Zeng L, An S, Goetzl E J
Department of Medicine, University of California Medical Center, San Francisco 94143-0711, USA.
Biochemistry. 1996 Jun 4;35(22):7159-64. doi: 10.1021/bi960036x.
Cell surface expression of multiple structurally and functionally distinct prostaglandin E2 (PGE2) receptors (Rs), designated the EP1, EP2, EP3, and EP4 Rs, is a principal determinant of the diverse cellular effects of PGE2. The RNK-16 line of rat large granular lymphocytes, which has served as a model for natural killer cells, coexpresses a mean of 1092 EP3 Rs and EP4 Rs per cell with a mean Kd of 2.7 nM. The presence of the EP3 and EP4 Rs and the absence of the EP1 and EP2 Rs were revealed by inhibition of [3H]PGE2 binding by the EP3/EP1R agonist sulprostone, the EP3/EP2/EP4R agonist M&B 28767, and the EP2/EP4/EP3R agonist misoprostol but not by the EP1R antagonist SC-19220 or the EP2R agonist butaprost. Functional EP4 R expression was confirmed by finding that PGE2 and misoprostol, but not butaprost or sulprostone, evoked increases in the intracellular concentration of cyclic AMP ([cAMP]) in RNK-16 cells. Matrix metalloproteinase (MMP)-1 and -3 were identified by zymography and Western blots as the principal MMPs secreted by RNK-16 cells. Secretion of both MMPs by RNK-16 cells attained a maximal level after 24 h of incubation and was enhanced significantly by 10(-9) to 10(-7) M PGE2, 10(-6) M misoprostol, and 10(-4) M dibutyryl cyclic AMP, but not by the EP3R agonist sulprostone. Thus, the effect of PGE2 on RNK-16 cell MMP secretion is mediated by an EP4 R-dependent mechanism involving increases in [cAMP]i. The migration of RNK-16 cells across micropore filters, without or with a layer of Matrigel, was stimulated chemokinetically by PGE2 and misoprostol. PGE2-elicited chemokinesis of RNK-16 cells across a Matrigel model basement membrane, but not across a microfilter alone, was suppressed by the GM 6001 inhibitor of MMP activities. Stimulation of MMP activities in RNK-16 cells by the EP4R thus facilitates migration of the NK cells across vascular basement membranes.
多种结构和功能各异的前列腺素E2（PGE2）受体（Rs），即EP1、EP2、EP3和EP4 Rs，在细胞表面的表达是PGE2多种细胞效应的主要决定因素。大鼠大颗粒淋巴细胞系RNK - 16作为自然杀伤细胞的模型，每个细胞平均共表达1092个EP3 Rs和EP4 Rs，平均解离常数（Kd）为2.7 nM。EP3/EP1R激动剂舒前列素、EP3/EP2/EP4R激动剂M&B 28767以及EP2/EP4/EP3R激动剂米索前列醇对[3H]PGE2结合的抑制作用揭示了EP3和EP4 Rs的存在以及EP1和EP2 Rs的缺失，而EP1R拮抗剂SC - 19220或EP2R激动剂布他前列素则无此作用。通过发现PGE2和米索前列醇而非布他前列素或舒前列素能引起RNK - 16细胞内环磷酸腺苷（[cAMP]）浓度升高，证实了功能性EP4 R的表达。通过酶谱分析和蛋白质印迹法鉴定出基质金属蛋白酶（MMP）-1和 -3是RNK - 16细胞分泌的主要MMP。RNK - 16细胞分泌这两种MMP在孵育24小时后达到最高水平，并且10(-9)至10(-7) M的PGE2、10(-6) M的米索前列醇和10(-4) M的二丁酰环磷酸腺苷能显著增强其分泌，但EP3R激动剂舒前列素则无此作用。因此，PGE对RNK - 16细胞MMP分泌的影响是由一种依赖EP4 R的机制介导的，该机制涉及细胞内[cAMP]的增加。PGE2和米索前列醇能趋化动力学地刺激RNK - 16细胞穿过微孔滤膜，无论有无基质胶层。GM 6001这种MMP活性抑制剂可抑制PGE2诱导的RNK - 16细胞穿过基质胶模型基底膜（而非仅穿过微孔滤膜）的趋化运动。EP4R对RNK - 16细胞MMP活性的刺激作用因此促进了自然杀伤细胞穿过血管基底膜的迁移。
