title: Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation.
abstract: Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts.
authors.affiliation: Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
authors.forename: Yuan-Tao
authors.initials: YT
authors.lastname: Liu
authors.forename: Lifang
authors.initials: L
authors.lastname: Song
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Templeton
mesh_terms: D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D008024:Ligands; D021962:Membrane Microdomains / Q000187:drug effects*; D009761:Nystatin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009761:Nystatin; D006493:Heparin; D002784:Cholesterol; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras)