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REGULATION OF UROKlNASE:PAI-1 INTERNALIZATION: COORDINATE EXPRESSION OF THE UROKINASE AND THE a2-MACROGLOBULIN RECEPTORS
1Soria MR and 1#qjJ5&. 1DIBIT, H.S. Raffaele, Milano, Italy and
‘Conese,‘Cavallaro,
2Department of Genetics and Biology of Microorganisms, University of Milano, Italy. Internalization and degradation of urokinase:PAl1 (plasminogen activator inhibitor type 1) complexes require binding to the urokinase receptor (uPAR) and the a Z-macroglobulin receptor (a2-MR). PMA (phorbol myristate acetate) treatment of U937 cells, while uPAR synthesis and increase in stimulating urokinase binding, was found to inhibit uPARdependent internalization. PMA treated ( 150 nM, 72 hours) and control cells were incubated with
1 2SI-urokinase:PAI-l
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then re-incubated for 2 hours at 37°C. While 34% of urokinase:PAl-1 was degraded by control cells, PMA treatment almost blocked degradation. Differentiated U937 also lost the abilitv to down regulate uPAR upon binding of urokinase:PAl-1 (4% vs 29% of control cells). PMA-treatment also blocked the uPAR-dependent killing by a conjugate of urokinase with the plant toxin saporin, resulting in toxin-resistance. PMA treatment results in differentiation in macrophages. This treatment caused a timedependent decrease of a2-MR as judged by FACS and ligand blotting analysis. All these effects were counteracted by the expression of PML-RARa, a leukemia-specific hybrid transcription factor with antidifferentiation action. These effects in general may shed light on the physiology of uPAR-mediated internalization.
4°C and
IDENTIFICATION OF TWO REPRESSOR REGIONS WITHIN THE PLASMINOGEN ACTIVATOR INHIBITOR 2 (PAI-2) GENE PROMOTER. Robert L. Medcalf, Anthonv Dear and Marlies Rueug# Monash University Department of Medicine, Box Hill Hospital, Box Hill, Victoria and #Central Hematology Laboratory, CHIJV, CH1011 Lausanne, Switzerland PAI- is a serine protease inhibitor which inhibits urokinase and tissue-type plasminogen activator. In HT-1080 tibrosarcoma cells, PAIgene transcription is stimulated by phorbol ester. tumour necrosis factor and okadaic acid. To identify cis-acting elements responsible for this expression, a series of PAI- promoter deletion mutants fused to the CAT reporter gene were prepared (range 1859 to -91) and transfected into HT-1080 cells. Cells transfccted with the -1859 PAI- promoter-CAT construct produced an increase in CAT expression when treated with either PMA, TNF or okadaic acid. Deletion to -1100 produced a signilicant increase in basal and PMA, TNF and okadaic acid-inducible CAT expression, suggesting the presence of a repressor element(s) between position -1859 and 1100. This elevated level of basal and inducible CAT activity was maintained when the promoter was truncated to -259. Further
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REGULATION OF THE MURINE VITRONECTIN GENE: - IDENTIFICATION OF REGULATORY SEQUENCES FOR INTERLEUKIN 6 ‘?Seiffert D, ‘Curriden SA, ‘Binder BR,‘Loskutoff DJ ‘The Scripps Research Institute, La Jolla, CA, USA, and *University of Vienna, Vienna, Austria.
Recent observations demonstrate- that expression of the murine vitronectin (Vn) gene is modulated during acute systemic inflammation. In order to define the cis-acting elements involved in this regulation, 1.8kB of the S-flanking region of the mouse Vn gene was isolated and sequenced. Primer extension experiments suggest that the transcription initiation site is heterogenous. Sequence analysis revealed a perfect “TATA box” at position -30 to -24 and an interleukin6 (IL-6) responsive element at -100 to -92. A 528-base pair fragment from the Vn promoter and S-flanking region was fused to the firefly luciferase reporter gene, and then transfected into human hepatoma cells (Hep3B). The transcriptional activity of this fragment was compared to that of an 800-base pair fragment of the human PAI- gene also transfected into Hep3B cells. Treatment of the cells with
deletion to position -216, however, produced an additional 6.fold increase in basal expression and a lo-fold increase in TNF-mediated expression, suggesting the presence of a second repressor region. A panel of overlapping 22bp oligomers complementary to the 43 bp region between position -259 and -216 were synthesised and used in gel shift experiments using HT.1080 nuclear proteins. Results indicated the presence of two specific binding regions (A & B). An 8 bp pallindromic sequence is present within region B. Gel shift analyses using labelled oligomers harbouring base substitutions within this pallindromic sequence suggested that this element was involved in binding activity. These results indicate that 1859 bp of the PA1-2 gene promoter directs PMA, TNF and okadaic acidmediated transcription, and that at least two repressor regions exists, the proximal one containing binding sites for two distinct nuclear proteins. Mutagcncsis of the proximal rcprcssor site and the pallidrl.)mic sequence within region B is being performed to dctcrmine its functional significance.
interleukin-6 (IL-6) induced the Vn construct by more than 20-fold, but had no effect on the PAI- promoter. Although the PAI- promoter construct was strongly stimulated by transforming growth factor B, the Vn promoter was not affected. Stimulation by these agents was relatively specific, since treatment with interleukin-lB, tumor necrosis factor a, and dexamethasone did not induce luciferase activity in either promoter. Transfection studies using Vn-constructs with serial 5’ deletions revealed that two sequences were important in the IL-6 response. The first was located in the distal 5’ flanking region (-399 to -391) and mediated a 3 to 4-fold response, while the second more proximal region (-100 to -92) which contained the IL-6 consensus sequence, accounted for another 4-fold response. Moreover, mutations in the proximal site reduced IL-6 inducibility by approximately 50%, while mutations in both sequences abolished the IL-6 response. Thus, the murine Vn gene is specifically regulated by IL-6, and the response is mediated by at least two different cisacting elements.