Arachidonate-preferential cytosolic phospholipases A_2 as novel signal transducers

我们检测了85 kDa胞浆磷脂酶A_2(CPLA_2)在哺乳动物细胞中的表达和功能。尽管CPLA_2在大多数哺乳动物细胞中普遍表达，但在不同类型的细胞中，CPLA_2的表达在细胞激活后的调节有所不同：(1)在成纤维细胞中，即使在促炎细胞因子激活后，CPLA_2的表达也几乎不变；(2)在成骨细胞和肥大细胞中，CPLA_2的蛋白表达在细胞因子刺激后显著增加，但没有伴随其mRNA水平的升高，这表明CPLA_2的表达在翻译后显著调节；(3)在巨噬细胞中，CPLA_2的mRNA和蛋白在脂多糖刺激后均增加。值得注意的是，我们首次发现，在小鼠成骨细胞中，CPLA_2表达的增加经历了PGA_2的正反馈增强，PGA_2是前列腺素生物合成途径的内源性产物。…的功能耦合在前列腺素生物合成途径的不同阶段，更多的CPLA_2和下游的环氧合酶(COX)酶被研究。(1)在细胞活化的即刻阶段，成纤维细胞、肥大细胞和巨噬细胞通过CPLA_2与COX-1的功能性偶联，分别产生PGE_2、PGD_2和TXA_2。相反，在细胞因子诱导的巨噬细胞或成骨细胞中，已经诱导了COX-2，在CPLA_2和诱导性COX-2功能相连的即时反应中，优先产生PGE_2。(2)在细胞激活延迟期，大多数细胞产生PGE_2，这是CPLA_2和COX-2功能偶联的结果。在巨噬细胞、肥大细胞和成纤维细胞中，分泌II型PLA2(通常是可诱导的)也是产生最佳延迟前列腺素所必需的，揭示了两种不同的PLA2酶之间的未知串扰。然而，这些研究表明，CPLA_2是前列腺素即刻和延迟生物合成的先决条件，其在不同细胞中的表达调控不同。最后，为了寻找与CPLA_2特异相互作用的蛋白，我们在几种细胞类型中鉴定了一个60 kDa的胞内蛋白。我们的目标是阐明这种60KDS的CPLA_2相互作用蛋白的结构和功能，它被认为是细胞内CPLA_2功能的调节因子。较少

We have examined the expression and function of 85-kDa cytosolic phospholipase A_2 (cPLA_2) in mammalian cells.1. Whereas cPLA_2 is ubiqitously and constitutively expressed in most mammalian cells, the regulation of its expression after cell activation was found to differ among cell types : (1) in fibroblasts, cPLA_2 expression was almost constant even after cell activation by proinflammatory cytokines ; (2) in osteoblasts and mast cells, cPLA_2 protein expression increased markedly after cytokine stimulation, without accompanied by concomitant increase in its mRNA level, revealing significant post-translational regulation of cPLA_2 expression ; and (3) in macrophages, both cPLA_2 mRNA and protein increased after lipopolysaccharide stimulation. Of note, we found for the first time that increase in cPLA_2 expression underwent positive-feedback augmentation by PGA_2, which is an endoproduct of the prostanoid biosynthetic pathway, in mouse osteoblastic cells.2. The functional coupling of … More cPLA_2 with downstream cyclooxygenase (COX) enzymes in the different phases of the prostanoid biosynthetic pathway was investigated. (1) Fibroblasts, mast cells and macrophages produced PGE_2, PGD_2 and TXA_2, respectively, in the immediate phase of cell activation through functional coupling of cPLA_2 with constitutive COX-1. In contrast, cytokine-primed macrophages or osteoblasts, in which COX-2 had been already induced, produced PGE_2 in preference to other prostanoids in the immediate response where cPLA_2 and inducible COX-2 were functionally linked. (2) In the delayd phase of cell activation, most cells produced PGE_2 as a consequence of the functional coupling of cPLA_2 and COX-2. In macrophages, mast cells and fibroblasts, secretory type II PLA_2, often inducible, was also required for the optimal delayd prostanoid generation, revealing an unexplored crosstalk between the two distinct PLA_2 enzymes. Nevertheless, these studies imply that cPLA_2 is prerequisite for both immediate and delayd prostanoid biosynthesis, irespective of the differential regulation of its expression in various cells.3. Finally, in search for the protein that specifically interacts with cPLA_2, we identified a 60-kDa intracellular protein in several cell types. We aim to clarify the structure and function of this 60-kDs cPLA_2-interacting protein, which is assumed to act as a regulator of cPLA_2 function in cells. Less