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Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

Malmström, Johan LU ; Marchese, Jason; Juhasz, Peter; Pukac, Laurel; Karnovsky, Morris; Marko-Varga, György LU and Westergren-Thorsson, Gunilla LU (2005) In Journal of Chromatography. B 815(1-2). p.333-342
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... (More)

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.

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keywords
Binding Sites, Cell Proliferation, Cells, Cultured, Down-Regulation, Fibroblasts, Glycosaminoglycans, Heparitin Sulfate, Humans, Isotope Labeling, Lung, Mass Spectrometry, Mitogen-Activated Protein Kinases, Nuclear Proteins, Phosphorylation, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Chromatography. B
volume
815
issue
1-2
pages
10 pages
publisher
Elsevier
external identifiers
  • pmid:15652821
  • wos:000226879700029
  • scopus:12344298497
ISSN
1873-376X
DOI
10.1016/j.jchromb.2004.11.054
language
English
LU publication?
yes
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c9b7d84f-887d-476d-b150-6a93343fb14c (old id 133114)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15652821&dopt=Abstract
date added to LUP
2007-06-27 10:40:21
date last changed
2017-09-11 15:23:27
@article{c9b7d84f-887d-476d-b150-6a93343fb14c,
  abstract     = {<p>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.</p>},
  author       = {Malmström, Johan and Marchese, Jason and Juhasz, Peter and Pukac, Laurel and Karnovsky, Morris and Marko-Varga, György and Westergren-Thorsson, Gunilla},
  issn         = {1873-376X},
  keyword      = {Binding Sites,Cell Proliferation,Cells, Cultured,Down-Regulation,Fibroblasts,Glycosaminoglycans,Heparitin Sulfate,Humans,Isotope Labeling,Lung,Mass Spectrometry,Mitogen-Activated Protein Kinases,Nuclear Proteins,Phosphorylation,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {02},
  number       = {1-2},
  pages        = {333--342},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography. B},
  title        = {Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate},
  url          = {http://dx.doi.org/10.1016/j.jchromb.2004.11.054},
  volume       = {815},
  year         = {2005},
}