Expression of calpain on apoptosis signaling in human rheumatoid synovial cells

Objectives-The effects of inhibitors for proteolysis on hydrogen peroxide（H_2O_2）-induced apoptosis was examined in cultured human synovial cells of rheumatoid arthritis（RA）patients.Methods-滑膜细胞从RA患者获得的时间全膝关节置换术。通过乳酸脱氢酶（LDH）释放到培养基中和Hoechst 33258核染色来评估细胞损伤。Western <WAF-1>blotting法检测RA滑膜细胞对H_2O_2的耐受性。在100 μM的N-乙酰-亮氨酰-亮氨酰-正亮氨酸（ALLN，又称calpain inhibitor 1）存在下，400 μM的H_2O_2可诱导细胞凋亡，但不诱导N-乙酰-亮氨酰-亮氨酰-蛋氨酸（ALLM）。ALLN可能通过抑制蛋白酶体而诱导抑癌基因p53蛋白和p21 ~+蛋白的表达<WAF-1>。H_2O_2可进一步增强p53的表达。H_2O_2可激活c-Jun N-末端激酶（JNK）、细胞外信号调节激酶（ERK）和Akt。这些参与细胞存活的激酶可能参与RA滑膜细胞对H_2O_2的抵抗。因此，JNK激活H_2O_2和p53诱导ALLN，单独是不够的，但它们的组合协同诱导RA滑膜cells. Conclusion这些结果表明，诱导p53可能是潜在的重要触发RA滑膜细胞的凋亡过程和ALLN，钙蛋白酶和蛋白酶体的抑制剂可能有治疗RA的潜力。

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.