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TOXICOLOGY
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
Received for publication
April 8, 2008
Accepted
June 23, 2008.
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Abstract |
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). Downstream MAPK substrates include transcription factors or their components, such as activator protein 1 (AP-1) and p53 (Turjanski et al., 2007), molecules involved in the detection and response to DNA damage, such as poly(ADP-ribose) polymerase 1 and 
H2AX (Caldini et al., 2005; Sluss and Davis, 2006), as well as a variety of pro- and antiapoptotic factors (Bogoyevitch and Kobe, 2006). Among the most widely studied MAPK families are extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPKs (
, β, , and 
), and c-Jun N-terminal kinases (JNKs; 1, 2, and 3) (Turjanski et al., 2007). The ERK1/2 pathways, which are thought to be triggered preferentially by growth factors and mitogens, play an important role during development in the regulation of cell proliferation and differentiation (Turjanski et al., 2007). Activation of the p38 and JNK MAPKs is triggered by stimuli ranging from growth factors to a variety of stress stimuli, including intracellular pH changes, ultraviolet irradiation, heat shock, DNA-damaging agents, hyperglycemia, ethanol, hypoxia, and oxidative stress (Kyriakis and Avruch, 1996; Ku et al., 2007). Stress-response MAPKs control defense responses that determine whether cells survive, differentiate, or apoptose.
MAPK pathways play crucial roles during normal embryo development. During mouse embryogenesis, extracellular signal-regulated kinase (ERK) is activated in discrete spatial and temporal domains, which are correlated with regions of fibroblast growth factor signaling (Corson et al., 2003). During limb development, ERK activation may transduce proliferation and differentiation signals to regulate growth, pattern formation, and skeletogenesis (Bobick et al., 2007). The p38 MAPK pathway is essential for cartilage formation in limb mesenchyme; inhibition of the p38 MAPK pathway leads to sustained Wnt7a signaling and inhibits precartilage condensation and chondrogenesis (Jin et al., 2006). JNK pathway signaling is of particular importance during neuronal development, because mice lacking both Jnk1 and 2 die during mid-gastrulation with neural tube defects (Sabapathy et al., 1999). In the limb, JNKs may function downstream of bone morphogenetic protein signals to sculpt the limb bud by triggering programmed cell death through induction of Dkk1, an inhibitor of Wnt (Grotewold and Ruther, 2002).
The extent to which disturbances in MAPK signaling pathways mediate teratogen-induced abnormal development remains to be elucidated. All three MAPK pathways were activated in organogenesis-stage mouse embryos exposed in vitro to heat shock, whereas exposure to cyclophosphamide or staurosporine activated only the p38 MAPK pathway (Mirkes et al., 2000). Induction of Dkk1, a downstream target of JNK-c-Jun, was associated with thalidomide-induced limb truncations (Knobloch et al., 2007). Cadmium-induced limb reduction defects in C57BL/6N mice were correlated with decreased activation of ERK1/2 phosphorylation, compared with the less sensitive SWV strain (Elsaid et al., 2007).
Reactive oxygen species modulate MAPK signaling (Torres and Forman, 2003). Oxidative stress, an imbalance between the rate of formation of reactive oxygen species and their detoxification by antioxidant defense systems, is induced by a number of developmental toxicants, including heat shock, cadmium, and thalidomide (Flanagan et al., 1998; Kovacic and Somanathan, 2006). Taken together, these findings have led us to propose that oxidative stress-induced modifications to DNA, proteins, and lipids in the embryo disrupt normal development, at least in part, by affecting MAPK signaling. In previous studies, we found that in utero exposure to a model teratogen, hydroxyurea (HU), disturbs skeletal development, depletes glutathione, increases the formation of 4-hydroxynonenal protein adducts, and induces AP-1 DNA-binding activity in embryos (Yan and Hales, 2005, 2006). AP-1 subunits, Jun, Fos, and activating transcription factor proteins are downstream targets of MAPK signaling (Turjanski et al., 2007). Furthermore, AP-1 plays a crucial role in the decision of cells to proliferate, differentiate, or die; tight regulation of AP-1 activity is necessary for normal limb development (Tufan et al., 2002).
The potential use of selective MAPK signaling pathway inhibitors as drugs has generated intense interest. To test the hypothesis that MAPK signaling pathways play an important role in determining the response of organogenesis-stage embryos to teratogen exposure, we investigated the impact of maternal exposure to teratogenic doses of HU on the activation of MAPKs (ERK1/2, JNK, and p38) in the embryo and evaluated the consequences of selectively blocking these MAPK pathways on the developmental toxicity of HU. Exposure of organogenesis-stage embryos to a model teratogen transiently activates p38 and JNK MAPKs; however, inhibition of this activation has adverse consequences to the embryo.
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Materials and Methods |
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SB203580 (EMD Biosciences, Inc., La Jolla, CA), a p38 MAPK inhibitor (Cuenda et al., 1995), L-JNKI1 (c-Jun N-terminal kinase peptide inhibitor 1, L-stereoisomer; Axxora, LLC, San Diego, CA), a JNK inhibitor (Bonny et al., 2001), or the JNK inhibitor L-TAT control peptide (Axxora LLC) was administered to the female mice before treatment with HU (400 or 600 mg/kg) on GD 9. SB203580 (chloride form) was dissolved in saline and given to the female mice by i.p. injection 30 min before HU exposure; L-JNKI1 or the L-TAT control peptide was dissolved in phosphate-buffered saline (PBS) and administered by tail vein injection immediately before HU treatment. Dams were euthanized on GD 9 at 0.5, 3, or 6 h after HU treatment or on GD 18 for the evaluation of developmental toxicity. On GD 9, the embryos were dissected out in Hanks' balanced salt solution for the subsequent assessment of MAPK activation. On GD 18, the uteri were removed, and the numbers of implantations, resorption sites, and live and dead fetuses were recorded. All of the live fetuses were weighed, inspected for external malformations, and then double-stained with Alcian Blue (cartilage) and Alizarin red S (bone) for the analysis of skeletal malformations, as described previously (Yan and Hales, 2005).
Western Blotting. At the time of collection on GD 9, embryos were pooled by litter. Whole-tissue lysates were prepared for phospho-c-Jun and MAPK determinations. Samples were placed in 40 µl of radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris, pH 7.5) containing 10 µl/ml protease inhibitor cocktail and 20 µl/ml phosphatase inhibitor mix (Active Motif Inc., Carlsbad, CA). The samples were homogenized with an ultrasonicator (Sonics and Materials Inc., Newtown, CT) and centrifuged at 10,000g for 15 min at 4°C. The supernatants were used for immunoblotting.
Proteins from each sample (7.5 or 15 µg, for 15-well or 10-well gels, respectively) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred onto equilibrated polyvinylidene difluoride membranes (BioSciences Inc., Baie d'Urfe, QC, Canada) by electroblotting. Membranes were blocked with 5% skim milk for 1 h at room temperature and then probed overnight at 4°C with primary antibodies against phospho-c-Jun (1:1000), phospho-JNK (1:1000), phospho-p38 (1:1000), phospho-ERK1/2 (1:1000), ERK2 (1:1000), or actin (1:5000). Rabbit polyclonal anti-phospho-c-Jun (Ser63), anti-phospho-JNK (Thr183/Tyr185), monoclonal anti-phospho-p38 (Thr180/Tyr182), and mouse monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). Rabbit polyclonal anti-ERK2 and goat polyclonal anti-actin (I-19) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
After incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000) for 2 h at room temperature, proteins were detected by enhanced chemiluminescence (GE Healthcare). The bands were quantified by densitometric analysis using a Chemi-Imager 400 imaging system (Alpha Innotech, San Leandro, CA); the peak area represents the intensity of the band. Each experiment was replicated five times with different litters (n = 5).
Statistical Analysis. Statistical analyses were done by 
2 tests or by two-way analysis of variance (ANOVA), one-way ANOVA, or one-way ANOVA on ranks, as appropriate, using the SigmaStat computer program (Systat Software, Inc., San Jose, CA), followed by a post hoc Holm-Sidak or Dunn's analysis. The a priori level of significance was p < 0.05.
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Results |
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), we determined the activation of MAPKs in embryos 0.5, 3, and 6 h post-treatment with low (L; 400 mg/kg) or high (H; 600 mg/kg) dose HU (Fig. 1). The phosphorylation of MAPKs (p38, JNK, and ERK1/2) was examined by immunoblot analysis by using phosphospecific MAPK antibodies. High-dose HU induced a transient but dramatic increase in the phosphorylation of p38 and JNK at 3 h (Fig. 1A). The phosphorylation of both p38 and JNK was significantly higher in the 600 mg/kg HU-treated groups than the control groups at 3 h post-treatment; by 6 h, the phosphorylation of p38 and JNK was not statistically different from controls (Fig. 1, B and C). Unlike p38 and JNK, the phosphorylated form of ERK1/2 was not detected by immunoblot analysis, even after maximally loading the samples. When the same membrane was probed with an antibody against ERK2, two distinct bands of nonphosphorylated ERK1/2, at 44 kDa and 42 kDa, were revealed (Fig. 1A). Therefore, in utero exposure to a teratogenic dose of HU triggered the activation of two major stress-responsive MAPKs, p38 and JNK, but did not activate the ERK1/2 pathway. Our next objective was to determine how HU-induced activation of p38 and JNK contributes to its embryotoxicity.
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). L-JNKI1 (L-stereoisomer), a cell-permeable peptide, is a substrate-based peptide inhibitor of the JNK-MAPK pathway (Bonny et al., 2001); L-JNKI1 competitively blocks the interaction between JNK and its substrates, but it does not influence JNK activation itself. Since, to the best of our knowledge, this is the first time that either MAPK inhibitor has been administered in an in vivo study to modify MAPK signaling in embryos, we determined the appropriate dosages needed to block these pathways. Timed-pregnant mice were given SB203580 (0.5, 1.0, or 3.0 mg/kg) or L-JNKI1 (1.5 or 3 µl/10 g) before treatment with HU at 600 mg/kg. Embryos were obtained 3 h after HU exposure, when 600 mg/kg HU activation of p38 and JNK had reached its peak. SB203580 reduced the phosphorylation of p38, compared with the saline control (Fig. 2, A and H, second bar), and prevented the 600 mg/kg HU-triggered phosphorylation of p38 MAPK in a dose-dependent manner; at 3.0 mg/kg, SB203580 reduced the phosphorylation of p38 to a level lower than that in saline controls (Fig. 2, B and H). Furthermore, 3.0 mg/kg SB203580 had no effect on the phosphorylation of JNK (Fig. 2, C, second lane, and J, second bar) or its downstream substrate, c-Jun (Fig. 2, E, last lane, and I, last bar). Based on these data, we decided to use SB203580 at 3 mg/kg dosage for our subsequent study.
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Effects of p38 MAPK Inhibition on HU-Induced Developmental Toxicity. HU at 600 mg/kg elevated the rate of fetal deaths, reduced the body weights of live fetuses, and increased the rate of external and skeletal malformations; in contrast, the only effect of maternal exposure to 400 mg/kg was a reduction in live fetal weights (Figs. 4 and 5). The major external malformations induced by HU exposure were hindlimb ectrodactyly, mainly observed as a completely or partially missing first digit (Tables 1, 2, 3, 4).
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A variety of skeletal malformations was observed in the high-dose HU-exposed fetuses (Fig. 3); among these, vertebral column defects (fused, partially ossified, or misaligned vertebrae) (Fig. 3C), hindlimb ectrodactyly and hemimelia (Fig. 3, C and D), and curly tail defects (Fig. 3C) were predominant; fused or mismatched sternebrae (Fig. 3E) and fused or forked ribs (Fig. 3, C and E) were observed at low incidences. The hindlimb defects observed were primarily missing or truncation of the first digit and tibia (Fig. 3, C and D), and partial or complete absence of more than the first digit, with the sequence from anterior (first digit) to posterior.
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Effects of JNK Inhibition on HU-Induced Developmental Toxicity. L-JNKI1 alone did not induce developmental toxicity, nor did it affect the susceptibility of embryos to HU-induced embryotoxicity, as assessed by the incidence of fetal mortality (Fig. 5A), the extent of growth retardation (Fig. 5B), or the overall incidence of external abnormalities (Fig. 5C). Nevertheless, L-JNKI1 pretreatment specifically enhanced the incidence of hindlimb malformations, both external and skeletal, in 600 mg/kg HU-exposed fetuses (Fig. 5D; Tables 3 and 4).
In the 600 mg/kg HU-treated group, the frequency of hindlimb defects was dramatically increased by L-JNKI1 (from 44.9 to 85.7%). In contrast to this observation, the incidences of other deformities in the HU-exposed fetuses, such as curly tail defects, forelimb defects, open eye, spina bifida, and gastroschisis, were not affected significantly by L-JNKI1 pretreatment. Evaluation of the fetal skeletons (Table 4) revealed similar results: blocking the JNK pathway increased the incidence of hindlimb defects by more than 2-fold (31.1 versus 74.1%, p < 0.01) in the 600 mg/kg HU-exposed group. Again, the frequency of other skeletal abnormalities, including vertebral, sternebrae, rib, and forelimb defects, in the HU-treated fetuses was not altered significantly by L-JNKI1 pretreatment. It is interesting to note that even though the incidence of hindlimb defects increased dramatically in fetuses exposed to HU plus L-JNKI1, neither their pattern nor their severity was significantly affected. Whether L-JNKI1 was administered or not, 600 mg/kg HU exposure mainly affected the preaxial skeletal elements of hindlimbs, which are shown as ablation or truncation of the tibia (31.1 versus 72.2%) or first digit (28.9 versus 72.2%); truncation of the femur was observed rarely (2.2 versus 5.6%).
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Discussion |
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), the ERK kinase pathway does not seem to play a role in the response of the embryo to HU exposure. The early cell death observed in HU-exposed embryos is thought to be due to the formation of free radical species (DeSesso and Goeringer, 1990); the timing of HU-induced oxidative stress (Yan and Hales, 2006) is coincident with the activation of p38 and JNK MAPKs, suggesting that it is these free radical species that trigger MAPK signaling.
The use of pharmacological inhibitors to selectively block activation of either the p38 or JNK MAPK pathways did not rescue the embryos from HU-induced embryotoxicity; rather, inhibition of MAPK activation augmented the embryotoxicity of HU. Thus, HU-induced activation of p38 and JNK MAPKs must stimulate defense responses in the embryo that protect it from this teratogen. It is interesting to note that the consequences of inhibiting MAPK signaling activation were pathway selective: inhibition of p38 MAPK activation enhanced fetal mortality, whereas inhibition of JNK signaling significantly increased the incidence of hindlimb malformations. Indeed, distinct and sometimes antagonistic effects have been reported for these two stress signaling pathways previously (Wada et al., 2007).
The conceptal deaths induced by exposure to HU on gestation day 9 were observed as resorption sites. We predict that these embryo deaths occurred shortly after HU exposure because no identifiable tissues remained by gestation day 18. Murine embryo development becomes dependent on p38 MAPKs at the 8 to 16 cell stage, when inhibition of p38 MAPKs disrupts the assembly and functions of filamentous actin (Paliga et al., 2005). During limb development, bone morphogenetic proteins activate p38 MAPK in interdigital tissues undergoing regression, up-regulating some of the genes that mediate programmed cell death (Zuzarte-Luís et al., 2004). However, p38 MAPK has an opposite role in neuronal cells in vitro: inhibitors of p38 promote cell survival (Horstmann et al., 1998) and have been reported to protect hippocampal cells against ethanol cytotoxicity (Ku et al., 2007). The mechanism by which inhibition of p38 MAPK activation is detrimental in one system, or at one time during development, and plays a protective role elsewhere remains to be determined.
Transient activation of JNK seems to specifically modulate the ability of cells in the hindlimb-forming region of the embryo to respond to stress. In a previous study, we demonstrated that depletion of glutathione elevated the incidence of hindlimb abnormalities induced by HU (Yan and Hales, 2006). This increase in hindlimb malformations was associated with an increase in immunoreactive 4-hydroxy-2-nonenal (HNE) protein adducts in hindlimb-forming regions. 4-HNE, a lipid peroxidation end product, increases the phosphorylation of JNK1 and c-Jun proteins in HBE1 cells and the levels of active phosphorylated forms of c-Jun in cultured neurons (Dickinson et al., 2002; Pugazhenthi et al., 2006). We hypothesize that HU-induced oxidative stress disrupts the redox homeostasis of the hindlimb-forming region of the embryo, increasing 4-HNE protein adducts; either the glutathione depletion or the 4-HNE protein adducts trigger activation of the JNK pathway and increase AP-1-binding activity. Increased AP-1-binding activity will induce the transcription and translation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, and restore the glutathione pool, thus limiting the extent of insult during this critical period of limb development. Redox homeostasis is critical during limb development. The administration of N-acetylcysteine, a glutathione precursor, ameliorated both the oxidative stress and hindlimb defects induced by another model teratogen, 5-bromo-2-deoxyuridine (Sahambi and Hales, 2006). Furthermore, the species specificity of the teratogenicity of thalidomide has been attributed to a difference between rabbit (susceptible) and mouse (resistant) embryo limbs: rabbit limbs have a lower antioxidant capacity and enhanced susceptibility to glutathione depletion (Hansen et al., 2002).
The limb defects induced by HU appeared specifically as the loss of preaxial elements, usually the tibia and first digit; in a few fetuses, more than one digit was missing. Inhibition of stress-activated JNK signaling in the embryo dramatically increased the incidence of hindlimb malformations but did not affect the type of defects observed. This is unexpected because JNK-dependent apoptosis plays a critical role in sculpting limb pattern formation in the chick limb bud (Grotewold et al., 2002) and the Drosophila leg (Manjón et al., 2007). In vitro cell culture studies have shown that the JNK pathway inhibits chondrogenesis (Hwang et al., 2005). The role of stress-activated JNK may be distinct from that of JNK signaling during normal development. When they become available, isozyme-specific JNK inhibitors might help to dissect out the contributions of JNKs 1, 2, and 3 to the stress response.
Although the downstream consequences of activation of the p38 and JNK MAPK pathways are clearly distinct, it is possible that they are regulated by a common upstream pathway. It has been proposed that MAP3Ks serve as "signal hubs" to regulate the specificity of MAPK activation (Cuevas et al., 2007). Apoptosis signal-regulating kinase 1 (ASK1) is one such MAP3K, upstream of JNK and p38 kinases; furthermore, ASK1 is preferentially activated by stress stimuli, specifically by oxidative stress (Cuevas et al., 2007). A dynamic and regional-specific expression of ASK1 was recently found in chick and mouse embryos (Ferrer-Vaquer et al., 2007), including the limb region. Mice deficient in ASK1 develop normally, but this may be due to gene redundancy (Tobiume et al., 2001). Although ASK1 was identified initially as a cell death inducer, evidence is emerging that moderate or transient activation promotes cell survival and differentiation (Takeda et al., 2000).
Selective MAPK signaling pathway inhibitors have potential as drugs for the treatment of human inflammatory diseases, prevention of acute ischemic damage, reduction of neurodegeneration, or inhibition of pancreatic β-cell death. It is interesting to note that there are also natural products, such as resveratrol, tangeretin, and ligustilide, to which pregnant women may be exposed that inhibit MAPK signaling in vitro (Malemud, 2007). In one study in which the impact of manipulation of MAPK activation on embryo development was evaluated, sorbitol-induced JNK activation mimicked the effects of hyperglycemia in inducing diabetic embryopathy (Yang et al., 2007).
It is clear that regulation of MAPK signaling in the conceptus exposed to a developmental toxicant is complex. We have demonstrated that inhibition of the transitory activation of MAPK signaling triggered by such an insult has adverse consequences to the conceptus: p38 MAPK and JNK signaling pathways stimulated defense responses that protected the embryo from damage. The therapeutic potential of MAPK pathway inhibitors is such that understanding the impact of even short-term exposure to these substances during pregnancy is a priority.
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Footnotes |
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Parts of this work were previously presented as follows: Yan J (2008) The Mechanisms of Hydroxyurea Induced Developmental Toxicity in the Organogenesis Stage Mouse Embryo, Ph.D. thesis, McGill University, Montreal, Quebec, Canada.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MAPK, mitogen-activated protein kinase(s); MAP3K, MAPK kinase kinases; AP-1, activator protein 1; ERK1/2, extracellular signal-regulated kinases 1 and 2; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; HU, hydroxyurea; GD, gestation day; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; L-JNKI1, c-Jun N-terminal kinase peptide inhibitor 1, L-stereoisomer; PBS, phosphate-buffered saline; ANOVA, analysis of variance; L, low; H, high; 4-HNE, 4-hydroxynonenal; ASK1, apoptosis signal-regulating kinase 1; HU600, HU at 600 mg/kg; HU400, HU at 400 mg/kg.
Address correspondence to: Dr. Barbara F. Hales, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. E-mail: barbara.hales{at}mcgill.ca
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bobick BE, Thornhill TM, and Kulyk WM (2007) Fibroblast growth factors 2, 4, and 8 exert both negative and positive effects on limb, frontonasal, and mandibular chondrogenesis via MEK-ERK activation. J Cell Physiol 211: 233-243.[CrossRef][Medline]
Bogoyevitch MA and Kobe B (2006) Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases. Microbiol Mol Biol Rev 70: 1061-1095.
Bonny C, Oberson A, Negri S, Sauser C, and Schorderet DF (2001) Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death. Diabetes 50: 77-82.
Caldini R, Barletta E, Del Rosso M, Giovannelli L, and Chevanne M (2005) FGF2-mediated upregulation of urokinase-type plasminogen activator expression requires a MAP-kinase dependent activation of poly(ADP-ribose) polymerase. J Cell Physiol 202: 125-134.[CrossRef][Medline]
Corson LB, Yamanaka Y, Lai KM, and Rossant J (2003) Spatial and temporal patterns of ERK signaling during mouse embryogenesis. Development 130: 4527-4537.
Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, and Lee JC (1995) SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364: 229-233.[CrossRef][Medline]
Cuevas BD, Abell AN, and Johnson GL (2007) Role of mitogen-activated protein kinase kinase kinases in signal integration. Oncogene 26: 3159-3171.[CrossRef][Medline]
DeSesso JM and Goeringer GC (1990) The nature of the embryo-protective interaction of proplyl gallate with hydroxyurea. Reprod Toxicol 4: 145-152.[CrossRef][Medline]
Dickinson DA, Iles KE, Watanabe N, Iwamoto T, Zhang H, Krzywanski DM, and Forman HJ (2002) 4-Hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells. Free Radic Biol Med 33: 974-987.[CrossRef][Medline]
Elsaid AF, Délot EC, and Collins MD (2007) Differential perturbation of the Fgf/Erk1/2 and Shh pathways in the C57BL/6N and SWV embryonic limb buds after mid-gestational cadmium chloride administration. Mol Genet Metab 92: 258-270.[CrossRef][Medline]
Ferrer-Vaquer A, Maurey P, Firnberg N, Leibbrandt A, and Neubüser A (2007) Expression of ASK1 during chick and early mouse development. Gene Expr Patterns 7: 808-816.[CrossRef][Medline]
Flanagan SW, Moseley PL, and Buettner GR (1998) Increased flux of free radicals in cells subjected to hyperthermia: detection by electron paramagnetic resonance spin trapping. FEBS Lett 431: 285-286.[CrossRef][Medline]
Grotewold L and Rüther U (2002) Bmp, Fgf and Wnt signalling in programmed cell death and chondrogenesis during vertebrate limb development: the role of Dickkopf-1. Int J Dev Biol 46: 943-947.[Medline]
Hansen JM, Harris KK, Philbert MA, and Harris C (2002) Thalidomide modulates nuclear redox status and preferentially depletes glutathione in rabbit limb versus rat limb. J Pharmacol Exp Ther 300: 768-776.
Horstmann S, Kahle PJ, and Borasio GD (1998) Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro. J Neurosci Res 52: 483-490.[CrossRef][Medline]
Hwang SG, Yu SS, Lee SW, and Chun JS (2005) Wnt-3a regulates chondrocyte differentiation via c-Jun/AP-1 pathway. FEBS Lett 579: 4837-4842.[Medline]
Jin EJ, Lee SY, Choi YA, Jung JC, Bang OS, and Kang SS (2006) BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway. Mol Cells 22: 353-359.[Medline]
Knobloch J, Shaughnessy JD Jr, and Rüther U (2007) Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway. FASEB J 21: 1410-1421.
Kovacic P and Somanathan R (2006) Mechanism of teratogenesis: electron transfer, reactive oxygen species, and antioxidants. Birth Defects Res C Embryo Today 78: 308-325.[CrossRef][Medline]
Ku BM, Lee YK, Jeong JY, Mun J, Han JY, Roh GS, Kim HJ, Cho GJ, Choi WS, Yi GS, et al. (2007) Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells. Neurosci Lett 419: 64-67.[CrossRef][Medline]
Kyriakis JM and Avruch J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271: 24313-24316.
Malemud CJ (2007) Inhibitors of stress-activated protein/mitogen-activated protein kinase pathways. Curr Opin Pharmacol 7: 339-343.[CrossRef][Medline]
Manjón C, Sánchez-Herrero E, and Suzanne M (2007) Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis. Nat Cell Biol 9: 57-63.[CrossRef][Medline]
Mirkes PE, Wilson KL, and Cornel LM (2000) Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos. Teratology 62: 14-25.[CrossRef][Medline]
Paliga AJ, Natale DR, and Watson AJ (2005) p38 mitogen-activated protein kinase (MAPK) first regulates filamentous actin at the 8–16-cell stage during preimplantation development. Biol Cell 97: 629-640.[CrossRef][Medline]
Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, and Cobb MH (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153-183.
Pugazhenthi S, Phansalkar K, Audesirk G, West A, and Cabell L (2006) Differential regulation of c-jun and CREB by acrolein and 4-hydroxynonenal. Free Radic Biol Med 40: 21-34.[CrossRef][Medline]
Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, and Wagner EF (1999) Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2. Mech Dev 89: 115-124.[CrossRef][Medline]
Sahambi SK and Hales BF (2006) Exposure to 5-bromo-2'-deoxyuridine induces oxidative stress and activator protein-1 DNA binding activity in the embryo. Birth Defects Res A Clin Mol Teratol 76: 580-591.[CrossRef][Medline]
Sluss HK and Davis RJ (2006) H2AX is a target of the JNK signaling pathway that is required for apoptotic DNA fragmentation. Mol Cell 23: 152-153.[CrossRef][Medline]
Takeda K, Hatai T, Hamazaki TS, Nishitoh H, Saitoh M, and Ichijo H (2000) Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells. J Biol Chem 275: 9805-9813.
Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T, and Ichijo H (2001) ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2: 222-228.[CrossRef][Medline]
Torres M and Forman HJ (2003) Redox signaling and the MAP kinase pathways. Biofactors 17: 287-296.[Medline]
Tufan AC, Daumer KM, DeLise AM, and Tuan RS (2002) AP-1 transcription factor complex is a target of signals from both WnT-7a and N-cadherin-dependent cell-cell adhesion complex during the regulation of limb mesenchymal chondrogenesis. Exp Cell Res 273: 197-203.[CrossRef][Medline]
Turjanski AG, Vaqué JP, and Gutkind JS (2007) MAP kinases and the control of nuclear events. Oncogene 26: 3240-3253.[CrossRef][Medline]
Wada T, Stepniak E, Hui L, Leibbrandt A, Katada T, Nishina H, Wagner EF, and Penninger JM (2007) Antagonistic control of cell fates by JNK and p38-MAPK signaling. Cell Death Differ 15: 89-93.[CrossRef][Medline]
Yan J and Hales BF (2005) Activator protein-1 (AP-1) DNA binding activity is induced by hydroxyurea in organogenesis stage mouse embryos. Toxicol Sci 85: 1013-1023.
Yan J and Hales BF (2006) Depletion of glutathione induces 4-hydroxynonenal protein adducts and hydroxyurea teratogenicity in the organogenesis stage mouse embryo. J Pharmacol Exp Ther 319: 613-621.
Yang P, Zhao Z, and Reece EA (2007) Involvement of c-Jun N-terminal kinases activation in diabetic embryopathy. Biochem Biophys Res Commun 357: 749-754.[CrossRef][Medline]
Zuzarte-Luís V, Montero JA, Rodriguez-León J, Merino R, Rodríguez-Rey JC, and Hurlé JM (2004) A new role for BMP5 during limb development acting through the synergic activation of Smad and MAPK pathways. Dev Biol 272: 39-52.[CrossRef][Medline]
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, a significant difference between the HU-treated group with or without SB203580 (
