Effect of retinoic acid on gene expression in human conjunctival epithelium:: Secretory phospholipase A2 mediates retinoic acid induction of MUC16
Effect of retinoic acid on gene expression in human conjunctival epithelium:: Secretory phospholipase A2 mediates retinoic acid induction of MUC16
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DOI：
10.1167/iovs.05-0627
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发表时间：
2005-11-01
影响因子：
4.4
通讯作者：
Gipson, IK
中科院分区：
文献类型：
--
作者：
Hori, Y;Spurr-Michaud, SJ;Gipson, IK
目的.维生素A如何有助于维持眼表的湿表面表型尚不清楚。本研究试图通过基因微阵列分析全反式维甲酸（RA）培养的人结膜上皮（HCjE）细胞系来鉴定眼表上皮中的维生素A反应基因。分析显示分泌型磷脂酶A（2）IIA组（sPLA（2）-IIA）是RA上调最多的基因，随后是在较晚时间点的膜相关粘蛋白MUC 16。由于类花生酸（PLA（2）家族产生的花生四烯酸的产物）已被证明能增加粘蛋白的产生，本研究试图确定sPLA（2）是否介导RA诱导MUC 16。HCjE细胞在有或没有RA的情况下培养3、6、24和48小时。将从HCjE细胞的RNA制备的互补RNA与人基因芯片杂交，并使用商业软件进行分析。通过实时PCR验证粘蛋白表达的微阵列数据。为了研究sPLA（2）是否与RA诱导的MUC 16上调相关，将HCjE细胞与RA和广谱PLA（2）抑制剂马兜铃酸（ArA）或特异性sPLA（2）-IIA抑制剂LY 315920孵育，然后通过实时PCR和Western印迹分析MUC 16 mRNA和蛋白。结果RA加入后3 h和6 h（早期）有28个转录本上调，6个转录本下调2倍以上（P < 0.01）。在24和48小时（晚期），80个基因转录物上调，45个下调。第IIA组sPLA（2）在24小时时显著上调，MUC 16在48小时时是RA上调最多的RNA。Western印迹分析证实RA上调sPLA（2）。当RA与ArA或sPLA（2）-IIA特异性抑制剂LY 315920共同孵育时，RA诱导的HCjE细胞MUC 16 mRNA表达明显降低（P < 0.01）。RA相关的膜相关粘蛋白MUC 16在晚期的上调似乎是通过sPLA（2）-IIA。这种亲水性膜相关粘蛋白的上调可能是维生素A促进维持眼表湿表面表型的重要机制之一。
PURPOSE. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A - responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid ( RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16.METHODS. HCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. RESULTS. After RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold ( P < 0.01) at both 3 and 6 hours ( early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours ( late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced ( P < 0.01).CONCLUSIONS. The RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface.