Zaniolo Karine, Gingras Marie-Eve, Audette Marie, Guérin Sylvain L
Oncology and Molecular Endocrinology Research Center, Universitaire de Québec and Laval University, Québec, Canada.
Invest Ophthalmol Vis Sci. 2006 Oct;47(10):4199-210. doi: 10.1167/iovs.06-0176.
Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme essential in several cellular functions such as DNA repair, DNA transcription, carcinogenesis, and apoptosis. Expression of the PARP-1 gene is mainly dictated by the transcription factor Sp1. Fibronectin (FN), a component from the extracellular matrix transiently expressed at high levels during wound healing of the corneal epithelium, was reported to exert a positive influence on expression of the alpha5 integrin subunit gene promoter by altering the state of Sp1 phosphorylation, a process that depended on the activation of the ERK signaling pathway. The present study was undertaken to investigate whether PARP-1 gene expression might be similarly regulated by FN through the same signaling pathways and attempted to link expression of this gene to corneal wound healing in vitro.
Expression of PARP-1, Sp1/Sp3, ERK1/2, phospho-ERK1/2, P38 and phospho-P38 was monitored by Western blot in cultures of rabbit corneal epithelial cells (RCECs) grown on FN in the presence of inhibitors of the MAPK, PI3K, and P38 signaling pathways. Electrophoretic mobility shift assays (EMSAs) were conducted to assess the binding of Sp1 and Sp3 in nuclear extracts from RCECs grown on FN in the presence of inhibitors. Plasmids bearing the PARP-1 promoter fused to the CAT reporter gene were also transfected into RCECs grown under similar culture conditions to assess the influence of these inhibitors on PARP-1 promoter activity.
Expression of PARP-1, Sp1, and Sp3 increased considerably in RCECs grown on FN and translated into increased binding of Sp1 and Sp3 to their DNA target sites. In addition, FN increased PARP-1 promoter activity in a cell-density-dependent manner. Inhibition of both the MAPK and the PI3K pathways entirely abolished these properties.
PARP-1 gene expression was strongly activated by FN through alterations in the phosphorylation state of Sp1 and Sp3 that resulted from the activation of the MAPK and PI3K signaling pathways, thereby suggesting that PARP-1 may play a critical function during the highly proliferative phase that characterizes wound healing of the corneal epithelium.
聚（ADP - 核糖）聚合酶（PARP）-1是一种核酶，在多种细胞功能如DNA修复、DNA转录、致癌作用和细胞凋亡中至关重要。PARP - 1基因的表达主要由转录因子Sp1决定。纤连蛋白（FN）是细胞外基质的一种成分，在角膜上皮伤口愈合过程中会短暂高水平表达，据报道它通过改变Sp1磷酸化状态对α5整合素亚基基因启动子的表达产生积极影响，这一过程依赖于ERK信号通路的激活。本研究旨在探讨PARP - 1基因表达是否可能通过相同信号通路受到FN的类似调控，并试图将该基因的表达与体外角膜伤口愈合联系起来。
在存在MAPK、PI3K和P38信号通路抑制剂的情况下，通过蛋白质免疫印迹法监测在FN上生长的兔角膜上皮细胞（RCECs）培养物中PARP - 1、Sp1/Sp3、ERK1/2、磷酸化ERK1/2、P38和磷酸化P38的表达。进行电泳迁移率变动分析（EMSA）以评估在存在抑制剂的情况下，在FN上生长的RCECs核提取物中Sp1和Sp3的结合情况。还将携带与CAT报告基因融合的PARP - 1启动子的质粒转染到在类似培养条件下生长的RCECs中，以评估这些抑制剂对PARP - 1启动子活性的影响。
在FN上生长的RCECs中，PARP - 1、Sp1和Sp3的表达显著增加，并转化为Sp1和Sp3与其DNA靶位点的结合增加。此外，FN以细胞密度依赖性方式增加PARP - 1启动子活性。MAPK和PI3K通路的抑制完全消除了这些特性。
FN通过激活MAPK和PI3K信号通路导致Sp1和Sp3磷酸化状态改变，从而强烈激活PARP - 1基因表达，这表明PARP - 1可能在角膜上皮伤口愈合特征性的高度增殖阶段发挥关键作用。
