- Email: [email protected]
15 Role of the low density lipoprotein receptorrelated protein (LRP) in the clearance and liver uptake of recombinant single chain urokinase-type plasminogen activator (rscu-PA) in rats
ORAL C O M M U N I C A T I O N S III-1 Cellular Receptors
13
14
THE CATION INDEPENDENT MANNOSE-6-PHOSPHATE RECEPTOR/ INSULIN-LIKE GROWTH FACTOR ll RECEPTOR (CIMPR) MODULATES THE SUBCELLULAR DISTRIBUTION OF THE UROKINASE RECEPTOR (uPAR).
Nykjaer A., Petersen C.M., Hager H., Nielsen M.S., Rasmussen, H.H., 2Vilhardt F., 3Shuman, M.A., 4Cohen R.L., 5Kornfeld S., 'Christcnsen E.I. and Gliemann J. Depts. of Medical Biochemistry and ~Cell Biology, University of Aarhus and 2Dept. of Anatomy, University of Copenhagen, Denmark; 3Cancer Research Institute, University of California San Francisco, CA; 4Genentech Inc., San Francisco, CA and 5Division of Hematology-Oncology, Washington School of Medicine, St. Louis, MO.
THE VERY LOW DENSITY LIPOPROTEIN RECEPTOR MEDIATES THE UPTAKE AND DEGRADATION OF THE A T H E R O G E N I C L I P O P R O T E I N , Lp(a) K,M. Ar~raves~:. C.D. MacCalman 'r . J.F. Strauss IIl'!. J.T. Fallon~. P.C. Harpel§. and D.K,, Strickland;t. :IHolland Laboratory, American Red Cross, Rockville Maryland 20855, ~University of Pennsylvania School o f Medicine, Philadelphia, Pennsylvania 19104-6142, §The Mount Sinai School of Medicine, N e w York, N Y 10029.
We have previously shown that the endocytic LDL receptor-related protein (LRP) can modulate cell surface expression ofuPAR by internalizing the ternary uPA:PAI-1 :uPAR complex. This uPA:serpin-induced uptake of uPAR is blocked by the 39-40 kDa LRP ligand RAP. Subsequent experiments showed that cells cultured in the presence of RAP exhibited significant lysosomal staining for uPAR, demonstrafing a sorting mechanism independent of LRP. By affinity chromatography using recombinant soluble uPAR (r-uPAR), we purified a 225 kDa protein which by aa microsequencing was identified as the ubiquitous lysnsomal sorting receptor CIMPR, also present at the cell surface. Binding of IGFI1 and I~-galactnsidase, and recognition by specific antibodies confirmed the identity. Both r-uPAR and purified wild-type uPAR bound to a CIMPR affinity column in a mannose-6-phophate (M6P) sensitive manner. Whereas the binding site in r-uPAR reside in N-linked M6P residues present in domains 2+3, the Pl-glycan of the GPl-anchor accounts for binding to CIMPR in wild-type uPAR Cells transfected with bovine CIMPR, but not receptor negative controls obtained from knock-out mice, targeted labeled ruPAR to lysosomes. Moreover, the ratio between intracellularly located and surfacebound endogenous uPAR was significantly higher in the CIMPR transfectants as compared to control cells. Finally, metabolically labeled uPAR in primary cultures of homan trophoblasts could be cross-linked to the endogenous CIMPR, demonstrating a direct contact between the two receptors. In conclusion, we have shown that the subcellular distribution of uPAR is modulated by the CIMPR, and we suggest that CIMPR may modulate the invasive phenotype of e.g. cancer cells, by regulating surface expression of uPAR.
Lp(a) is a major inherited risk lhctor associated with premature coronary heart disease and stroke. The m e c h a n i s m o f Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle following receptor-mediated uptake. The mechanisms responsible for the cellular catabolism of Lp(a) are currently a topic of debate. We demonstrate using adenovirus mediated gene transfer, that the V L D L receptor is responsible for the endocytosis of Lp(a) leading to its degradation within lysosomes. Lp(a) degradation was prevented by antibodies against the ¥ L D L receptor, and by RAP, an antagonist of ligand binding. The catabolism of Lp(a) was also inhibited by apolipoprotein(a), indicating that apolipoprotein(a) mediates the binding of Lp(a) to this receptor. We also demonstrate the expression of the V L D L receptor in macrophages present in atherosclerotic lesions. The ability of the V L D L receptor to nrediate endocytosis of Lp(a) could lead to cellular accumulation o f lipid within macrophages, and may represent a molecular basis for the atherogenic effects o f Lp(a).
15
16
ROLE OF THE LOW DENSITY LIPOPROTEIN RECEPTORRELATED PROTEIN (LRP) IN THE CLEARANCE AND LIVER UPTAKE OF RECOMBINANT SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (RSCU-PA) IN RATS, ~van der Kaaden ME 2Riiken DC, ~Kruiit JK, ~van Berkel ThJC and ~Kuiper J. 1LACDR, Division of Biopharmaceutics, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden and the 2Gaubius Laboratory TNO P.G., P.O. Box 430, 2300 AK Leiden, the Netherlands. Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, E.coli produced rscu-PA is recognized by LRP on parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA. A preinjection of the LRP inhibitor GSTRAP reduced the maximal liver uptake of 12Sl-rscu-PAfrom 50 to 30 % of the injected dose and decreased the clearance from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10 % of the liver uptake, respectively. The 40 % reduction in liver uptake of rscu-PA by preinjection of GST-RAP was caused by a 9 1 % and 62 % reduction in the uptake by the parenchymal and Kupffer cells, respectively. Deletion of residue 11-135 from rscu-PA (=delta125-rscu-PA) resulted in a 80 % reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for the uptake of 60.2, 32.9 and 7.0 % of ~2Sl-delta125-rscu-PA in the liver. Preinjection of GST-RAP completely reduced the liver uptake and reduced the clearance to 0.79 ml/min. Treatment of Kupffer cells with pI-PLC reduced the binding of rscu-PA by 40 %, indicating the involvement of u-PAR in the recognition of rscu-PA by the Kupffer cells. Our results demonstrate that in vivo LRP is responsible for the parenchymal liver cell mediated uptake of rscu-PA and for 60 % of the Kupffer cell interaction. It is also shown that u-PAR is involved in the Kupffer cell recognition of rscu-PA.
INHIBITION OF PLASMIN GENERATION ON CELL SURFACES BY MONOCLONAL ANTIBODIES AGAINST A 55-60 kDa PLASMINOGEN
RECEPTOR Lopez-Alemany R. ~, Longstaff C], Ffthregas P.~, JardI M. ~, Merton E) and Ftlez J.~ Institut de Recerca Otlcoltgica 0RO), Hospital Duran i Reynals, Barcelona, SPAIN; 2 Department of Haematology, National Institute for Biological Standards and Control, Blanche Lane, S. Minmls, Hertfordshire, UK, EN6 3QG. Several proteins able to bind plasminogen have been described in a variety of cell types. We have isolated a hydrophobic protein of 55-60 kDa from the human breast tumor cell line, MCF7, that binds specifically plasminogen and plasmin. Our initial amino acid sequencing data showed a partial identity with enolase isoforms. We have previously described that plasminogen receptor (PIg-R) occupancy on leukocyte cell lines induced a 80-fold increase in plasminogen activation rate. We have identified a monoclonal antibody, 11GI, against the 55-60 kDa plasminogen receptor, that was able to inhibit plasminogen activation by uPA or tPA in a dose-dependent mamler on several leukocyte cell titles. Plasmiuogen activation was inlfibited by 85-95% with 1 ,~M final concentration of 11GI. Interestingly, with this antibody concentration, binding of '~q-plasminogen (100 nM input concentration), was only inhibited by a 10-20%. In contrast, binding of '2q-plasminogen to the purified PIg-R was inhibited by a 85-90 % by llG1. Pericellular proteolysis activity was also blocked by IlGI as measured by the caseinolytic plaque assay. We have also measured the ability of IlG1 to interfere on file lysis of a fibrin clot due to plasminogen activation in the presence of tPA. l l G I has no effect in that system. These data allow us to conclude that although cells display several proteins capable of plasminogen binding only a sub-set of these molecules, the 55-60 kDa enolase-related plasminogen receptor, is responsible for the promotion of plasmiu generation by leukocyte ceils. The interaction of plasminogen with cells and fibrin occurs in a different way, and only in the cell it involves the specific receptor recognized by 11GI.
5
13
14
THE CATION INDEPENDENT MANNOSE-6-PHOSPHATE RECEPTOR/ INSULIN-LIKE GROWTH FACTOR ll RECEPTOR (CIMPR) MODULATES THE SUBCELLULAR DISTRIBUTION OF THE UROKINASE RECEPTOR (uPAR).
Nykjaer A., Petersen C.M., Hager H., Nielsen M.S., Rasmussen, H.H., 2Vilhardt F., 3Shuman, M.A., 4Cohen R.L., 5Kornfeld S., 'Christcnsen E.I. and Gliemann J. Depts. of Medical Biochemistry and ~Cell Biology, University of Aarhus and 2Dept. of Anatomy, University of Copenhagen, Denmark; 3Cancer Research Institute, University of California San Francisco, CA; 4Genentech Inc., San Francisco, CA and 5Division of Hematology-Oncology, Washington School of Medicine, St. Louis, MO.
THE VERY LOW DENSITY LIPOPROTEIN RECEPTOR MEDIATES THE UPTAKE AND DEGRADATION OF THE A T H E R O G E N I C L I P O P R O T E I N , Lp(a) K,M. Ar~raves~:. C.D. MacCalman 'r . J.F. Strauss IIl'!. J.T. Fallon~. P.C. Harpel§. and D.K,, Strickland;t. :IHolland Laboratory, American Red Cross, Rockville Maryland 20855, ~University of Pennsylvania School o f Medicine, Philadelphia, Pennsylvania 19104-6142, §The Mount Sinai School of Medicine, N e w York, N Y 10029.
We have previously shown that the endocytic LDL receptor-related protein (LRP) can modulate cell surface expression ofuPAR by internalizing the ternary uPA:PAI-1 :uPAR complex. This uPA:serpin-induced uptake of uPAR is blocked by the 39-40 kDa LRP ligand RAP. Subsequent experiments showed that cells cultured in the presence of RAP exhibited significant lysosomal staining for uPAR, demonstrafing a sorting mechanism independent of LRP. By affinity chromatography using recombinant soluble uPAR (r-uPAR), we purified a 225 kDa protein which by aa microsequencing was identified as the ubiquitous lysnsomal sorting receptor CIMPR, also present at the cell surface. Binding of IGFI1 and I~-galactnsidase, and recognition by specific antibodies confirmed the identity. Both r-uPAR and purified wild-type uPAR bound to a CIMPR affinity column in a mannose-6-phophate (M6P) sensitive manner. Whereas the binding site in r-uPAR reside in N-linked M6P residues present in domains 2+3, the Pl-glycan of the GPl-anchor accounts for binding to CIMPR in wild-type uPAR Cells transfected with bovine CIMPR, but not receptor negative controls obtained from knock-out mice, targeted labeled ruPAR to lysosomes. Moreover, the ratio between intracellularly located and surfacebound endogenous uPAR was significantly higher in the CIMPR transfectants as compared to control cells. Finally, metabolically labeled uPAR in primary cultures of homan trophoblasts could be cross-linked to the endogenous CIMPR, demonstrating a direct contact between the two receptors. In conclusion, we have shown that the subcellular distribution of uPAR is modulated by the CIMPR, and we suggest that CIMPR may modulate the invasive phenotype of e.g. cancer cells, by regulating surface expression of uPAR.
Lp(a) is a major inherited risk lhctor associated with premature coronary heart disease and stroke. The m e c h a n i s m o f Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle following receptor-mediated uptake. The mechanisms responsible for the cellular catabolism of Lp(a) are currently a topic of debate. We demonstrate using adenovirus mediated gene transfer, that the V L D L receptor is responsible for the endocytosis of Lp(a) leading to its degradation within lysosomes. Lp(a) degradation was prevented by antibodies against the ¥ L D L receptor, and by RAP, an antagonist of ligand binding. The catabolism of Lp(a) was also inhibited by apolipoprotein(a), indicating that apolipoprotein(a) mediates the binding of Lp(a) to this receptor. We also demonstrate the expression of the V L D L receptor in macrophages present in atherosclerotic lesions. The ability of the V L D L receptor to nrediate endocytosis of Lp(a) could lead to cellular accumulation o f lipid within macrophages, and may represent a molecular basis for the atherogenic effects o f Lp(a).
15
16
ROLE OF THE LOW DENSITY LIPOPROTEIN RECEPTORRELATED PROTEIN (LRP) IN THE CLEARANCE AND LIVER UPTAKE OF RECOMBINANT SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (RSCU-PA) IN RATS, ~van der Kaaden ME 2Riiken DC, ~Kruiit JK, ~van Berkel ThJC and ~Kuiper J. 1LACDR, Division of Biopharmaceutics, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden and the 2Gaubius Laboratory TNO P.G., P.O. Box 430, 2300 AK Leiden, the Netherlands. Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, E.coli produced rscu-PA is recognized by LRP on parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA. A preinjection of the LRP inhibitor GSTRAP reduced the maximal liver uptake of 12Sl-rscu-PAfrom 50 to 30 % of the injected dose and decreased the clearance from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10 % of the liver uptake, respectively. The 40 % reduction in liver uptake of rscu-PA by preinjection of GST-RAP was caused by a 9 1 % and 62 % reduction in the uptake by the parenchymal and Kupffer cells, respectively. Deletion of residue 11-135 from rscu-PA (=delta125-rscu-PA) resulted in a 80 % reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for the uptake of 60.2, 32.9 and 7.0 % of ~2Sl-delta125-rscu-PA in the liver. Preinjection of GST-RAP completely reduced the liver uptake and reduced the clearance to 0.79 ml/min. Treatment of Kupffer cells with pI-PLC reduced the binding of rscu-PA by 40 %, indicating the involvement of u-PAR in the recognition of rscu-PA by the Kupffer cells. Our results demonstrate that in vivo LRP is responsible for the parenchymal liver cell mediated uptake of rscu-PA and for 60 % of the Kupffer cell interaction. It is also shown that u-PAR is involved in the Kupffer cell recognition of rscu-PA.
INHIBITION OF PLASMIN GENERATION ON CELL SURFACES BY MONOCLONAL ANTIBODIES AGAINST A 55-60 kDa PLASMINOGEN
RECEPTOR Lopez-Alemany R. ~, Longstaff C], Ffthregas P.~, JardI M. ~, Merton E) and Ftlez J.~ Institut de Recerca Otlcoltgica 0RO), Hospital Duran i Reynals, Barcelona, SPAIN; 2 Department of Haematology, National Institute for Biological Standards and Control, Blanche Lane, S. Minmls, Hertfordshire, UK, EN6 3QG. Several proteins able to bind plasminogen have been described in a variety of cell types. We have isolated a hydrophobic protein of 55-60 kDa from the human breast tumor cell line, MCF7, that binds specifically plasminogen and plasmin. Our initial amino acid sequencing data showed a partial identity with enolase isoforms. We have previously described that plasminogen receptor (PIg-R) occupancy on leukocyte cell lines induced a 80-fold increase in plasminogen activation rate. We have identified a monoclonal antibody, 11GI, against the 55-60 kDa plasminogen receptor, that was able to inhibit plasminogen activation by uPA or tPA in a dose-dependent mamler on several leukocyte cell titles. Plasmiuogen activation was inlfibited by 85-95% with 1 ,~M final concentration of 11GI. Interestingly, with this antibody concentration, binding of '~q-plasminogen (100 nM input concentration), was only inhibited by a 10-20%. In contrast, binding of '2q-plasminogen to the purified PIg-R was inhibited by a 85-90 % by llG1. Pericellular proteolysis activity was also blocked by IlGI as measured by the caseinolytic plaque assay. We have also measured the ability of IlG1 to interfere on file lysis of a fibrin clot due to plasminogen activation in the presence of tPA. l l G I has no effect in that system. These data allow us to conclude that although cells display several proteins capable of plasminogen binding only a sub-set of these molecules, the 55-60 kDa enolase-related plasminogen receptor, is responsible for the promotion of plasmiu generation by leukocyte ceils. The interaction of plasminogen with cells and fibrin occurs in a different way, and only in the cell it involves the specific receptor recognized by 11GI.
5