Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: Co-operation with Src tyrosine kinase
Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: Co-operation with Src tyrosine kinase
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DOI：
10.1016/j.cardiores.2006.05.026
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发表时间：
2006-09-01
影响因子：
10.8
通讯作者：
Heads, Richard J.
中科院分区：
文献类型：
--
作者：
Obasanjo-Blackshire, Kofo;Mesquita, Rul;Heads, Richard J.
目的：探讨钙调神经磷酸酶(calcineurin，CaN)和Src酪氨酸激酶在心肌细胞诱导型一氧化氮合酶(INOS)表达调控和保护中的作用。方法：采用体外培养的新生大鼠心肌细胞，观察细菌脂多糖(LPS)刺激、钙调神经磷酸酶(Calcineurin)和Src基因转染后心肌细胞中NOS表达的变化，以及野生型和钙调素Aβ基因敲除小鼠心肌细胞中NOS的表达。通过高表达活性钙调神经磷酸酶，研究模拟缺血再灌注对细胞的损伤作用。应用启动子-荧光素酶报告基因和染色质免疫共沉淀法研究钙调神经磷酸酶对一氧化氮合酶基因启动子的调节作用。结果：钙调神经磷酸酶或酪氨酸酶的药理作用可抑制钙调神经磷酸酶对一氧化氮合酶基因启动子的调节作用。脂多糖还诱导酪氨酸激酶依赖但不依赖于钙调神经磷酸酶的Src Tyr(418)的磷酸化。脂多糖诱导野生型但不能诱导钙调神经磷酸酶A基因敲除小鼠心肌一氧化氮合酶的表达。在分离的心肌细胞中过表达活性钙蛋白可引起活化T细胞核因子CL亚型(NFATc1)的沉积和核积聚，诱导NOS表达增强，并诱导对心肌肥大前模拟缺血再灌注损伤的NOS依赖的保护。将小鼠一氧化氮合酶启动子-荧光素酶报告基因与活性的钙调神经磷酸酶和野生型或显性负性的Src基因共同转染，证实了钙调神经磷酸酶的结构性激活足以进行反式激活。染色质免疫沉淀证实了NFATc1依赖于钙调神经磷酸酶与一氧化氮合酶启动子内的共同位点的体内结合。结论：这些结果支持了钙调神经磷酸酶通过依赖于NFAT诱导一氧化氮合酶表达以及钙调神经磷酸酶与Src之间的协同作用而发挥的心脏保护作用。(C)2006年欧洲心脏病学会。爱思唯尔出版，版权所有。
Objective: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (NOS) expression and protection in cardiomyocytes.Methods: NOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin A beta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the NOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays.Results: Overexpression of constitutively active Src co-operated with [Ca2+](c) elevation to induce NOS expression, and LPS-induced NOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr(418). LPS induced myocardial NOS expression in wild-type but not calcineurin A knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused deposphorylation and nuclear accumulation of the cl isoform of nuclear factor of activated T-cells (NFATc1), induced strong NOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse NOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the NOS promoter.Conclusions: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of NOS expression and co-operativity between calcineurin and Src. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.