ABSTRACT
During restenosis following arterial injury, vascular smooth muscle cells (VSMCs) form a neointimal layer in arteries by changing from a differentiated, contractile phenotype to a dedifferentiated, migratory, and proliferative phenotype. Several growth factors, cytokines, and extracellular matrix components released following injury have been implicated in these phenotypic changes. We have recently detected the expression of laminin-5, an ECM protein found predominantly in epithelial tissues, in the arterial vasculature. Here we report that ln-5 expression by VSMC is upregulated by platelet-derived growth factor (PDGF-BB), epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta1. Adhesion to ln-5 specifically enhances PDGF-BB-stimulated VSMC proliferation and migration. PD98059, a specific inhibitor of the ERK1/2 members of the Mitogen Activated Protein kinase family, increases both VSMC adhesion to ln-5 and blocks PDGF-BB-stimulated VSMC migration on ln-5. These results suggest that adhesion to ln-5 mediates a PDGF-BB-stimulated VSMC response to vascular injury via an ERK1/2 signaling pathway.
Subject(s)
Cell Adhesion Molecules/pharmacology , Cell Movement , Mitogen-Activated Protein Kinases/physiology , Muscle, Smooth, Vascular/physiology , Platelet-Derived Growth Factor/pharmacology , Animals , Becaplermin , Blotting, Western , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Chemotaxis , Enzyme Inhibitors/pharmacology , Extracellular Matrix Proteins/pharmacology , Flavonoids/pharmacology , Growth Substances/pharmacology , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Proto-Oncogene Proteins c-sis , Rats , KalininABSTRACT
The laminin family of extracellular matrix (ECM) proteins plays crucial roles in regulating cellular growth, migration, and differentiation. We report here that laminin-5 is expressed in the tunica media of the rat aorta and pulmonary arteries. Using indirect immunofluorescence microscopy, Western blots, and RT-PCR analysis, we found that primary cultures of rat arterial smooth muscle cells express laminin-5 and deposit it into their insoluble ECM. These cells also attach strongly to laminin-5 via beta1 integrin receptors in 30 min adhesion assays. Laminin-5 expression in these cells is upregulated by growth factors in vitro and platelet-derived growth factor (PDGF-BB) stimulation reduces adhesion to laminin-5. As laminin-5 promotes enhanced migration of other cell types, the production of and adhesion to laminin-5 by vascular smooth muscle cells may play a role in the pathological growth and migration of these cells associated with restenosis following vascular injury.
Subject(s)
Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/pharmacology , Cell Adhesion , Muscle, Smooth, Vascular/physiology , Platelet-Derived Growth Factor/pharmacology , Animals , Aorta/cytology , Becaplermin , Cell Adhesion Molecules/genetics , Cells, Cultured , Drug Synergism , Extracellular Matrix Proteins/pharmacology , Growth Substances/pharmacology , Immunohistochemistry , In Situ Hybridization , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins c-sis , RNA, Messenger/biosynthesis , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation , KalininABSTRACT
Large-scale screening strategies aimed at finding anticancer drugs traditionally focus on identifying cytotoxic compounds that attack actively dividing cells. Because progression to malignancy involves acquisition of an aggressively invasive phenotype in addition to hyperproliferation, simple and effective screening strategies for finding compounds that target the invasive aspects of cancer progression may prove valuable for identifying alternative and preventative cancer therapies. Here, we describe a fluorescence-based automated assay for identifying antimigratory compounds, with the ability to discern cytotoxic from noncytotoxic modes of action. With this assay, we analyzed the effects of two drugs on tumorigenic (MDA-MB-435) and nontumorigenic (MCF-10A) human breast cell lines. We chose to compare carboxyamidotriazole (CAI), an experimental compound shown to inhibit migration of various cell types, with tamoxifen, a common preventative and therapeutic anticancer compound. Our assay demonstrated that both these compounds inhibit migration at sublethal concentrations. Furthermore, CAI was more effective than tamoxifen at inhibiting chemotactic and haptotactic migration of both cell lines at all concentrations tested.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Neoplasm Metastasis/prevention & control , Tamoxifen/pharmacology , Triazoles/pharmacology , Drug Screening Assays, Antitumor/methods , Fluoresceins , Fluorescent Dyes , Humans , Propidium , Spectrometry, Fluorescence , Tumor Cells, CulturedABSTRACT
The beta1 integrin-stimulating antibody TS2/16 induces cAMP-dependent migration of MCF-10A breast cells on the extracellular matrix protein laminin-5. TS2/16 stimulates a rise in intracellular cAMP within 20 min after plating. Pertussis toxin, which inhibits both antibody-induced migration and cAMP accumulation, targets the Galphai3 subunit of heterotrimeric G proteins in these cells, suggesting that Galphai3 may link integrin activation and migration via a cAMP signaling pathway.
