Expression of calpain on apoptosis signaling in human rheumatoid synovial cells

目的：观察蛋白水解物对过氧化氢(H_2O_2)诱导的类风湿关节炎(RA)患者滑膜细胞凋亡的影响。细胞损伤通过乳酸脱氢酶释放到培养液中和Hoechst 33258细胞核染色来评估。结果RA滑膜细胞对H_2O_2有较强的抵抗力。然而，在100μM-N-乙酰亮氨酰-去亮氨酸(ALLN，称为钙蛋白酶抑制剂1)存在时，400μM_2O_2可诱导细胞死亡，而N-乙酰亮氨酰-亮氨酰-甲硫氨酸(ALLM)则不能。ALLN可诱导抑癌基因P53蛋白和p21~+WAF-1~GT；蛋白表达，其机制可能与抑制蛋白酶体有关。H_2O_2进一步增强ALLN诱导的P53表达。H_2O_2可激活c-jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)和Akt。因此，H_2O_2诱导的JNK活化和ALLN诱导的P53单独作用不足以诱导RA滑膜细胞的凋亡。结论这些结果提示，P53的诱导可能在RA滑膜细胞的凋亡过程中起重要作用，而AlLN作为钙蛋白和蛋白酶体的抑制剂可能对RA具有治疗作用。

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.