Wang Ting, Brown Mary E, Kelly Gabriel T, Camp Sara M, Mascarenhas Joseph B, Sun Xiaoguang, Dudek Steven M, Garcia Joe G N
1 Department of Medicine, University of Arizona Health Sciences, Tucson, AZ, USA.
2 Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA.
Pulm Circ. 2018 Apr-Jun;8(2):2045894018764171. doi: 10.1177/2045894018764171. Epub 2018 Feb 26.
Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y and Y phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.
1-磷酸鞘氨醇（S1P）是一种具有强大生物活性的内源性脂质，它通过调节非肌肉肌球蛋白轻链激酶同工型（nmMLCK）来信号传导肌动蛋白细胞骨架的重排。S1P诱导关键的nmMLCK Y和Y磷酸化，导致nmMLCK易位至外周，在那里肌球蛋白轻链（MLC）磷酸化和张力的空间定向增加导致片状伪足突出、细胞间粘附增加以及血管屏障完整性增强。MYLK是编码nmMLCK的基因，是肺部炎症性疾病中一个已知的候选基因，其编码的遗传变异（Pro21His、Ser147Pro、Val261Ala）会增加炎症性肺损伤的风险并影响疾病严重程度。这些MYLK编码单核苷酸多态性（SNP）影响生物学过程以增加疾病风险和严重程度的功能机制仍不清楚。在本研究中，我们利用可量化的细胞免疫荧光测定法来确定MYLK编码SNP对S1P介导的nmMLCK磷酸化和易位至人肺内皮细胞（EC）外周的影响。这些与疾病相关的MYLK变异导致S1P诱导的Y磷酸化水平降低，Y磷酸化是nmMLCK酶调节和激活的关键位点。Y磷酸化水平降低导致nmMLCK蛋白向细胞外周的易位减弱。我们进一步进行了EC运动学测定，证实携带三个MYLK编码SNP的MYLK转基因的表达会抑制对S1P刺激的片状伪足突出。这些数据表明，与急性呼吸窘迫综合征/重度哮喘相关的MYLK SNP通过直接改变nmMLCK酪氨酸磷酸化水平、空间定位和片状伪足突出，在功能上影响血管屏障调节的细胞骨架反应。
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