title: Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate.
abstract: Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.
authors.affiliation: Department of Cell and Molecular Biology, C13, BMC, Lund University, S-221 84 Lund, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Malmström
authors.forename: Jason
authors.initials: J
authors.lastname: Marchese
authors.forename: Peter
authors.initials: P
authors.lastname: Juhasz
authors.forename: Laurel
authors.initials: L
authors.lastname: Pukac
authors.forename: Morris
authors.initials: M
authors.lastname: Karnovsky
authors.forename: György
authors.initials: G
authors.lastname: Marko-Varga
authors.forename: Gunilla
authors.initials: G
authors.lastname: Westergren-Thorsson
mesh_terms: D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006025:Glycosaminoglycans / Q000494:pharmacology; D006497:Heparan Sulfate / Q000494:pharmacology*; D006801:Humans; D007553:Isotope Labeling; D008168:Lung / Q000166:cytology / Q000196:embryology; D013058:Mass Spectrometry; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D009687:Nuclear Proteins / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D020928:Mitogen-Activated Protein Kinases; D009687:Nuclear Proteins