Extracellular matrix gene alternative splicing by trabecular meshwork cells in response to mechanical stretching
Extracellular matrix gene alternative splicing by trabecular meshwork cells in response to mechanical stretching
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DOI：
10.1167/iovs.06-0875
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Acott, Ted S.
中科院分区：
文献类型：
--
作者：
Keller, Kate E.;Kelley, Mary J.;Acott, Ted S.
目的.眼内压（IOP）升高，被小梁网（TM）细胞感知为机械拉伸，触发细胞外基质（ECM）重塑。除了基因表达的变化外，mRNA的选择性剪接可能会改变ECM蛋白的亚型。研究机械牵张对TM细胞4种ECM分子mRNA表达和选择性剪接的影响。将猪TM细胞机械拉伸12、24或48小时。分离RNA，并使用侧翼为可变剪接结构域的引物进行RT-PCR。PCR产物经DNA测序鉴定。使用位于基因的非剪接和剪接区域内的引物进行定量RT-PCR（qRT-PCR）。腱生蛋白C、胶原蛋白XII型和CD 44 mRNA的总水平增加，而多功能蛋白聚糖mRNA水平响应于机械拉伸而降低：此外，这些基因中的每一个表达交替的mRNA同种型。包含腱生蛋白C的纤连蛋白III型结构域D、胶原蛋白XII型的长NC 3同种型、多功能蛋白聚糖的VI同种型以及CD 44的外显子v7和v8的转录物均响应于机械拉伸而增加。观察到胶原蛋白XII型的一种新的同种型，其导致两个外显子的缺失、移码和提前终止密码子。该亚型仅在拉伸的TM细胞中表达。这些选择性剪接事件导致潜在的GAG附着位点和其他ECM结合基序的调节。这些变化应影响ECM重塑期间TM细胞-ECM和/或蛋白质-蛋白质相互作用，ECM重塑与IOP恢复正常的稳态同时发生。
PURPOSE. Elevated intraocular pressure (IOP), sensed as mechanical stretching by trabecular meshwork (TM) cells, triggers extracellular matrix (ECM) remodeling., In addition to changes in gene expression, alternative mRNA splicing may alter ECM protein isoforms. Changes in mRNA expression and alternative splicing of four ECM molecules in response to mechanical stretching of TM cells were investigated.METHODS. Porcine TM cells were mechanically stretched for 12, 24, or 48 hours. RNA was isolated, and RT-PCR was performed with primers that flanked alternatively spliced domains. PCR products were identified by DNA sequencing. Quantitative RT-PCR (qRT-PCR) was performed with primers positioned within nonspliced and spliced regions of the genes.RESULTS. Total levels of tenascin C, Collagen type XII, and CD44 mRNA were increased, whereas versican mRNA levels were decreased in response to the mechanical stretch : In addition, each of these genes expressed alternate mRNA isoforms. Transcripts containing the fibronectin type III domain D of tenascin C, the long NC3 isoform of collagen type XII, the VI isoform of versican, and exons v7 and v8 of CD44 all increased in response to mechanical stretching. A novel isoform of Collagen type XII was observed that resulted in deletion of two exons, a frameshift, and a premature stop codon. This isoform was expressed only by stretched TM cells.CONCLUSIONS. These alternative splicing events led to the modulation of potential GAG attachment sites and other ECM-binding motifs. These changes should affect TM cell-ECM and/or protein-protein interactions during the ECM remodeling that occurs coincident with homeostatic restoration of IOP to normal.