Characterization of Site-Specific Phosphorylation of NF-κB p65 in Retinal Cells in Response to High Glucose and Cytokine Polarization.
Characterization of Site-Specific Phosphorylation of NF-κB p65 in Retinal Cells in Response to High Glucose and Cytokine Polarization.
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DOI：
10.1155/2018/3020675
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发表时间：
2018
影响因子：
4.6
通讯作者：
Berger EA
中科院分区：
文献类型：
--
作者：
Shi H;Berger EA
炎症是糖尿病视网膜病变（DR）发病机制的重要因素。 NF-κB 是许多炎症基因的主要转录调节因子。尽管NF-κB由多个亚基组成，但p65最受关注。然而，p65 亚基可以在许多位点被磷酸化，而 DR 相关条件的影响尚未得到很好的表征。由于 NF-κB 失调与慢性炎症有关，因此当前的研究检查了暴露于高葡萄糖的视网膜细胞中的位点特异性 p65 磷酸化，并研究了细胞因子极化的影响。 在暴露于高葡萄糖 (HG) 和促炎或抗炎细胞因子后，在人原代视网膜内皮细胞 (HREC) 和 MIO-M1 Müller 细胞中检查了 NF-κB p65 位点的磷酸化。通过实时 RT-PCR、ELISA 和/或蛋白质印迹选择性测量相关下游基因激活。 HG 暴露导致 HREC 和 Müller 细胞之间 p65 亚基位点的磷酸化差异。促炎细胞因子进一步增加了这些位点以及 HG 中未改变的其他位点的磷酸化。相反，IL-4 对两种细胞类型中 p65 位点的磷酸化表现出抑制作用，并促进 IκBα 表达。与HG暴露相比，促炎细胞因子治疗的下游炎症介质增加。 IL-4 抑制促炎细胞因子，而尽管暴露于 HG，IL-10 仍得到增强。 目前的研究首次描述了细胞因子极化后 HG 诱导的 NF-κB p65 磷酸化。通过了解高血糖条件下 NF-κB 磷酸化和细胞因子的影响，可以确定 DR 早期治疗的干预点。
Inflammation is an important contributor to the pathogenesis of diabetic retinopathy (DR). NF-κB is a master transcriptional regulator for numerous inflammatory genes. Although NF-κB is comprised of multiple subunits, p65 has received the most attention. However, the p65 subunit can be phosphorylated at numerous sites, for which the effects of DR-related conditions are not well characterized. Since dysregulation of NF-κB has been linked to chronic inflammation, the current study examines site-specific p65 phosphorylation in retinal cells exposed to high glucose and investigates the effects of cytokine polarization. Phosphorylation of NF-κB p65 sites was examined in human primary retinal endothelial cells (HREC) and MIO-M1 Müller cells after exposure to high glucose (HG) and pro- or anti-inflammatory cytokines. Related downstream gene activation was selectively measured by real-time RT-PCR, ELISA, and/or Western blot. HG exposure resulted in differential phosphorylation of p65 subunit sites between HREC and Müller cells. Proinflammatory cytokines further increased phosphorylation of these sites and additional sites that were not altered in HG. In contrast, IL-4 exhibited a suppressive effect on the phosphorylation of p65 sites in both cell types and promoted IκBα expression. Downstream inflammatory mediators were increased in response to proinflammatory cytokine treatment versus HG exposure. IL-4 inhibited proinflammatory cytokines, while IL-10 was enhanced despite HG exposure. The current study is the first to characterize HG-induced NF-κB p65 phosphorylation after cytokine polarization. By understanding NF-κB phosphorylation and cytokine influence during hyperglycemic conditions, intervention points can be identified for early-stage treatment of DR.
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