Protein Analytics Group, Institute of Biochemistry, Justus Liebig University Giessen, 35392 Giessen, Germany.
Laboratory for Experimental Orthopedics, Department of Orthopedics, Justus Liebig University Giessen, 35392 Giessen, Germany.
Cells. 2023 Jun 29;12(13):1743. doi: 10.3390/cells12131743.
Synovial fluid (SF) from human knee joints with osteoarthritis (OA) has elevated levels of lysophosphatidylcholine (LPC) species, but their functional role is not well understood. This in vitro study was designed to test the hypothesis that various LPCs found elevated in OA SF and their metabolites, lysophosphatidic acids (LPAs), modulate the abundance of proteins and phospholipids (PLs) in human fibroblast-like synoviocytes (FLSs), with even minute chemical variations in lysophospholipids determining the extent of regulation. Cultured FLSs ( = 5-7) were treated with one of the LPC species, LPA species, IL-1β, or a vehicle. Tandem mass tag peptide labeling coupled with LC-MS/MS/MS was performed to quantify proteins. The expression of mRNA from regulated proteins was analyzed using RT-PCR. PL synthesis was determined via ESI-MS/MS, and the release of radiolabeled PLs was determined by means of liquid scintillation counting. In total, 3960 proteins were quantified using multiplexed MS, of which 119, 8, and 3 were significantly and reproducibly regulated by IL-1β, LPC 16:0, and LPC 18:0, respectively. LPC 16:0 significantly inhibited the release of PLs and the synthesis of phosphatidylcholine, LPC, and sphingomyelin. Neither LPC metabolite-LPA 16:0 nor LPA 18:0-had any reproducible effect on the levels of each protein. In conclusion, small chemical variations in LPC species can result in the significantly altered expression and secretion of proteins and PLs from FLSs. IL-1β influenced all proteins that were reproducibly regulated by LPC 16:0. LPC species are likely to modulate FLS protein expression only in more advanced OA stages with low IL-1β levels. None of the eight proteins being significantly regulated by LPC 16:0 have been previously reported in OA. However, our in vitro findings show that the CD81 antigen, calumenin, and B4E2C1 are promising candidates for further study, focusing in particular on their potential ability to modulate inflammatory and catabolic mechanisms.
关节滑液（SF）来自患有骨关节炎（OA）的人类膝关节，具有升高的溶血磷脂酰胆碱（LPC）种类，但它们的功能作用尚不清楚。这项体外研究旨在测试以下假设：在 OA SF 中发现的各种升高的 LPC 及其代谢物溶血磷脂酸（LPA）调节人成纤维样滑膜细胞（FLS）中蛋白质和磷脂（PL）的丰度，即使是溶血磷脂中的微小化学变化决定了调节的程度。用一种 LPC 种类、LPA 种类、IL-1β或载体处理培养的 FLS（=5-7）。采用串联质量标签肽标记与 LC-MS/MS/MS 联用的方法定量蛋白质。使用 RT-PCR 分析受调节蛋白质的 mRNA 的表达。通过 ESI-MS/MS 测定 PL 合成，通过液体闪烁计数测定放射性标记的 PL 的释放。总共使用多重 MS 定量了 3960 种蛋白质，其中 119、8 和 3 种蛋白质分别受 IL-1β、LPC 16:0 和 LPC 18:0 显著且可重复调节。LPC 16:0 显著抑制 PL 的释放和磷脂酰胆碱、LPC 和神经鞘磷脂的合成。LPC 代谢物-LPA 16:0 和 LPA 18:0 均未对每种蛋白质的水平产生任何可重复的影响。总之，LPC 种类中的微小化学变化可导致 FLS 中蛋白质和 PL 的表达和分泌显著改变。IL-1β 影响所有可重复调节的 LPC 16:0 调节的蛋白质。LPC 种类可能仅在具有低 IL-1β 水平的更晚期 OA 阶段调节 FLS 蛋白表达。在 OA 中以前没有报道过被 LPC 16:0 显著调节的八种蛋白质中的任何一种。然而，我们的体外发现表明 CD81 抗原、钙调蛋白和 B4E2C1 是进一步研究的有希望的候选物，特别关注它们调节炎症和分解代谢机制的潜在能力。
