Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line.
Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line.
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DOI：
10.1042/bj2850629
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发表时间：
1992-07
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影响因子：
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通讯作者：
William;Hollas;Emilia Soravia;Andrew P. Mazar;Jack Henkin;T. F. Blasi;Douglas BOYDt
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文献类型：
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作者：
William;Hollas;Emilia Soravia;Andrew P. Mazar;Jack Henkin;T. F. Blasi;Douglas BOYDt
现在有充分的证据表明，尿激酶（UK）的蛋白水解作用是由一个特定的细胞表面受体增强。本研究旨在确定UK作为其结合位点调节剂的作用。GEO结肠细胞分泌低水平的UK（每10（6）个细胞每72小时约2.5ng/ml），并显示约2.5ng/ml的UK。用RSV长末端重复序列（LTR）启动子驱动的外源UK基因（pRSVUK）转染每个细胞10（4）个受体，其中大部分是空的。通过e.l.i.s.a.鉴定了几个UK过表达的pRSVUK克隆，北方印迹和南方印迹，并在酸预处理解离受体结合的UK后分析受体数量。表达高水平UK的pRSVUK GEO克隆始终比仅携带选择性标记基因neo的克隆或对照GEO细胞结合放射性二异丙基氟磷酸（DFP）-UK少50-75%。用与受体结合的UK的放射性N-末端片段进行的交联实验显示，结合蛋白的量减少约10%。在代表性pRSVUK转染细胞中为51 kDa。饱和度和Scatchard分析表明，放射性配体结合的减少反映了UK受体数量减少40-70%，而不是解离常数的变化。受体展示的减少可以通过编码受体的稳态mRNA的量的减少来解释。放射性DFP-UK与pRSVUK GEO克隆的结合，其显示比其neo对应物少三分之二的受体，可以通过在抑制UK与其受体相互作用的抗体存在下培养它们来恢复到对照水平（携带neo的未处理细胞）。这些数据表明，至少对于一个结肠细胞系，UK通过其细胞表面受体的自分泌刺激降低其自身结合位点的表达。
There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to determine the role of UK as a modulator of its binding site. GEO colonic cells, which secrete low levels of UK (approximately 2.5 ng/ml per 72 h per 10(6) cells) and display approx. 10(4) receptors per cell, the majority of which are vacant, were transfected with an exogenous UK gene driven by the RSV long terminal repeat (LTR) promoter (pRSVUK). Several UK-overexpressing pRSVUK clones were identified by an e.l.i.s.a., Northern blotting and Southern blotting, and analysed for receptor numbers after an acid pretreatment which dissociates receptor-bound UK. pRSVUK GEO clones, expressing high levels of UK, consistently bound 50-75% less radioactive di-isopropylfluorophosphate (DFP)-UK than clones harbouring the selectable marker gene neo only or control GEO cells. Cross-linking experiments with a radioactive N-terminal fragment of UK which binds to the receptor showed a decreased amount of a binding protein of approx. 51 kDa in representative pRSVUK-transfected cells. Saturation and Scatchard analysis indicated that this reduction in radioligand binding reflected a 40-70% decrease in the number of UK receptors, rather than a change in the dissociation constant. The reduction in receptor display could be accounted for by a decrease in the amount of steady-state mRNA encoding the receptor. Radioactive DFP-UK binding to pRSVUK GEO clones, which display two-thirds less receptors than their neo counterparts, could be restored to control levels (untreated cells harbouring neo) by cultivating them in the presence of an antibody which inhibits the interaction of UK with its receptor. These data suggest that for one colonic cell line at least, UK reduces the expression of its own binding site via an autocrine stimulation of its cell surface receptor.