Preexisting maternal diabetes is a high-risk factor of diabetic embryopathy, such as neural tube defects and congenital heart defects. Maternal diabetes significantly increases the production of reactive oxygen species, resulting in oxidative stress and diabetic embryopathy. Multiple cellular and metabolic factors contribute to these processes. Forkhead box O (FoxO)-3a has been demonstrated as a key transcription factor in the signaling transduction pathways responsible for maternal diabetes-induced birth defects. Apoptosis signal-regulating kinase 1 (ASK1) activated by oxidative stress stimulates nuclear translocation of FoxO3a, resulting in the overexpression of tumor necrosis factor receptor 1-associated death domain protein, which, in turn, leads to caspase-8 activation and apoptosis. Maternal diabetes–activated c-Jun N-terminal kinase (JNK)-1/2, downstream effectors of ASK1, can be blocked by superoxide dismutase-1 overexpression, suggesting that oxidative stress is responsible for JNK1/2 signaling activation. Deletion of JNK1/2 significantly suppressed the activity of FoxO3a. These observations indicate that maternal diabetes–induced oxidative stress stimulates the activation of ASK1, JNK1/2, FoxO3a, tumor necrosis factor receptor 1-associated death domain protein, caspase-8 cleavage, and finally, apoptosis and diabetic embryopathy.
Pregnancy with preexisting maternal diabetes significantly increases the risk of excess apoptosis occurs in target tissues of the developing embryos resulting in diabetes-induced birth defects, such as neural tube defects (NTDs) and congenital heart defects. Each year, 10% of babies of diabetic mothers are born with a major congenital malformation. Mechanistic studies demonstrate that maternal diabetes alters multiple cellular and metabolic factors contributing to diabetic embryopathy. We propose that these cellular and metabolic aberrations occur through a single transcriptional mechanism, a transcription factor, and its responsive gene, leading to apoptosis in embryonic cells.
We have determined that the transcription factor, Forkhead box O (FoxO)-3a, is activated in diabetic embryopathy. FoxO factors are functionally diversified in the induction of apoptosis-related pathogenesis. The transcription factor FoxO3a is a key target of phosphatidylinositol-3 kinase (PI3K)/AKT pathway, in which AKT inactivates FoxO3a by phosphorylation. Maternal diabetes activates FoxO3a by using several different manners: inhibited AKT function and the activated apoptosis signal-regulating kinase 1 (ASK1)–c-Jun N-terminal kinase (JNK)-1/2 pathway.
The expression of tumor necrosis factor receptor 1–associated death domain protein (TRADD), an apoptotic gene, is up-regulated in diabetic embryopathy, and we propose that TRADD is a FoxO3a-responsive gene that initiates caspase-dependent apoptosis in diabetic embryopathy. Maternal diabetes–induced embryonic cell apoptosis is caspase dependent.
Previous work by our group and others has suggested that the proapoptotic JNK pathway, which is downstream of the ASK1 pathway, plays a causative role in the induction of diabetic embryopathy. Activated ASK1 stimulates JNK1/2 activation and subsequent mitochondrial dysfunction and cell apoptosis, resulting in diabetic embryopathy. Deletion of both the JNK1 and JNK2 gene could inhibit nuclear translocation of FoxO3a. Thus, we propose a link between the JNK pathway and FoxO3a activation.
In this review, we will discuss the general function, possible clinical application, and cross talk relationship of molecules downstream of oxidative stress–induced kinase signaling in diabetic embryopathy.
Pathogenesis in the induction of diabetic embryopathy
Hyperglycemia-induced oxidative stress
A variety of antioxidants have been shown to effectively suppress hyperglycemia-induced dysmorphogenesis both in vivo and in vitro. Conversely, the induction of oxidative stress by the depletion of glutathione, by exposure to xathine/xanthine, which directly generate reactive oxygen species (ROS), or by treatment with antimycin, a mitochondrial complex III inhibitor that stimulates superoxide production, significantly increase embryonic anomalies. Therefore, we hypothesize that oxidative stress is the primary cause of diabetic embryopathy because of the enhanced ROS production and weakening of the cellular antioxidant systems ( Figure 1 ).
In maternal diabetes, increased levels of cellular glucose in embryonic tissues may enhance mitochondrial oxidative glucose metabolism and thus increase mitochondrial ROS production. Enhanced ROS production facilitates lipid peroxidation and protein carboxylation, contributing to overall oxidative stress in embryos under maternal diabetic conditions. Markers of lipid peroxidation, 8-iso-prostaglandin E2 and malondialdehyde, are dramatically elevated in embryos cultured in vitro under hyperglycemic conditions as well as in diabetic patients ( Figure 1 ).
Cells possess a wide range of antioxidant systems to protect themselves from the toxic effects of excessive levels of ROS. Diabetic conditions profoundly influence cellular antioxidant potential. A significant decrease in the intracellular ROS scavenging enzyme activities of superoxide dismutase (SOD) and catalase (CAT) are seen when rat embryos and their yolk sacs are maintained under diabetic condition. In addition, the levels of SOD and CAT mRNA decrease under maternal hyperglycemic conditions correlating inversely to an increase in embryonic anomalies. The evidence cited in the previous text supports our assertion that cellular antioxidant defense systems are severely compromised in embryos and the yolk sac in response to maternal hyperglycemia, thereby contributing to cellular oxidative stress during the critical stages of organogenesis ( Figure 1 ).
The role of nitric oxide
Nitric oxide (NO), a critical signaling molecule involving in many processes, is produced from L-arginine by a family of 3 nitric oxide synthases. NO plays an important role in early embryonic development by regulating cell survival, apoptosis, and differentiation. Because NO synthesis and function are critical during period of organogenesis, appropriate intracellular NO concentrations is a prerequisite for normal embryonic development and deregulated NO concentrations has been linked to abnormal embryonic outcomes. NO production that is elevated during early organogenesis in embryos from rat models of mild and severe diabetes leading to malformations.
Elevated NO may directly interact with ROS generated under hyperglycemic conditions to form potent oxidant peroxynitrite leading to nitrosative stress ( Figure 1 ). The peroxynitrite anion inhibits mitochondrial electron transport, oxidizes important proteins, and initiates lipid peroxidation, thus affecting many signal transduction pathways. The mechanism underlying maternal diabetes-increased NO production is not clear and needs to be investigated further. Nitrosative stress resulting from elevated NO levels may be one of the mechanisms in the induction of diabetic embryopathy through the JNK pathway because nitrosative stress leads to JNK activation. The role of JNK in diabetic embryopathy will be discussed later in this review.
The role of nitric oxide
Nitric oxide (NO), a critical signaling molecule involving in many processes, is produced from L-arginine by a family of 3 nitric oxide synthases. NO plays an important role in early embryonic development by regulating cell survival, apoptosis, and differentiation. Because NO synthesis and function are critical during period of organogenesis, appropriate intracellular NO concentrations is a prerequisite for normal embryonic development and deregulated NO concentrations has been linked to abnormal embryonic outcomes. NO production that is elevated during early organogenesis in embryos from rat models of mild and severe diabetes leading to malformations.
Elevated NO may directly interact with ROS generated under hyperglycemic conditions to form potent oxidant peroxynitrite leading to nitrosative stress ( Figure 1 ). The peroxynitrite anion inhibits mitochondrial electron transport, oxidizes important proteins, and initiates lipid peroxidation, thus affecting many signal transduction pathways. The mechanism underlying maternal diabetes-increased NO production is not clear and needs to be investigated further. Nitrosative stress resulting from elevated NO levels may be one of the mechanisms in the induction of diabetic embryopathy through the JNK pathway because nitrosative stress leads to JNK activation. The role of JNK in diabetic embryopathy will be discussed later in this review.
Aberrant signaling pathways
The protein kinase C (PKC) family of serine/threonine protein kinases consists of 12 members, involved in a number of cellular activities, including proliferation, migration, apoptosis, differentiation, and secretion. Each member plays its own unique role in cell physiology, although overlapping functions may exist for some isoenzymes. Deregulated PKC activity may be mechanistically involved in diabetic embryopathy.
The diacylglycerol (DAG)-PKC pathway has been implicated in diabetic embryopathy. Maternal hyperglycemia stimulates DAG production in embryonic cells, which, in turn stimulates PKC activity. Some PKC isoforms (α, β2, and δ) are up-regulated, whereas others (ε and ξ) are down-regulated in diabetic embryopathy. Inhibiting the activity of some PKC isoforms significantly decreases the malformation rate ( Figure 2 ).
We have also found that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is down-regulated in diabetic embryopathy. The activity of a prosurvival kinase, Akt, is reduced in diabetic embryopathy. Akt is the key mediator in the PI3K pathway, a central regulator of the mammalian target of rapamycin. The down-regulation of Akt by maternal diabetes results in the activation of FoxO3a and downstream TRADD and caspase-8 cleavage, contributing to diabetic embryopathy ( Figure 2 ).
Our work has shown that ASK1-JNK1/2 signaling pathway plays important role in diabetic embryopathy. Under hyperglycemic condition, ASK1 is phosphorylated and activated through its dissociation from oxidized thioredoxin. On the one hand, ASK1 phosphorylation at Thr845 activates JNK1/2 kinase by phosphorylation, which initiates proapoptotic signaling pathways, which play key roles in the diabetic embryopahthy ; on the other hand, ASK1 phosphorylation initiates the unfolded protein response and endoplasmic reticulum (ER) stress, which induces diabetic embryopathy by triggering beta-cell dysfunction and apoptosis ( Figure 2 ).
The cross talk between the deregulated PKC and ASK1 signaling pathways with the JNK pathway seems to contribute to the induction of diabetic embryopathy.
Altered glucose metabolic pathways
Activity of the hexosamine biosynthetic pathway (HBP) is increased in embryos during diabetic pregnancy, which may contribute to hyperglycemia-induced oxidative stress. Increased glycolytic flux can stimulate glucose flux through the HBP pathway, in which fructose-6-phosphate and glutamine are converted to glucosamine-6-phosphate and glutamate ( Figure 3 ). Experimental activation of the HBP pathway by glucosamine administration mimics the effects of maternal diabetes to inhibit the pentose shunt pathway, a main glucose metabolism pathway in early embryonic development, and to induce oxidative stress by inhibiting information of reduced glutathione ( Figure 3 ).
We propose that all these hyperglycemia-triggered upstream events converge on the FoxO3a central transcription mechanism, leading to hyperglycemia-induced apoptosis in the embryonic neural epithelium cells.
Maternal diabetes–induced apoptosis: primary mechanism of diabetic embryopathy
Compelling evidence demonstrates that maternal hyperglycemia increases apoptosis in the embryo. Apoptosis is specifically seen in neuroepithelial cells, which are particularly susceptible to hyperglycemic damage. Multiple studies have confirmed that excess cell death, at least in the central nervous system, contributes to the abnormal development of structures in the embryos of diabetic animals. Hyperglycemia-induced apoptosis involves the altered regulation of Bcl-2 family members and caspase activation, critical events in the mitochondrial apoptotic pathway ( Figure 4 ).
In addition, hyperglycemia-associated oxidative stress increases the Bax:Bcl-2 ratio, which is associated with cytochrome c release and activation of caspase-3 in embryonic cells ( Figure 4 ). An increase of Bax expression and release of cytochrome c, and activated caspase 3 are characteristics of embryonic cells undergoing apoptosis under maternal hyperglycemic conditions ( Figure 4 ).
These observations strongly suggest that high glucose concentrations cause damage to the neural progenitor cells, leading to excessive apoptosis and abnormal organogenesis. Moreover, our recent studies, in which we used antioxidants to neutralize oxidative stress, suggest a direct connection between hyperglycemia-induced oxidative stress and apoptosis.
Induction of apoptosis by the ASK1-mitogen-activated protein kinase (MKK)-4-JNK signaling pathway
Mitogen-activated protein kinase (MAPK) mediates a range of cellular processes, including apoptotic cell death. MAPKs are members of a superfamily of serine/threonine kinases that are activated in response to a variety of extracellular stimuli, including ROS. Three distinct MAPK pathways regulate extracellular signal–regulated kinases (ERK1 and ERK2), c-Jun NH2-terminal kinases (JNK1, JNK2, and JNK3), and p38 MAPKs (p38α, p38β, p38γ, and p38δ). These pathways control a variety of cellular functions, including gene expression, mitosis, and apoptosis through the phosphorylation of specific serine and/or threonine residues of target proteins.
The basic assembly of the MAPK signaling pathway is a 3-component module: via sequential activation of MAPK kinase (MAP3K), MAPK kinase (MAPKK), and MAPK. MAP3K phosphorylates and activates MAPKK, and activated MAPKK phosphorylates and activates MAPK. Because the activation status of MAPKs is largely dependent on MAP3Ks, it is important to understand how MAP3Ks are regulated. Fourteen different MAP3Ks have been identified. Among them, several MAP3Ks, including ASK1, transforming growth factor-[beta]–activated kinase-1, and mixed-lineage protein kinase-3, are known to activate the JNK pathway in response to diverse stimuli. Those MAP3Ks phosphorylate and activate the dual specific kinases, MKK4 (SEK1) and MKK7, which in turn, phosphorylate and activate JNKs.
Oxidative stress is one of the most potent activators of ASK1, which is essential for oxidative stress-induced apoptosis through the activation of MKK4/MKK7-JNK cascade. Oxidative stress induces the phosphorylation of Thr-845 in the activation loop of ASK1, correlating with enhanced ASK1 activity and increased apoptosis ( Figure 5 ). ASK1-mediated apoptosis is involved in pathogenesis of several oxidative stress-related diseases such as brain ischemia, ischemic heart disease, and Alzheimer’s disease.
indicates dephosphorylation; Most recently it has been shown that high-glucose–activated ASK1 mediates hyperglycemia-induced endothelial cell senescence. These findings are consistent with our hypothesis that ASK1 functions as a mediator of diabetes-related embryo malformation. Indeed, we found that the deletion of the Ask1 gene significantly reduces maternal diabetes–induced caspase cleavage, neuroepithelial cell apoptosis, and NTD formation. This suggests that ASK1 plays an essential role in the induction of apoptosis, leading to NTD formation ( Figure 5 ).
The JNK pathway specifically responds to stress-induced signals that drive apoptosis. JNK has 3 isoforms (JNK1, JNK2, and JNK3) encoded by 3 different genes. The Jnk1 and Jnk2 genes are ubiquitously expressed, whereas Jnk3 is found to be neural tissue specific. The specific molecular targets of JNK include transcription factor activator protein-1 (mainly c-Jun, JunB, and activating transcription factor-2) and FoxO factors as well as many other nontranscription factors, such as Bcl-2 proteins, which are closely related to apoptotic cell death factors.
Both exogenous and endogenous ROS increase JNK1/2 activity. Substantial genetic and pharmacological evidence suggests that JNK serves as a key proapoptotic mediator during oxidative stress. Mice having null mutations in any single JNK gene develop normally, as do JNK1/JNK3 or JNK2/JNK3 double mutants. Although JNK1/JNK2 null mutants die in utero because of abnormal apoptosis in the brain. These mice are useful models for delineating apoptotic pathways involving JNKs.
The ASK1-JNK pathway appears to play mutual causation role with ER stress signaling. Maternal diabetes induces ER stress and ASK1-JNK signaling pathway ( Figure 5 ). ASK1-JNK is a key component of the unfolded protein response signalosome, which leads to ER stress. We also found the deletion of the Ask1 , Jnk1 , or Jnk2 gene could abolish maternal diabetes–induced ER stress and subsequent apoptosis in the neuroepithelial cell.
JNK1/2 activation: a critical role in diabetic embryopathy
MAPK activity is altered in diabetic patients and in cells cultured in high glucose, suggesting that MAPKs may be involved in hyperglycemia-induced complications. Less, however, is known about the activity of MAPKs in embryos under maternal diabetic conditions. An increase in JNK1/2 activity is associated with increased apoptosis in the yolk sacs of malformed embryos from diabetic mothers.
We have reported that supplementation with antioxidants reduces JNK1/2 activity and the embryonic malformation rate in embryos, suggesting that hyperglycemia-induced oxidative stress is responsible for JNK1/2 activation. These observations indicate that increased JNK1/2 activity may play an important role in diabetic embryopathy ( Figure 5 ). In addition, an increase in phosphorylated MKK4 coincides with JNK1/2 activation in diabetic embryopathy. Treatment with a JNK1/2 inhibitor, SP600125, prevents hyperglycemia-induced embryopathy.
Additionally, maternal diabetes-induced embryonic anomalies are significantly reduced in the JNK2 null background. It is also revealing that sorbitol, a potent JNK1/2 activator, mimics the teratogenic effect of hyperglycemia. This evidence strongly suggests that JNK1/2 activation is crucial for diabetic embryopathy. The neural tube and yolk sac of the conceptus are extremely vulnerable to the negative effects of maternal hyperglycemia. We have demonstrated that hyperglycemia induces yolk sac vasculopathy and embryo malformation and that blockade of JNK1/2 activation reverses these effects. Thus, pharmacological and genetic evidences strongly suggest that JNK1/2 activation mediates the deleterious effect of hyperglycemia on embryonic development and yolk sac vasculature.
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