Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Cell Transplant. 2013;22(10):1859-68. doi: 10.3727/096368912X657206. Epub 2012 Oct 2.
Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.
乳腺癌耐药蛋白 1（BCRP1/ABCG2）用于鉴定细胞群体中的侧群（SP），该群体富含不同组织中的干细胞和祖细胞。在这里，我们研究了细胞外信号调节激酶（ERK）1/2 在肾脏 SP 细胞缺血/缺氧条件下信号机制中的作用。使用 Hoechst 33342 染料介导的荧光素激活细胞分选分离肾脏 SP 细胞，然后在缺氧/复氧（H/R）下孵育，同时或不使用维拉帕米（一种选择性 BCRP1/ABCG2 抑制剂）。在 H/R 处理后测试 ABCG2 表达、ERK 活性、细胞活力、代谢活性和膜损伤。为了评估 ERK 1/2 在 ABCG2 的表达和功能中的作用，通过转染重组有意义表达载体 pcDNA3.1-MEK 上调丝裂原激活蛋白激酶（MAPK）/ERK 激酶（MEK）的表达，MEK 优先激活 ERK，并用特异性 MEK 抑制剂 U0126 预处理下调。我们发现，体外和体内缺氧均激活肾脏 SP 细胞中的 ERK 活性，但不激活非 SP 细胞。MEK 的过表达模拟了缺氧诱导的 ABCG2 表达。相反，U0126 抑制了缺氧和 MEK 上调的 ABCG2 表达。此外，H/R 诱导 SP 细胞和非 SP 细胞的核、代谢和膜损伤均显著增加；然而，非 SP 细胞中的这种 H/R 诱导的细胞毒性比 SP 细胞严重得多。值得注意的是，MEK 的过表达增强了肾脏 SP 细胞的活力，而 U0126 抑制了该活力。维拉帕米处理逆转了 MEK 诱导的肾脏 SP 细胞活力。当将其全身给予肾缺血/再灌注损伤的动物时，SP 细胞显著改善了肾功能，加速了有丝分裂反应，并减少了细胞凋亡。然而，SP 细胞的这种改善治疗潜力在维拉帕米预处理后显著降低。总之，这些发现为 MEK/ERK-ABCG2 通路在保护肾脏 SP 细胞免受缺血/缺氧损伤中的关键作用提供了证据。
