Mascarenhas Joseph B, Tchourbanov Alex Y, Fan Hanli, Danilov Sergei M, Wang Ting, Garcia Joe G N
1 Department of Medicine, and.
2 Arizona Research Laboratory, University of Arizona, Tucson, Arizona; and.
Am J Respir Cell Mol Biol. 2017 Jan;56(1):29-37. doi: 10.1165/rcmb.2016-0053OC.
The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y and Y) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.
由MYLK基因编码的非肌肉（nm）肌球蛋白轻链激酶同工型（MLCK）是调节血管屏障对机械和炎症刺激反应的重要参与者。我们确定MYLK存在可变剪接，产生功能不同的nmMLCK剪接变体，包括nmMLCK2，这是一种在血管内皮细胞（EC）中高度表达的剪接变体，与EC屏障完整性降低有关。我们之前证明nmMLCK2变体缺少外显子11，该外显子编码一个关键调节区域，包含两个磷酸化程度不同的酪氨酸残基（Y和Y），它们在炎症过程中影响血管屏障功能。在本研究中，我们使用小基因构建体和逆转录-聚合酶链反应（RT-PCR）来探究可能参与调节MYLK可变剪接和nmMLCK2产生的生物物理因素（机械应力）和基因变体（MYLK单核苷酸多态性[SNPs]）。暴露于病理性机械应力（18%周期性拉伸）的人肺EC相对于nmMLCK1水平产生了增加的nmMLCK2表达，可变剪接受MYLK SNPs rs77323602和rs147245669的显著影响。计算机分析预测这些变体将改变外显子11的可变剪接供体和受体位点，小基因研究证实了这些计算机预测。rs77323602的引入有利于野生型nmMLCK表达，而rs147245669有利于外显子11的可变剪接和缺失，导致nmMLCK2表达增加。最后，选择性携带这些MYLK SNPs（rs77323602和rs147245669）的淋巴母细胞系直接验证了SNP对MYLK可变剪接和nmMLCK2产生的特异性影响。总之，这些研究表明机械应力和MYLK SNPs调节MYLK可变剪接以及一种剪接变体nmMLCK2的产生，该变体导致炎症损伤的严重程度增加。
