Motta Carla, D'Angeli Floriana, Scalia Marina, Satriano Cristina, Barbagallo Davide, Naletova Irina, Anfuso Carmelina Daniela, Lupo Gabriella, Spina-Purrello Vittoria
Department of Biomedical Sciences and Biotecnology, University of Catania, Via Santa Sofia 64, 95125 Catania, Italy.
Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy.
Eur J Pharmacol. 2015 Aug 15;761:55-64. doi: 10.1016/j.ejphar.2015.04.026. Epub 2015 Apr 28.
Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.
聚（ADP - 核糖）聚合酶 -1（PARP -1）抑制剂通过与烟酰胺腺嘌呤二核苷酸（NAD⁺）竞争发挥作用，NAD⁺是该酶的生理底物，在许多以PARP -1过度激活为特征的病理状况中起保护作用。研究表明，PARP -1还通过其DNA修复活性促进肿瘤生长和进展。由于血管生成是这些活动的必要条件，我们试图确定PARP抑制是否会影响由C6胶质瘤条件培养基（CM）刺激的大鼠脑微血管内皮细胞（GP8.3）的迁移。通过伤口愈合实验和MTT分析，我们证明PARP -1抑制剂PJ -34 [N -（6 - 氧代 -5,6 - 二氢菲啶 -2 - 基） -N，N - 二甲基乙酰胺]消除了GP8.3细胞的迁移反应并降低了它们的活力。PARP -1还以不依赖DNA的方式在细胞外调节激酶（ERK）信号级联反应中起作用，该信号级联反应调节细胞增殖和分化。通过蛋白质免疫印迹分析和共聚焦激光扫描显微镜（LSM），我们分析了PJ -34对PARP -1表达、磷酸化ERK和磷酸化Elk -1激活的影响。通过逆转录 - 聚合酶链反应（RT -PCR）分析了丝裂原活化蛋白激酶激酶（MEK）抑制剂PD98059 [2 -（2 - 氨基 -3 - 甲氧基苯基） -4H -1 - 苯并吡喃 -4 - 酮]对未刺激和CM刺激的GP8.3细胞中PARP -1表达的影响。用PJ -34或PD98059处理GP8.3细胞可显著降低PARP -1表达和磷酸化ERK激活。通过LSM，我们进一步证明PARP -1和磷酸化ERK在GP8.3细胞中共表达，并在细胞质和核质中具有相同的亚细胞定位。基于这些数据，我们提出PARP -1和磷酸化ERK在细胞质中相互作用，然后转移到细胞核，在那里它们引发增殖反应。我们还提出PARP -1抑制通过干扰ERK信号转导途径来阻断CM诱导的内皮细胞迁移。
