Regulated expression and intracellular localization of cystatin F in human U937 cells
Regulated expression and intracellular localization of cystatin F in human U937 cells
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DOI：
10.1046/j.1432-1033.2002.03252.x
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发表时间：
2002-11-01
期刊：
影响因子：
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通讯作者：
Abrahamson, M
中科院分区：
文献类型：
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作者：
Nathanson, CM;Wassélius, J;Abrahamson, M
半胱氨酸蛋白酶抑制剂F是最近通过cDNA克隆在造血细胞中发现的半胱氨酸肽酶抑制剂。为了进一步研究天然人抑制剂的表达、分布和性质，研究了早幼粒细胞系U937。细胞表达相对大量的胱抑素F，这是发现分泌和细胞内。对于分泌的半胱氨酸蛋白酶抑制剂，细胞内水平异常高（在2天或4天的培养基中约为半胱氨酸蛋白酶抑制剂F的25%）。相比之下，U937细胞仅含有3-4%的相关抑制剂半胱氨酸蛋白酶抑制剂C。从U937细胞裂解物中纯化的半胱氨酸蛋白酶抑制剂F显示三种主要形式，携带两个，一个或没有碳水化合物链。免疫细胞化学显示细胞质中存在明显的颗粒状半胱氨酸蛋白酶抑制剂F染色。内质网标记物的双重染色显示半胱氨酸蛋白酶抑制剂F没有共定位。CystatinF基因（CystatinF，CST 7）与CystatinC基因（CystatinC，CST 3）一样，不含典型的TATA盒和CAAT盒元件。与半胱氨酸蛋白酶抑制剂C启动子相反，它不包含多个Sp1结合位点，但具有C/EBPalpha的独特位点，这可能解释了半胱氨酸蛋白酶抑制剂F基因的受限表达。用全反式视黄酸刺激细胞以使其向粒细胞途径分化，显示出细胞内半胱氨酸蛋白酶抑制剂F的强烈（约18倍）下调，并且几乎消除了抑制剂的分泌水平。十四烷酰佛波醇乙酸酯刺激，引起单核细胞分化，也导致半胱氨酸蛋白酶抑制剂F的表达下调（2倍至3倍），而半胱氨酸蛋白酶抑制剂C的表达基本上是不变的，在这两个实验。结果表明，半胱氨酸蛋白酶抑制剂F作为一种细胞内半胱氨酸肽酶抑制剂，具有容易调节的表达，可能是一个候选人，以控制半胱氨酸肽酶活性已知是必不可少的抗原呈递在不同的血细胞谱系。
Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin (approximate to 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the romoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong (approximate to 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation ( two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.