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doi: 10.1242/10.1242/jcs.00081

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Role of ß₃-endonexin in the regulation of NF-B-dependent expression of urokinase-type plasminogen activator receptor

¹ 1. Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Lazarettstraße 36, 80636 München, Germany
² Institut für Klinische Chemie und Pathobiochemie, Technische Universität München, Ismaningerstraße 22, 81675 München, Germany
³ Institut Albert Bonniot, Joseph Fourier University of Grenoble, Faculty of Medicine, Domaine de la Merci, 38706 La Tronche Cedex, France
⁴ Laboratorium für Molekulare Biologie, Genzentrum der Universität München, Feodor Lynen Strasse 25, 81377 München, Germany
⁵ Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco Comprehensive Cancer Center, San Francisco, CA 94143-0875, USA

View larger version (37K): [in a new window]   Fig. 1. The cytoplasmic protein ß3-endonexin downregulates uPAR protein expression in endothelial cells. (A) Immunoblot of endogenous ß3-endonexin. Lysates of platelets, HUVECs and human placenta tissue were evaluated by immunoblotting with the help of a specific polyclonal anti-ß3-endonexin antiserum. A purified recombinant His-tagged ß3-endonexin (14 kDa) served as antigen control. (B) FACS. Confluent monolayers of endothelial cells were transiently transfected with GFP/En-S, GFP/En-L, or GFP control vector. After 48 hours cells were stained with anti-uPAR mAb and evaluated by flow cytometry. The mean intensity±s.e.m. of uPAR immunofluorescence of GFP-positive cells in four experiments was used as an index of uPAR protein expression.

View larger version (45K): [in a new window]   Fig. 2. ß3-endonexin downregulates uPAR promoter activity. (A) CAT-assay. CHO cells were transiently co-transfected with the -398 bp fragment of the uPAR promoter (uPAR-CAT -398) (1 µg), a luciferase vector for normalization and various concentrations of expression vectors encoding the short or long form of ß3-endonexin or the mock expression vector (GFP). Cell extracts normalized for luciferase activity were incubated with [14C]chloramphenicol, extracted with ethyl acetate, and subjected to thin-layer chromatography. The conversion of [14C]chloramphenicol to acetylated derivatives was quantified using BioRad GelDoc scanning software. (B) Histobars depict the results (mean±s.e.m.) of the CAT-assays performed as described in A with 3 µg of the expression vector. The data shown is representative of six independently performed experiments.

View larger version (35K): [in a new window]   Fig. 3. ß3-endonexin localizes to the cell nucleus of endothelial cells. (A) Confocal laser microscopy. HUVECs were transiently transfected with GFP control vector, GFP/En-S, GPF/En-S-K62RKK or GFP/En-L and the cellular localization of GFP fluorescence was studied by confocal laser scanning microscopy. (B) Immunoblot. Endothelial cells were transiently transfected with GFP/En-S, GPF/En-S-K62RKK, or GFP/En-L, and cytosolic and nuclear extracts were probed by immunoblotting with anti-ß3-endonexin polyclonal antiserum.

View larger version (51K): [in a new window]   Fig. 4. The NF-B-binding site of the uPAR promoter is required for ß3-endonexin-mediated downregulation of the uPAR gene transcription. (A) CAT-assay. CHO cells were transiently co-transfected with either the uPAR promoter construct (uPAR-CAT -398) described in Fig. 2A or a CAT-reporter construct driven by the -398 by uPAR promoter sequence mutated at the NF-B motif at -45 bp (uPAR-CAT mt-45). Additionally, a luciferase vector for normalization, and 3 µg of expression vectors encoding either the short or long form of ß3-endonexin or the mock expression vector (GFP) were transfected. The results are representative of three independent experiments. (B) EMSA. Recombinant GST-fusion proteins, GST/En-S and GST/En-L, or GST control were added in increasing ratios (1:2, 1:3, 1:4) to nuclear extracts of IL-1ß-activated HUVECs. Binding of B oligonucleotides was evaluated by EMSA.

View larger version (43K): [in a new window]   Fig. 5. ß3-endonexin binds to p50/p65 complex and inhibits p50-DNA binding. (A) Immunoprecipitation. Confluent monolayers of endothelial cells were transfected with GFP/En-S, GFP/En-L or control vector. Immunoprecipitation was performed with anti-p65 or anti-GFP mAb and Agarose A. Thereafter, immunoprecipitate was probed by anti-GFP or anti-p65, by immunoblotting. (B) Pull-down. Recombinant p50 was incubated with GST/En-S, GST/En-L or GST, which were immobilized on Sepharose. The precipitate and the supernatant were probed by immunoblotting using anti-p50 mAb. Anti-GST-blotting showed equal expression of the fusion proteins. (C) EMSA. Recombinant p50 was incubated with increasing ratios (1:2, 1:3, 1:4) of GST-En-S, GST-En-L or GST, and binding of B-DNA was determined by EMSA. (D) Pulldown. Recombinant p50 was incubated with double-stranded consensus or mutated B oligonucleotides and subsequently precipitated with immobilized GST/En-S or GST/En-L proteins. The precipitate was evaluated for p50 binding by immunoblotting. Equal amounts of GST proteins was confirmed with an anti-GST pAb. (E) Pull-down. As described in D, p50 protein was incubated with double-stranded B consensus or B uPAR oligonucleotides, which contained the specific NF-B sequence of the uPAR promoter and GST/En-L. B uPAR was used in raising ratios.