Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

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. 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. Heparan sulfate (HS) is a linear, highly sulfated polysaccharide composed of glucosamine and hexauronic acid disaccharide repeating units (1Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1267) Google Scholar). HS covalently attaches to core proteins to form HS proteoglycans (HSPG). Dictated by the location of the core proteins, HS chains present on cell surfaces, such as linking to syndecans and glypicans, and in the basement membrane by attaching to perlecan and agrin (1Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1267) Google Scholar, 2Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Functions of cell surface heparan sulfate proteoglycans.Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2319) Google Scholar, 3Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1241) Google Scholar). HS biosynthesis is initiated by heterodimers formed by copolymerases Exostosin-1 (Ext1) and Exostosin-2 (Ext2) that elongate HS chains by alternatively adding glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues from their respective UDP-sugar nucleotide precursors. N-deacetylase/N-sulfotransferase (Ndst) initiates modification reactions that occur on newly assembled HS chains, including N-, 3-O, and 6-O sulfation of GlcNAc units (NS, 3S, 6S, respectively), epimerization of GlcA to iduronic acid (IdoA), and 2-O-sulfation of IdoA (2S). These modification reactions are incomplete, resulting in enormous structural diversity in mature HS and form a variety of ligand-binding sites to interact with a large number of protein ligands (1Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1267) Google Scholar, 2Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Functions of cell surface heparan sulfate proteoglycans.Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2319) Google Scholar, 3Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1241) Google Scholar). protein ligand-binding sites in HS of of sulfated glucosamine and acid the is composed of a M. a in the of the the binding PubMed Scopus Google Scholar). binding is a sulfated with and 2-O-sulfation J. M. of Heparan with for and PubMed Scopus Google Scholar). Intriguingly, the of the ligand-binding sites is and that the regulatory of HS occur in a and J. M. Selleck Heparan sulfate in with heparan sulfate PubMed Scopus Google Scholar, of heparan sulfate as a of and PubMed Scopus Google Scholar). cells are of the cellular of that form the of endothelial cells and to to inflammatory and coagulation, and of in and 71: PubMed Scopus Google Scholar). cells are also in that is by the and J. and of the PubMed Scopus Google Scholar). role in such as and as as in such as inflammatory M. and of PubMed Scopus Google Scholar). the mechanisms that endothelial cell the of for related HS is in the vasculature. studies that HS is for endothelial cell function and J. Schuksz M. J.R. Esko J.D. of endothelial heparan sulfate 2007; PubMed Scopus Google Scholar). previously that of and in M. Esko J.D. heparan sulfate deficiency and inflammatory PubMed Scopus Google revealing that endothelial HS is to This confirmed by the by J. J. Esko J.D. of in and PubMed Scopus Google Scholar, Esko J.D. of endothelial heparan PubMed Scopus Google and by of M. heparan sulfate in of and cells to PubMed Scopus Google HS J. Esko J.D. of heparan sulfate in PubMed Scopus Google Scholar). also that deficiency of endothelial J. Schuksz M. J.R. Esko J.D. of endothelial heparan sulfate 2007; PubMed Scopus Google showing that endothelial HS critically Recent studies by also revealed that endothelial HS to Esko J.D. Heparan sulfate and PubMed Scopus Google and to J. A role for endothelial heparan sulfate in PubMed Scopus Google Scholar). These studies HS to be a in the underlying molecular mechanisms of HS on endothelial cell we examined the of endothelial HS with HS-binding ligands known to be in the functions. we examined the of endothelial HS with and in M. Esko J.D. heparan sulfate deficiency and inflammatory PubMed Scopus Google and the signaling in J. Schuksz M. J.R. Esko J.D. of endothelial heparan sulfate 2007; PubMed Scopus Google Scholar). and signaling were examined for their regulatory role of endothelial HS in Esko J.D. Heparan sulfate and PubMed Scopus Google and were in the of to J. A role for endothelial heparan sulfate in PubMed Scopus Google Scholar). This to underlying molecular However, in a large number of HS-binding growth and are to the examined and HS interact with a of HS-binding protein ligands to endothelial J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1241) Google Scholar, heparan sulfate in PubMed Scopus Google Scholar). molecular analysis is to the and regulatory of HS on endothelial cell function and related signaling. modification a cellular signaling mechanism that cells to by and a role in signaling in a PubMed Scopus Google Scholar). phosphoproteomics and signaling a analysis of acid with PubMed Scopus Google Scholar, signaling in 2007; PubMed Scopus Google Scholar). In this we the by used cell growth growth growth factor growth growth factor binding growth tyrosine tyrosine factor by growth endothelial growth endothelial growth factor used cell growth growth growth factor growth growth factor binding growth tyrosine tyrosine factor by growth endothelial growth endothelial growth factor global quantitative phosphoproteomics analysis to examine HS-dependent endothelial signaling by comparing wild type and HS-deficient endothelial cells that were cultured in serum-containing medium. A total of 7222 corresponding to 1179 proteins were identified. analysis of the identified proteins determined that HS deficiency altered cellular that are highly related to cell morphology, cellular assembly and organization, cellular function and maintenance, inflammatory response and disorder, cell growth and proliferation, cellular survival and consistent with cell function which found that HS deficiency endothelial cell growth and mobility. Mining for the underlying molecular mechanisms revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, and found that HS critically modulates IGF-1 signaling. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling including both previously known and such as IGF-1, erythropoietin, angiopoietin/Tie, and growth hormones. Twelve of the identified 22 HS-dependent signaling were alternatively confirmed by phospho-receptor tyrosine kinase array analysis. our revealed a and regulatory role of HS on endothelial signaling. acid cell growth factor glucuronic acid N-acetylglucosamine growth factor heparan sulfate growth factor growth growth factor binding protein cells N-deacetylase/N-sulfotransferase growth factor tyrosine kinase tyrosine kinase factor acid by growth factor endothelial growth factor endothelial growth factor acid cell growth factor glucuronic acid N-acetylglucosamine growth factor heparan sulfate growth factor growth growth factor binding protein cells N-deacetylase/N-sulfotransferase growth factor tyrosine kinase tyrosine kinase factor acid by growth factor endothelial growth factor endothelial growth factor experimental were and a as previously Heparan sulfate is for cells to from PubMed Scopus Google Scholar, Heparan and to of PubMed Scopus Google Scholar). by the and of the of endothelial cell were from as previously M. 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J. for protein PubMed Scopus Google Scholar). were as acid and A of by to the with to be and PubMed Scopus Google Scholar). of the identified proteins the and analysis of large Scopus Google Scholar). were by core analysis. molecular and pathways were on in the of the the and the in A of the number of from the that to the by the total number of that to the is used to a the that the the in the and the is by the regulatory role of HS on endothelial cell function and related signaling, we to endothelial cells that are in HS this we HS to with wild type SILAC-based quantitative phosphoproteomic analysis out to that were by in the were by in and the cell the were with by cell and of to for to HS biosynthesis by the HS and previously that of HS in cells Heparan sulfate is for cells to from PubMed Scopus Google Scholar, Heparan and to of PubMed Scopus Google Scholar, M. Esko J.D. of heparan sulfate the of cells to 2007; PubMed Scopus Google Scholar). HS in the the and cells were with and by cells cell surface HS corresponding to a much signaling of with In is in the cells and the showing that HS biosynthesis in the that the cells are endothelial the cells were with to the endothelial cell the and cells of and on their cell surface that both cell are endothelial the and cells both in not which is for endothelial cells and that the cell are endothelial and cell we HS and their wild type the cells were from the both cell the genetic the and a genetic and experimental to the regulatory role of HS on endothelial cell function and related signaling. 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J. J. of protein protein by quantitative PubMed Scopus Google Scholar). in the for the the we A total of 7222 phosphopeptides, which to 1179 proteins, were identified in both and were corresponding to the proteins the the 7222 identified phosphopeptides, and of to to and with the M. M. of protein the 2002; PubMed Scopus Google Scholar). of the number in the identified revealed that of the are and of the are A were identified with sites and were of the corresponding has been in a and be the the of the proteins we analysis of the identified are cellular molecular and the and revealed that of the identified proteins are with and identified protein location and signaling and cell cell and with the analysis of molecular further revealed that the identified proteins were related to the binding of various kinase and endothelial signaling by we core analysis of the the In 25 functional networks were identified to be HS top five identified functional networks cell morphology, cellular assembly and organization, cellular function and maintenance, inflammatory response and disorder, cell growth and proliferation, cellular survival and In to the networks, signaling pathways that been known to be modulated by HS were also such as and signaling, the of the analysis of the the top HS-dependent signaling identified is IGF-1 signaling, which previously not been known to be In signaling also identified in the HS-dependent signaling, including signaling and signaling HS identified to signaling critically related to endothelial cell including actin cytoskeleton and cell junction the thrombin signaling, a in coagulation, to be altered that HS critically of endothelial networks from and and and and and and and and in a growth such as VEGF, the cellular of endothelial cells as in this to examine growth factor signaling modulated by HS. analysis determined with 22 growth factor/cytokine/growth hormone signaling pathways were altered in the including the HS-dependent signaling such as VEGF, and This analysis also revealed that not been known to be including IGF-1, erythropoietin, angiopoietin/Tie, and growth alternatively we out array analysis. array examined and signaling pathways that were identified in our phosphoproteomics analysis to be HS-dependent and of the growth factor that were altered in in phosphoproteomics were in the array analysis and growth factor such as that were in the phosphoproteomic analysis and in the kinase array alternatively showing that the analysis is and to HS-dependent growth factor signaling in endothelial of growth factor signaling determined by phosphoproteomics and array present as signaling altered in to the signaling in is altered in phosphoproteomic and in the array the were to be the signaling in is altered in phosphoproteomic and in the array the were and the were in a surface HS has been to function as a to a large variety of growth factor signaling. our findings in phosphoproteomics and array and to the newly identified HS-dependent growth factor signaling a we used analysis to examine HS deficiency cell surface binding of HGF and in HGF cell surface the HGF binding to cells is the cell expresses of the HGF This that HS deficiency HGF binding to the HGF on the cell surface and to the of HGF signaling in HGF is known to HS M. of growth factor with heparan of the heparan sulfate structural PubMed Google this also that HS as a to HGF signaling. IGF-1 to and and the to IGF-1 signaling. analysis that the binding of IGF-1 to is also However, IGF-1 is a protein J. M. in the growth factor growth factor binding protein PubMed Scopus Google and not the cell surface IGF-1 binding that cell surface HS with to IGF-1 signaling. this we further examined the cell surface binding of the protein are a protein and to function mechanisms to signaling, including of from and the and of the in and PubMed Scopus Google Scholar). is by endothelial cells and on the endothelial cell surface M. J. and with endothelial of binding Google Scholar). and both binding to M. J. and with endothelial of binding Google Scholar). IGF-1, and been to form J. M. in the growth factor growth factor binding protein PubMed Scopus Google that endothelial cell surface HS function in a to IGF-1 signaling. this cell surface binding examined by analysis. cell surface binding that endothelial cell surface HS binding of to IGF-1 signaling. Recent genetic studies that HS critically modulates endothelial cell including of and heparan sulfate in PubMed Scopus Google Scholar, Esko J.D. Heparan PubMed Scopus Google Scholar). However, elucidation of their underlying cellular and molecular mechanism has been This is because in endothelial cells in with a large number of HS-binding protein ligands J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1241) Google Scholar). HS-binding proteins the to endothelial signaling in a HS-dependent endothelial cell functions. studies revealed the of endothelial HS with a protein ligands that are known to be critically in M. Esko J.D. heparan sulfate deficiency and inflammatory PubMed Scopus Google M. heparan sulfate in of and cells to PubMed Scopus Google J. Schuksz M. J.R. Esko J.D. of endothelial heparan sulfate 2007; PubMed Scopus Google Esko J.D. Heparan sulfate and PubMed Scopus Google and J. A role for endothelial heparan sulfate in PubMed Scopus Google Scholar). These studies to the underlying cellular and molecular In this report, we SILAC-based quantitative phosphoproteomic analysis in with genetic of HS to examine global HS-dependent and signaling in endothelial cells conditions. that HS modulates 25 functional networks, and the top five are critically related to cell morphology, cellular assembly and organization, cellular function and maintenance, inflammatory response and disorder, cell growth and proliferation, cellular survival and death. analysis for the underlying molecular mechanisms revealed that HS modulates pathways related to cell adhesion, migration, and coagulation, such as signaling, signaling, signaling related to actin cytoskeleton organization, and tight junction and thrombin signaling. our molecular signaling analysis unexpectedly identified that the top HS-dependent is IGF-1 signaling, that HS critically IGF-1 cell functions. analysis of growth factor signaling revealed 22 growth factor/cytokine/growth hormone signaling pathways that were modulated by including signaling pathways that not been known to be including IGF-1, erythropoietin, angiopoietin/Tie, and growth hormones. In summary, this SILAC-based phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, our of the regulatory role of HS on endothelial function and the underlying molecular mechanisms from a In this pathways were identified to be HS-dependent the of the regulatory of HS on endothelial cell function and signaling This is consistent with the HS structural that HS to interact with a large number of growth growth factor binding proteins, and the identified signaling pathways revealed that the top HS-dependent signaling pathways were IGF-1 signaling, signaling, and signaling related to actin cytoskeleton and cell These top signaling pathways are critically related to cell proliferation, cell adhesion, and and are with the cell in the are the growth cell and These to that HS deficiency cell proliferation, cell adhesion, and signaling, resulting in cell and of the as in our cell function studies studies are in with a that HS and a highly sulfated form of a large number of proteins to form a highly which is to and A for the of the PubMed Scopus Google Scholar). In this we identified 22 growth factor/cytokine/growth hormone signaling pathways that were by which previously known growth factor signaling pathways such as VEGF, and J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1241) Google and signaling pathways such as IGF-1, angiopoietin/Tie, erythropoietin, IL-17A and growth hormone signaling the signaling pathways in we identified that endothelial HS also modulates the signaling of protein including and as as the These findings that HS modulated the signaling of a large number of and the by cellular the of in the in our the identified signaling pathways were in revealing that HS as a of pathways, including the tyrosine kinase signaling pathway, to cell adhesion, and Recent studies showing that of tyrosine the HGF and signaling, cell of a novel to M. for PubMed Scopus Google Scholar, and pathways to PubMed Scopus Google Scholar, of to Rev. PubMed Scopus Google Scholar). findings that of tyrosine be by HS a to to growth that the of the identified HS-dependent signaling pathways, their their HS. we that of identified HS-dependent signaling pathways are modulated by HS. However, has been to sulfate which has the to signaling also in of of heparan sulfate to sulfate and the PubMed Scopus Google Scholar). we also that of the identified HS-dependent signaling not be modulated by are by the of the studies with be to this HS has been in mechanisms to the signaling of protein ligands (1Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1267) Google Scholar, Esko J.D. Heparan PubMed Scopus Google Scholar). studies with and their tyrosine documented the function for cell surface HS and a in which HS with both and to the of signaling on the cell to and of kinase is that cell surface HS as a by with ligand with both ligands and for growth and including VEGF, and the function a mechanism by which HS on cell and on the of signaling HS has also been to protein to the related signaling (1Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1267) Google Scholar, Esko J.D. Heparan PubMed Scopus Google Scholar, Heparan sulfate as a protein by PubMed Scopus Google Scholar). we that HS signaling of protein in Heparan and to of PubMed Scopus Google Scholar). In this we examined the regulatory mechanism of HS on HGF and IGF-1 signaling. the cell surface binding of that the cell surface as a to HGF our of signaling a regulatory mechanism in which endothelial cell surface HS with not with the ligand IGF-1 to IGF-1 signaling. IGF-1 is protein and to and the to IGF-1 signaling. IGF-1 has been to be both in and in Fitzgerald M. J. growth factor as in and studies with growth PubMed Scopus Google Scholar, Fitzgerald J. growth factor as in and in PubMed Scopus Google and of to in endothelial signaling PubMed Scopus Google the to critically In this we unexpectedly that of IGF-1 signaling, revealing a novel molecular mechanism by which endothelial HS to studies also that endothelial HS with to IGF-1 signaling. However, further such as of cell surface of the and on signaling, are to HS modulates IGF-1 signaling. These further studies a to the regulatory mechanism of HS in endothelial cell function and in such as the of our this the the regulatory role of HS on endothelial signaling a a studies of endothelial cells and endothelial for and mechanisms of endothelial PubMed Scopus Google Scholar). to the in in to SILAC-based and in because of of the in in this we used by and HS-deficient and the genetic a to the regulatory role of HS on endothelial cell and the underlying signaling. our phosphoproteomic are consistent with the known HS-dependent signaling mechanisms found in the and were by array analysis in we are that our are and that the are from cells as as that endothelial cells from their we also that the of HS on endothelial cell signaling and the signaling of endothelial cells from be for of the and for with with