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Phosphatase-1 and -2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production
  1. M J López-Armada1,
  2. B Caramés1,
  3. B Cillero-Pastor1,
  4. M Lires-Deán1,
  5. E Maneiro1,
  6. I Fuentes2,
  7. C Ruíz1,
  8. F Galdo1,2,
  9. F J Blanco1
  1. 1Laboratory of Investigation, Rheumatology Division, Juan Canalejo Hospital, Xubias 84. 15006-A Coruña, Spain
  2. 2Department of Medicine, Universidade da Coruña, Spain
  1. Correspondence to:
    Dr F J Blanco


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.

Conclusion: PP1/2A modulate apoptosis in human OA chondrocytes; this is independent of NO production but dependent on caspases.

  • DAPI, 4′,6-dianidino-2-phenylindole dihydrochloride
  • ELISA, enzyme linked immunosorbent assay
  • iNOS, inducible nitric oxide synthase
  • MTT, ???
  • NO, nitric oxide
  • OA, osteoarthritis
  • OKA, okadaic acid
  • PBS, phosphate buffered saline
  • PP1/2A, phosphatase-1 and -2A
  • PPase, protein phosphatases
  • chondrocytes
  • phosphatases
  • apoptosis
  • nitric oxide
  • osteoarthritis

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  • Presented in part at the 67th Annual Scientific Meeting of the American College of Rheumatology, Orlando, FL, October 2003.