An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo
An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo
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DOI：
10.1167/iovs.04-1285
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发表时间：
2005-04-01
影响因子：
4.4
通讯作者：
Eckert, RL
中科院分区：
文献类型：
--
作者：
Adhikary, G;Crish, JF;Eckert, RL
目的.角膜缘干细胞的细胞分裂产生瞬时扩增细胞，其最终分化形成多层角膜上皮。在这个过程中调节基因表达变化的机制还不清楚。在本研究中，involucrin基因被用作一个模型来研究这种调控。使用体内转基因小鼠模型和培养的原代人角膜上皮细胞评估人外皮蛋白基因表达和启动子活性的调节。人外皮蛋白（hINV）是一种结构蛋白，在分化的角膜上皮细胞中选择性表达。结果表明，激活蛋白1（AP 1）的DNA结合位点是必要的适当的基础和刺激依赖的hINV启动子活性。该位点AP 1 -5的突变导致hINV基因在体内角膜上皮和培养的角膜上皮细胞中表达的丧失。凝胶迁移率超位移分析显示的AP 1因子，Fra-1和JunB，与这个元素的相互作用。用显性阴性形式的AP 1抑制AP 1功能也抑制表达。用12-O-十四烷酰佛波醇-13-乙酸酯（TPA）（一种蛋白激酶C激活剂）治疗，增加hINV基因表达，这一反应与增加的AP 1因子（Fra-1和JunB）结合到hINV基因AP 1 -5反应元件相关。这些研究结果指出，AP 1转录因子，通过远端调控区AP 1 -5元件，在角膜上皮细胞分化过程中的外皮蛋白基因表达的调节中发挥重要作用。
PURPOSE. Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation.METHODS. Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells.RESULTS. Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element.CONCLUSIONS. These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.