Department of Biomedical Sciences and Morphological and Functional Imaging, University of Messina, AOU Policlinico "G. Martino", Via C. Valeria, 98125, Messina, Italy.
Amino Acids. 2014 Mar;46(3):759-66. doi: 10.1007/s00726-013-1569-y. Epub 2013 Aug 3.
Several experimental approaches have demonstrated that transglutaminase 2 (TG2) increased activity is involved in monocyte activation and inflammatory response. Preliminary results also demonstrate a TG-mediated post-translational modification of phospholipase A2 (PLA2), which catalyzes the release of arachidonic acid from its lipid storage sites. The control of PLA2-mediated production of eicosanoids has been found to be of great benefit for inflammatory disease treatment. However, the identification of the mechanisms of PLA2 activation is a very complex issue, because of the presence of multiple PLA2 forms. The aim of this study was to characterize the interactions between TG2 and sPLA2 in LPS-stimulated THP-1 cells, which were treated with TPA to induce early differentiated macrophage-type model. We demonstrated that increases in TG2 enzyme activity and protein expression may be considered an early event in monocyte/macrophage activation by LPS. Under these conditions, TG2 protein was co-immunoprecipitated with PLA2 by monoclonal antibody directed against the secretory form of the enzyme (sPLA2-V). Concomitantly, the PLA2 enzyme activity increased in TPA-treated cells exposed to LPS; these high levels of enzyme activity were significant reduced by R283, a site-specific inhibitor of TG2. Moreover, confocal laser scanning microscopy analysis of double-immunostained cytochemical specimens confirmed a co-localization of BAPA-labeled proteins and sPLA2-V in LPS-treated cells. These findings give evidence of a complex TG2/sPLA2-V, suggesting the possibility that sPLA2-V is a substrate for TG2. These results demonstrated that TG2 increases produced a sustained activation of PLA2 activity, suggesting a functional interaction between these enzymes in the regulation of inflammatory response.
几种实验方法已经证明，转谷氨酰胺酶 2（TG2）活性增加与单核细胞激活和炎症反应有关。初步结果还表明 TG2 介导的磷脂酶 A2（PLA2）的翻译后修饰，该修饰催化花生四烯酸从其脂质储存部位释放。已经发现控制 PLA2 介导的类二十烷酸的产生对于炎症性疾病的治疗非常有益。然而，由于存在多种 PLA2 形式，PLA2 激活的机制的鉴定是一个非常复杂的问题。本研究的目的是表征 LPS 刺激的 THP-1 细胞中 TG2 和 sPLA2 之间的相互作用，用 TPA 处理这些细胞以诱导早期分化的巨噬细胞样模型。我们证明，TG2 酶活性和蛋白表达的增加可被认为是 LPS 诱导单核细胞/巨噬细胞激活的早期事件。在这些条件下，TG2 蛋白与针对酶的分泌形式（sPLA2-V）的单克隆抗体共免疫沉淀 PLA2。同时，在 LPS 暴露于 TPA 处理的细胞中，PLA2 酶活性增加；这些高水平的酶活性通过 R283 显著降低，R283 是 TG2 的特异性抑制剂。此外，用双免疫染色细胞化学标本进行共聚焦激光扫描显微镜分析证实了在 LPS 处理的细胞中 BAPA 标记蛋白和 sPLA2-V 的共定位。这些发现为 TG2/sPLA2-V 的复合物提供了证据，表明 sPLA2-V 可能是 TG2 的底物。这些结果表明 TG2 增加产生了 PLA2 活性的持续激活，表明这些酶在调节炎症反应中存在功能相互作用。
