Libert Sarah, Willermain François, Weber Célia, Bryla Angélic, Salik Dany, Gregoire Françoise, Bolaky Nargis, Caspers Laure, Perret Jason, Delporte Christine
Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium; Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
I.R.I.B.H.M., campus Erasme, Brussels, Belgium; Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
Mol Vis. 2016 Jan 31;22:100-15. eCollection 2016.
Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation.
ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites.
In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38α and p38β) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation.
Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.
黄斑水肿是糖尿病视网膜病变（DR）常见的并发症，由血视网膜屏障（BRB）改变引起，并导致视网膜色素上皮（RPE）功能改变。周围介质的渗透压变化可能是导致RPE功能改变进而破坏视网膜内环境稳态的原因。本研究在源自人RPE的ARPE - 19细胞中，研究了关键的高渗反应因子渗透压增强结合蛋白（TonEBP，也称为活化T细胞核因子5 - NFTA5）的表达、激活及功能，以响应高渗刺激。
将ARPE - 19细胞暴露于高渗培养基中。通过qRT - PCR和半定量蛋白质印迹法对TonEBP mRNA和蛋白水平进行定量。通过免疫荧光研究TonEBP的核转位。使用含有TonEBP结合位点的报告质粒测量TonEBP的反式激活活性。
在对ARPE - 19细胞进行高渗刺激后，观察到TonEBP mRNA和蛋白水平呈剂量依赖性增加，以及TonEBP核转位。使用含有TonEBP结合位点的报告质粒进一步证实了TonEBP的反式激活活性。TonEBP的显性负性形式消除了NaCl诱导的TonEBP反式激活活性增加，并抑制了靶基因醛糖还原酶和钠依赖性牛磺酸转运体mRNA水平的升高。SB203580，一种p38蛋白激酶的两种同工型（p38α和p38β）的抑制剂，抑制了暴露于高渗刺激的ARPE - 19细胞中TonEBP的核转位和反式激活活性。
我们的数据表明TonEBP参与了RPE细胞对高渗应激的渗透适应机制。鉴于TonEBP在不同病理途径中的新作用，这些数据为分析黄斑水肿期间RPE功能改变所涉及的机制开辟了新的视角。
