Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein.
Nitric oxide (NO) plays a crucial role in the physiological and pathophysiological regulations of osteoblast functions. This study is designed to evaluate the toxic effects of NO released by sodium nitroprusside (SNP), an NO donor, on neonatal Wistar rat calvarial osteoblasts from the analyses of cell viability, alkaline phosphatase (ALP) activity, cell morphology, apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end-label (TUNEL) assay, DNA ladder, and immunocytochemistry and Western blot for proapoptotic Bax protein. SNP increased the levels of nitrite, an oxidative product of NO, in the culture medium of osteoblasts in concentration- and time-dependent manners, and altered cell morphologies to round and shrinkage shapes. Administration of osteoblasts with SNP resulted in concentration- and time-dependent decreases of cell viability and ALP activity. Analysis of apoptotic cells revealed that SNP increased the percentages of osteoblasts processing apoptosis. Analyses of TUNEL and DNA ladder showed that SNP caused DNA fragmentation. Pretreatment with cycloheximide, an inhibitor of protein synthesis, partially blocked SNP-induced osteoblast apoptosis. Imunocytochemical and immunoblotting analyses revealed that SNP increased Bax protein in osteoblasts. This study suggests that SNP could increase the levels of NO in osteoblasts, and cause osteoblast apoptosis possibly through the de novo synthesis of proapoptotic Bax protein.
Chen
Ruei-Ming
RM
Department of Anesthesiology, Taipei Medical College, Wan-Fang Hospital, Taiwan, ROC.
Liu
Hwa-Chang
HC
Lin
Yi-Ling
YL
Jean
Wen-Chyi
WC
Chen
Jui-Shui
JS
Wang
Jyh-Horng
JH
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004347:Drug Interactions; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017208:Rats, Wistar; D012886:Skull / Q000166:cytology; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D009599:Nitroprusside; D009569:Nitric Oxide; D003513:Cycloheximide; D000469:Alkaline Phosphatase