Advanced Biotechnology Center, Genova, Italy.
Stem Cells Dev. 2011 Jul;20(7):1183-98. doi: 10.1089/scd.2010.0295. Epub 2010 Dec 22.
In several cell types, a regulated efflux of NAD(+) across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD(+) (NAD(+)(e)) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD(+) through Cx43 HC. NAD(+) efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca(2+) (5-6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD(+)(e) activates the purinergic receptor P2Y(11) and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/Ca(2+) signaling cascade, involving the opening, unique to MSC, of L-type Ca(2+) channels. Extracellular NAD(+) enhanced nuclear translocation of cAMP/Ca(2+)-dependent transcription factors. Moreover, NAD(+), either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E(2) and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD(+)(e). Controls included Cx43-siRNA transfected and/or NAD(+)-glycohydrolase-treated MSC (autocrine effects), and NAD(+)-untreated or P2Y(11)-siRNA-transfected MSC (exogenous NAD(+)). These findings suggest a potential beneficial role of NAD(+)(e) in modulating MSC functions relevant to MSC-based cell therapies.
在几种细胞类型中，NAD(+)可通过连接蛋白 43 半通道（Cx43 HC）进行调节外排，细胞外 NAD(+)（NAD(+)(e)）会影响细胞特异性功能。我们研究了骨髓间充质干细胞（MSC）通过 Cx43 HC 释放细胞内 NAD(+)的能力。通过灵敏的酶循环测定法对 NAD(+)外排进行定量分析，结果表明，低细胞外 Ca(2+)（5-6 倍）、切应力（13 倍）和炎症条件（用脂多糖（LPS）孵育的细胞分别增加 3.1 倍和 2.5 倍，在 39°C 下孵育的细胞分别增加 2.5 倍），与未处理的细胞相比，NAD(+)外排显著上调，而在 Cx43-siRNA 转染的 MSC 中（下调 53%）和细胞间接触（下调 45%）则下调。此外，我们发现 NAD(+)(e)激活嘌呤能受体 P2Y(11)和环腺苷单磷酸（cAMP）/环 ADP-核糖/Ca(2+)信号级联反应，涉及到 MSC 特有的 L 型 Ca(2+)通道开放。细胞外 NAD(+)增强了 cAMP/Ca(2+)-依赖性转录因子的核转位。此外，与对照组相比，细胞外添加或自分泌释放的 NAD(+)可刺激 MSC 功能，包括增殖、迁移、前列腺素 E(2)和细胞因子的释放以及 T 淋巴细胞增殖的下调。与对照组相比，NAD(+)(e)没有检测到对 MSC 标志物和脂肪细胞或成骨细胞分化的修饰。对照组包括 Cx43-siRNA 转染和/或 NAD(+)-糖基水解酶处理的 MSC（自分泌效应），以及未处理的 NAD(+)或 P2Y(11)-siRNA 转染的 MSC（外源性 NAD(+)）。这些发现表明，NAD(+)(e)在调节与基于 MSC 的细胞治疗相关的 MSC 功能方面可能具有潜在的有益作用。
