Hypoxia induces apoptosis by caspase activation accompanying cytochrome C release from mitochondria in MC3T3E1 osteoblasts. p38 MAPK is related in hypoxia-induced apoptosis.
The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphologystained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase- activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anti-caspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.
Chae
H J
HJ
Department of Dental Pharmacology and Wonkwang Dental Reseaarch Institute, School of Dentistry, Wonkwang University, South Korea.
Kim
S C
SC
Han
K S
KS
Chae
S W
SW
An
N H
NH
Kim
H M
HM
Kim
H H
HH
Lee
Z H
ZH
Kim
H R
HR
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505409:Casp3 protein, mouse; C505516:Casp6 protein, mouse; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; C093642:SB 203580