肿瘤坏死因子α介导人骨关节炎软骨细胞中SirT1的裂解和失活。
Tumor necrosis factor α-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes.
作者信息
Dvir-Ginzberg Mona, Gagarina Viktoria, Lee Eun Jin, Booth Richard, Gabay Odile, Hall David J
机构信息
National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland, USA.
出版信息
Arthritis Rheum. 2011 Aug;63(8):2363-73. doi: 10.1002/art.30279.
OBJECTIVE
The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression.
METHODS
Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNFα-treated extracts was identified by mass spectroscopy, and its amino-terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα.
RESULTS
TNFα-treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full-length 110-kd protein and a smaller 75-kd fragment. The 75-kd SirT1 fragment was found to lack the carboxy-terminus. Cathepsin B was identified as the TNFα-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75-kd SirT1 fragment.
CONCLUSION
These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B-mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage-specific gene expression evident in these TNFα-treated cells.
目的
蛋白质脱乙酰酶SirT1正向调节软骨特异性基因表达，而促炎细胞因子肿瘤坏死因子α（TNFα）则负向调节这些相同基因。本研究旨在验证SirT1受TNFα负面影响从而导致基因表达改变这一假说。
方法
在未处理或经TNFα处理的人骨关节炎软骨细胞的RNA、蛋白质提取物和细胞核中，测定软骨特异性基因表达、SirT1活性以及α2（I）型胶原增强子位点的染色质免疫沉淀分析结果。用SirT1和表位特异性抗体对未处理或经TNFα处理的、转染了表位标记SirT1的人软骨细胞的蛋白质提取物进行免疫印迹分析。通过质谱鉴定TNFα处理提取物中存在的75-kd SirT1反应性蛋白，并通过埃德曼测序确定其氨基末端切割位点。在体外组织蛋白酶B切割反应后测定SirT1活性。将组织蛋白酶B小干扰RNA（siRNA）转染到未处理或经TNFα处理的软骨细胞中。
结果
经TNFα处理的软骨细胞中SirT1酶活性受损，并呈现出该酶的两种形式：全长110-kd蛋白和较小的75-kd片段。发现75-kd SirT1片段缺少羧基末端。组织蛋白酶B被鉴定为在残基533处切割SirT1的TNFα反应性蛋白酶。TNFα暴露后通过siRNA降低组织蛋白酶B水平可阻止75-kd SirT1片段的产生。
结论
这些数据表明，TNFα作为一种介导关节炎关节炎症的细胞因子，可诱导组织蛋白酶B介导的SirT1切割，导致SirT1活性降低。这种降低的SirT1活性与这些经TNFα处理的细胞中明显降低的软骨特异性基因表达相关。
Zotero 插件