Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling
Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling
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DOI：
10.1074/mcp.m112.026609
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发表时间：
2013-08-01
影响因子：
7
通讯作者：
Wang, Lianchun
中科院分区：
文献类型：
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作者：
Qiu, Hong;Jiang, Jun-Lin;Wang, Lianchun
硫酸乙酰肝素（HS）是一种线性、丰富、高度硫酸化的多糖，在血管系统中表达。最近的遗传学研究表明，HS严重调节各种内皮细胞功能。然而，阐明的潜在的分子机制一直具有挑战性，因为在检查的实验条件下发现了大量的HS-结合配体的存在。在这份报告中，我们使用定量磷酸化蛋白质组学来检查全球HS依赖性信号通过比较野生型和HS缺陷的内皮细胞，在含血清的培养基中培养。共鉴定出7222个磷酸肽，对应于1179个蛋白质。功能相关性分析确定了25个HS依赖性功能网络，并且前五个与细胞形态、细胞组装和组织、细胞功能和维持、细胞间通讯、炎症反应和紊乱、细胞生长和增殖、细胞运动以及细胞存活和死亡相关。这与显示HS缺乏改变内皮细胞生长和移动性的细胞功能研究一致。对潜在分子机制的挖掘进一步揭示了HS调节与细胞粘附、迁移和凝血密切相关的信号通路，包括ILK、整合素、肌动蛋白细胞骨架组织、紧密连接和凝血酶信号通路。有趣的是，该分析出乎意料地确定了顶部HS依赖性信号传导是IGF-1信号传导途径，其尚未被HS调节。对生长因子信号传导的深入分析鉴定了22种HS依赖性生长因子/细胞因子/生长激素信号传导途径，包括先前已知的那些，如HGF和VEGF，以及那些未知的，如IGF-1、促红细胞生成素、促血管生成素/Tie、IL-17 A和生长激素。12所确定的22个生长因子/细胞因子/生长激素信号传导途径，包括IGF-1和血管生成素/Tie信号传导，在磷酸受体酪氨酸激酶阵列分析中被交替确认。总之，我们基于SILAC的定量磷酸化蛋白质组学分析证实了先前的发现，也发现了新的HS依赖性功能网络和信号传导，揭示了HS对内皮信号传导的更广泛的调节作用。
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.