Department of Physiology, University of Tübingen, Tübingen, Germany.
FASEB J. 2012 Jul;26(7):3049-58. doi: 10.1096/fj.12-204024. Epub 2012 Apr 2.
In dendritic cells (DCs), chemotactic chemokines, such as CXCL12, rapidly increase cytosolic Ca(2+)concentrations (Ca(2+)) by triggering Ca(2+) release from intracellular stores followed by store-operated Ca(2+) (SOC) entry. Increase of Ca(2+) is blunted and terminated by Ca(2+) extrusion, accomplished by K(+)-independent Na(+)/Ca(2+) exchangers (NCXs) and K(+)-dependent Na(+)/Ca(2+) exchangers (NCKXs). Increased Ca(2+) activates energy-sensing AMP-activated protein kinase (AMPK), which suppresses proinflammatory responses of DCs and macrophages. The present study explored whether AMPK participates in the regulation of DC Ca(2+) and migration. DCs were isolated from AMPKα1-deficient (ampk(-/-)) mice and, as control, from their wild-type (ampk(+/+)) littermates. AMPKα1, Orai1-2, STIM1-2, and mitochondrial calcium uniporter protein expression was determined by Western blotting, Ca(2+) by Fura-2 fluorescence, SOC entry by inhibition of endosomal Ca(2+) ATPase with thapsigargin (1 μM), Na(+)/Ca(2+) exchanger activity from increase of Ca(2+), and respective whole-cell current in patch clamp following removal of extracellular Na(+). Migration was quantified utilizing transwell chambers. AMPKα1 protein is expressed in ampk(+/+) DCs but not in ampk(-/-) DCs. CXCL12 (300 ng/ml)-induced increase of Ca(2+), SOC entry, Orai 1 protein abundance, NCX, and NCKX were all significantly higher in ampk(-/-) DCs than in ampk(+/+) DCs. NCX and NCKX currents were similarly increased in ampk(-/-) DCs. Moreover, CXCL12 (50 ng/ml)-induced DC migration was enhanced in ampk(-/-) DCs. AMPK thus inhibits SOC entry, Na(+)/Ca(2+) exchangers, and migration of DCs.
在树突状细胞 (DCs) 中，趋化因子趋化因子，如 CXCL12，通过触发细胞内储存的 Ca(2+)释放，随后通过 SOC 进入来快速增加细胞溶质 Ca(2+)浓度 (Ca(2+))。通过 K(+)非依赖性 Na(+)/Ca(2+)交换器 (NCXs) 和 K(+)依赖性 Na(+)/Ca(2+)交换器 (NCKXs) 进行 Ca(2+)外排，从而使 Ca(2+) 增加的幅度减小并终止。增加的 Ca(2+) 激活能量感应 AMP 激活蛋白激酶 (AMPK)，其抑制 DCs 和巨噬细胞的促炎反应。本研究探讨了 AMPK 是否参与调节 DC Ca(2+) 和迁移。从 AMPKα1 缺陷型 (ampk(-/-)) 小鼠中分离 DC，并作为对照，从其野生型 (ampk(+/+)) 同窝仔中分离 DC。通过 Western blot 测定 AMPKα1、Orai1-2、STIM1-2 和线粒体钙单向转运蛋白的表达，通过 Fura-2 荧光测定 Ca(2+)，通过抑制内体 Ca(2+)ATP 酶用 thapsigargin (1 μM) 测定 SOC 进入，通过增加 Ca(2+) 测定 Na(+)/Ca(2+)交换器活性，以及在用细胞外 Na(+)去除后，通过在膜片钳中进行的相应全细胞电流来测定。通过 Transwell 室定量迁移。AMPKα1 蛋白在 ampk(+/+) DC 中表达，但在 ampk(-/-) DC 中不表达。在 ampk(-/-) DC 中，CXCL12(300ng/ml)诱导的 Ca(2+)增加、SOC 进入、Orai 1 蛋白丰度、NCX 和 NCKX 均明显高于 ampk(+/+) DC。NCX 和 NCKX 电流在 ampk(-/-) DC 中也同样增加。此外，在 ampk(-/-) DC 中，CXCL12(50ng/ml)诱导的 DC 迁移增强。AMPK 因此抑制 SOC 进入、Na(+)/Ca(2+)交换器和 DC 的迁移。
