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首页> 外文期刊>American Journal of Physiology >Cofilin mediates ATP depletion-induced endothelial cell actin alterations.
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Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

机译：Cofilin介导ATP耗竭诱导的内皮细胞肌动蛋白改变。

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Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.
机译：缺血和败血症导致内皮细胞损伤，导致许多器官的微血管流量受损。关于导致内皮细胞功能障碍的细胞内结构事件，还有很多待确定。为了研究潜在的肌动蛋白细胞骨架相关机制，在小鼠胰腺微血管内皮细胞（MS1）中诱导ATP耗竭。荧光成像和生化研究表明，F-肌动蛋白在60分钟时迅速且逐渐增加，而G-肌动蛋白则下降。共聚焦显微镜分析表明，ATP耗竭会导致肌动蛋白应力纤维的破坏和F-肌动蛋白聚集体的积累。我们假设这些肌动蛋白改变是继之于肌动蛋白解聚因子（ADF）/ cofilin蛋白的去磷酸化/激活。 Cofilin是MS1细胞中表达的主要同种型，在ATP消耗过程中被迅速去磷酸化/激活。为了直接研究缺血期间cofilin激活对肌动蛋白细胞骨架的作用，将MS1细胞感染了含有野生型非洲爪蟾ADF / cofilin绿色荧光蛋白[XAC（wt）-GFP]，GFP和其组成型的cDNA的腺病毒活性和非活性同工型XAC（S3A）-GFP和XAC（S3E）-GFP。在表达ATP的XAC（wt）-GFP-和XAC（S3A）-GFP表达的细胞中，皮质肌动蛋白破坏和肌动蛋白聚集体形成的速率和程度增加，而在XAC（S3E）-GFP中观察到肌动蛋白应激纤维增加。表达细胞。为了研究ADF / cofilin的上游信号通路，在MS1细胞中表达了LIM激酶1-GFP（LIMK1-GFP）。表达LIMK1-GFP蛋白的细胞在ATP耗竭期间具有较高水平的磷酸化ADF / cofilin，增加的应力纤维和延迟的F-肌动蛋白细胞骨架破坏。这些结果强烈支持cofilin调节在缺血诱导的内皮细胞肌动蛋白细胞骨架改变，导致细胞损伤和微血管功能障碍中的重要性。

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