Thompson Melissa D, Mei Yu, Weisbrod Robert M, Silver Marcy, Shukla Praphulla C, Bolotina Victoria M, Cohen Richard A, Tong Xiaoyong
From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
the Ion Channel and Calcium Signaling Unit, Boston University School of Medicine, Boston, Massachusetts 02118, and.
J Biol Chem. 2014 Jul 18;289(29):19907-16. doi: 10.1074/jbc.M114.554451. Epub 2014 Jun 11.
The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.
肌浆网/内质网Ca(2+)ATP酶（SERCA）是钙稳态的关键，在体外，其半胱氨酸（C）674硫醇上的可逆谷胱甘肽（GSH）加合物对其进行氧化还原调节，这些加合物可刺激Ca(2+)摄取活性和内皮细胞血管生成反应。我们发现小鼠后肢肌肉缺血会在全肌肉组织和内皮细胞中诱导SERCA上形成S-谷胱甘肽加合物。为了确定S-谷胱甘肽化的作用，我们使用了一种SERCA 2 C674S杂合子敲入（SKI）小鼠，该小鼠缺少一半关键硫醇。后肢缺血后，SKI动物的SERCA S-谷胱甘肽加合物减少，血流恢复受损。我们研究了总SERCA 2表达未改变的SKI微血管内皮细胞。与野生型（WT）相比，培养的SKI微血管内皮细胞显示迁移和网络形成受损。Ca(2+)研究表明，SKI细胞中一氧化氮（·NO）诱导的(45)Ca(2+)摄取进入内质网（ER）减少，而Fura-2研究显示Ca(2+)储存较低，血管内皮生长因子（VEGF）和·NO诱导的Ca(2+)内流减少。内质网Ca(2+)结合蛋白钙网蛋白的腺病毒过表达增加了离子霉素可释放的储存、VEGF诱导的Ca(2+)内流和内皮细胞迁移。综上所述，这些数据表明SERCA 2上对氧化还原敏感的半胱氨酸-674硫醇对于正常内皮细胞Ca(2+)稳态和缺血诱导的血管生成反应是必需的，揭示了一种通过Ca(2+)储存对血管生成的新型氧化还原控制。
