Nathanson Carl-Michael, Wassélius Johan, Wallin Hanna, Abrahamson Magnus
Department of Clinical Chemistry, Institute of Laboratory Medicine, and Department of Ophtalmology, University of Lund, University Hospital, Lund, Sweden.
Eur J Biochem. 2002 Nov;269(22):5502-11. doi: 10.1046/j.1432-1033.2002.03252.x.
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 ( approximately 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 promoter 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 ( approximately 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.
胱抑素F是最近通过cDNA克隆在造血细胞中发现的一种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、分布和特性，对早幼粒细胞系U937进行了研究。这些细胞表达了相对大量的胱抑素F，在细胞外和细胞内均有发现。对于一种分泌型胱抑素而言，其细胞内水平异常高（在2天或4天培养基中约占胱抑素F的25%）。相比之下，U937细胞仅含有3%-4%的相关抑制剂胱抑素C。从U937细胞裂解物中纯化的胱抑素F显示出三种主要形式，分别带有两条、一条或没有糖链。免疫细胞化学显示在细胞质中有明显的颗粒状胱抑素F染色。用内质网标志物进行双重染色显示胱抑素F没有共定位。对胱抑素F基因（CST7）启动子区域的分析表明，它与胱抑素C基因（CST3）的启动子一样，缺乏典型的TATA盒和CAAT盒元件。与胱抑素C启动子不同，它不包含多个Sp1结合位点，但有一个独特的C/EBPα结合位点，这可能解释了胱抑素F基因表达受限的原因。用全反式维甲酸刺激细胞使其向粒细胞途径分化，结果显示细胞内胱抑素F强烈下调（约18倍），且抑制剂的分泌水平几乎消失。用十四酰佛波醇乙酸酯刺激导致单核细胞分化，也导致胱抑素F表达下调（2至3倍），而在这两个实验中胱抑素C的表达基本未改变。结果表明，胱抑素F作为一种细胞内半胱氨酸肽酶抑制剂，其表达易于调节，可能是控制已知在不同血细胞谱系中抗原呈递所必需的半胱氨酸肽酶活性的候选物质。
