Altered regulation of cytosolic Ca2+ concentration in dendritic cells from klotho hypomorphic mice

Abstract

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 ( klotho hm). The present study explored whether Klotho deficiency modifies [Ca2+]i regulation in DCs. DCs were isolated from the bone marrow of klotho hm 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 klotho hm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klotho hm DCs. The [Ca2+]i increase upon acute application of LPS (1 μg/ml) was significantly lower in klotho hm 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 klotho hm 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 klotho hm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca2+]i, and enhanced migration of klotho hm DCs, thus dissipating the differences between klotho hm 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.