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

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 uPArich, 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.

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

“…In contrast, the migration of murine embryonic fibroblasts lacking LRP/␣ 2 MR (MEF-2) is enhanced (71). Increased migration of the latter cells might be explained, in part, by their increased expression of uPAR, and the higher levels of u-PA present in their conditioned medium (71). Nevertheless, reconciliation of these contrasting observations will require more detailed evaluation of the effect of LRP/␣ 2 MR on specific cellular processes, including uPAR-dependent adhesion (72,73), activation of u-PA/uPAR-initiated signaling pathways (33), and, as suggested by the current study, cell surface plasmin generation.…”

“…For example, a plasmin-dependent pathway of migration, which is inhibited by anti-LRP/ ␣ 2 MR antibodies, has been demonstrated on smooth muscle cells (31). In contrast, the migration of murine embryonic fibroblasts lacking LRP/␣ 2 MR (MEF-2) is enhanced (71). Increased migration of the latter cells might be explained, in part, by their increased expression of uPAR, and the higher levels of u-PA present in their conditioned medium (71).…”

Regulation of leukocyte adherence and migration by glycosylphosphatidyl-inositol-anchored proteins

“…of uPA-PAI complexes [35] make it difficult to understand the relationships between this system and cell migration [36] as well as cell adhesion, as already mentioned. However, infiltration of inflammatory leukocytes, especially macrophages, neutrophils, and T lymphocytes, into Cryptococcus neoformans-infected lung is severely impaired in uPAdeficient mice [37], clearly suggesting a requirement for the uPAR-uPA-PAI system for leukocyte migration in certain conditions.…”

Soluble Urokinase-type Plasminogen Activator Receptor Inhibits Cancer Cell Growth and Invasion by Direct Urokinase-independent Effects on Cell Signaling

“…MEF-2 cells are murine and LRP-1-deficient. These cells have increased levels of uPA and uPAR and establish autocrine signaling in serum-free medium so that the basal level of activated ERK is elevated (13,52). Similarly, MDA-MB 231 breast cancer cells express large amounts of uPA and uPAR, which form an autocrine signaling loop to maintain an elevated level of activated ERK under serum-free conditions (53).…”

“…Because SuPAR is less effective at triggering signaling than membrane-anchored uPAR-uPA complex, competitive displacement of the membrane-anchored complex results in a decrease in the level of activated ERK. Murine uPA does not bind to human uPAR (47), precluding the alternative model in which SuPAR inhibits ERK activation by binding uPA produced endogenously by the MEF-2 cells (52).…”