Objective
Neural tube defects in diabetic embryopathy are associated with increased protein kinase C (PKC)β2 activity and programmed cell death (apoptosis). The apoptosis is triggered by caspase 8, which activates members of the Bcl-2 and caspase families, such as Bid and caspase 3. Whether PKCβ2 regulates caspase 8–induced apoptosis remains to be addressed.
Study Design
Mouse embryos at embryonic day 8.5 were cultured in a high concentration of glucose (22 mmol/L) and treated with PKCβ2 inhibitor (50 nmol/L) for 48 hours. The levels of apoptosis and activation of apoptotic factors were quantified using the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and Western blot assays, respectively.
Results
Reduction in the rate of neural tube defect by PKCβ2 inhibition is associated with significant decreases in the levels of apoptosis, and caspase 8, caspase 3, and Bid activation, and cytochrome C release from mitochondria, to the similar levels as in euglycemic controls (8.3 mmol/L; P < .05).
Conclusion
PKCβ2 influences a caspase 8–regulated apoptotic pathway in diabetic embryopathy.
Diabetes mellitus in pregnancy is largely associated with a variety of birth defects in infants, including malformations in the central nervous system. However, the precise mechanism of neural tube defect (NTD) has not been fully elucidated, although increased oxidative stress and programmed cell death (apoptosis) have been found to be involved.
NTD is a result of a failure in neural tube closure along the dorsal edges of the neural groves during neurulation. Increased apoptosis in the neuroepithelium has been seen in the embryos of diabetic animals and demonstrated to be a major factor in preventing the closure of the neural tube in diabetic embryopathy.
Apoptosis is a complex process regulated by a number of factors in which caspases play the key roles. Caspases are cysteine proteases and divided into initiator and effector caspases. The effector caspases (eg, caspase 3, 6, and 7) are directly responsible for the execution of apoptosis. However, the initiator caspases including caspase 8 and 9 are carrying apoptotic signals by triggering a molecular cascade to activate effector caspases.
Activated caspase 8 can cleave Bid to form truncated Bid (tBid). tBid triggers a release of cytochrome C from mitochondria. Cytochrome C further activates caspase 9, leading activation of effector caspases and execution of apoptosis. In addition, caspase 8 can also directly target effector caspases, depending on the cell type and pathological conditions. In hyperglycemia (HG)-induced NTDs, caspase 8 has been shown to play a key role in apoptotic regulation. It is activated by HG in the embryos. A blockade of caspase 8 activity significantly reduces NTD rate in the embryos exposed to high glucose.
Maternal diabetes disturbs intracellular signaling involving the protein kinase C (PKC) family. The PKC serine/threonine protein kinase family comprises at least 12 isoforms and can be activated by various factors including diacylglycerol (DAG), inositol 1,4,5-trisphosphate, and calcium.
Specifically, PKCα, PKCβ2, and PKCδ have been found to be associated with NTDs in diabetic embryopathy. In addition, PKCβ2 has been shown to be responsible for apoptosis through increased oxidative stress. Therefore, it is hypothesized that PKCβ2 plays a role in diabetic embryopathy via regulating apoptosis. In the present study, the role of PKCβ2 in apoptosis and caspase regulation has been investigated in embryos exposed to HG.
Materials and Methods
Embryo culture
The use of animals was approved by Institutional Animal Care and Use Committee of University of Maryland Baltimore. Female mice (C57BL/6J) were mated in the afternoon. The next day was designated as embryonic day (E) 0.5 if a vaginal plug was present.
Mouse embryos were dissected in cold phosphate-buffered saline (pH 7.4). The parietal yolk sac was removed, and the visceral yolk sac was left intact. Embryos were cultured for 48 hours in a bottle containing 5 mL of heat-inactivated rat serum at 38°C with rotation. Embryos were cultured in euglycemic control (CON) (8.3 mmol/L glucose) and HG (22 mmol/L) media in the presence or absence of PKCβ2 inhibitor (PKCβ2I) [3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] (50 nmol/L), EMD Biosciences, San Diego, CA. The bottles were gassed with 5% oxygen (O 2 )-5% carbon dioxide (CO 2 )-90% nitrogen (N 2 ) at first 24 hours and then in 20% O 2 -5% CO 2 -75% N 2 for another 24 hours. At the end of culture, embryos were harvested for examination of NTDs. A malformation rate is calculated as a percentage of the embryos with NTDs in total number of embryos in each experiment.
To examine the effects of PKCβ2 inhibition on apoptosis and protein changes, comparisons were made among normal embryos in the CON and HG + PKCβ2 inhibitor groups and malformed embryos in the HG group.
TUNEL assay
The terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay was used to detect fragmented DNA, which is a product of apoptosis. Embryos were embedded in OCT tissue-embedding medium (Sakura Scientific, Torrance, CA) and frozen on dry ice. Sections were cut in 20 μm. The sections were incubated with fluorescein-conjugated deoxyuridine triphosphate and terminal transferase for 60 minutes at 37°C (Roche, Mannheim, Germany) and counterstained with DAPI (4′,6-diamindino-2-phenylendole). TUNEL-positive apoptotic bodies in green fluorescence and all nuclei in blue fluorescence were observed under a fluorescent microscope.
Western blot assay
The neural tube tissues of the brain and the anterior part of spinal cord of the embryos were dissected using a pair of fine scissors. The ectoderm was carefully removed. Tissues were homogenized in RIPA lysis buffer (Millipore Corp, Temecula, CA) containing protease inhibitor cocktail (Thermo Scientific, Waltham, MA) and then centrifuged at a speed of 14,000 rpm/min for 20 minutes to obtain supernatants. After denaturing for 3 minutes at 95°C, equal amount of the proteins were loaded on 8% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, separated electrophoretically, and blotted to Immobilon-P nylon membranes (Millipore Corp). The membranes were incubated with primary antibodies anti-β-actin (Sigma-Aldrich, St. Louis, MO), anticytochrome C (Santa Cruz Biotechnology, Santa Cruz, CA), anticaspase 8 (Alexis Biochemicals, Tracy, CA), anticaspase 3, and anti-Bid (Cell Signaling, Danvers, MA) in a buffer (20 mmol/L Tris·HCl, 136 mmol/L NaCl, 0.1% Tween 20, pH 7.6) containing 5% nonfat milk. Detection of specific proteins was carried out using peroxidase-conjugated secondary antibodies and a chemiluminescent substrate (Amersham Biosciences, Piscatacay, NJ). The bands of fluorescence were captured and fluorescence intensity was analyzed using UVP Bioimaging Systems (Upland, CA) with VisionWork software (UVP Bioimaging Systems). Data were expressed as ratio of the fluorescence intensity of target proteins to that of β-actin.
Statistical analysis
Data are presented as mean ± SE. Student t test was used to compare significance between 2 groups. The criterion for statistical significance was P < .05 in all tests.
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