Peixin Yang

Activation of oxidative stress signaling that is implicated in apoptosis with a mouse model of diabetic embryopathy

OBJECTIVE A mouse model of diabetic embryopathy in C57BL/6J background was established to use the resources of genetically engineered mice in which a specific gene is deleted or overexpressed. To test whether our previous fundamental findings in the rat model of diabetic embryopathy are transferable to this mouse model of diabetic embryopathy, levels of phosphorylated-JNK1/2 (c-Jun N-terminal kinase 1 and 2) and apoptotic markers (cleaved caspase 3) were determined. To establish a link between oxidative stress signaling and diabetic embryopathy, levels of phosphorylated-p66Shc (which is a key signaling molecule that mediates oxidative stress-induced apoptosis) were evaluated. STUDY DESIGN Diabetes mellitus was induced in female C57BL/6J mice by an intravenous injection of streptozotocin (75 mg/kg). Glucose levels were controlled by the subcutaneous implantation of insulin pellets. The female mice were mated with normal male mice. At gestation day 5 or embryonic day 5 (E5), the insulin pellets were removed from a group of animals, which made them hyperglycemic (> 250 mg/dL glucose). The animals with retained insulin pellets served as controls. On embryonic day 11, mice were killed, and embryos were dissected from the uteri for examination. Embryos and yolk sacs from individual conceptus were collected. Levels of phosphorylated-JNK1/2, phosphorylated-p66Shc, and cleaved caspase 3 were determined in the embryos and yolk sacs. RESULTS Malformation rates in embryos from diabetic mice were 3-fold higher than those in embryos from nondiabetic or diabetic/euglycemic control groups. JNK1/2, especially p54 JNK isoform, which is predominantly expressed by jnk2 gene, was activated in malformed embryos and their respective yolk sacs from diabetic mice and was significantly higher than those in normally developed embryos and their respective yolk sacs from nondiabetic and diabetic mice. Correlating to JNK1/2 activation, phosphorylated-p66Shc was also significantly increased in malformed embryos and their respective yolk sacs from diabetic mice than in normally developed embryos and their respective yolk sacs from nondiabetic and diabetic mice. Cleaved caspase 3 was observed in malformed embryos from diabetic mice. CONCLUSION The present study shows that maternal hyperglycemia is able to induce embryonic dysmorphogenesis in C57BL/6J mice that is comparable with that seen in the rat model of diabetic embryopathy. Like the well-studied rat model of diabetic embryopathy, activation of JNK1/2 and p66Shc and the increase of apoptotic markers are manifested in this mouse model of diabetic embryopathy. These findings suggest that the activation of oxidative stress signaling in diabetic embryopathy leads to excessive embryonic cell apoptosis and ultimately embryonic dysmorphogenesis. To apply the powerful genetic approach to the research of diabetic embryopathy, a mouse is a better animal model than a rat because all gene knockout (deletion) and gene transgenic (gene overexpression) animals are made in the mouse. The mouse model of diabetic embryopathy that was established in the present study may serve as a suitable substitute for the rat model of diabetic embryopathy, thus enabling us and other investigators to use genetically engineered technologies in the study of diabetic embryopathy.

Expression of ER-α and ER-β in the Hamster Ovary: Differential Regulation by Gonadotropins and Ovarian Steroid Hormones

Spatiotemporal expression patterns of ER-α and ER-β protein and mRNA in hamster ovarian cells during the estrous cycle and following hypophysectomy and selective hormone replacement were evaluated by immunofluorescence, immunoblotting and in situ hybridization analyses. Whereas ER-β mRNA and protein expression predominated in granulosa cells and ER-α expression was in interstitial and thecal cells, overlap in receptor subtype expression across cell types was evident. Both ER subtypes were present from primordial follicle stage onward. ER-α mRNA levels and immunoreactivity started increasing from D3:0900 h in intersitial and granulosa cells and peaked on the proestrous (D4:0900 h). Regionalized higher expression of ER-α in granulosa cells in and around the forming antrum was evident. Surface epithelial cells were also positive. ER-β mRNA and protein expression increased markedly in granulosa and interstitial cells on D2:0900 h, reached a peak on D3:0900 h, and then declined sharply on D4:0900 h. No change ...

c-Jun NH2-Terminal Kinase 1/2 and Endoplasmic Reticulum Stress as Interdependent and Reciprocal Causation in Diabetic Embryopathy

Embryos exposed to high glucose exhibit aberrant maturational and cytoarchitectural cellular changes, implicating cellular organelle stress in diabetic embryopathy. c-Jun-N-terminal kinase 1/2 (JNK1/2) activation is a causal event in maternal diabetes–induced neural tube defects (NTD). However, the relationship between JNK1/2 activation and endoplasmic reticulum (ER) stress in diabetic embryopathy has never been explored. We found that maternal diabetes significantly increased ER stress markers and induced swollen/enlarged ER lumens in embryonic neuroepithelial cells during neurulation. Deletion of either jnk1 or jnk2 gene diminished hyperglycemia-increased ER stress markers and ER chaperone gene expression. In embryos cultured under high-glucose conditions (20 mmol/L), the use of 4-phenylbutyric acid (4-PBA), an ER chemical chaperone, diminished ER stress markers and abolished the activation of JNK1/2 and its downstream transcription factors, caspase 3 and caspase 8, and Sox1 neural progenitor apoptosis. Consequently, both 1 and 2 mmol/L 4-PBA significantly ameliorated high glucose–induced NTD. We conclude that hyperglycemia induces ER stress, which is responsible for the proapoptotic JNK1/2 pathway activation, apoptosis, and NTD induction. Suppressing JNK1/2 activation by either jnk1 or jnk2 gene deletion prevents ER stress. Thus, our study reveals a reciprocal causation of ER stress and JNK1/2 in mediating the teratogenicity of maternal diabetes.

Oxidative Stress–Induced JNK1/2 Activation Triggers Proapoptotic Signaling and Apoptosis That Leads to Diabetic Embryopathy

Oxidative stress and apoptosis are implicated in the pathogenesis of diabetic embryopathy. The proapoptotic c-Jun NH2-terminal kinases (JNK)1/2 activation is associated with diabetic embryopathy. We sought to determine whether 1) hyperglycemia-induced oxidative stress is responsible for the activation of JNK1/2 signaling, 2) JNK1 contributes to the teratogenicity of hyperglycemia, and 3) both JNK1 and JNK2 activation cause activation of downstream transcription factors, caspase activation, and apoptosis, resulting in neural tube defects (NTDs). Wild-type (WT) embryos from nondiabetic WT dams and WT, superoxide dismutase (SOD)1–overexpressing, jnk1+/−, jnk1−/−, and jnk2−/− embryos exposed to maternal hyperglycemia were used to assess JNK1/2 activation, NTDs, activation of transcription factors downstream of JNK1/2, caspase cascade, and apoptosis. SOD1 overexpression abolished diabetes-induced activation of JNK1/2 and their downstream effectors: phosphorylation of c-Jun, activating transcription factor 2, and E twenty-six–like transcription factor 1 and dephosphorylation of forkhead box class O3a. jnk1−/− embryos had significantly lower incidences of NTDs than those of WT or jnk1+/− embryos. Either jnk1 or jnk2 gene deletion blocked diabetes-induced activation of JNK1/2 signaling, caspases 3 and 8, and apoptosis in Sox1+ neural progenitors of the developing neural tube. Our results show that JNK1 and JNK2 are equally involved in diabetic embryopathy and that the oxidative stress–JNK1/2–caspase pathway mediates the proapoptotic signals and the teratogenicity of maternal diabetes.

Maternal Hyperglycemia Activates an ASK1-FoxO3a-Caspase 8 Pathway That Leads to Embryonic Neural Tube Defects

Interference with a cell death–promoting signaling pathway could prevent neural tube defects in the children of diabetic women. Preventing Sugar-Induced Birth Defects The central nervous system initially develops from an embryonic structure called the neural tube. Neural tube defects result when the neural tube does not close properly and can range in severity from spina bifida to anencephaly, which is fatal. Maternal hyperglycemia is associated with the development of neural tube defects. Yang et al. uncovered a signaling pathway initiated by apoptosis signal–regulating kinase 1 (ASK1) that was associated with an increased incidence of neural tube defects due to cell death in the embryos of diabetic mice. The incidence of neural tube defects was decreased by genetic ablation of ASK1 or the downstream components of this pathway, or by injecting pregnant mice with an inhibitor of ASK1. Thus, interfering with activation of the ASK1 signaling pathway could help to limit the development of neural tube defects in babies born to diabetic women. Neural tube defects result from failure to completely close neural tubes during development. Maternal diabetes is a substantial risk factor for neural tube defects, and available evidence suggests that the mechanism that links hyperglycemia to neural tube defects involves oxidative stress and apoptosis. We demonstrated that maternal hyperglycemia correlated with activation of the apoptosis signal–regulating kinase 1 (ASK1) in the developing neural tube, and Ask1 gene deletion was associated with reduced neuroepithelial cell apoptosis and development of neural tube defects. ASK1 activation stimulated the activity of the transcription factor FoxO3a, which increased the abundance of the apoptosis-promoting adaptor protein TRADD, leading to activation of caspase 8. Hyperglycemia-induced apoptosis and the development of neural tube defects were reduced with genetic ablation of either FoxO3a or Casp8 or inhibition of ASK1 by thioredoxin. Examination of human neural tissues affected by neural tube defects revealed increased activation or abundance of ASK1, FoxO3a, TRADD, and caspase 8. Thus, activation of an ASK1–FoxO3a–TRADD–caspase 8 pathway participates in the development of neural tube defects, which could be prevented by inhibiting intermediates in this cascade.

Birth defects in pregestational diabetes: Defect range, glycemic threshold and pathogenesis.

Currently, 60 million women of reproductive age (18-44 years old) worldwide, and approximately 3 million American women have diabetes mellitus, and it has been estimated that this number will double by 2030. Pregestational diabetes mellitus (PGD) is a significant public health problem that increases the risk for structural birth defects affecting both maternal and neonatal pregnancy outcome. The most common types of human structural birth defects associated with PGD are congenital heart defects and central nervous system defects. However, diabetes can induce birth defects in any other fetal organ. In general, the rate of birth defects increases linearly with the degree of maternal hyperglycemia, which is the major factor that mediates teratogenicity of PGD. Stringent prenatal care and glycemic control are effective means to reduce birth defects in PGD pregnancies, but cannot reduce the incidence of birth defects to the rate of that is seen in the nondiabetic population. Studies in animal models have revealed that PGD induces oxidative stress, which activates cellular stress signalling leading to dysregulation of gene expression and excess apoptosis in the target organs, including the neural tube and embryonic heart. Activation of the apoptosis signal-regulating kinase 1 (ASK1)-forkhead transcription factor 3a (FoxO3a)-caspase 8 pathway causes apoptosis in the developing neural tube leading to neural tube defects (NTDs). ASK1 activates the c-Jun-N-Terminal kinase 1/2 (JNK1/2), which leads to activation of the unfolded protein response and endoplasmic reticulum (ER) stress. Deletion of the ASK1 gene, the JNK1 gene, or the JNK2 gene, or inhibition of ER stress by 4-Phenylbutyric acid abrogates diabetes-induced apoptosis and reduces the formation of NTDs. Antioxidants, such as thioredoxin, which inhibits the ASK1-FoxO3a-caspase 8 pathway or ER stress inhibitors, may prevent PGD-induced birth defects.

Ask1 gene deletion blocks maternal diabetes-induced endoplasmic reticulum stress in the developing embryo by disrupting the unfolded protein response signalosome

Apoptosis signal–regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose– or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis.

The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression

The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes‐associated protein (YAP). Here, we show that YAP plays a central role in controlling the progression of cervical cancer. Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer. TGF‐α and amphiregulin (AREG), via EGFR, inhibit the Hippo signaling pathway and activate YAP to induce cervical cancer cell proliferation and migration. Activated YAP allows for up‐regulation of TGF‐α, AREG, and EGFR, forming a positive signaling loop to drive cervical cancer cell proliferation. HPV E6 protein, a major etiological molecule of cervical cancer, maintains high YAP protein levels in cervical cancer cells by preventing proteasome‐dependent YAP degradation to drive cervical cancer cell proliferation. Results from human cervical cancer genomic databases and an accepted transgenic mouse model strongly support the clinical relevance of the discovered feed‐forward signaling loop. Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer.

Epigallocatechin-3-gallate ameliorates hyperglycemia-induced embryonic vasculopathy and malformation by inhibition of Foxo3a activation

OBJECTIVE Maternal hyperglycemia increases the risk of congenital malformations. Epigallocatechin-3-gallate (EGCG), a natural antioxidant purified from green tea, inhibits oxidative stress signaling. We propose that EGCG prevents hyperglycemia-induced malformation via inhibition of oxidative stress signaling. The objective of this study is to examine the effect of EGCG on hyperglycemia-induced adverse effects during embryonic development. STUDY DESIGN Day-9 rat conceptuses were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (300 mg/dL glucose) conditions in the presence or absence of 1 or 10 micromol/L of EGCG. RESULTS Both 1 and 10 micromol/L of EGCG significantly ameliorated hyperglycemia-induced embryonic vasculopathy and malformations. Hyperglycemia inactivated protein kinase B (Akt) by reducing phosphorylated Akt levels. EGCG reversed the inhibitory effect of hyperglycemia on Akt activation. EGCG also prevented hyperglycemia-reduced phosphorylated Forkhead transcription factor 3a levels. CONCLUSION EGCG prevented hyperglycemia-induced embryopathy through inhibition of Forkhead transcription factor 3a activation. This may have been mediated via the activation of Akt. These findings offer the potential for a possible pharmacological prophylaxis for hyperglycemia-induced embryonic malformations.

Hyperglycemia induces inducible nitric oxide synthase gene expression and consequent nitrosative stress via c-Jun N-terminal kinase activation.

OBJECTIVE Maternal diabetes has an adverse impact on embryonic development. We tested the hypothesis that hyperglycemia-induced c-Jun N-terminal kinases (JNK) 1/2 activation mediates inducible nitric oxide synthase (iNOS) induction. STUDY DESIGN Levels of iNOS messenger ribonucleic acid (mRNA) and nitrosylated protein were determined in cultured C57BL/6J conceptuses exposed to hyperglycemia (300 mg/dL glucose) and C57BL/6J embryos exposed to streptozotocin-induced diabetes. The iNOS-luciferase activity and endogenous reactive nitrogen species were determined in transfected PYS-2 (mouse teratocarcinoma) cells exposed to hyperglycemia (450 mg/dL glucose). RESULTS Hyperglycemia increased iNOS mRNA, and SP600125, a potent JNK1/2 inhibitor, abolished this effect. Hyperglycemia increased iNOS-luciferase activities, and SP600125 blocked this effect. Diabetes increased iNOS mRNA and jnk2 gene deletion abrogated this effect. Correlated with iNOS gene induction, both hyperglycemia in vitro and diabetes in vivo enhanced the production of reactive nitrogen species and increased protein nitrosylation. The jnk2 gene deletion blocked diabetes-induced protein nitrosylation. CONCLUSION JNK1/2 activation mediates hyperglycemia-induced iNOS gene expression and consequent nitrosative stress in diabetic embryopathy.