Liu Rui, Wang Yi, Li Bingxin, Wang Hui, Guan Feng, Tan Zengqi, Li Xiang
1Joint International Research Laboratory of Glycobiology and Medicinal Chemistry, College of Life Science, Northwest University, 229 Taibai North Road, Xi'an, 710069 Shaanxi China.
Department of Hematology, Provincial People's Hospital, Xi'an, Shaanxi China.
Clin Proteomics. 2019 Jul 18;16:32. doi: 10.1186/s12014-019-9249-x. eCollection 2019.
Bone marrow stromal cells protect hematopoietic cells and provide drug resistance by delivering bunch of variable proteins. Thus, alterations of protein expression are typically associated with cell-cell signal transduction and regulation of cellular functions.
Co-culture models of bone marrow stromal cells and hematopoietic cells are often used in studies of their crosstalk. Studies of altered protein expression initiated by stromal cell/hematopoietic cell interactions are an important new trend in microenvironmental research. There has been no report to date of global quantitative proteomics analysis of crosstalk between hematopoietic cells and stromal cells. In this study, we analyzed quantitative proteomes in a co-culture system of stromal HS5 cells and hematopoietic KG1a cells, and simultaneously tracked differentially expressed proteins in two types of cells before and after co-culture by stable isotope labeling by amino acids in cell culture (SILAC) method.
We have shown that in co-cultured KG1a, 40 proteins (including CKAP4, LMNA, and SERPINB2) were upregulated and 64 proteins (including CD44, CD99, and NCAM1) were downregulated relative to KG1a alone. We utilized IPA analysis to discover that the NOD-like receptor signaling pathway was upregulated, whereas platelet activation was downregulated in co-cultured KG1a cells. Furthermore, 95 proteins (including LCP1, ARHGAP4, and UNCX) were upregulated and 209 proteins (including CAPG, FLNC, and MAP4) were downregulated in co-cultured HS5 relative to HS5 alone. The tight junction pathway was downregulated and glycolysis/gluconeogenesis pathway was dysfunctional in co-cultured HS5. Most importantly, the significantly differentially expressed proteins can also be confirmed using different co-cultured cell lines.
Altogether, we recommend such quantitative proteomics approach for the studies of the hematopoietic-stroma cross-talk, differentially expressed proteins and related signaling pathways identification. The differentially expressed proteins identified from this current SILAC method will provide a useful basis for ongoing studies of crosstalk between stromal cells and hematopoietic cells in co-culture systems. All these result suggested our ongoing studies can focus on the mechanisms underlying CKAP4 increase and CD44 decrease in co-cultured hematopoietic cells, and the increase of LCP1 and decrease of CAPG in co-cultured stromal cell. The proteomic profiles from the KG1a/stromal cell co-culture system give new molecular insights into the roles of these cells in MDS pathophysiology and related bone disease.
骨髓基质细胞通过传递多种可变蛋白来保护造血细胞并提供耐药性。因此，蛋白质表达的改变通常与细胞间信号转导和细胞功能调节相关。
骨髓基质细胞与造血细胞的共培养模型常用于研究它们之间的相互作用。由基质细胞/造血细胞相互作用引发的蛋白质表达改变的研究是微环境研究中的一个重要新趋势。迄今为止，尚无关于造血细胞与基质细胞之间相互作用的全球定量蛋白质组学分析的报道。在本研究中，我们分析了基质HS5细胞与造血KG1a细胞共培养系统中的定量蛋白质组，并通过细胞培养中氨基酸稳定同位素标记（SILAC）方法同时追踪共培养前后两种细胞中差异表达的蛋白质。
我们发现，与单独的KG1a相比，共培养的KG1a中有40种蛋白质（包括CKAP4、LMNA和SERPINB2）上调，64种蛋白质（包括CD44、CD99和NCAM1）下调。我们利用IPA分析发现，共培养的KG1a细胞中NOD样受体信号通路上调，而血小板活化下调。此外，与单独的HS5相比，共培养的HS5中有95种蛋白质（包括LCP1、ARHGAP4和UNCX）上调，209种蛋白质（包括CAPG、FLNC和MAP4）下调。共培养的HS5中紧密连接通路下调，糖酵解/糖异生途径功能失调。最重要的是，使用不同的共培养细胞系也可以证实显著差异表达的蛋白质。
总之，我们推荐这种定量蛋白质组学方法用于造血-基质相互作用、差异表达蛋白质及相关信号通路鉴定的研究。通过当前SILAC方法鉴定出的差异表达蛋白质将为共培养系统中基质细胞与造血细胞之间相互作用的 ongoing 研究提供有用的基础。所有这些结果表明，我们 ongoing 的研究可以聚焦于共培养造血细胞中CKAP4增加和CD44减少以及共培养基质细胞中LCP1增加和CAPG减少的潜在机制。KG1a/基质细胞共培养系统的蛋白质组学图谱为这些细胞在骨髓增生异常综合征病理生理学和相关骨病中的作用提供了新的分子见解。
