Dvoriantchikova Galina, Ivanov Dmitry
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, 33136, USA.
Eur J Neurosci. 2014 Oct;40(8):3171-8. doi: 10.1111/ejn.12710. Epub 2014 Aug 27.
Tumor necrosis factor-alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF-κB, nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF-regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF-treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF-κB subunits in RGCs and astrocytes were evaluated by quantitative RT-PCR (qRT-PCR) and Western blot (WB) analysis. NF-κB and JNK activity in TNF-treated cells was determined in a time-dependent manner using ELISA and WB. Gene expression in TNF-treated astrocytes was measured by qRT-PCR. We found that NF-κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF-κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF-κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro-inflammatory factors. Our findings suggest that, in the presence of TNF, NF-κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.
肿瘤坏死因子-α（TNF）是视网膜先天免疫反应的重要介质。TNF可激活多种信号级联反应，包括核因子κB（NF-κB）和c-Jun氨基末端激酶（JNK）信号通路。这些信号通路以及TNF的有害作用先前已在包括青光眼和视网膜缺血在内的几种视网膜神经退行性疾病中得到证实。然而，TNF和TNF调节的信号级联反应不仅能够介导神经毒性，还具有保护作用。我们进行这项研究以阐明TNF信号在视网膜中的有益和有害作用。为此，我们使用了经TNF处理的原代视网膜神经节细胞（RGC）和星形胶质细胞培养物。通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹（WB）分析评估RGC和星形胶质细胞中NF-κB亚基的表达水平。使用酶联免疫吸附测定（ELISA）和WB以时间依赖性方式测定TNF处理细胞中的NF-κB和JNK活性。通过qRT-PCR测量TNF处理的星形胶质细胞中的基因表达。我们发现NF-κB家族成员在RGC和星形胶质细胞中以mRNA和蛋白质水平存在。在TNF存在下，RGC无法激活NF-κB，这一现象与JNK持续激活和RGC死亡有关。然而，TNF在星形胶质细胞中启动了NF-κB的激活并介导了JNK的短暂激活。这些事件与神经胶质细胞存活和神经毒性促炎因子表达增加有关。我们的研究结果表明，在TNF存在下，NF-κB和JNK信号级联反应在RGC和星形胶质细胞中以相反的方式被激活。这些事件可直接或间接促进RGC死亡。
