Abstract
Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.
Highlights
From the ‡Complex Carbohydrate Research Center, and §Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602; ¶Department of Pathology and Microbiology, ʈMass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, Nebraska 68198-5900
We examined the interactions of endothelial Heparan sulfate (HS) with L-selectin and three chemokines involved in leukocyte trafficking [12], and the VEGF signaling in tumor angiogenesis [11]
Recent genetic studies showed that HS critically modulates endothelial cell functions, including mediation of leukocyte trafficking, inflammation, angiogenesis, vascular permeability, and tumor metastasis [19, 46]
Summary
Mouse and Cell Line—The experimental mice harboring conditionally targeted Ext (Ext1f/f mice) were bred onto C57BL/6J background and housed at a specific pathogen-free facility as reported previously [23, 24]. After removing debris by PBS washing, the cells were cultured in DMEM medium supplemented with 10% FBS. The mouse p-RTK array analysis was initiated by blocking the test membranes with Array Buffer 1 for 1 h and incubating the membrane overnight at 4 °C with 500 g sample protein prepared from Ext1f/f or Ext1Ϫ/Ϫ MLECs. Following, the membrane was washed and incubated with a HRP-conjugated pan antiphospho-tyrosine antibody (1:5000, Cell Signaling Technology, MA). Search parameters are set as sample type [SILAC (Lysϩ, Argϩ6)], cys alkylation (Iodoacetamide), digestion (Trypsin), instrument (TripleTOF 5600), special factors (phosphorylation emphasis and urea denaturation), species (Mus musculus), ID Focus (Biological modifications), database (uniprot_sprot.fasta), search effort (Thorough), false discovery rate (FDR) analysis (Yes), and user modified parameter files (No). The significance of the association between the data set and the canonical pathway was measured in two ways: 1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. 2) Fisher’s exact test was used to calculate a p value determining the probability that the association between the genes in the data set and the canonical pathway is explained by chance alone
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