Study On The Abnormal Neural Progenitor Cells Apoptosis In Gestational Diabetic Embryos

Maternal diabetes increases the risk for congenital malformations. Maternal hyperglycemia perturbs organogenesis of the embryo during the early developmental stages, resulting in malformations of many major systems, including the central nervous, cardiovascular, gastrointestinal, urinary, skeletal and reproductive system. Neural tube defects (NTDs), the failure in closure of the neural tube, are among the most common birth defects observed in infants of diabetic mothers. Observation of excessive apoptosis in neuroepithelium of malformed neural tubes renders the hypothesis that maternal hyperglycemia-induced apoptosis in neural tube cells results in NTDs. During embryo development, the neuroepithelium is composed of NPCs. Excessive apoptosis in NPCs by hyperglycemia disturbs the normal development of neural tube, which results in NTDs. However, the precise mechanisms underlying the regulation of apoptotic pathways in NPCs remain largely undetermined.Apoptosis is a multistage, genetically controlled process of selective cell deletion. Protein kinases regulate the early stages of apoptosis by phosphorylating key protein. Previous studies have showed that the protein kinase C (PKC) family plays an important role in mammalian neurulation, and increased activity of PKC was correlated with diabetic embryopathy. The PKC family of serine-threonine kinases plays important roles in a variety of cellular functions, such as cell growth, differentiation, and apoptosis. Based on their structures and cofactor regulation, PKC isozymes have been subdivided into 3 categories:i) the conventional PKCs (α,βⅠ,βⅡ, and y), which are activated by diacylglycerol (DAG) but not by calcium;ⅱ) the novel PKCs (δ,ε,θandμ), which are calcium-independent and activated by DAG;and iii) the atypical PKCs (ζandλ), which are calcium-independent and not activated by DAG. The PKCδhas been studied extensively because it appears to play an important role in cell apoptosis in a cellular and stimulus-dependent manner.PKCδregulates cell apoptosis of different cell types in response to H2O2, UV radiation, anti-cancer agents and reactive oxygen species (ROS). Molecular mechanisms such as tyrosine phosphorylation and PKCδtranslocation are of importance for the pro-apoptotic role for PKCδactivation. Tyrosine phosphorylation of PKCδis one of the earliest events and it has been shown to play a major role in the apoptotic function of PKCδ. Another important aspect that regulates the apoptotic function of PKCδis via its translocation during apoptotic responses. In response to various apoptotic stimuli, PKCδtranslocates to different subcellular components, including plasma membrane, mitochondria, Golgi complex, endoplasmic reticulum and nucleus.In our previous study, we have shown that c-Abl, a src-related non-receptor tyrosine kinase is involved in apoptotic pathways in apoptosis of NPCs induced by high glucose. c-Abl expression is up-regulated in NPCs exposed to high glucose, especially in the nucleus. Moreover, c-Abl has been implicated in the phosphorylation of PKCδin response to genotoxic and oxidative stress. PKCδis constitutively associated with c-Abl via the SH3 domain on c-Abl. Thus, c-Abl seems to act as a communicator between the PKCδand apoptosis.There is ample evidence showing that p53-mediated apoptosis is a key point for neural tube abnormalities during embryogenesis. Inhibition of p53 accumulation in neuroepithelium could prevent cell apoptosis and allow neural tube closure in wild-type embryos. Meanwhile, neuroepithelial cell apoptosis can not be detected in p53-/- embryos. All these indicate that p53 is a key factor in apoptosis of NPCs. Some studies have reported that c-Abl and p53 respond to similar genotoxic stresses. p53 interacts with c-Abl and plays a crucial role in oxidative stress-induced apoptosis. Thus, it is surmised that p53 may be an important factor in PKCδ-c-Abl pathway involving in NPCs apoptosis exposed to high glucose.In order to determine the PKCδand it's sinal pathway during the apoptosis of NPCs induced by high glucose, we performed our tests form the following three parts:Ⅰ. High glucose induced apoptosis of NPCs in vivo and in vitroIn vivo:Diabetes was induced in 8-week-old female Kunming mice with streptozotocin (STZ) at a dose of 75mg/kg body weight by intraperitoneal injection on three continuous days. Blood glucose levels were examined seven days after STZ injection. The mice with non-fasting blood glucose level exceeding 16.7 mmol/1 (300 mg/dl) were considered as diabetic mice. Timed mating was carried out at noon on the day, and when a copulation plug was observed it was considered as embryonic day 0.5 (E0.5). Mice were killed on embryonic day 11.5 (E11.5). All embryos were examined to identify NTD with stereoscope. The experimental results show that the rate of NTD or unclosed neural tube is approximately 32.7% of the total examined embryos, indicating mat the animal model of maternal diabetes-induced NTD has been successfully established. Apoptosis in neuroepithelium of the developing ventricular zone (VZ) area of E11.5 brain from DM and normal mice was detected by TUNEL labeling. Results of TUNEL labeling showed that the number of apoptotic cells in the neuroepithelium of the VZ area from DM mice was significantly increased compared with that of embryos from controls.In vitro:Embryos were recovered from pregnant mice on day 11.5 of gestation. The brain was dissected out from the embryo and mechanically dissociated into single cells. The dissociated cells were plated in Dulbecco's modified Eagle's medium/F12 (1:1) medium. After 5 days of incubation, neurospheres were harvested by centrifugation, dissociated using trypsin and EDTA and re-plated. After 5-6 days of growth, new neurospheres formed again and second passage was performed. NPCs were expanded as neurospheres, and experimental procedures were carried out with monolayers of NPCs. To prepare monolayers, the neurospheres were resuspended and plated on poly-d-lysine-coated culture plates in DMEM/F12 supplemented with B27 and bFGF and grown as monolayers for 24 h. In order to investigate the effects of high glucose on apoptosis in primary mouse NPCs, cells were exposed to normal glucose (5 mM D-glucose) or high glucose (35 mM D-glucose),or mannitol (5 mM D-glucose+30 mM mannitol). Mannitol was used as an osmotic pressure-matched control for high glucose medium. DAPI staining was used to observe the internucleosome DNA degradation. Significant internucleosome DNA degradation in NPCs was observed in cells after exposed to high glucose 24 h or 48 h, whereas normal glucose caused little internucleosome DNA degradation. In the control NPCs, there were 5%±1.3% apoptotic cells at 24 h, and 7%±1.9% at 48 h; in the mannitol NPCs, there were 8%±2.3% apoptotic cells at 24 h, and 9%±2.1% at 48 h; however, the frequency of apoptotic cells increased to 28%±3.9% at 24 h, and 35%±4.2% at 48 h following treatment with HG.Ⅱ.High glucose induces apoptosis of NPCs through a PKCδ-dependent mechanismTo evaluate whether elevating blood glucose of pregnant mice causes the activation of PKCδin the brain of embryo, immunoprecipitation-based kinase assays were used. In DM mouse, we observed that the basal level of PKCδactivity was much stronger than that in control group. Consistent with in vivo test, HG induced the increased PKCδactivity in NPCs. In order to determine whether PKCδwas involved in HG-induced apoptosis in vitro, we utilized rottlerin, which has been reported to be a PKC8 selective inhibitor. Treatment of NPCs with rottlerin (5μM) for 24 h did not affect the basal level of cell apoptosis; however, rottlerin inhibited the apoptotic effect of HG, reducing cell apoptosis by approximately 25% when exposed to HG for 24 h. To ascertain a possible role for PKCδin HG induced NPCs apoptosis, we evaluated the PKCδmRNA and protein levels of NPCs exposed to HG. In the presence of HG, there were no significant changes in the levels of PKCδmRNA after 9,16 and 24 h incubation. The protein levels of PKC8 were consistent with the mRNA levers. These results suggest that effects of hyperglycemia on PKCδactivity were due to stimulation of enzymatic activity rather than elevated mRNA or protein synthesis. Because tyrosine phosphorylation is important for the pro-apoptotic role for PKCδactivation, we then examined the effects of HG on tyrosine phosphorylation of PKCδ. NPCs were treated with HG for different duration. PKCδwas immunoprecipitated, and the membrane was blotted with anti-phosphotyrosine antibody. The rusult shows a low basal level of tyrosine phosphorylation was observed in untreated cells. HG induced tyrosine phosphorylation of PKCδin a time-dependent manner. Phosphorylation was first observed at 3 h, peaking at 6 h but it decreased thereafter. We next determined whether PKCδphosphorylation occurred in vivo. In DM mouse induced by STZ injection, it was difficult to ascertain the exact point time for blood glucose increase. In view of this, we had used hyperglycemic mouse given subcutaneous injections of glucose. The results were consistent with that in cultured NPCs. At 6 h after glucose injections, tyrosine phosphorylation in the embryo brain tissue was evidently increased.Activation of PKC8 by different stimuli results in distinct patterns of translocation, which are cell and stimulus dependent. To examine the effect of HG on the translocation of PKCδ, the subcellular distribution of PKCδin NPCs exposed to HG was analyzed. By immunofluorescence labeling, PKC8 was present in both the cytoplasm and nucleus of the control NPCs; however, HG treatment induced translocation of PKCδto the nucleus. Subcellular fractionation analysis confirmed these results showing that the PKCδlevel was significantly increased in the nuclear fraction of the NPCs exposed to HG. The cytoplasmic PKCδlevels were concomitantly and markedly decreased. In parallel to the findings for cultured NPCs, at 12 and 24 h after glucose injections, nuclear translocation of PKCδin the embryonic brain was increased.Ⅲ.The signal pathway of PKCδinvolved in NPCs apoptosis induced by high glucosePrevious studies have demonstrated that c-Abl, a non-receptor tyrosine kinase, interacts with PKCδin the cellular response to genotoxic stress and oxidative stress. Moreover, in our previous studies, we have shown that c-Abl expression notably the nuclear c-Abl signal was up-regulated in NPCs exposed to high glucose. Here we show that the time course increase of c-Abl in the nucleus coincided with that of PKCδ. We then determined whether PKCδand c-Abl had direct interaction when induced with HG. Analysis of anti-c-Abl immunoprecipitates by immunoblotting with anti-PKCδantibody demonstrated a significant increase in the association of PKCδwith c-Abl in the cytoplasm fraction 3 h after HG treatment. In reciprocal experiments, immunoblot analysis of anti-PKCδimmunoprecipitates using anti-c-Abl antibody confirmed the association of PKCδwith c-Abl. Under the same conditions, the nuclear fractions were also subjected to immunoprecipitations with anti-PKC8 and anti-c-Abl antibodies. Interestingly, a significant amount of the complex was found in nuclear fractions after 9 h, but not after 3 h following HG treatment. These data suggest that under HG stress, PKCδand c-Abl first interacted in the cytoplasm and then translocated to the nucleus, perhaps as a pre-formed complex. We then investigated whether PKC8-c-Abl interaction occurred also in vivo in the glucose injection mouse model. Increased interaction of PKCδand c-Abl in the embryo brain was observed at 6 h of glucose injections. These in vitro and in vivo data collectively support our hypothesis that hyperglycemia induces binding of PKC8 to c-Abl.To assess the effect of c-Abl on PKCδtranslocation to the nucleus, we inhibited the cytoplasmic c-Abl levels by transfecting NPCs with small interfering RNAs (siRNAs) targeting c-Abl; non-silencing siRNA was transfected into NPCs as the control. Translocation of PKCδto the nucleus at 16 h after HG treatment was significantly reduced when c-Abl levels were knocked down when compared with the control-siRNA transfected cells. We next determined whether PKCδis required for c-Abl translocation to the nucleus. c-Abl translocation to the nucleus which was evident at 16 h after HG induction, was almost completely abolished following PKCδknockdown.As a non-receptor tyrosine kinase, c-Abl phosphorylates PKCδin the tyrosine residues, which represents an early event in the apoptotic pathways and plays an important role in the pro-apoptotic effects of PKCδin response to various stimuli, such as H2O2, ionizing irradiation, UV radiation and ceramide. Therefore, we then tested whether the tyrosine phosphorylation of PKCδis mediated by c-Abl in response to high glucose. Here, we found that tyrosine phosphorylation of PKCδin the cytoplasmic fractions was almost completely abrogated in cells in which c-Abl was knocked down. Pretreatment of the NPCs with STI571 (5μM), an inhibitor of c-Abl, significantly decreased the phosphorylation of tyrosine of PKCδlikewise. These results indicate that, c-Abl, either directly or indirectly, are responsible for tyrosine phosphorylation of PKCδin the cytoplasm.Many studies have shown that p53-mediated apoptosis is responsible for neural abnormalities during embryogenesis. Inhibition of p53 accumulation in neuroepithelium could prevent cell death. We reported previously that in hyperglycemia induced apoptosis, p53 accumulated in nucleus of NPCs. In the light of this, we tested whether c-Abl-PKCδinteraction would play a role in the p53 accumulation in NPCs exposed to hyperglycemia. Indeed, silencing either c-Abl or PKCδreduced the p53 accumulation. Furthermore, treatment of cells with siRNA targeting PKCδor c-Abl decreased the incidence of apoptosis of NPCs as evaluated by DAPI staining. These data suggest that hyperglycemia-induced apoptosis mediated by PKCδand c-Abl is dependent on p53 signal.Conclusion:Here, we investigate the possible involvement of PKCδin apoptosis of NPCs exposed to high glucose both in vitro and in vivo. We show here that PKCδis phosphorylated by c-Abl, and translocation of PKC8-c-Abl complex from the cytoplasm to nucleus as well as the interaction between them plays a crucial role in p53 accumulation in nucleus. The series of events would ultimately lead to apoptosis of NPCs in response to high glucose.