Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression
Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression
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DOI：
10.1016/j.exer.2008.06.006
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发表时间：
2008-09-01
影响因子：
3.4
通讯作者：
Tamm, Ernst R.
中科院分区：
文献类型：
--
作者：
Paper, Walter;Kroeber, Markus;Tamm, Ernst R.
肌球蛋白是一种55-57 kDa的分泌性糖蛋白，是嗅觉调节蛋白家族的成员，在某些形式的原发性开角型青光眼中发生突变。为了评估体内系统中升高量的肌球蛋白对房水流出动力学的影响，已经开发了在它们的眼睛中强烈过表达肌球蛋白的转基因β B1-stac-MYOC小鼠。转基因过表达myocilin导致β B1-stac-MYOC小鼠房水中分泌的正常myocilin增加近5倍。在本研究中，我们希望使用β B1-stac-MYOC作为一种工具，以确定眼组织对高于正常量的myocilin的反应，并确定基因表达的变化，这可能有助于揭示myocilin的体内功能特性。从β B1-stac-MYOC小鼠和野生型同窝仔的眼组织中分离RNA。基因表达的变化通过基因微阵列杂交确定，并通过真实的时间RT-PCR和蛋白质印迹证实。已发现在β B1-stac-MYOC小鼠中差异调节的基因的表达在用重组肌球蛋白处理的培养的人小梁网（HTM）细胞中进一步分析。虽然β B1-stac-MYOC小鼠没有明显的表型，但与野生型同窝仔中的基因表达相比，发现了几个不同基因的统计学显著上调和下调。在被发现差异调节的基因中有Was 1、Ceacam 1和Spon 2，它们参与细胞粘附和细胞-基质相互作用。表达差异也被发现为六1编码的转录因子，和Pftk 1的基因产物是一个cdc 2相关的蛋白激酶。这些基因的表达也被发现在体外调节与重组myocilin处理的HTM细胞。在β B1-stac-MYOC小鼠的眼组织中，发现连接蛋白46和α B-晶状体蛋白的含量显著较高。此外，编码嗅觉调节蛋白的几个基因表现出明显的表达变化。Olfml 3显著下调，而Lphn 1、Lphn 2和Lphn 3显著上调。我们的研究结果支持myocilin在调节细胞粘附中的作用，并建议涉及嗅觉介导蛋白家族其他蛋白质的功能过程，（C）2008 Elsevier Ltd.保留所有权利。
Myocilin is a 55-57 kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic beta B1-crystallin-MYOC mice have been developed that strongly overexpress myocilin in their eyes. The transgenic overexpression of myocilin results in an almost five-fold increase of secreted normal myocilin in the aqueous humor of beta B1-crystallin-MYOC mice. In the present study, we wanted to use beta B1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of myocilin. RNA was isolated from ocular tissues of beta B1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in beta B1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant myocilin. Although beta B1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six 1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant myocilin. Substantially higher amounts in ocular tissues of beta B1-crystallin-MYOC mice were found for connexin 46 and alpha B-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family, (C) 2008 Elsevier Ltd. All rights reserved.