Melo Carolina Soares Barros, Arantes Faria Jerusa Araújo Quintão, Corrêa Natássia Caroline Resende, de Andrade Carolina, Carvalho Juliana Lott, Goes Alfredo M, Rodrigues Michele A, Gomes Dawidson Assis
Stem Cell Res Ther. 2013 Aug 8;4(4):92. doi: 10.1186/scrt291.
Multipotent mesenchymal stromal cells (MSCs) have gained considerable interest because of their potential use in the treatment of a variety of diseases and injuries. Although remarkable advancements have been made in clinical studies, substantial concerns still regard the safety of MSCs. Some evidence suggests that MSCs can spontaneously generate a population of cells with tumorigenic potential. Thus, studying the molecular mechanisms that control the proliferation of MSCs may be a necessary step toward the development of strategies for safe clinical practice. Ca(2+) is a second messenger that mediates a wide range of cellular responses, including the regulation of cell proliferation, but little is known about its function in MSCs. The aim of this study was to investigate the effects of targeted Ca(2+) buffering on MSCs proliferation in vitro.
Here, we used an adenoviral (Ad) vector encoding the Ca(2+) chelator protein parvalbumin (PV) fused to a nuclear exclusion signal (NES) and the Discosoma red fluorescent protein (DsRed) to investigate the function of cytoplasmic Ca(2+) signals on MSC proliferation. Confocal microscopy was used to demonstrate that PV-NES-DsRed was expressed in the cytoplasm. Ca(2+) signaling was monitored by using Fluo-4-AM. Fluorescence-activated cell sorting (FACS) analysis of cells that were stained with propidium iodide was used as a quantitative measure of cell death. The mitotic index was assessed by immunofluorescence, and the expression of cyclins was examined with Western blot.
Our results show that the Ad-PV-NES-DsRed fusion protein decreased serum-induced Ca(2+) signaling and blocked the proliferation of rat adipose-derived MSCs (AT-MSCs) in prophase. FACS analysis revealed that Ad-PV-NES-DsRed did not induce cell death in AT-MSCs. Furthermore, Western blot analysis demonstrated that Ad-PV-NES-DsRed reduced extracellular signal-regulated kinase (Erk1/2) phosphorylation and cyclin B1 expression. Buffering cytosolic Ca(2+) did not alter the expression of cyclins A/D1/D2/D3/E and E2.
Our results show that cytoplasmic Ca(2+) signals are important for AT-MSCs progression beyond prophase because of their effects on Erk phosphorylation and cyclin B1 expression.
多能间充质基质细胞（MSCs）因其在治疗多种疾病和损伤方面的潜在用途而备受关注。尽管临床研究取得了显著进展，但对MSCs安全性的担忧仍然很大。一些证据表明，MSCs可自发产生具有致瘤潜力的细胞群体。因此，研究控制MSCs增殖的分子机制可能是制定安全临床实践策略的必要步骤。Ca(2+)是一种第二信使，介导多种细胞反应，包括细胞增殖的调节，但对其在MSCs中的功能了解甚少。本研究的目的是探讨靶向Ca(2+)缓冲对体外MSCs增殖的影响。
在此，我们使用编码与核输出信号（NES）融合的Ca(2+)螯合蛋白小白蛋白（PV）和盘基网柄菌红色荧光蛋白（DsRed）的腺病毒（Ad）载体，研究细胞质Ca(2+)信号对MSC增殖的功能。共聚焦显微镜用于证明PV-NES-DsRed在细胞质中表达。使用Fluo-4-AM监测Ca(2+)信号。用碘化丙啶染色的细胞的荧光激活细胞分选（FACS）分析用作细胞死亡的定量测量。通过免疫荧光评估有丝分裂指数，并用蛋白质印迹法检测细胞周期蛋白的表达。
我们的结果表明，Ad-PV-NES-DsRed融合蛋白降低了血清诱导的Ca(2+)信号，并在前期阻断了大鼠脂肪来源的MSCs（AT-MSCs）的增殖。FACS分析显示，Ad-PV-NES-DsRed在AT-MSCs中未诱导细胞死亡。此外，蛋白质印迹分析表明，Ad-PV-NES-DsRed降低了细胞外信号调节激酶（Erk1/2）的磷酸化和细胞周期蛋白B1的表达。缓冲细胞质Ca(2+)并未改变细胞周期蛋白A/D1/D2/D3/E和E2的表达。
我们的结果表明，细胞质Ca(2+)信号对AT-MSCs前期后的进展很重要，因为它们对Erk磷酸化和细胞周期蛋白B1表达有影响。
