A novel activation mechanism of cellular Factor XIII in zebrafish retina after optic nerve injury
A novel activation mechanism of cellular Factor XIII in zebrafish retina after optic nerve injury
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视神经损伤后斑马鱼视网膜细胞因子 XIII 的新激活机制
DOI：
10.1016/j.bbrc.2019.07.003
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发表时间：
2019
影响因子：
3.1
通讯作者：
Koriyama Yoshiki
中科院分区：
文献类型：
--
作者：
Sugitani Kayo;Ogai Kazuhiro;Muto Haruka;Onodera Keisuke;Matsuoka Ayaka;Sugita Takahira;Koriyama Yoshiki
视神经损伤 (ONI) 后，鱼视网膜中的细胞因子 XIII (cFXIII) mRNA 迅速上调。在这里，我们利用 cFXIII A 亚基 (cFXIII-A) 的遗传信息研究了 cFXIII 基因激活的分子机制。扩增 cFXIII-A 活性位点（外显子 7-8）的实时 PCR 显示 ONI 后视网膜中 cFXIII-A mRNA 增加，而扩增激活肽（外显子 1-2）的 PCR 显示没有变化。扩增外显子 1-8 的 RT-PCR 分析显示两条带，对照视网膜中的微弱长带和受伤视网膜中的致密短带。因此，我们得出结论，ONI 后激活的 cFXIII-A mRNA 比对照视网膜的激活的 cFXIII-A mRNA 短。蛋白质印迹分析还证实，与对照 84kDa 蛋白相比，受损视网膜中存在活性形式的 65kDa cFXIII-A 蛋白。使用受损视网膜进行的 5'-RACE 分析显示，短的 cFXIII-A mRNA 缺少外显子 1、2 和部分外显子 3。外显子 3 有两个热休克因子 1 (HSF-1) 结合共有序列位点。 ONI 后 1 天，眼内注射 HSF 抑制剂可将视网膜中 cFXIII-A mRNA 的表达抑制至 ONI 后正常水平的 40%。染色质免疫沉淀提供了与 HSF-1 结合的 cFXIII-A 基因组 DNA 富集的直接证据。目前的数据表明，在不使用凝血酶的 ONI 后，HSF-1 与 cFXIII-A 基因的快速结合导致斑马鱼视网膜中激活肽和短 cFXIII-A mRNA 和蛋白质的活性形式裂解。
Cellular Factor XIII (cFXIII) mRNA is rapidly upregulated in the fish retina after optic nerve injury (ONI). Here, we investigated the molecular mechanism of cFXIII gene activation using genetic information from the A-subunit of cFXIII (cFXIII-A). Real-time PCR that amplified the active site (exons 7–8) of cFXIII-A showed increased cFXIII-A mRNA in the retina after ONI, whereas the PCR that amplified the activation peptide (exons 1–2) showed no change. RT-PCR analysis that amplified exons 1–8 showed two bands, a faint long band in the control retina and a dense short band in the injured retina. Therefore, we conclude that activated cFXIII-A mRNA after ONI is shorter than that of the control retina. Western blot analysis also confirmed an active form of 65 kDa cFXIII-A protein in the injured retina compared to the control 84 kDa protein. 5′-RACE analysis using injured retina revealed that the short cFXIII-A mRNA lacked exons 1, 2 and part of exon 3. Exon 3 has two sites of heat shock factor 1 (HSF-1) binding consensus sequence. Intraocular injection of HSF inhibitor suppressed the expression of cFXIII-A mRNA in the retina 1 day after ONI to 40% of levels normally seen after ONI. Chromatin immunoprecipitation provides direct evidence of enrichment of cFXIII-A genomic DNA bound with HSF-1. The present data indicate that rapid HSF-1 binding to the cFXIII-A gene results in cleavage of activation peptide and an active form of short cFXIII-A mRNA and protein in the zebrafish retina after ONI without thrombin.