsk3-inhibitor IX for 1 h.

Cartilage staining
Cartilage staining was done as described elsewhere (6) .

Plasmids
The ß-catenin S45A plasmid encodes a stable and constitutively active form of human ß-catenin under the control of a cytomegalovirus (CMV) promoter (19) . A construct encoding the green fluorescent protein (GFP) downstream of a CMV promoter was used as a control. After transfection with Lipofectamine (Invitrogen) for 24 h, thalidomide treatment and measuring of dead cells were done as described above or below, respectively.

TOP/FOPflash assay
Plasmids with wild-type (TOPflash) or mutated (FOPflash) LEF/TCF binding sites (20) upstream of a luciferase reporter gene were transiently transfected into chicken embryonic fibroblasts (CEFs) using Lipofectamine (Invitrogen). After transfection for 24 h, the cells were exposed to Wnt3a-conditioned medium or to control medium (21) with or without thalidomide or recombinant human DKK1 (150 ng/ml, R&D Systems) for 24 before luciferase assay. Values were normalized for transfection efficiency by cotransfection with a pSV-ß-galactosidase vector and for the number of living cells. Luciferase activity was measured using the Dual-Light System (Applied Biosystems, Foster City, CA, USA).

Subcellular localization
CEFs or human embryonic fibroblasts (HEFs) attached on cover slips were incubated with Wnt3a-conditioned medium including thalidomide or solvent or with control medium for 5 h. ß-catenin was detected by using a mouse anti-ß-catenin antibody (1:500, BD Transduction Laboratories, Franklin Lakes, NJ, USA), an anti-mouse Cy3-coupled secondary antibody (Dianova, Hamburg, Germany), and fluorescence microscopy. Afterward, a 4',6'-diam idino-2-phenylidole (DAPI) staining was performed as a control. Quantification was done by counting the nuclei with a clear fluorescent signal in relation to the number of DAPI-stained nuclei.

Detection of transcriptional activity
RNA isolation, semiquantitative RT-PCRs, and whole mount in situ hybridizations were performed as described previously 6 ).

Detection of cell death
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining was performed as described previously (6) with minor modifications. Cell death in PLBCs, CEFs, embryonic fibroblasts of mice (MEFs), and HEFs was detected by staining with trypan blue (0.1% (w/v) in PBS; Sigma) for 90 s. Cells were counted in a counting chamber to determine the ratio of dead to live cells.

Statistical analyses
Two-tailed students t-tests and {chi}2 tests were done using Microsoft Excel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Thalidomide induces limb truncations and microphthalmia in chicken embryos
By performing dose-response and time-course experiments, we found that racemic thalidomide induces a maximum of limb truncations and microphthalmia in chicken embryos when applied at 750 µg/kg egg wt shortly after the primitive streak had fully formed at HH stages 4/5 (Fig. 1 A–C). Therefore, these parameters were used to analyze thalidomide effects on chicken embryonic limb and eye development in detail. Thalidomide-exposed as well as solvent-treated control embryos were analyzed when the three major limb segments, the stylopod, the zeugopod, and the autopod could be demarcated. As a further control, we also analyzed untreated embryos in windowed eggs. About 10% of the embryos of each group died during the first 7 days of incubation due to the breeding conditions. Importantly, in comparison to solvent controls, thalidomide did not significantly increase embryonic lethality. Forty-seven (15%) of a total of 309 thalidomide-treated embryos that survived until day 7 revealed uni- or bilateral limb truncations including amelia (Fig. 1D-F ). Besides amelia, we most frequently found limbs with a complete lack of zeugopodal and autopodal elements and a dramatically shortened stylopod. However, we also observed more mildly affected limbs with missing autopods, truncated zeugopods, and complete or nearly complete stylopods. Of note, both proximal and distal structures were affected in all cases (Supplemental Table 1). Furthermore, thalidomide caused uni- or bilateral microphthalmia in 21% of the embryos (Fig. 1G, H ; Supplemental Table 2). Only ~5% of the embryos showed both limb abnormalities and microphthalmia, suggesting that these defects develop independent from each other. In most cases, a severe form of microphthalmia was observed (Fig. 1H ); however, milder forms were also detected (Fig. 1G ). Untreated or solvent-treated control embryos showed no or little evidence of limb malformations (0 of 102; 4 of 212, 2%) or microphthalmia (0 of 102; 6 of 212, 3%), respectively. Increasing the amount of thalidomide up to 3 mg/kg egg wt influenced neither the quality nor the quantity of limb truncations or microphthalmia (Fig. 1A ; data not shown). The application of thalidomide at later stages clearly reduced the frequency of limb truncations and microphthalmia (Fig. 1B, C ). Moreover, in relation to the total number of limb defects, the numbers of amelia and of severe truncations were reduced relative to the milder forms described above (data not shown). Accordingly, the proportion of milder eye defects was increased (data not shown). Consistent with effects of thalidomide exposure being specific to a developmental window, we were unable to detect embryonic malformations in response to thalidomide applied before HH stage 4 or beyond HH stage 12 (Fig. 1B, C ). As a control reagent, we used the thalidomide-derivative phthalimide. Thalidomide is composed of a glutarimide and a phthalimide ring, the latter one assumed to be essential for teratogenicity (22) . However, phthalimide itself has reported to be nonteratogenic (23) . As expected, phthalimide did not cause limb or eye defects when applied to chicken embryos (Fig. 1A-C, I, J ).


Figure 1
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  		Figure 1. Thalidomide but not phthalimide induces limb truncations and microphthalmia in chicken embryos. A–C) Thalidomide or phthalimide was applied at HH stages 4/5 for dose-response (A) or at 0.75 mg/kg egg wt (~50 µg per embryo) for time-course experiments (B, C). Ratios of embryos with limb truncations or microphthalmia are indicated. A) 50–60 embryos were analyzed for each drug dosage in 1 experiment. Diagram represents mean values and SD from 3 independent experiments. Limb and eye defects were induced by thalidomide at an EC50 of 50 µg/kg egg wt. B, C) 100–120 embryos were analyzed for each stage and each kind of treatment. D–F) Skeletal preparations were made from thalidomide-treated (Th+) and solvent-treated (Th-) day 6–7 embryos and stained for cartilage. Developing wings are shown in D and E. Note that in F (whole embryo, dorsal view) only left hind limb is truncated, whereas right hind limb and the wings appear to be normal. a, autopod; sc, scapula; st, stylopod; z, zeugopod. G, H) Heads of day 7 embryos are shown. A mild (D, right) and a severe (E) form of microphthalmia is presented. I, J) Embryos were treated as indicated and ratio of embryos showing limb truncations (I) or microphthalmia (J) is given. For 1 single experiment, between 50 and 60 embryos were analyzed for each kind of treatment. Any diagram represents mean values and SD from 4 independent experiments. {chi}2 test: *P < 0.001, related to solvent-treated control embryos. phthal, phthalimide; rac, racemic thalidomide; S-thal, (S)-(–) thalidomide; R-thal, (R)-(+) thalidomide.

Conflicting data exist about potential differences in the teratogenic properties of the two thalidomide enantiomers. Blaschke et al. (24) reported that only the (S)-(-)-enantiomer acts as a teratogen. In contrast, another study did not reveal any differences in the teratogenic potentials of the two enantiomers (25) . To investigate the teratogenic effects of the two optical isomers in our model system, we applied 750 µg/kg egg wt of (S)-(–)-, (R)-(+)-, or racemic thalidomide at HH stages 4/5 to chicken embryos. As the racemic mixture, the (S)-(–)-enantiomer as well as the (R)-(+)-enantiomer induced limb deformities in ~18% and eye defects in ~22% of the embryos (Fig. 1I, J ). Thus, we used racemic thalidomide at 750 µg/kg egg wt for the following experiments.

Thalidomide induces Dkk1 and Bmp expression and apoptosis in limb buds
To determine if thalidomide induces Dkk1 expression during early embryonic limb development, we performed semiquantitative reverse transcriptase-polymerase chain reaction (sqRT-PCR) analyses using RNA of limb buds of HH stages 23/24 chicken embryos. In comparison to controls, Dkk1 expression was dramatically enhanced in limb buds of thalidomide-treated embryos (Fig. 2 A). Since Dkk1 expression is governed by Bmp signaling during early limb development (6 , 8) , we looked for the impact of thalidomide on the expression of Bmp genes. These data showed that the expression of Bmp4, Bmp5, and Bmp7 was clearly up-regulated by the drug (Fig. 2A ). However, the expression level of Bmp2, another Bmp family member with important functions in limb development, was not affected (Fig. 2A ). Notably, Bmp and Dkk1 expression was not altered in response to treatment with phthalimide (data not shown). In limb buds, Bmp4 and Dkk1 are coexpressed in the apical ectodermal ridge (AER), including the underlying mesenchyme and in the anterior and posterior necrotic zones (ANZ, PNZ; ref 6 , 26 ). Whole mount in situ hybridizations revealed that thalidomide induces the transcription of these genes exclusively in their normal expression domains. In 20 of 36 limb buds from thalidomide-treated HH stages 23/24 embryos, Bmp4 expression was dramatically up-regulated in all three domains compared to the controls (Fig. 2B ). Furthermore, in 19 of 64 limb buds, Dkk1 expression was also clearly enhanced in the AER and in the underlying mesenchyme as well as in the ANZ. However, expression in the PNZ appeared almost normal (Fig. 2B , data not shown).


Figure 2
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  		Figure 2. Thalidomide induces Dkk1 and Bmp expression as well as apoptosis in limb buds. Analyses were performed with HH stages 23/24 chicken embryos that were treated as indicated. A) sqRT-PCRs were carried out with RNA isolated from pooled wing and hind limb buds of 60 untreated, solvent treated, or thalidomide-treated embryos. Gapdh was used for normalization. One representative set of sqRT-PCRs of 3 independent experiments is shown. Whole mount in situ hybridizations with Dkk1 and Bmp4 probes (B) and whole mount TUNEL staining (C) were carried out on wing and hind limb buds. Representative sets of hind limb buds (Dkk1, distal view; TUNEL, distal view) and wing buds (Bmp4, dorsal view) are shown. Anterior is always on top. D) TUNEL staining of wing bud cross sections. Dark spots indicate PCD. Dorsal is on right. dmc, distal mesenchyme; pmc, proximal mesenchyme.

Since both Bmp4 and Dkk1 are linked to PCD in the developing limb (6 , 26 , 27) , we analyzed the pattern of apoptosis in limb buds of thalidomide-treated HH stages 23/24 embryos. By performing whole mount TUNEL staining, we found the ANZ, but not the PNZ, to be enlarged in 22 of 40 limb buds. Furthermore, the number of TUNEL-positive cells within the AER was also clearly increased in these limb buds compared to controls (Fig. 2C ). In agreement with the distribution of limb anomalies, enhanced PCD was most frequently observed unilaterally in wing buds and bilaterally in hind limb buds. Since these studies could not establish that thalidomide induces cell death in the mesenchyme, we performed TUNEL staining on wing bud cross sections. Here we found that thalidomide clearly enhances PCD in the distal mesenchyme, which includes the progress zone (PZ) underlying the AER (Fig. 2D ).

Blocking of Bmps, Dkk1, or Gsk3ß counteracts thalidomide-induced cell death
To obtain further insights, we investigated the sensitivity of embryonic cells from multiple species to thalidomide-induced cell death in vitro. First, we tested primary embryonic cells isolated from the mesenchyme of chicken limb buds (PLBCs). Time-course and dose-response experiments revealed that racemic thalidomide caused a maximum of cell death (~6-fold above control) in PLBCs after 6–8 h of treatment at a concentration of 38.7 µM (10 µg/ml; Fig. 3 A, B, E). With the use of these parameters, thalidomide also induced PCD 6-fold in primary CEFs compared to controls (Fig. 3E ). However, the drug did not induce cell death in primary MEFs, a species resistant to thalidomide teratogenicity (Fig. 3E ). Notably, longer incubation times as well as increasing thalidomide concentrations up to 100 µg/ml did not significantly induce cell death above controls (data not shown). In contrast, the potential of thalidomide to induce PCD in HEFs was very similar to that in chicken embryonic cells (PLBCs, CEFs; Fig. 3C-E ). The nonteratogenic thalidomide-derivative phthalimide did not induce PCD in PLBCs or HEFs (Fig. 3A-D ). We also tested HEFs for differences in responses to the thalidomide enantiomers. In agreement with our results from the chicken embryos, we did not find significant differences in the potentials of the two enantiomers to induce PCD in HEFs (Fig. 3F ). As a consequence, we treated primary embryonic cells for 6 h with racemic thalidomide at 10 µg/ml in the following experiments.


Figure 3
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  		Figure 3. Thalidomide induces cell death in primary embryonic cells of thalidomide-sensitive species. A–D) For time-course experiments in PLBCs (A) or HEFs (C), thalidomide or phthalimide was applied at 38.7 µM (thalidomide, 10 µg/ml; phthalimide, 5.7 µg/ml). For dose-response experiments, PLBCs (B) or HEFs (D) were treated with drugs for 6 h. Ratios of dead to live cells were determined after incubation. For dose-response curves, these numbers were normalized to solvent controls. Each diagram represents 4 individual experiments. SD are indicated. PCD was induced by thalidomide at EC50 values of 5.0 µM (PLBCs) or 8.9 µM (HEFs). E) Cells as indicated were treated with thalidomide (+) or solvent (–). Shown are ratios of dead to live cells. Data represent in each case eight individual experiments. SD are indicated. Student’s t test (2-tailed): *P < 10–5, related to solvent controls. F) HEFs were treated with thalidomide forms as indicated at 10 µg/ml and ratios of dead to live cells were determined. Data represent in each case 4 individual experiments. Values were normalized to solvent controls exhibiting basal level of cell death under given conditions. SD are indicated. Student’s t test (2-tailed): *P < 10–5, related to solvent controls.

We next asked whether enhanced Dkk1 and/or Bmp4 expression might be essential for thalidomide-induced cell death. As in whole limb buds, thalidomide induced the expression of both Bmp4 and Dkk1 in PLBCs and in HEFs but not in MEFs (Fig. 4 A). Furthermore, phthalimide did not influence the expression of these genes in PLBCs and HEFs (data not shown). Interestingly, when thalidomide was applied to PLBCs either together with recombinant Noggin, an inhibitor of Bmp signaling (28) , or together with an antibody against Dkk1, thalidomide-induced cell death was clearly reduced (Fig. 4B ). Moreover, the simultaneous addition of Noggin and anti-Dkk1 antibody completely neutralized thalidomide-induced cell death (Fig. 4B ). These results demonstrate that thalidomide acts via Bmps and Dkk1 to induce cell death in PLBCs.


Figure 4
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  		Figure 4. Blocking of Bmps, Dkk1, or Gsk3ß antagonizes thalidomide-induced cell death in embryonic cells of thalidomide-sensitive species. PLBCs, MEFs, or HEFs were treated with thalidomide (+) or solvent (-) and reagents as indicated. The relative transcription levels of the given genes (A) or ratios of dead to live cells (B, C) were determined after incubation. For standardization in A, GAPDH (PLBCs) or Hprt (MEFs, HEFs) was used. For each cell type, 1 representative set of sqRT-PCRs of three independent experiments is shown. B, C) For concentrations of reagents, see Material and Methods. Triangles indicate increasing concentrations. Data represent in each case at least 8 individual experiments. Values were normalized to negative controls (cells exclusively treated with solvent). SD are indicated. Student’s t test (2-tailed): *P < 10–5, related to thalidomide-treated cells. BIO, Gsk3-inhibitor IX

In early limb development, Bmp4 activation leads to up-regulation of Dkk1 with subsequent inhibition of Wnt/ß-catenin signaling and PCD (6 , 29) . Hence, we investigated whether thalidomide could induce cell death in PLBCs in the presence of Wnt signaling activators that operate downstream of Dkk1. Both LiCl and (2Z,3E)-6-bromoindirubin-3-oxime (BIO, Gsk3-Inhibitor IX) are known to block Gsk3ß activity, thus, preventing ß-catenin phosphorylation and degradation (30 , 31) . When applied to thalidomide-treated cells, both LiCl and Gsk3-inhibitor IX were individually able to completely abrogate thalidomide-induced cell death (Fig. 4B ). Taken together, these results suggest that thalidomide causes cell death in PLBCs by blocking Wnt/ß-catenin signaling. Moreover, Noggin, anti-Dkk1 antibody, or LiCl was also able to block thalidomide-induced cell death in CEFs (data not shown) and in HEFs (Fig. 4C ). These data demonstrate that the effects of thalidomide on Bmp/Dkk1/Wnt signaling are not restricted to limb bud cells and that thalidomide affects the same signaling pathways in chicken and human embryonic cells.

Thalidomide inhibits ß-catenin activity
Since Gsk3ß participates in a number of signaling pathways, we performed additional experiments to focus on the impact of thalidomide on canonical Wnt signaling. We transfected CEFs with a TOPflash reporter construct that contains Tcf/Lef binding sites and the activity of which is a quantitative read-out for Wnt/ß-catenin signaling. Treatment with Wnt3a-conditioned medium caused a dramatic induction of TOPflash activity, which was clearly counteracted by recombinant DKK1 (positive control) or thalidomide (Fig. 5 A). We next performed ß-catenin subcellular localization studies. If cells are not exposed to a Wnt signal, ß-catenin remains in the cytoplasm and becomes degraded. However, if a canonical Wnt signal is transmitted through Frizzled and low-density-lipoprotein-receptor-related-protein (LRP) receptors, stabilized ß-catenin enters the nucleus and converts Tcf/Lef to a transcriptional activator (32) . In CEFs and HEFs, ß-catenin accumulated in the nucleus as a response to stimulation with Wnt3a (data not shown, Fig. 5B ). Quantification revealed that ß-catenin was predominantly localized in the nucleus in ~70% of the cells (Fig. 5B ). Treatment with thalidomide in the presence of Wnt3a, however, caused a dramatic reduction in the number of CEFs and HEFs positive for nuclear ß-catenin (data not shown, Fig. 5B ). In HEFs, the number was reduced to 10% (Fig. 5B ). Furthermore, transient expression of constitutively activated ß-catenin counteracts thalidomide-induced cell death in CEFs and HEFs (Fig. 5C ). Taken together, these data demonstrate that thalidomide dramatically inhibits canonical Wnt/ß-catenin signaling.


Figure 5
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  		Figure 5. Thalidomide blocks canonical Wnt signaling. A) CEFs were transfected with TOPflash or FOPflash (negative control) and incubated with Wnt3a-conditioned- or with control medium. Values were normalized to TOPflash-transfected cells that were incubated with control medium and to number of living cells; n = 8, SD are indicated. Student’s t test (2-tailed): *P < 3 x 10–9 related to Wnt3a-treated TOPflash-transfected cells. B) Subcellular localization of ß-catenin in Wnt3a- or Wnt3a- and thalidomide-treated HEFs. DAPI was used for nuclear staining (control). Diagram summarizes proportion of nuclei with a clear fluorescent signal in relation to total number of nuclei of three independent experiments (C) CEFs or HEFs were transfected with constructs encoding constitutively active ß-catenin (S45A) or GFP and treated with thalidomide (+) or solvent (–). After incubation, ratios of dead to live cells were determined. Values were normalized to GFP/solvent controls; n = 9, SD are indicated. Student’s t test (2-tailed): *P < 10–6 related to GFP-transfected thalidomide-treated cells.

Thalidomide-induced ROS formation is a prerequisite for enhanced Bmp expression and inhibition of Wnt signaling
Thalidomide has been shown to induce oxidative stress through the formation of ROS in rabbit model systems (4 , 17) . The free-radical spin trapping agent PBN, which antagonizes oxidative stress, counteracts thalidomide-induced embryopathy in rabbits (17) . In line with these results, thalidomide-induced cell death was completely inhibited by PBN in PLBCs (Fig. 6 A). This demonstrates that thalidomide also induces oxidative stress in the chicken model system. We then asked whether there is a correlation between the impact of thalidomide on oxidative stress and on Bmp/Wnt signaling. Treatment with juglone, a potent inducer of ROS, provokes increased cell death in PLBCs (Fig. 6A ). Juglone-induced cell death could be inhibited by PBN, Noggin, anti-Dkk1 antibody, LiCl, and Gsk3-inhibitor IX (Fig. 6A ). Furthermore, counteracting oxidative stress through pretreatment with PBN abolishes the up-regulation of Bmp4 and Dkk1 expression in thalidomide-treated PLBCs and HEFs (data not shown, Fig. 6B ). From these data, we conclude that thalidomide initially causes oxidative stress leading to alterations of Bmp and subsequently of Wnt/ß-catenin signaling.


Figure 6
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  		Figure 6. Up-regulation of Bmp signaling is a consequence of thalidomide-induced oxidative stress. A) PLBCs were treated as indicated, and after incubation ratios of dead to live cells were determined. Values were normalized to solvent controls; n = 8, SD are indicated. Student’s t test (2-tailed): *P < 10–8 related to thalidomide- or to juglone-treated cells. B) HEFs were treated as indicated and relative transcription levels of given genes were investigated by sq-RT-PCR. HPRT was used for standardization. 1 representative set of sqRT-PCRs of 2 independent experiments is shown. C) PLBCs were treated as indicated. Data summarize ratios of dead to live cells of 4 individual experiments. Values were normalized to the negative control (untreated cells). SD are indicated. Student’s t test (2-tailed): *P < 10–3, **P < 10–5, related to Bmp4-treated cells. Triangle indicates increasing concentrations of BIO (Gsk3-inhibitor IX; see Material and Methods). It should be noted that pretreatment with 2 mM PBN completely reduces thalidomide- and juglone-induced cell death (A), whereas it has no significant effect on Bmp4-induced cell death (C).

To support the hypothesis that the inhibition of canonical Wnt signaling is a consequence of up-regulated Bmp signaling, we induced PCD in PLBCs by treatment with recombinant BMP4. The activation of ß-catenin through the addition of Gsk3-inhibitor IX or LiCl to BMP4-treated PLBCs counteracted BMP4-induced PCD in a dose-dependent manner (Fig. 6C ). In contrast, treatment with PBN did not influence BMP4-induced PCD. These results confirmed that Bmp signaling acts upstream of canonical Wnt signaling but downstream of ROS formation in thalidomide-induced molecular pathology responsible for PCD.

Inhibition of Bmps, Dkk1, or Gsk3ß counteracts thalidomide-induced teratogenicity
Following the insights obtained from the in vitro data, we treated chicken embryos with a combination of thalidomide and LiCl (0.6 mg per embryo) and performed TUNEL staining on whole limb buds. Clearly, enhanced PCD was observed in 53% (42 of 79) of the individual limb buds isolated from thalidomide-treated HH stage 23/24 embryos. This number was reduced to 24% (19 of 80) when LiCl was applied to thalidomide-treated embryos at HH stages 17–19. Although our analyses did not permit the evaluation of small quantitative differences in the levels of PCD between different limb buds, they nevertheless indicated that the suppression of thalidomide-induced cell death by LiCl is rather an all-or-nothing than a gradual effect.

We next analyzed embryos for embryopathy. Thalidomide treatment caused limb truncations in 19% of the embryos that survived to day 7 of embryonic development. This number was reduced to 5% when Noggin (100 ng/embryo) or LiCl (0.6 mg/embryo) was applied at HH stages 17–19 to thalidomide-treated embryos (Fig. 7 A). Inhibitor application at later stages (HH stages 22–24) did not prevent limb deformities anymore (data not shown). To ask whether enhanced Dkk1 expression is responsible for thalidomide-induced embryopathy, we treated thalidomide-exposed HH stages 17–19 embryos with a Dkk1 specific goat IgG. Limb truncations were found in 15% of the thalidomide-treated control embryos and in 3% of the embryos treated with both thalidomide and anti-Dkk1 antibody (50 ng/embryo; Fig. 7B ).


Figure 7
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  		Figure 7. Noggin, LiCl and Dkk1 antiserum antagonize thalidomide-induced limb truncations and microphthalmia in chicken embryos. Embryos were treated as indicated (goat serum: serum of nonimmunized goats; {alpha}Dkk1: Dkk1 specific goat IgG) and the ratio of embryos showing limb truncations (A, B) or microphthalmia (C, D) is given. Triangles indicate increasing Noggin, {alpha}Dkk1 antibody, or LiCl dosages (see Material and Methods). For 1 single experiment, between 35 and 45 embryos were analyzed for each kind of treatment. Any diagram represents mean values and SD from 4 independent experiments. {chi}2 test: *P < 0.01, related to thalidomide- (A, D) or to thalidomide- and goat serum- (B, C) treated embryos.

We next analyzed the embryos for microphthalmia. Small eyes were found in 23% of the thalidomide-treated control embryos. This number was reduced to 10% through the application of the Dkk1 antibody to thalidomide-treated embryos (Fig. 7C ). In contrast, the application of 0.6 mg LiCl did not rescue thalidomide-induced microphthalmia. However, increasing the dosage to 3.2 mg LiCl per embryo caused a reduction in the number of thalidomide-induced microphthalmia to 9% (Fig. 7D ).

It should be noted that although the inhibitors reduced the quantity of limb truncations and eye defects, they did not influence their spectrum of intensities. Thus, in agreement with the effect of the inhibitors on thalidomide-induced PCD in limb buds, the phenotypic rescue is an all-or-nothing effect. Importantly, no additional anomalies due to inhibitor-treatment were observed. In summary, these results demonstrate that the thalidomide-induced molecular pathologies responsible for limb truncations and microphthalmia are essentially identical.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here demonstrate that thalidomide-induced PCD, limb truncations, and microphthalmia are a result, at least in part, of a cascade of events that includes ROS generation, enhanced Bmp signaling, activation of the Wnt antagonist Dkk1, and suppression of canonical Wnt/ß-catenin signaling. The fact that thalidomide-induced embryopathy could be traced to increased level of ROS is consistent with previous studies (17) . One important question that remains unresolved is whether and how these key events in thalidomide-induced molecular pathology are connected. In line with our study, it has been shown that Bmps promote PCD during normal embryonic limb and eye development (26 , 33) . Furthermore, we have previously demonstrated that Bmp4 and Dkk1 are coexpressed at the sites of PCD in limb buds (6 ; also this study), that Dkk1 is a Bmp4 target gene, and that Dkk1 induces PCD during embryonic limb development (6) . In our cell culture system, both neutralizing ROS and activating ß-catenin by inhibiting Gsk3ß completely counteract thalidomide-induced PCD. However, Bmp4-induced cell death is completely counteracted by Gsk3ß inhibitors but remains unaffected when ROS is neutralized. Furthermore, counteracting ROS abolishes thalidomide-induced up-regulation of Bmp4 and Dkk1 expression. Taking all these data in consideration, we propose a model whereby thalidomide initially induces ROS formation that in turn causes enhanced Bmp signaling. This leads to the hyperexpression of the Wnt antagonist Dkk1 and subsequent suppression of Wnt signaling. The diminished ß-catenin activity ultimately leads to increased PCD that is responsible for the limb and eye defects during embryonic development. The negative impact of oxidative stress on canonical Wnt signaling has also been provided by studies reporting the up-regulation of Dkk1 expression and the down-regulation of ß-catenin activity by hydrogen peroxide treatment (6 , 34) .

It is important to note that thalidomide-induced molecular pathologies likely involve a spectrum of events of which only a few have been elucidated here. For example, individual use of either Noggin or anti-Dkk1 antibody alone causes only a partial neutralization of thalidomide-induced PCD in cell culture, whereas the combined use these agents results in complete inhibition. Moreover, we observed enhanced Bmp expression and PCD in 55% of the limb buds, whereas Dkk1 expression was only increased in 30%. Thus, thalidomide-induced Bmp signaling might cause PCD also independent of Dkk1. However, the mechanisms by which Bmps trigger PCD independent of Dkk1 are unclear (35) . Dkk1 expression is induced by the ROS-sensitive transcription factors c-Jun and p53 (6 , 36) . It has been suggested that Bmps induce PCD in limb buds through c-Jun-mediated induction of Dkk1 expression (6) . However, the apoptosis-inducing ability of Bmps appears to act independent of p53 (37 38 39) . Hence, a Bmp-independent activation of Dkk1 through p53 on thalidomide-induced ROS generation cannot be ruled out and could explain why Noggin does not completely block thalidomide-induced PCD. Noggin blocks Bmp signaling by direct binding to Bmp4, -5, -7 or other Bmps (28 , 40) . However, the affinity of the Noggin/Bmp interaction varies for different Bmps. For example, Bmp4 is more efficiently bound by Noggin than Bmp7 (28) . Furthermore, it should be noted that Noggin does not bind all Bmp family members and that it is unclear if thalidomide enhances Dkk1 expression through Noggin-insensitive Bmps.

Several teratogens are proposed to generate ROS as a prerequisite for their teratogenic activity; however, the spectrum of birth defects induced by these compounds are distinctly different (41) . A multiplicity of ROS modulates the activity of many signaling pathways including Ras, tyrosine kinase/phosphatase, and phosphatidylinositide 3-OH kinase (PI3K) signaling (41 42 43 44) . We propose that thalidomide metabolites mainly generate a specific kind or a specific subset of ROS that affects a limited number of signaling pathways in specific cell (sub-) types resulting in the thalidomide-specific spectrum of birth defects. This assumption is supported by the observation that at most ~10% of primary embryonic fibroblasts or limb bud cells undergo PCD in tissue culture on thalidomide exposure. Consistently, the drug seems to induce gene expression and PCD exclusively in specific domains of the limb buds. Nevertheless, due to pathway crosstalk, most likely in connection with Bmp and Wnt signaling, other pathways might also be involved in thalidomide-induced molecular pathology. Gsk3ß inhibition completely blocks thalidomide-induced PCD in cell culture and dramatically reduces the number of embryos showing limb and eye malformations. Beside its crucial role in canonical Wnt signaling, Gsk3ß is a direct downstream target of the protein kinase Akt (protein kinase B). Akt activity is stimulated through Wnt signaling and activated Akt blocks Gsk3ß, thereby preventing ß-catenin degradation (45) . This suggests a potential role for Akt signaling in the molecular mechanism of thalidomide-induced embryopathy.

Conflicting results have been obtained on the teratogenicity of the enantiomers of thalidomide. In one study, only the (S)-(–)-enantiomer was claimed to be teratogenic in mice and rats (24) . However, it should be noted that these animals are widely accepted as thalidomide insensitive, and therefore, they are unsuited for such investigations. In contrast, no differences in the teratogenic properties of the two enantiomers have been observed in the thalidomide-sensitive species rabbit (25) and chicken (this study). Furthermore, both enantiomers induce cell death to comparable levels in HEFs. However, it has been reported that thalidomide enantiomers rapidly convert into each other in solution. The half-life periods of both enantiomers are about <20 min in human blood serum or in albumin containing phosphate buffer (46) . These data explain why we have not found any differences in the potentials of the two enantiomers to induce cell death or birth defects. Furthermore, this chemical explanation essentially invalidates this kind of analyses.

In chicken embryos, thalidomide primarily causes severe distal limb truncations affecting stylo-, zeugo-, and autopods, thus proximal as well as distal structures. We have shown that drug-induced PCD occurs in the distal tip of the chicken limb bud, including the AER. Enhanced PCD within the AER mimics AER depletion, and it has been reported that AER depletion disturbs proximo-distal outgrowth resulting in severe distal limb truncations (47 , 48) . In the chicken limb bud, thalidomide also enhances PCD in the ANZ, which is responsible for patterning. We think that ANZ enlargement is not reflected by a prevalence of anterior defects in the resulting anatomy because AER disruption is dominant.

One outstanding question arising from our study is why, unlike humans, thalidomide-treated chicken embryos do not display phocomelia? It has been reported that proximal limb truncations in chickens are inducible by driving mesenchymal cells of the distal part of limb buds into apoptosis when simultaneously the ectodermal cells of the AER remain intact (49) . The distal mesenchyme includes the PZ that assigns positional information to mesenchymal cells, determining the structures they will develop into (50) . Our studies show that thalidomide induces apoptosis in distal mesenchymal cells of the limb bud as well as in the AER. Consequently, proximo-distal outgrowth is disturbed resulting in truncated limbs lacking both proximal and distal structures. However, phocomelia is frequent in human thalidomide embryopathy, suggesting that in human embryos drug-induced PCD occurs in the distal mesenchymal cells and rarely in the AER. This putative discrepancy between chicken and human could be explained by differences in the expression pattern of Dkk1: Dkk1 is strongly expressed in the AER of chicken limb buds, but it is only weakly expressed in the AER of mice, a species closely related to humans (J. Knobloch, unpublished observation). However, it will be a challenge for future studies to elucidate theses differences between chicken and human thalidomide embryopathy.

In this study, we provide evidence that thalidomide induces Bmp signaling and the (subsequent) expression of Dkk1. The resulting down-regulation of Wnt/ß-catenin signaling is important for the teratogenic properties of the drug. Identical molecular consequences were observed in HEFs, demonstrating that the insights gained from our studies in the chicken model system are relevant to humans. Importantly, the effect of thalidomide on Bmp/Dkk1/Wnt-signaling is not only restricted to limb bud cells and limb development but has also been shown for fibroblasts isolated from whole embryos and for eye development. Thus, our insights into thalidomide-induced pathology might be of importance for all tissues affected teratogenically by the drug. Given the strong relationship between embryogenesis and carcinogenesis, the insights into the molecular mechanisms of thalidomide-induced embryopathy described here may also apply to the antineoplastic effects of the drug, which is now being used in the treatment of multiple myeloma. If true, this may form the basis for the development of a newer generation of more potent antineoplastic drugs lacking the teratogenic risks of thalidomide.


   ACKNOWLEDGMENTS
 
We thank Wioletta Hörschken for excellent technical support and Celgene Corporation for providing thalidomide. We are grateful to Ya-Wei Qiang, Matthias Maass, and Sven Schinner for providing cells, antibodies, and constructs; to Christof Niehrs and David Stirling for helpful suggestions; and to Patrick Hill and Renate Dildrop for critical reading of the manuscript. This research was supported by the Fund to Cure Myeloma (J. D. Shaughnessy, Jr.) and by National Institutes of Health grants CA-55819 and CA-97513 (J. D. Shaughnessy, Jr.) from the National Cancer Institute, Bethesda, MD.

Received for publication November 24, 2006. Accepted for publication December 25, 2006.


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