Kotelevets L, Noë V, Bruyneel E, Myssiakine E, Chastre E, Mareel M, Gespach C
INSERM U482 and IFR 65, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France.
J Biol Chem. 1998 Jun 5;273(23):14138-45. doi: 10.1074/jbc.273.23.14138.
This study was designed to characterize platelet-activating factor receptor (PAF-R) expression and function in normal and cancerous human colonic epithelial cells. PAF-R gene transcripts were analyzed by reverse transcription-polymerase chain reaction and Southern blot, using three sets of primers corresponding either to the coding region of the human PAF-R sequence (polymerase chain reaction product: 682 base pairs (bp)) or to the leukocyte- and tissue-type transcripts of 166 and 252 bp, respectively. An elongated splice variant was identified in the 5'-untranslated region of the tissue-type PAF-R transcript (334 bp) in colonic epithelial crypts and tumors. In human colonic PCmsrc cells transformed by c-src oncogene, the hepatocyte growth factor (HGF)-dependent invasiveness of collagen gels was abolished by 0.1 microM PAF and restored by the PAF-R antagonists WEB2086 and SR27417. PAF blocked HGF-induced tyrosine phosphorylation of p125 focal adhesion kinase. The phosphatidylinositol 3'-kinase (PI3'-K) inhibitors wortmannin and LY294002 totally blocked the HGF-induced invasion. Similar effects were observed in ts-srcMDCK kidney epithelial cells transformed by a v-Src temperature-sensitive mutant: (i) PAF and wortmannin exerted additive inhibitory effects on Src-induced invasion and (ii) activated and dominant negative forms of p110alpha PI3'-K, respectively, amplified and abrogated the Src- and HGF-dependent invasiveness of parental and ts-srcMDCK cells. We also provided the first evidence for the contribution of rapamycin-insensitive, pertussis toxin-dependent G-protein pathways to the integration of the signals emerging from activated Met and PAF receptors. These results indicate that PI3'-K is a critical transducer of invasiveness and strongly suggest that PAF exerts a negative control on invasion by inhibiting this signaling pathway. A possible beneficial role of PAF analogs on tumor invasion is therefore proposed.
本研究旨在表征血小板活化因子受体（PAF-R）在正常和癌性人结肠上皮细胞中的表达及功能。采用逆转录-聚合酶链反应和Southern印迹法，使用三组引物分析PAF-R基因转录本，这三组引物分别对应于人PAF-R序列的编码区（聚合酶链反应产物：682个碱基对（bp）），或分别对应于166 bp和252 bp的白细胞型和组织型转录本。在结肠上皮隐窝和肿瘤的组织型PAF-R转录本（334 bp）的5'-非翻译区鉴定出一种延长的剪接变体。在由c-src癌基因转化的人结肠PCmsrc细胞中，0.1 microM的PAF消除了肝细胞生长因子（HGF）依赖的胶原凝胶侵袭性，并被PAF-R拮抗剂WEB2086和SR27417恢复。PAF阻断了HGF诱导的p125粘着斑激酶的酪氨酸磷酸化。磷脂酰肌醇3'-激酶（PI3'-K）抑制剂渥曼青霉素和LY294002完全阻断了HGF诱导的侵袭。在由v-Src温度敏感突变体转化的ts-srcMDCK肾上皮细胞中观察到类似的效应：（i）PAF和渥曼青霉素对Src诱导的侵袭具有累加抑制作用；（ii）p110alpha PI3'-K的活化形式和显性负性形式分别增强和消除了亲本细胞和ts-srcMDCK细胞的Src和HGF依赖的侵袭性。我们还首次证明了对雷帕霉素不敏感、百日咳毒素依赖的G蛋白途径对来自活化的Met和PAF受体的信号整合的贡献。这些结果表明PI3'-K是侵袭性的关键转导因子，并强烈提示PAF通过抑制该信号通路对侵袭发挥负调控作用。因此，提出了PAF类似物对肿瘤侵袭可能具有的有益作用。
