Prevention of hypoxia-induced neuronal apoptosis through histone deacetylase inhibition.
BACKGROUND
We have recently discovered that administration of valproic acid (VPA), a histone deacetylase inhibitor, enhances nuclear histone acetylation and improves survival after lethal hemorrhage in rats. In the present study, neurons were subjected to severe hypoxic condition in vitro to test whether VPA would prevent hypoxia-induced apoptosis, and to explore the possible mechanisms.


METHODS
Primary hippocampal and cortical cultures dissociated from E18 rat embryos were plated in quadruplicate at a density of 2 x 10/well in neurobasal medium supplemented with B-27 on glass cover-slips coated with poly-l-lysine. On the 10th day after plating, cells were incubated in a hypoxia chamber (0.5% O2, 10% CO2, 89.5% N2) at 37 degrees C for 6 hour and 16 hour in the presence or absence of VPA (1 mmol/L). The cells were then fixed, stained with antiactivated caspase-3 and antiacetyl histone H3 lysine 9 (Ac H3 K9) antibodies and visualized under confocal microscope. The caspase-3 positive cells were counted as apoptotic. Ratio of the apoptotic to total cells stained with 4',6-diamidino-2-phenylindole was determined. Numerical data were subjected to t test analysis. p < 0.05 was considered statistically significant. Western blot was performed to determine the level of acetylation of nuclear factor-kappa B (NF-kappaB) and phospho-JNK (c-Jun N-terminal kinase) in cells treated with or without VPA. Luciferase report assay was employed to analyze the activation of NF-kappaB after the cells were transfected with NF-kBLuc with or without VPA treatment.


RESULTS
Exposure of neurons to VPA prevented apoptotic cell death under hypoxic conditions (20% apoptosis). In contrast, about 95% cells underwent apoptosis at the same level of hypoxia. VPA treatment induced acetylation of histone H3 K9 and NF-kappaB lysine 310. NF-kappaB was activated at the same time as the protein acetylation. Moreover, JNK phosphorylation was inhibited after the cells were treated with VPA under hypoxia condition.


CONCLUSIONS
VPA enhances acetylation of histone 3 at lysine 9 and NF-kappaB at 310, induces NF-kappaB activation, reduces JNK activation, and protects the neurons from hypoxia-induced apoptosis in vitro.
Li
Yongqing
Y
Department of Surgery, Division of Trauma, Emergency Surgery and Surgical Critical Care, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA.
Yuan
Zengqiang
Z
Liu
Baoling
B
Sailhamer
Elizabeth A
EA
Shults
Christian
C
Velmahos
George C
GC
Demoya
Marc
M
Alam
Hasan B
HB
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian; D005260:Female; D006655:Histone Deacetylases / Q000187:drug effects* / Q000378:metabolism; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D000860:Hypoxia / Q000150:complications; D048031:JNK Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D011247:Pregnancy; D011270:Pregnancy, Animal; D011336:Probability; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014635:Valproic Acid / Q000494:pharmacology*
D006657:Histones; D016328:NF-kappa B; D014635:Valproic Acid; D048031:JNK Mitogen-Activated Protein Kinases; D020169:Caspases; D006655:Histone Deacetylases