Hydrogen peroxide induces apoptosis of chondrocytes; involvement of calcium ion and extracellular signal-regulated protein kinase.
OBJECTIVE
Recent observations demonstrated that reactive oxygen species facilitate cartilage degradation. We demonstrated that hydrogen peroxide (H2O2) caused inhibition of proteoglycan synthesis, induction of apoptosis and stimulation of extracellular signal-regulated protein kinase (ERK) of the chondrocytes (Inflamm Res 48: 399-403, 1999). To determine whether activation of ERK is involved in the induction of chondrocyte apoptosis, we examined the signal transduction pathways in this hydrogen peroxide induced apoptosis.


METHODS
Bovine articular chondrocytes were cultured. To determine the induction of apoptosis, Annexin V staining and terminal deoxynucleotidyl transferase were used. The activity of caspase-3 was measured using an apopain assay kit. Intracellular Ca2+ imaging was observed after fura2-AM loading.


RESULTS
Hydrogen peroxide enhanced annexin V positive apoptotic cells and caspase-3 activity, which is an executor of apoptosis. Hydrogen peroxide also enhanced intracellular Ca2+ and preincubation with the intracellular Ca2+ chelator protected chondrocytes against hydrogen peroxide-induced cell apoptosis, indicating that an increase in the cytosolic Ca2+ plays a decisive role in this action. When ERK activity was blocked with geldanamycin and PD098059, increased apoptosis was evident.


CONCLUSIONS
Hydrogen peroxide induces chondrocyte apoptosis via Ca2+ signaling, and ERK is involved in these signal transduction pathways.
Department of Orthopaedic Surgery, Kinki University School of Medicine, Osaka-sayama, Osaka, Japan.
S
S
Asada
K
K
Fukuda
F
F
Nishisaka
M
M
Matsukawa
C
C
Hamanisi
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D019902:Chondrocytes / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D047029:Lactams, Macrocyclic; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D015398:Signal Transduction
D017304:Annexin A5; D016227:Benzoquinones; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005419:Flavonoids; D047029:Lactams, Macrocyclic; D011809:Quinones; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D006861:Hydrogen Peroxide; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3; D020169:Caspases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium; C001277:geldanamycin