Expression of calpain on apoptosis signaling in human rheumatoid synovial cells

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Objectives-The effects of inhibitors for proteolysis on hydrogen peroxide (H_2O_2)-induced apoptosis were examined in cultured human synovial cells of rheumatoid arthritis (RA) patients.Methods- Synovial cells from RA patients were obtained at the time of total knee replacement. Cellular damages were assessed by the release of lactate dehydrogenase (LDH) into the culture medium and nuclear staining with Hoechst 33258. Processing of procaspase-3, expression of p53 and p21^<WAF-1>, and phosphorylation (activation) of protein kinases were examined by Western blotting.Results-RA synovial cells were relatively resistant to H_2O_2. However, in the presence of 100 μM N-acetyl-leucyl-leucyl-norleucinal (ALLN, known as calpain inhibitor 1), but not N-acetyl-leucyl-leucyl-methioninal (ALLM) apoptotic cell death was elicited by 400 μM H_2O_2, at a concentration which alone never induced cell death. ALLN induced the expression of tumor suppressor p53 protein and p21^<WAF-1> protein, probably through inhibition of proteasome. H_2O_2 further potentiated ALLN-induced p53 expression. H_2O_2 appeared to activate c-Jun N-terminal kinase (JNK), and also extracellular signal-regulated kinase (ERK) and Akt. The latter kinases implicated in cell survival may be engaged in resistance of RA synovial cells to H_2O_2. Therefore, JNK activation by H_2O_2 and p53 induction by ALLN, either one alone is insufficient but their combinations synergistically induce apoptosis of RA synovial cells.Conclusion-These results suggest that induction of p53 may be potentially important for triggering of apoptosis processes in RA synovial cells and that ALLN, an inhibitor of both calpain and proteasome may have a therapeutic potential to RA.