Tencer Lilach, Burgermeister Elke, Ebert Matthias P, Liscovitch Mordechai
Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
Anticancer Res. 2008 Mar-Apr;28(2A):895-906.
Caveolin-1, a key component of plasma membrane caveolae, has been implicated in the regulation of cancer cell growth and survival. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor which plays a pivotal role in many cellular processes. Activation of PPARgamma by its ligand rosiglitazone upregulates caveolin-1 mRNA and protein in human carcinoma cells.
We have used specific signaling inhibitors to dissect the mechanisms of caveolin-1 mRNA and protein induction by rosiglitazone, determined by RT-PCR and Western blotting, respectively. ROS generation was measured by flow cytometry and cell survival was determined by the MTT assay.
We show that in HT-29 human colon cancer cells the induction ofcaveolin-1 by rosiglitazone is inhibited by the EGF receptor (EGFR) blocker AG1478. Moreover, rosiglitazone stimulates EGFR phosphorylation, while direct activation of EGFR by EGF up-regulates caveolin-1 mRNA. Inhibitors of Src and the Mek1-Erk1/2 and p38 MAP kinase pathways also inhibit up-regulation of caveolin-1 by rosiglitazone. Furthermore, rosiglitazone stimulates formation of superoxide anions, whereas induction of caveolin-1 expression by rosiglitazone is attenuated by the antioxidant N-acetyl-cysteine. Finally, rosiglitazone increases the resistance of HT-29 cells to doxorubicin and to hydrogen peroxide. The caveolin-1 gene promoter lacks a canonical PPARgamma response element (PPRE) and a PPRE-reporter construct is not sensitive to EGF or EGFR inhibition.
Our findings indicate that up-regulation of caveolin-1 by rosiglitazone requires superoxide formation and the activation of Src, EGFR, and the Mek1-Erk1/2 and p38 MAP kinase pathways. We suggest a novel mode of action of PPARgamma ligands in the regulation of caveolin-1, and possibly other genes devoid of a PPRE in their promoters, which involves the coordinate activation of PPARgamma and intracellular signaling pathways.
小窝蛋白-1是质膜小窝的关键组成部分，与癌细胞的生长和存活调节有关。过氧化物酶体增殖物激活受体γ（PPARγ）是一种配体激活的核受体，在许多细胞过程中起关键作用。其配体罗格列酮激活PPARγ可上调人癌细胞中小窝蛋白-1的mRNA和蛋白质水平。
我们使用特异性信号抑制剂来剖析罗格列酮诱导小窝蛋白-1 mRNA和蛋白质的机制，分别通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法进行测定。通过流式细胞术检测活性氧（ROS）的生成，通过MTT法测定细胞存活率。
我们发现，在HT-29人结肠癌细胞中，表皮生长因子受体（EGFR）阻断剂AG1478可抑制罗格列酮对小窝蛋白-1的诱导作用。此外，罗格列酮可刺激EGFR磷酸化，而表皮生长因子（EGF）直接激活EGFR可上调小窝蛋白-1 mRNA水平。Src、Mek1-Erk1/2和p38丝裂原活化蛋白激酶（MAPK）途径的抑制剂也可抑制罗格列酮对小窝蛋白-1的上调作用。此外，罗格列酮可刺激超氧阴离子的形成，而抗氧化剂N-乙酰半胱氨酸可减弱罗格列酮对小窝蛋白-1表达的诱导作用。最后，罗格列酮可增加HT-29细胞对阿霉素和过氧化氢的抗性。小窝蛋白-1基因启动子缺乏典型的PPARγ反应元件（PPRE），且PPRE报告基因构建体对EGF或EGFR抑制不敏感。
我们的研究结果表明，罗格列酮上调小窝蛋白-1需要超氧阴离子的形成以及Src、EGFR、Mek1-Erk1/2和p38 MAPK途径的激活。我们提出了PPARγ配体在调节小窝蛋白-1以及可能在其启动子中缺乏PPRE的其他基因方面的一种新作用模式，这涉及PPARγ和细胞内信号通路的协同激活。
