Zhang Wei, Zhang Xuexin, González-Cobos José C, Stolwijk Judith A, Matrougui Khalid, Trebak Mohamed
From the The State University of New York College of Nanoscale Science and Engineering, Albany, New York 12203,; Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, and.
From the The State University of New York College of Nanoscale Science and Engineering, Albany, New York 12203.
J Biol Chem. 2015 Feb 20;290(8):5015-5027. doi: 10.1074/jbc.M114.625822. Epub 2014 Dec 24.
Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca(2+) (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.
白三烯-C4合成酶（LTC4S）通过花生四烯酸代谢生成LTC4。LTC4是一种促炎因子，作用于质膜半胱氨酰白三烯受体。然而，最近我们发现LTC4也是一种胞质第二信使，可激活血管平滑肌细胞（VSMC）中由Orai1/Orai3异源多聚体编码的不依赖于钙库的LTC4调节性Ca(2+)（LRC）通道。我们发现血管损伤后，中膜和新生内膜VSMC中的Orai3和LRC电流上调，且Orai3基因敲低可抑制LRC电流和新生内膜增生。然而，LTC4S在新生内膜形成中的作用仍不清楚。在此我们表明，LTC4S基因敲低可抑制VSMC中的LRC电流。我们进行了体内实验，用球囊血管成形术损伤大鼠左颈动脉以引起新生内膜增生。新生内膜形成与VSMC中LTC4S蛋白表达上调有关。通过编码短发夹RNA的慢病毒颗粒抑制损伤颈动脉中LTC4S的表达可抑制新生内膜形成以及血管的内向和外向重塑。LRC电流激活不会导致VSMC中活化T细胞核因子（NFAT）的核转位。令人惊讶的是，LTC4S或Orai3基因敲低在血清刺激后在Ser-473/Ser-474位点产生更强且持续时间更长的Akt1和Akt2磷酸化。LTC4S和Orai3基因敲低在体外抑制VSMC迁移，但对增殖无影响。损伤后2周，损伤血管的新生内膜和中膜VSMC中的Akt活性受到抑制，但当通过短发夹RNA阻止LTC4S或Orai3上调时，Akt活性得以恢复。我们得出结论，LTC4S和Orai3改变Akt信号传导以促进VSMC迁移和新生内膜形成。
