Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin*
- From the ‡Departments of Pathology and Biochemistry, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 and ¶Abbott Laboratories, Abbott Park, Illinois 60064
Abstract
Low density lipoprotein receptor-related protein (LRP) mediates the endocytosis of diverse ligands, including urokinase plasminogen activator (uPA) and its receptor, uPAR, which have been implicated in cellular migration. The purpose of this study was to determine whether LRP affects cellular migration. Murine embryonic fibroblasts (MEF) that are LRP-deficient due to targeted gene disruption and exotoxin selection (MEF-2), heterozygous fibroblasts (PEA-10), and wild-type fibroblasts (MEF-1) were compared. When cultures were denuded of cells in a 1-mm-wide strip, all three cell types migrated into the denuded area. The MEF-2 cells migrated nearly twice as rapidly as the MEF-1 cells or PEA-10 cells. The difference in migration velocity was duplicated in culture wells that were precoated with serum or vitronectin and partially duplicated in wells coated with fibronectin but not in wells coated with type I collagen or Matrigel. uPA was detected in MEF-2 conditioned medium (CM) at a concentration of 0.30 ± 0.02 nm, which was 13-fold higher than the level detected in MEF-1 CM or PEA-10 CM, suggesting one potential mechanism for the enhanced migration of MEF-2 cells. uPAR was also increased on MEF-2 cells by 4–5-fold, as determined by PI-PLC release, and by 2.5-fold, as determined by a uPA/uPAR activity assay. Mannosamine treatment, which down-regulates cell-surface uPAR, decreased MEF-2 migration by 40% without significantly affecting MEF-1 migration. MEF-2 CM, which is uPA-rich, increased the rate of MEF-1 migration, and MEF-1 CM did not. These studies demonstrate alterations in cellular migration and in the activity of the uPA/uPAR system which accompany complete deficiency of LRP expression in fibroblasts. We propose that uPA and uPAR form an autocrine loop for promoting fibroblast migration and that LRP counteracts the activity of this system.
Footnotes
-
↵* This work was supported in part by Grant CA-53462 from the National Institutes of Health (to S. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵§ Recipient of a fellowship from the Medical Scientist Training Program, National Institutes of Health Grant GM07267.
-
↵‖ To whom correspondence should be addressed: University of Virginia Health Sciences Center, Dept. of Pathology, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-924-8060.
-
↵1 The abbreviations used are: ECM, extracellular matrix; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor; GPI, glycosylphosphatidylinositol; LRP, low density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor-1; MEF, murine embryonic fibroblasts; scuPA, single-chain uPA; tcuPA, two-chain uPA; VLK-pNA, H-d-valyl-l-leucyl-l-lysyl-p-nitroanilide; PI-PLC, phosphatidylinositol-specific phospholipase C; RAP, receptor-associated protein; PGAD, 3-phosphoglyceraldehyde dehydrogenase; CM, conditioned medium; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum.
-
- Received December 17, 1996.
