Department of Physiology, University of Tübingen, Tübingen, Germany.
Am J Physiol Cell Physiol. 2013 Jul 1;305(1):C70-7. doi: 10.1152/ajpcell.00355.2012. Epub 2013 Apr 17.
The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]i). [Ca²⁺]i is increased by store-operated Ca²⁺ entry and decreased by K⁺-independent (NCX) and K⁺-dependent (NCKX) Na⁺/Ca²⁺ exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃], the biologically active form of vitamin D. Formation of 1,25(OH)₂D₃ is inhibited by the antiaging protein Klotho. Thus 1,25(OH)₂D₃ plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²⁺]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²⁺]i increase upon acute application of LPS (1 μg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 μM). CCL21-dependent migration was significantly less in klothohm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)₂D₃ the first 2 days after isolation from bone marrow. Feeding klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²⁺]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Na⁺/Ca²⁺-exchange activity, thus blunting the increase of [Ca²⁺]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)₂D₃ formation.
树突状细胞 (DCs) 的功能是调节初始 T 细胞，作为抗原呈递细胞，其细胞内钙离子浓度 ([Ca²⁺]i) 的调节受到影响。[Ca²⁺]i 的增加是由储存操作钙内流引起的，而减少则是由 K⁺-非依赖性 (NCX) 和 K⁺-依赖性 (NCKX) Na⁺/Ca²⁺交换器引起的。NCKX 交换器受免疫抑制 1,25-二羟基维生素 D3 [1,25(OH)₂D₃] 刺激，1,25(OH)₂D₃ 是维生素 D 的生物活性形式。衰老蛋白 Klotho 会抑制 1,25(OH)₂D₃ 的形成。因此，Klotho 缺乏的 Klotho 缺陷型小鼠 (klothohm) 中 1,25(OH)₂D₃ 血浆水平过高。本研究探讨了 Klotho 缺乏是否会改变 DCs 中的 [Ca²⁺]i 调节。从 klothohm 小鼠和野生型小鼠 (klotho+/+) 的骨髓中分离出 DCs，并在粒细胞-巨噬细胞集落刺激因子的作用下培养 7-9 天。根据主要组织相容性复合物 II (MHC II) 和 CD86 的表达，klothohm DCs 和 klotho+/+ DCs 的分化和脂多糖 (LPS) 诱导成熟相似。然而，klothohm DCs 中的 NCKX1 膜丰度和 NCX/NCKX 活性明显增强。Klothohm DCs 中 LPS (1 μg/ml) 急性应用后 [Ca²⁺]i 的增加明显低于 klotho+/+ DCs，而 NCKX 阻滞剂 3',4'-二氯苯甲酰基 (DBZ; 10 μM) 可逆转这种差异。klothohm DCs 的 CCL21 依赖性迁移明显低于 klotho+/+ DCs，但可通过 DBZ 恢复。klotho+/+ DC 前体在分离后前 2 天用 1,25(OH)₂D₃ 预处理可增强 NCKX 活性。用缺乏维生素 D 的饮食喂养 klothohm 小鼠可降低 NCKX 活性，增加 LPS 诱导的 [Ca²⁺]i 增加，并增强 klothohm DCs 的迁移，从而消除了 klothohm DCs 和 klotho+/+ DCs 之间的差异。总之，Klotho 缺乏会增加 NCKX1 膜丰度和 Na⁺/Ca²⁺交换活性，从而减轻 LPS 暴露和 CCL21 介导的迁移后 [Ca²⁺]i 的增加。这些影响在很大程度上归因于过多的 1,25(OH)₂D₃ 形成。
