Obasanjo-Blackshire Kofo, Mesquita Rui, Jabr Rita I, Molkentin Jeffery D, Hart Stephen L, Marber Michael S, Xia Yang, Heads Richard J
Cardiovascular Division, King's College London School of Medicine, Department of Cardiology, The Rayne Institute, St Thomas's Hospital, Lambeth Palace Road, London SE1 7EH, UK.
Cardiovasc Res. 2006 Sep 1;71(4):672-83. doi: 10.1016/j.cardiores.2006.05.026. Epub 2006 Jun 3.
To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes.
iNOS 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 Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays.
Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS 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 iNOS promoter.
These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.
确定钙调神经磷酸酶和Src酪氨酸激酶在调节心肌细胞中诱导型一氧化氮合酶（iNOS）表达及保护作用中的作用。
在分离的新生大鼠心室肌细胞培养物中，研究iNOS表达对细菌脂多糖（LPS）的反应，或在转染组成型活性钙调神经磷酸酶或Src后，以及在从野生型或钙调神经磷酸酶Aβ基因敲除小鼠分离的心脏中进行研究。在活性钙调神经磷酸酶过表达后，研究对模拟缺血再灌注的细胞损伤反应。使用启动子-荧光素酶报告基因和染色质免疫沉淀试验研究钙调神经磷酸酶对iNOS基因启动子的调节。
组成型活性Src的过表达与[Ca2+]c升高协同诱导iNOS表达，LPS诱导的iNOS表达可通过钙调神经磷酸酶或酪氨酸激酶的药理学抑制作用而消除。LPS还诱导Src Tyr418的酪氨酸激酶依赖性但钙调神经磷酸酶非依赖性磷酸化。LPS在野生型小鼠而非钙调神经磷酸酶Aβ基因敲除小鼠中诱导心肌iNOS表达。在分离的心肌细胞中组成型活性钙调神经磷酸酶的过表达导致活化T细胞核因子c1亚型（NFATc1）的去磷酸化和核积累，诱导强烈的iNOS表达，并在心肌细胞肥大之前诱导对模拟缺血再灌注的NOS依赖性保护。将小鼠iNOS启动子-荧光素酶报告基因与活性钙调神经磷酸酶和野生型或显性负性Src共转染证实，钙调神经磷酸酶的组成型激活足以进行反式激活。染色质免疫沉淀证实了钙调神经磷酸酶依赖性的体内NFATc1与iNOS启动子内共有位点的结合。
这些结果支持钙调神经磷酸酶通过NFAT依赖性诱导iNOS表达以及钙调神经磷酸酶与Src之间的协同作用发挥心脏保护作用。
