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First published online 16 December 2003
doi: 10.1242/jcs.00861

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Reciprocal regulation of urokinase receptor (CD87)-mediated cell adhesion by plasminogen activator inhibitor-1 and protease nexin-1

¹ Institute for Biochemistry, Justus-Liebig-University, 35392 Giessen, Germany
² Department of Internal Medicine, Justus-Liebig-University, 35392 Giessen, Germany
³ Department of Clinical Chemistry, Justus-Liebig-University, 35392 Giessen, Germany
⁴ Friedrich Miescher Institute, Basel, Switzerland

View larger version (36K): [in a new window]   Fig. 1. Characteristics of the interactions between uPA, VN, suPAR and PN-1 using ELISA. (A) Binding of VN (1 µg/ml) to suPAR-coated wells (5 µg/ml) in the absence (open circle) or presence (open square) of uPA (20 nM) and increasing concentrations of PN-1. (B) Binding of VN to suPAR-coated wells in the absence (open circle) or presence (open square) of PN-1 (250 nM) and increasing concentrations of uPA. VN binding was detected with the mAb 13H1. (C) Binding of VN (1 µg/ml) to suPAR (D2 + D3; 5 µg/ml)-coated wells (dotted bars) or Vß3 (5 µg/ml)-coated wells (hatched bars) in the absence or presence of uPA (100 nM), PN-1 (100 nM), PAI-1 (100 nM) or a combination of uPA and PN-1. VN binding was detected with the mAb 13H1. (D) Binding of uPA (20 nM) to VN-coated wells (5 µg/ml) was tested in the absence (control) or presence of either PN-1 (100 nM), suPAR (50 nM) or a combination of the two as indicated. uPA binding was detected with the mAb 4D1E8. (E) Binding of VN (2 µg/ml) to uPA-coated wells (5 µg/ml) was tested in the absence (control) or presence of either PN-1 (100 nM), suPAR (50 nM) or a combination of the two as indicated. VN binding was detected with the mAb VN-7. (F) Binding of VN (1 µg/ml) to suPAR-coated wells (5 µg/ml) was measured in the absence (control) or presence of PN-1 (200 nM) or PAI-1 (100 nM), without any further addition (dotted bars) or together with enzymatically active uPA (20 nM; hatched bars) or enzymatically inactive Gly158-ScuPA (20 nM; chequered bars), respectively. VN binding was detected with the mAb 13H1. (G) Binding of uPA (20 nM) to VN-coated wells (5 µg/ml) in the presence of suPAR (50 nM) and increasing concentrations of PN-1 was tested in the absence (open square) or presence of thrombin (20 nM) (solid square) as indicated. Binding was detected with the mAb 4D1E8. (H) At the end of the incubation period in the above experiment the supernatants were removed and then analyzed for proteolytic activity using the chromogenic substrate S-2444, which is specific for uPA but not for thrombin. Results from a typical experiment are shown (mean absorbance ± s.e.m. of triplicate wells), and similar data were obtained in three separate experiments. Absence of error bars indicates that the s.e.m. is smaller than the size of the symbol.

View larger version (31K): [in a new window]   Fig. 2. Interaction between uPA, VN, suPAR and PN-1 in the solution phase. PN-1, VN, suPAR, uPA, PAI-1 and ATF (4 µg/ml each) were incubated in different combinations, as indicated, in TBS with 1% (w/v) BSA for 2 hours at 22°C. Thereafter, immunoprecipitation was performed with control mAb (lanes 1 and 8), anti-VN, 13H1 (lanes 2, 6 and 7), anti-uPAR, R4 (lane 5), anti-uPA, 4D1E8 (lane 4) or anti-PN-1, 4B3 (lanes 3 and 9) all at 4 µg/ml. The immune complexes were captured by adding protein A/G Sepharose and the immunoprecipitates were analyzed by western blotting with a rabbit polyclonal anti-uPAR followed by densitometric analysis. Similar results were obtained in three independent experiments.

View larger version (13K): [in a new window]   Fig. 3. Influence of PAI-1 on uPA-PN-1-induced VN binding to immobilized suPAR. Binding of VN (1 µg/ml) to suPAR-coated wells (5 µg/ml) was tested in the presence of increasing concentrations of PN-1 in the absence (solid triangle) or presence of uPA (20 nM; solid circle). For comparison, the binding of VN in the presence of uPA (20 nM) and PN-1 (250 nM) was determined in the presence of increasing concentrations of PAI-1 (open square). Binding was detected with the mAb VN-7, and data represent mean absorbance ±s.e.m. (n=3) of a typical experiment. Similar results were obtained in three separate experiments.

View larger version (21K): [in a new window]   Fig. 4. Influence of PN-1 on uPAR-dependent cell adhesion on VN. (A) Adhesion of uPAR-transfected BAF3 cells to VN-coated wells (2 µg/ml) in the presence of uPA (50 nM) as well as different concentrations of PN-1. The dotted line indicates the level of basal adhesion in the absence of any test substances. (B) Adhesion of uPAR-BAF3 cells to VN-coated wells in the absence (control) or presence of uPA, ATF or Gly158ScuPA (each at 50 nM) in the absence of any further additions (dotted bars) or the presence of PN-1 (100 nM) (hatched bars). (C) Adhesion of uPAR-BAF3 cells to VN-coated wells in the absence (control) or presence of uPA (50 nM), PN-1 (50 nM) or a combination of uPA (50 nM) and PN-1 (50 nM) in the absence of any further additions (dotted bars) or the presence of thrombin (50 nM) (hatched bars). uPA and PN-1 or uPA, PN-1 and thrombin were preincubated for 60 minutes at room temperature to allow complex formation before addition to the cell adhesion assay. (D) U937 cells were differentiated overnight with vitamin D3 and TGFß prior to the assay. Time-dependent adhesion of cells to VN in the presence of uPA (50 nM; solid circle) or uPA/PN-1 (50 nM each; solid square). Cell adhesion was measured as absorbance at 590 nm and is expressed as mean ± s.e.m. (n=3) of triplicate wells. Similar results were obtained in three separate experiments.

View larger version (51K): [in a new window]   Fig. 5. Localization of uPAR and PN-1 to sites of cell adhesion in monocytic cells. (A) U937 cells were differentiated into monocytic lineage and preincubated with a combination of uPA (50 nM) and rhodamine labeled PN-1 (3 µg/ml). After extensive washing the cells were allowed to adhere to VN- or FN-coated slides. Adherent cells were fixed and immunostained with anti-uPAR (rabbit polyclonal) antibody followed by FITC-coupled anti-rabbit IgG. Confocal microscopy was used to determine the location of the antigens in serial sections of the cells. The top, middle and bottom sections from adherent cells on VN and FN are shown. Left column shows uPAR labeling, the middle column, PN-1 and the right column shows the overlay of the two. Similar results were obtained in three separate experiments. (B) The fluorescence intensity of uPAR and PN-1 on VN- or FN-coated slides was quantified in 0.2 µm serial sections. Relative fluorescence with anti-uPAR IgG on a VN (open triangle) or FN substratum (solid triangle) and PN-1-Rhodamine on a VN (open square) or FN substratum (solid square) is shown.

View larger version (128K): [in a new window]   Fig. 6. Localization of PN-1 in atherosclerotic vessels. Immunohistochemistry of arterosclerotic carotid artery, visualised through streptavidin-alkaline phosphatase conjugate, Fast Red stain and Mayer's hemalum counterstain. (A) General view (x34) of the endarterectomy specimen showing VN immunostaining. The extension of the neointima is indicated by a dotted line, the luminal border is marked by white arrows, the border of the neointima with the media is marked by black arrows. (B-F) The VN-positive foam cell accumulation, marked by a rectangle, shown at higher magnification. Serial sections (x272) showing PN-1 (B), uPAR (C), uPA (D), macrophage-antigen CD68 (E) and smooth muscle -actin (F) immunostaining. Note that PN-1 is expressed in VN-, uPAR- and uPA-positive areas (red stain). The foam cell accumulation is composed of smooth muscle cells and macrophages. Note the distribution of PN-1 that can be attributed to cytoplasmatic localization and matrix-associated protein. (G) A negative IgG control of PN-1 immunostaining of the same area (x272) was completely negative, underlining the specificity of the PN-1 immunoreactivity. (H) PN-1 was also demonstrated in a thrombus at the edge of a ruptured abdominal aortic aneurysm (x272). The dark arrows point to accumulations of PN-1-positive thrombocytes.