Activation of c-Jun N-Terminal Kinase (JNK) during Mitosis in Retinal Progenitor Cells

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.现有针对视网膜组织中c-Jun氨基末端激酶（c-Jun N-terminal Kinase, JNK）激活的相关研究，大多围绕神经退行性变展开。本研究旨在探讨新生大鼠视网膜祖细胞有丝分裂过程中JNK的活性变化。实验将新生大鼠的视网膜外植体置于体外培养3小时，并施加不同处理条件。通过免疫组织化学法检测视网膜外植体切片或新鲜固定的视网膜组织中的JNK磷酸化水平，并分别采用荧光显微镜与共聚焦显微镜进行成像观察。研究通过染色质形态、组蛋白H3磷酸化水平以及视网膜组织中的定位特征来识别有丝分裂细胞。通过DNA标记物与对应蛋白抗体的双染色法，分析靶蛋白的亚细胞定位情况。此外，采用蛋白质免疫印迹（Western Blot）法检测JNK的磷酸化水平。实验结果显示，在出生后1天（P1）的新生大鼠视网膜中，JNK在祖细胞有丝分裂过程中发生磷酸化，且主要集中于有丝分裂早期阶段。有丝分裂细胞中优先发生磷酸化的亚型为JNK1和/或JNK2。抑制JNK活性可引发细胞周期阻滞，且该阻滞特异性发生于有丝分裂阶段。施加JNK抑制剂处理后，处于后期的细胞数量显著减少，但前/早前期以及中期的细胞数量未发生明显改变。此外，JNK抑制剂处理后可观察到染色质形态异常的细胞。本研究数据首次证实，发育中视网膜组织的有丝分裂祖细胞内存在JNK激活现象，这表明JNK在视网膜祖细胞增殖调控中发挥了此前未被发现的新功能。