Fujibayashi Tatsuya, Kurauchi Yuki, Hisatsune Akinori, Seki Takahiro, Shudo Koichi, Katsuki Hiroshi
Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Priority Organization for Innovation and Excellence, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan; Program for Leading Graduate Schools "HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program", Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
J Pharmacol Sci. 2015 Oct;129(2):119-26. doi: 10.1016/j.jphs.2015.09.001. Epub 2015 Sep 11.
We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.
我们之前已经表明，在大鼠中脑切片培养物中，激动剂Am80对维甲酸受体（RAR）的刺激通过增加神经元型一氧化氮合酶（nNOS）的表达来募集一氧化氮依赖性信号传导。在这里，我们使用神经母细胞瘤SH-SY5Y细胞研究了RAR诱导nNOS表达的机制，以及nNOS表达与神经突生长之间的关系。Am80促进了神经突生长，而磷酸肌醇3-激酶（PI3K；LY294002）、c-Jun氨基末端激酶（JNK；SP600125）和p38丝裂原活化蛋白激酶（p38 MAPK；SB203580）的抑制剂减弱了这种促进作用。一种选择性nNOS抑制剂3-溴硝基吲唑也抑制了Am80诱导的神经突生长。LY294002、SP600125和SB203580减弱了Am80诱导的nNOS蛋白表达增加，而nNOS mRNA表达增加仅被LY294002减弱。LY294002阻断了Am80诱导的JNK和p38 MAPK激活，表明这些激酶在PI3K下游起作用。我们还证实，据报道可调节nNOS表达的核受体DAX1在对Am80的反应中上调。小干扰RNA介导的DAX1敲低消除了Am80诱导的nNOS表达和神经突生长。这些结果首次揭示，nNOS表达对于RAR介导的神经突生长至关重要，并且JNK和p38 MAPK等非基因组信号传导参与了RAR介导的nNOS表达。
