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

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.