Kammouni Wafa, Wong Keng, Ma Guoping, Firestein Gary S, Gibson Spencer B, El-Gabalawy Hani S
University of Manitoba, Winnipeg, Manitoba, Canada.
Arthritis Rheum. 2007 Sep;56(9):2854-63. doi: 10.1002/art.22853.
Rheumatoid arthritis (RA) synovial hyperplasia is related in part to a resistance to apoptosis exhibited by fibroblast-like synoviocytes (FLS). Since hypoxia is a regulator of apoptosis, and since RA synovium is hypoxic, we conducted this study to examine the effects of hypoxia on the Bcl-2 pathway and the role this may play in regulating apoptosis in FLS.
Synovium samples from RA patients, osteoarthritis (OA) patients, and normal subjects were used for immunohistologic assessments and for generating FLS lines in vitro. FLS were stimulated under conditions of hypoxia (1% O(2)) and using 100 microM CoCl(2) to simulate the effects of severe hypoxia. Changes in the gene expression profile of FLS were evaluated using microarrays and were confirmed by quantitative polymerase chain reaction (PCR). Changes in protein expression were detected by Western blotting. The effect of transient transfection with a BNIP3 plasmid on the apoptosis of FLS was evaluated in the presence and absence of cytokines.
Gene expression profiling demonstrated that BNIP3 was unique among the BCL2 family, in that it was induced by hypoxia in FLS. Quantitative PCR indicated a 2-3-fold induction of BNIP3 messenger RNA, and Western blotting showed a 3-5-fold increase in the 30-kd Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3 (BNIP-3) monomer. BNIP-3 was widely expressed in RA synovium and was prominent in FLS from the lining layer. Overexpression of BNIP3 increased FLS apoptosis under hypoxic conditions, an effect that was inhibited by tumor necrosis factor alpha and interleukin-1beta.
The proapoptotic protein BNIP-3 is induced in FLS by hypoxia and is widely expressed in RA synovium, but its proapoptotic effects may be inhibited in vivo by proinflammatory cytokines. Since overexpression of BNIP3 in FLS increases apoptosis, this may provide a novel approach for controlling synovial hyperplasia in RA.
类风湿关节炎（RA）滑膜增生部分与成纤维样滑膜细胞（FLS）表现出的抗凋亡能力有关。由于缺氧是细胞凋亡的调节因子，且RA滑膜处于缺氧状态，我们开展本研究以检测缺氧对Bcl-2通路的影响及其在调节FLS细胞凋亡中可能发挥的作用。
使用来自RA患者、骨关节炎（OA）患者及正常受试者的滑膜样本进行免疫组织学评估，并在体外建立FLS系。FLS在缺氧条件（1% O₂）下以及使用100 μM氯化钴（CoCl₂）模拟严重缺氧的作用下受到刺激。使用微阵列评估FLS基因表达谱的变化，并通过定量聚合酶链反应（PCR）进行确认。通过蛋白质印迹法检测蛋白质表达的变化。在有或无细胞因子存在的情况下，评估用BNIP3质粒瞬时转染对FLS凋亡的影响。
基因表达谱显示，BNIP3在BCL2家族中是独特的，因为它在FLS中由缺氧诱导。定量PCR表明BNIP3信使核糖核酸诱导了2至3倍，蛋白质印迹法显示30-kd Bcl-2/腺病毒E1B 19-kd蛋白相互作用蛋白3（BNIP-3）单体增加了3至5倍。BNIP-3在RA滑膜中广泛表达，在衬里层的FLS中尤为突出。BNIP3的过表达在缺氧条件下增加了FLS凋亡，但该作用被肿瘤坏死因子α和白细胞介素-1β抑制。
促凋亡蛋白BNIP-3在FLS中由缺氧诱导且在RA滑膜中广泛表达，但其促凋亡作用在体内可能被促炎细胞因子抑制。由于FLS中BNIP3的过表达增加了细胞凋亡，这可能为控制RA中的滑膜增生提供一种新方法。
