Coordinate activation of HIF-1 and NF-κB DNA binding and COX-2 and VFGF expression in retinal cells by hypoxia
Coordinate activation of HIF-1 and NF-κB DNA binding and COX-2 and VFGF expression in retinal cells by hypoxia
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DOI：
10.1167/iovs.02-0655
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发表时间：
2003-10-01
影响因子：
4.4
通讯作者：
Bazan, NG
中科院分区：
文献类型：
--
作者：
Lukiw, WJ;Ottlecz, A;Bazan, NG
目的.促炎信号传导机制与缺血性视网膜病变期间视网膜新生血管（NV）的诱导有关。本研究检测了缺氧触发的猴脉络膜视网膜（RF/6A）内皮细胞中转录因子（FF）AP- 1、HIF-1和NF-κ B DNA结合与环氧合酶（考克斯）-2和VEGF RNA和蛋白水平的关系。测试了羧酰胺CGP 43182对考克斯-2和VEGF活化以及前列腺素（PG）E-2释放的影响。RF/6A细胞经受缺氧1小时和3小时，此时分离RNA和蛋白质。使用DNA序列搜索算法鉴定潜在的AP-1、缺氧诱导因子（HIF）-1和NF-κ B DNA结合位点，并使用凝胶迁移试验进行分析。RT-PCR、Western分析和酶免疫分析分别定量检测考克斯-2和VEGF的RNA、蛋白和PGE（2）水平。用相差成像显微镜分析肾小管形态发生。缺氧1小时后，核AP-1、HIF-1和NF-κ B启动子DNA结合分别增加1.5倍、4倍和3倍。考克斯-2 RNA分别在缺氧1小时和3小时后升高5倍和4倍。VEGF RNA和蛋白质丰度滞后于考克斯-2诱导，但在缺氧后3小时各自增加2 - 3倍。CGP 43182可抑制NF-κ B DNA结合、考克斯-2和VEGF基因表达、PGE 2释放和缺氧诱导的肾小管形态发生。在考克斯-2表达之前，HIF-1和NF-κ B DNA结合达到最大值，表明这些TF是低氧RF/6A细胞中考克斯-2诱导的重要调节因子。IL-1 β模拟AP-1、HIF-1和NF-kappaB在缺氧时的DNA结合，可能是NV的一种新的细胞因子触发因子。CGP 43182似乎是NV的有效抑制剂。VEGF的表达似乎是通过HIF-1直接和间接通过NF-κ B介导的考克斯-2表达和PGE（2）产生的双重相互依赖机制调节的。
PURPOSE. Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (FF) AP- 1, HIF-1, and NF-kappaB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E-2 release.METHODS. RF/6A cells were subjected to hypoxia for I and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy.RESULTS. Nuclear AP-1, HIF-1 and NF-kappaB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after I and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-kappaB DNA binding, COX-2 and VEGF gene expression, PGE2 release, and hypoxia-induced tubular morphogenesis.CONCLUSIONS. Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-kappaB-mediated COX-2 expression and PGE(2) production.