Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line

W. Hollas, E. Soravia, A. Mazar, J. Henkin, F. Blasi, D. Boyd*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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 (~ 2.5 ng/ml per 72 h per 106 cells) and display approx. 104 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 Or UK, consistently bound 50-75% less radioactive di-isopropylfluorophosphate (DFP)-UK than clones harbouring thc selectable marker gene neo only or control GEO cells. Cross-linking experiments with a radioactivc N-terminal fragment of UK which binds to the receptor showed a decreased amount of a binding protein of approx. 51 kDa in represcntative 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.

Original languageEnglish (US)
Pages (from-to)629-634
Number of pages6
JournalBiochemical Journal
Volume285
Issue number2
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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