Department of Pathology and Lab Medicine, University of Cincinnati, Cincinnati, OH 45220, USA.
Cardiovasc Res. 2010 Nov 1;88(2):277-86. doi: 10.1093/cvr/cvq293. Epub 2010 Sep 10.
We previously reported that preconditioning of stem cells with insulin-like growth factor-1 (IGF-1) translocated connexin-43 (Cx-43) into mitochondria, causing cytoprotection. We posit that these preconditioning effects could be simulated by mitochondria-specific overexpression of Cx-43.
During IGF-1-induced preconditioning of C57black/6 mouse bone marrow stem cell antigen-1(+) (Sca-1(+)) cells, Cx-43 was mainly translocated onto the mitochondrial inner membrane, which was abrogated by an extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker PD98059. To investigate the role of mitochondrial Cx-43, we successfully designed a vector coding for full-length mouse Cx-43 with a mitochondria-targeting sequence (mito-Cx-43) and cloned into a shuttle vector (pShuttle-IRES-hrGFP-1) for mitochondria-specific overexpression of Cx-43 (mito-Cx-43). Sca-1(+) cells with mito-Cx-43 reduced cytosolic accumulation of cytochrome c, lowered caspase-3 activity, and improved survival during index oxygen-glucose deprivation as determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling and lactate dehydrogenase assays. Computational analysis revealed a B-cell lymphoma-2 (Bcl-2) homology domain-3 (BH3) motif in Cx-43 with a conserved pattern of amino acids consistent with the Bcl-2 family that regulated cytochrome c release. Moreover, computational secondary structure prediction indicated an extended α-helix in this region, a known condition for BH3-driven protein-protein interactions.
Cx-43 translocation into mitochondria during preconditioning was ERK1/2-dependent. Expression of mito-Cx-43 simulated the cytoprotective effects of preconditioning in stem cells. Structural features of Cx-43 were shared with the Bcl-2 family as determined by computational analysis.
我们之前报道过，胰岛素样生长因子-1（IGF-1）预处理可将连接蛋白-43（Cx-43）转位到线粒体，从而产生细胞保护作用。我们假设这种预处理效应可以通过线粒体特异性过表达 Cx-43 来模拟。
在 IGF-1 诱导 C57black/6 小鼠骨髓干细胞抗原-1（+）（Sca-1（+））细胞预处理过程中，Cx-43 主要转位到线粒体的内膜上，而这一过程可被细胞外信号调节激酶 1 和 2（ERK1/2）抑制剂 PD98059 阻断。为了研究线粒体 Cx-43 的作用，我们成功设计了一个带有线粒体靶向序列（mito-Cx-43）的全长小鼠 Cx-43 编码载体，并克隆到穿梭载体（pShuttle-IRES-hrGFP-1）中，用于线粒体特异性过表达 Cx-43（mito-Cx-43）。mito-Cx-43 的 Sca-1（+）细胞可减少细胞色素 c 的细胞质积累，降低半胱天冬酶-3 的活性，并通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记和乳酸脱氢酶测定法改善指数氧葡萄糖剥夺期间的细胞存活率。计算分析显示 Cx-43 中存在 B 细胞淋巴瘤-2（Bcl-2）同源结构域-3（BH3）结构域，其氨基酸序列具有与调节细胞色素 c 释放的 Bcl-2 家族一致的保守模式。此外，计算二级结构预测表明该区域存在延伸的α-螺旋，这是 BH3 驱动蛋白-蛋白相互作用的已知条件。
预处理过程中 Cx-43 向线粒体的转位依赖于 ERK1/2。mito-Cx-43 的表达模拟了预处理对干细胞的细胞保护作用。通过计算分析，Cx-43 的结构特征与 Bcl-2 家族具有相似性。
