Phosphatase-1 and -2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production.
OBJECTIVE
To characterise the role of phosphatase-1 and -2A (PP1/2A) in the modulation of apoptosis in human osteoarthritis (OA) chondrocytes.


METHODS
Human OA chondrocytes were isolated from cartilage obtained from the femoral heads of patients undergoing joint replacement surgery. Cell viability was evaluated by MTT assay. Apoptosis was quantified by ELISA, which measures DNA fragmentation. Nitric oxide (NO) production was evaluated by the Greiss method, and inducible nitric oxide synthase (iNOS) protein synthesis was studied by western blotting.


RESULTS
Inhibition of PP1/2A by the specific inhibitor okadaic acid (OKA) dose and time dependently caused a reduction of cell viability (OKA at 50 nmol/l: a reduction to 60% and 43% at 48 and 72 hours, respectively). Genomic DNA from chondrocytes treated with OKA at 50 and 100 nmol/l for 48 hours displayed increased internucleosomal DNA fragmentation by 11 and 13 fields, respectively. Light microscopy and DAPI studies showed that OKA induced DNA condensation and fragmentation, typical of death by apoptosis. The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK increased cell viability, reduced by OKA at 50 nmol/l to 87% and 73%, respectively. OKA did not increase iNOS protein synthesis or NO production.


CONCLUSIONS
PP1/2A modulate apoptosis in human OA chondrocytes; this is independent of NO production but dependent on caspases.
López-Armada
M J
MJ
Laboratory of Investigation, Rheumatology Division, Juan Canalejo Hospital, Xubias 84. 15006-A Coruña, Spain.
Caramés
B
B
Cillero-Pastor
B
B
Lires-Deán
M
M
Maneiro
E
E
Fuentes
I
I
Ruíz
C
C
Galdo
F
F
Blanco
F J
FJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002358:Cartilage, Articular; D061945:Caspase Inhibitors; D018929:Cell Culture Techniques; D019902:Chondrocytes / Q000201:enzymology* / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D020287:In Situ Nick-End Labeling; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D019319:Okadaic Acid / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D015207:Osteoarthritis, Hip / Q000201:enzymology* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054645:Protein Phosphatase 1
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019319:Okadaic Acid; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1