The Very Low Density Lipoprotein Receptor Regulates Urokinase Receptor Catabolism and Breast Cancer Cell Motility in Vitro*
- From the Departments of ‡Pathology and¶Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Abstract
The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA)
and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the
internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged
with uPA·PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr
was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the
catabolism of [35S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t
was 8.2 h. uPA·PAI-1 complex accelerated uPAR catabolism (t
to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t
of 7.8 h) and absence (t
of 16.9 h) of uPA·PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell
surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the
medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 ± 0.1-fold and by 4.4 ± 0.3-fold when
1.0 nm uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents
uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein
kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures.
These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular
motility.
Footnotes
-
↵* This work was supported in part by National Institutes of Health Grant CA-53462 and Department of the Army Breast Cancer Research Program Grant 94-J-4447.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.
-
↵§ Fellow of the American Heart Association, Virginia Affiliate.
-
↵‖ To whom correspondence should be addressed: University of Virginia Health Sciences Center, Depts. of Pathology and Biochemistry and Molecular Genetics, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-982-0283; E-mail: SLG2T{at}VIRGINIA.EDU.
-
↵2 D. H. D. Nguyen et al., unpublished results.
- Abbreviations:
- VLDLr
-
very low density lipoprotein receptor
- LDL
-
low density lipoprotein receptor
- LRP
-
low-density lipoprotein receptor-related protein
- RAP
-
receptor-associated protein
- uPA
-
urokinase-type plasminogen activator
- uPAR
-
urokinase receptor
- MEF
-
murine embryonic fibroblasts
- PAI-1
-
plasminogen activator inhibitor-1
- ERK
-
extracellular signal-regulated kinase
- GST
-
glutathione S-transferase
- FBS
-
fetal bovine serum
- CM
-
conditioned medium
- VSMC
-
vascular smooth muscle cells
- scuPA
-
single-chain uPA
- tcuPA
-
two-chain uPA
- DIP-μPA
-
diisopropylphospho-uPA
- MAP
-
mitogen-activated protein kinase
- PAGE
-
polyacrylamide gel electrophoresis
- VLK-AMC
-
Val-Leu-Lys-7-amido-4-methylcoumarin
-
- Received July 9, 1998.
- Revision received January 6, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
