Faculty of Pharmacy, Laboratory of Pharmacology, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tonda-bayashi, Osaka 584-8540, Japan.
Neurochem Int. 2013 Jul;63(1):25-34. doi: 10.1016/j.neuint.2013.04.004. Epub 2013 Apr 22.
Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), a selective agonist of the ETB receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ETB antagonist (1 μM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 μM PD98059), p38 (20 μM SB203580) and JNK (1 μM SP600125), but not inhibitors of NF-κB (10 μM SN50 and 100 μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ETB receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.
内皮素（ETs）是一种血管收缩肽家族，在大脑的几种病理状态下上调，并诱导星形胶质细胞增殖。我们之前观察到 ET-1 增加了 cyclin D1 蛋白的表达。因此，我们证实了 ET-1 在大鼠培养的星形胶质细胞中对内源性 cyclin D1 的上调作用。实时 PCR 分析表明，ET-1（100 nM）和 Ala（1,3,11,15）-ET-1（100 nM），一种选择性的 ETB 受体激动剂，诱导 cyclin D1 mRNA 产生时间依赖性和短暂的增加。ET-1 的作用被 ETB 拮抗剂（1 μM BQ788）或 Sp1 抑制剂（500 nM 米托蒽醌）、ERK 抑制剂（50 μM PD98059）、p38 抑制剂（20 μM SB203580）和 JNK 抑制剂（1 μM SP600125）减弱，但不被 NF-κB 抑制剂（10 μM SN50 和 100 μM 吡咯烷二硫代氨基甲酸盐）减弱。Sp1 的结合实验表明，ET-1 增加了 Sp1 与共有序列的结合活性，并且包含 Sp1 结合位点的 cyclin D1 启动子的两个寡核苷酸减弱了 ET-1 的作用。Western blot 分析表明，ET-1 通过 ETB 受体诱导 Sp1 的 Thr453 和 Thr739 发生时间依赖性和短暂的磷酸化。PD98059 和 SP600125 减弱了 ET-1 诱导的 Sp1 磷酸化。此外，ET-1 增加了培养的星形胶质细胞中溴脱氧尿苷（BrdU）的掺入，并且在 PD98059、SP600125 和米托蒽醌存在的情况下，BrdU 阳性细胞的数量减少。这些结果表明，ET-1 通过激活 Sp1 增加 cyclin D1 的表达，并诱导星形胶质细胞增殖。
