Thompson Brian, Ancellin Nicolas, Fernandez Salvador M, Hla Tim, Sha'afi Ramadan I
Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030-3505, USA.
Prostaglandins Other Lipid Mediat. 2006 Jul;80(1-2):15-27. doi: 10.1016/j.prostaglandins.2006.03.002. Epub 2006 Jul 7.
Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCalpha and PKCepsilon activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P(3) stimulates PKCalpha and S1P1 leads to PKCepsilon activity. Infection of endothelial cells with dominant negative (DN)PKCalpha adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCalpha, as infection with DN PKCepsilon does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCalpha reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCalpha.
蛋白激酶C（PKC）介导的信号转导通路将细胞外刺激转化为多种细胞功能。然而，各种PKC亚型在1-磷酸鞘氨醇（S1P）刺激的内皮细胞中的作用尚不清楚。在内皮细胞培养物中PKCα和PKCε的活性增加，并且S1P受体转染研究表明S1P(3)刺激PKCα，而S1P1导致PKCε活性。用显性负性（DN）PKCα腺病毒感染内皮细胞会降低细胞迁移，并极大地抑制S1P刺激的细胞形态发生。这种作用对PKCα具有特异性，因为用DN PKCε感染不会改变迁移或形态发生。PKC特异性化学抑制剂GF109203X也会抑制这两种反应。用显性负性PKCα感染内皮细胞会降低S1P诱导的钙升高。这种最大升高需要钙摄取，但不需要激酶的酶活性。用PKC特异性抑制剂GF109203X预处理这些细胞不会抑制S1P诱导的钙升高。S1P诱导的形态发生而非细胞迁移严重依赖于细胞外钙。用佛波醇12-肉豆蔻酸酯13-乙酸酯预处理内皮细胞5分钟可消除S1P刺激的钙升高，但对迁移几乎没有影响。PKC抑制剂或用显性负性PKCα感染可预防PMA抑制的钙升高。
