Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil.
PLoS One. 2012;7(4):e34483. doi: 10.1371/journal.pone.0034483. Epub 2012 Apr 4.
Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.
大多数关于 c-Jun N 末端激酶 (JNK) 在视网膜组织中激活的研究都是在神经退行性变的背景下进行的。在这项研究中，我们研究了 JNK 在新生大鼠视网膜祖细胞有丝分裂过程中的行为。将新生大鼠的视网膜外植体在体外培养 3 小时，并进行不同的处理。通过免疫组织化学检测视网膜外植体切片或新鲜固定的视网膜组织中 JNK 的磷酸化，并通过荧光和共聚焦显微镜进行检查。有丝分裂细胞通过染色质形态、组蛋白 H3 磷酸化和在视网膜组织中的位置来鉴定。通过用 DNA 标记物和针对每种蛋白质的抗体双重染色来分析蛋白质的亚细胞定位。还通过 Western blot 检查 JNK 的磷酸化。结果表明，在新生大鼠（P1）的视网膜中，JNK 在祖细胞的有丝分裂过程中被磷酸化，主要在有丝分裂的早期阶段。JNK1 和/或 JNK2 在有丝分裂细胞中优先被磷酸化。JNK 的抑制诱导细胞周期停滞，特别是在有丝分裂中。用 JNK 抑制剂处理会减少后期的细胞数量，但不会改变前期/前中期或中期的细胞数量。此外，在用 JNK 抑制剂处理后发现了染色质形态异常的细胞。这些数据首次表明 JNK 在发育中视网膜组织的有丝分裂祖细胞中被激活，提示 JNK 在控制视网膜祖细胞增殖方面具有新的作用。
