NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis
NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis
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DOI：
10.1002/art.20680
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发表时间：
2004-12-01
影响因子：
--
通讯作者：
Pope, RM
中科院分区：
文献类型：
--
作者：
Bai, SC;Liu, HT;Pope, RM
Objective.在类风湿关节炎（RA）滑膜组织中几乎没有观察到细胞凋亡。肿瘤坏死因子α（TNF α）在RA患者的关节中表达，但RA滑膜成纤维细胞对TNF α诱导的细胞凋亡具有相对抗性。最近，我们证明FLIP在RA关节中高度表达。进行这些研究以确定TNF α诱导的NF-κ B是否控制RA滑膜成纤维细胞中FLIP长（FLIPL）和FLIP短（FLIPS）的表达，并确定FLIP在TNF α诱导的滑膜炎控制中的作用。从RA滑膜组织中分离RA滑膜成纤维细胞，并在第3代和第9代之间使用。将RA滑膜或对照成纤维细胞假感染或用对照腺病毒载体或表达超阻遏物IkappaB α（srIkappabalpha）的载体感染。用TNF α或对照载体刺激细胞，并通过同种型特异性实时聚合酶链反应和Western印迹分析测定FLIP和FLIPS的表达。XTT裂解法检测细胞活力，Annexin V染色、DNA片段化、caspase 8和caspase 3活化检测细胞凋亡。TNF α诱导两种异构体的FLIP信使RNA（mRNA）在RA滑膜成纤维细胞的表达，然而，FLIPL是占主导地位的异构体检测蛋白质印迹分析。在对照成纤维细胞中，TNFa诱导FLIPL和FLIPS mRNA和蛋白的表达。TNF α诱导的FLIP表达受NF-κ B调控，而基础FLIP表达不受NF-κ B调控。当NF-κ B活化被srIkappaB α的表达抑制时，TNF α介导的细胞凋亡被诱导。TNF α诱导的凋亡性细胞死亡是通过caspase 8激活介导的，并被FLIP或caspase 8抑制剂CrmA的异位表达所阻止。在RA滑膜成纤维细胞中，TNF α诱导的FLIP表达受NF-κ B调节，而非基础表达。RA滑膜成纤维细胞对TNF α诱导的凋亡的抵抗是由NF-κ B调节的FLIP表达介导的。这些观察结果支持NF-κ B和FLIP作为RA有吸引力的治疗靶点的作用。
Objective. Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor a (TNFalpha) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFa. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFalpha-induced NF-kappaB controls the expression of FLIP long (FLIPL) and FLIP short (FLIPS) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFa-induced apoptosis.Methods. RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super-repressor IkappaBalpha (srIkappabalpha). The cells were stimulated with TNFalpha or a control vehicle, and expression of FLIP, and FLIPS was determined by isoform-specific real-time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3.Results. TNFalpha induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIPL was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFa induced the expression of FLIPL and FLIPS mRNA and protein. The TNFalpha-induced, but not the basal, expression of FLIP was regulated by NF-kappaB. When NF-kappaB activation was suppressed by the expression of srIkappaBalpha, TNFalpha-mediated apoptosis was induced. TNFa-induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP, or the caspase 8 inhibitor CrmA.Conclusion. The TNFalpha-induced, but not the basal, expression of FLIP is regulated by NF-kappaB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFa-induced apoptosis is mediated by the NF-kappaB-regulated expression of FLIP. These observations support the role of NF-kappaB and FLIP as attractive therapeutic targets in RA.