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.
Maternal diabetes is a significant risk factor for adverse pregnancy outcomes including miscarriages and congenital malformations. These adverse pregnancy outcomes that are associated with diabetic women are significant clinical problems.
The recent rise of diabetes in reproductive-age women makes these pregnancy complications a continuing issue. Because glycemic control in diabetic women is difficult to control and maintain and malformation rates of children of diabetic women are at least 6 times higher than those in nondiabetic women, therapeutic interventions in addition to insulin are needed for diabetes-associated adverse pregnancy outcomes. Mechanistic studies are essential steps for the development of accessible, convenient, and effective prevention strategies.
Hyperglycemia resulting from diabetes mellitus has an adverse impact on embryonic development through induction of apoptosis in embryonic tissues. The mechanisms underlying hyperglycemia-induced apoptosis are not completely understood. We have found that the proapoptotic c-Jun N-terminal kinases 1 and 2 (JNK1/2) is activated in embryonic tissues exposed to maternal diabetes in vivo and hyperglycemic embryo cultures of both Sprague Dawley rats and C57BL/B6J mice in vitro. JNK1/2 agonist mimics the teratogenic effect of hyperglycemia to induce embryonic malformations, whereas targeted deletion of the jnk2 gene significantly ameliorates diabetes-induced malformations. Thus, JNK1/2 plays a causative role in the induction of diabetic embryopathy.
JNK has 3 isoforms (JNK1, JNK2, and JNK3) encoded by 3 different genes. The jnk1 and jnk2 genes are ubiquitously expressed. The specific molecular targets of JNK include transcription factor activator protein -1 (mainly c-Jun, JunB, and activating transcription factor-2), Forkhead box O factors as well as many other nontranscription factors such as Bcl-2 proteins.
The JNK pathway specifically responds to stress-induced signals that drive apoptosis. Activation of transcription factors downstream of JNK1/2 activation relays the proapoptotic signals to the nucleus, thus inducing apoptotic gene expression that leads to apoptosis. Mice having null mutations in a single JNK gene develop normally. However, the double-JNK1/JNK2 null mutants are embryonic lethal because of abnormal apoptosis in the brain. The single JNK gene null mice including jnk1 –/– (JNK1KO) and jnk2 –/– (JNK2KO) mice are useful models for delineating proapoptotic effects emanating from JNK1/2 activation.
Increased levels of nitric oxide (NO) are associated with the adverse impact of maternal diabetes on embryonic development. NO is synthesized from oxidation of L-arginine by 3 distinct NO synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS and eNOS are constitutively expressed at low levels. When iNOS is induced, it generates very high concentrations of NO.
NO has been shown to be involved in cell differentiation, proliferation, and apoptosis. Although NO is of physiological importance, it can also be cytotoxic. iNOS and eNOS are expressed during early embryonic development. Hyperglycemia increases NO production in embryonic tissues inducing the production of reactive nitrogen species that leads to nitrosative stress.
We hypothesized that hyperglycemia-induced JNK1/2 activation mediates iNOS induction. To test this hypothesis, we investigated the relationship between JNK1/2 activation and iNOS gene expression in diabetic embryopathy. By using pharmacological inhibitors of JNK1/2 activation (SP600125) in vitro and target deletion of jnk2 in mice, we have demonstrated that JNK1/2 activation is responsible for hyperglycemia-induced iNOS gene expression and consequent nitrosative stress.
Materials and Methods
Animals and reagents
C57BL/6J mice (median body weight 22 g) and jnk2 –/– (JNK2KO) mice on the same background were purchased from Jackson Laboratory (Bar Harbor, ME). Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). The PYS-2 cell line was purchased from the American Type Culture Collection (catalog no. CRL-2745; Manassas, VA). Streptozotocin (STZ) from Sigma (St Louis, MO) was dissolved in sterile 0.1 M citrate buffer (pH 4.5). Sustained-release insulin pellets were purchased from Linplant (Linshin, Canada).
Whole-conceptus culture
C57BL/6J mice were paired overnight. The next morning was designated embryonic day (E) 0 if a vaginal plug was present. Mouse conceptuses at E8 were dissected out of the uteri in phosphate-buffered saline (PBS; Invitrogen, La Jolla, CA). The parietal yolk sac was removed using a pair of fine forceps, and the visceral yolk sac was left intact.
Conceptuses (4/bottle) were cultured in 4 mL of rat serum at 38°C in 30 revolutions/min rotation in the roller bottle system. For the initial 24 hours of culture, the bottles were gassed with 5% O 2 /5% CO 2 /90% N 2 . For the following 12 hours, the bottles were gassed with 20% O 2 /5% CO 2 /75% N 2 , and in the last 12 hours, 40% O 2 /5% CO 2 /75% N 2 were applied. Conceptuses were cultured under euglycemic (150 mg/dL of glucose, a value close to the blood glucose level of nondiabetic mice) and hyperglycemic (300 mg/dL of glucose) conditions in the presence and absence of SP600125, a pharmacological JNK1/2 inhibitor.
Real-time polymerase chain reaction (RT-PCR)
Total ribonucleic acid (RNA) was isolated from embryonic tissues of cultured conceptuses or conceptuses retrieved from nondiabetic or diabetic mice using an RNeasy minikit (Qiagen, Valencia, CA). RT-PCR for iNOS, eNOS, and β-actin were performed using ABI TaqMan gene expression assays (assay ID: Mm01309897_m1, Mm01164908_m1, and Mm00607939_s1, respectively; Applied Biosystems, Foster City, CA). Briefly, RNA was reverse transcribed by using the high-capacity cDNA archive kit (Applied Biosystems). RT-PCR and subsequent calculations were performed by a 7700 ABI PRISM sequence detector system (Applied Biosystems), which detected the signal emitted from fluorogenic probes during the polymerase chain reaction.
Cell culture, transient transfection, and luciferase assay
PYS-2 cells were cultured in DMEM (Invitrogen) plus 2% fetal bovine serum. Cells were plated overnight to reach about 80% confluency and were transfected with 0.8 μg mouse iNOS promoter luciferase constructor (iNOS-luc) using Lipofectamine 2000 (Invitrogen). The iNOS-luc that contains the mouse iNOS promoter from –1588 to +165 plus the luciferase coding sequence was provided by Dr Sang Geon Kim (Seoul National University, Seoul, South Korea).
After cotransfection with Renilla-luc (normalization control; Promega, Madison, WI), PYS-2 cells were incubated for 24 hours under euglycemic (90 mg/dL of glucose, a value commonly used in cell culture studies) or hyperglycemic (300, 450, 800, or 1000 mg/dL of glucose) conditions in the presence or absence of 400 nM SP600125. Osmotic controls were not used in this study. Luciferase activities were measured by a dual-luciferase kit (Promega) according to the manufacturer’s instructions.
Mouse models of diabetic embryopathy
The procedures for animal use were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Eight week old JNK2KO mice on a C57BL/6J background and wild-type (WT) mice of the same strain were intravenously injected daily with 75 mg/kg STZ over 3 days to induce diabetes. Insulin pellets were subcutaneously implanted in diabetic mice to restore euglycemia prior to mating.
WT and JNK2KO homozygous female mice were mated with male mice of the same respective genotype. On day 5 of pregnancy (E5), insulin pellets were removed to permit frank hyperglycemia (>250 mg/dL glucose level), so the developing conceptuses would be exposed to a hyperglycemic environment during organogenesis (E7-11). Nondiabetic WT mice with vehicle injections were served as nondiabetic WT controls. On E9, mice were euthanized, and conceptuses were dissected out of the uteri for analysis.
Detection of nitrosylated proteins using Western blotting
Western blotting was performed as described by Yang at el. Briefly, embryonic samples were sonicated in 80 μL of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 10 mM NaF; 2 mM Na orthovanadate; 1 mM phenylmethylsulfonyl fluoride; and 1% Triton 100) containing a protease inhibitor cocktail (Sigma).
Equal amounts of protein (50 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Membranes were incubated for 18 h at 4°C with the following primary antibodies at 1:1000 to 1:2000 dilutions in 5% nonfat milk: rabbit antinitrotyrosine (CHEMICON, Temecula, CA). Membranes were exposed to goat antirabbit and antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies.
To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and probed with a mouse antibody against β-actin (Abcam, Cambridge, MA). Signals were detected using an Amersham enhanced chemiluminescence Advance detection kit (GE Healthcare, Piscataway, NJ), and chemiluminescence emitted from the bands was directly captured using a UVP Bioimage EC3 system (UVP, Upland, CA). Densitometric analysis of chemiluminescence signals were performed by VisionWorks LS software (UVP). Images of representative immunoblots were arranged using Adobe Photoshop (San Jose, CA) and Microsoft PowerPoint software (Richmond, CA). All experiments were repeated 3 times with the use of independently prepared tissue lysates.
Detection of endogenous reactive nitrogen species by immunofluorescence
Reactive nitrogen species production was measured by 2 μM DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, Molecular Probes, Eugene, OR). PYS-2 cells after different treatments were washed with PBS and subsequently incubated with DAF-FM diacetate in PBS at 37°C for 30 minutes.
After washing PYS-2 cells with PBS, chamber slide–containing cells were mounted in 4′,6′-diamino-2-phenylindole/antifade solution (CHEMICON), and the fluorescence was visualized under an Olympus BX41 microscope (Optical Elements Corp., Dulles, VA) equipped with an Olympus DP70 digital camera with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and FITC/TRITC filter sets. Cell nuclei were labeled as blue and DAF-FM diacetate emitted green signals.
Statistics
Data are presented as means ± SE. One-way analysis of variance (ANOVA) or Student t test was performed using SigmaStat 3.5 software (Systat Software, San Jose, CA). In ANOVA analysis, a Tukey test was used to estimate the significance of the results. Statistical significance was accepted at P < .05.
Results
Hyperglycemia increases iNOS but not eNOS mRNA, and pharmacological inhibition of JNK1/2 blocks this effect
Because hyperglycemia increased embryonic levels of NO, we tested whether hyperglycemia affected iNOS expression, and JNK1/2 activation played a role. E7 mouse conceptuses were cultured under euglycemic and hyperglycemic conditions in the presence or absence of 800 nM SP600125 for 24 hours. Yolk sacs were harvested for assessment of iNOS messenger ribonucleic acid (mRNA) levels. Hyperglycemia significantly increased iNOS mRNA expression in yolk sacs, and inhibition of JNK1/2 activation by SP600125 blocked hyperglycemia-induced iNOS expression ( Figure 1 , A).
