Yang Ping, McKay Brian S, Allen Janice B, Roberts Wendy L, Jaffe Glenn J
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1339-47. doi: 10.1167/iovs.02-0878.
The nuclear transcription factor (NF)-kappaB is a central regulator of multiple inflammatory cytokines. The current study was conducted to determine whether infection of human retinal pigment epithelial (RPE) cells by adenovirus carrying a mutant inhibitory (I)-kappaB (IkappaB) transgene inhibits cytokine-induced activity of NF-kappaB and expression of NF-kappaB-dependent cytokines by preventing degradation of IkappaB. The persistence of recombinant protein expression and function after the viral infection was also examined.
Cultured human RPE cells were infected with adenovirus encoding either beta-galactosidase (LacZ) or mutant IkappaB and were treated with interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha. IkappaB protein expression was determined by Western blot. NF-kappaB nuclear translocation was evaluated by immunofluorescence, and functional NF-kappaB activation was determined by luciferase reporter assay. NF-kappaB-dependent cytokine gene expression was determined by reverse transcription-polymerase chain reaction. IL-1beta-induced monocyte chemoattractant protein (MCP)-1 protein secretion was measured by enzyme-linked immunosorbent assay.
Stimulation of RPE cells with IL-1beta or TNF-alpha caused rapid degradation of the endogenous, but not mutant, IkappaB protein. Expression of the mutant IkappaB isoform inhibited cytokine-stimulated NF-kappaB nuclear translocation, NF-kappaB transcriptional activity, NF-kappaB-dependent gene expression, and secretion of MCP-1. Significant levels of mutant IkappaB protein were expressed for at least 7 weeks after infection.
Infection of human RPE by an adenoviral vector carrying a mutant IkappaB transgene blocks NF-kappaB activation and expression of multiple NF-kappaB-dependent cytokine genes over an extended period. This technique will be useful to determine the role of NF-kappaB in experimental proliferative vitreoretinopathy (PVR), and may offer a novel approach to treatment of PVR with a gene therapy approach.
核转录因子（NF）-κB是多种炎性细胞因子的核心调节因子。本研究旨在确定携带突变型抑制性（I）-κB（IkappaB）转基因的腺病毒感染人视网膜色素上皮（RPE）细胞是否通过阻止IkappaB的降解来抑制细胞因子诱导的NF-κB活性以及NF-κB依赖性细胞因子的表达。同时还检测了病毒感染后重组蛋白表达和功能的持续性。
用编码β-半乳糖苷酶（LacZ）或突变型IkappaB的腺病毒感染培养的人RPE细胞，并用白细胞介素（IL）-1β或肿瘤坏死因子（TNF）-α处理。通过蛋白质印迹法测定IkappaB蛋白表达。通过免疫荧光评估NF-κB核转位，通过荧光素酶报告基因测定法确定功能性NF-κB激活。通过逆转录-聚合酶链反应测定NF-κB依赖性细胞因子基因表达。通过酶联免疫吸附测定法测量IL-1β诱导的单核细胞趋化蛋白（MCP）-1蛋白分泌。
用IL-1β或TNF-α刺激RPE细胞导致内源性而非突变型IkappaB蛋白迅速降解。突变型IkappaB异构体的表达抑制了细胞因子刺激的NF-κB核转位、NF-κB转录活性、NF-κB依赖性基因表达以及MCP-1的分泌。感染后至少7周仍表达显著水平的突变型IkappaB蛋白。
携带突变型IkappaB转基因的腺病毒载体感染人RPE可在较长时间内阻断NF-κB激活和多种NF-κB依赖性细胞因子基因的表达。该技术将有助于确定NF-κB在实验性增殖性玻璃体视网膜病变（PVR）中的作用，并可能为基因治疗PVR提供一种新方法。
