Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release

Abstract

Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

Fig. 1.

NO derived from iNOS activity induces intranuclear Zn²⁺ release in MAEC. MAEC from WT (A–D), iNOS-deficient (E and F), or MT-deficient mice (G and H) were activated with IL-1β + TNF-α + IFN-γ for 24 h in the absence or presence of 250 μM of the iNOS-specific inhibitor l-NIO. Cells were then labeled with Zinquin, which specifically becomes fluorescent after reaction with labile, but not protein-bound, Zn²⁺. In activated WT MAEC, a bright Zn²⁺-specific fluorescence signal is located almost exclusively in the nuclei, with a punctate-staining pattern against a more diffuse nuclear background staining (A and B). In contrast, no Zn²⁺-specific fluorescence signal can be detected in activated WT MAEC cultured in the presence of l-NIO (C and D)or in MAEC deficient for either iNOS (E and F)orfor MT-1 + MT-2 (G and H). Thus, iNOS-derived NO, as well as MT, is essential for intranuclear Zn²⁺ release. (Scale bar = 5 nm.)

Fig. 2.

NO derived from iNOS activity induces a punctate nuclear Zn²⁺-specific fluorescence activity. Fluorescence and transillumination micrographs of nuclei of cytokine-activated WT MAEC cultured in the absence (A–C) or presence (E)of250 μM of the iNOS-specific inhibitor l-NIO. A line scan through the nuclei was performed at 500 nm (D and F), confirming the nuclear punctate-staining pattern against a more diffuse nuclear background. [Scale bars represent 0.5 nm (A and B) or 0.75 nm (C and E).]

Fig. 3.

Both proinflammatory cytokines as well as iNOS-derived NO are essential for MT nuclear translocation. Micrographs of resting (A) or cytokine-activated WT MAEC cultured for 24 h in the absence (B) or presence (C)of250 μM of the iNOS-specific inhibitor l-NIO or resting WT MAEC cultured for 24 h in the presence of 200 μM of the NO donor DETA/NO (D). Cells were labeled with a mAb specific for MT-1 as well as MT-2, and staining was visualized with the peroxidase-diaminobenzidine method. (Scale bar = 5 nm.)

Fig. 4.

NO derived from iNOS activity increases constitutive MT-1 and MT-2 mRNA expression. Wt MAEC were cultured for6hinthe absence or presence of IL-1β + TNF-α + IFN-γ (CM) with or without 250 μM of the iNOS-specific inhibitor l-NIO. Total cellular RNA was isolated, and MT-1-, MT-2-, and GAPDH-specific RT-PCR were performed. The CM in an NO-dependent manner increases constitutive MT-1 and MT-2 mRNA expression. Values are mean ± SD of six independent experiments. *, P < 0.005 as compared with cytokine-treated cells.

Fig. 5.

NO exogenously applied by an NO donor as well as Zn²⁺ increase constitutive MT-1 and MT-2 mRNA expression. Wt MAEC were cultured for 6 h in the absence (lanes 1 and 4) or presence (lane 2) of 200 μM of the NO donor DETA/NO or of decomposed DETA/NO (DETA/NO_–NO) (lane 3), or of 100 μM Zn²⁺ (lane 5). Total cellular RNA was isolated, and MT-1-, MT-2-, and GAPDH-specific RT-PCR were performed. NO derived from DETA/NO as well as Zn²⁺ increase constitutive MT-1 and MT-2 mRNA expression. Values are mean ± SD of three independent experiments. *, P < 0.02 as compared with lane 2.

Fig. 6.

Intracellular Zn²⁺ released after exogenously applied NO upregulates constitutive MT-1 as well as MT-2 mRNA expression. Wt MAEC were cultured in the absence (lane 1) or presence (lanes 2 and 3) of 200 μM DETA/NO with or without 5 μM of the intracellular Zn²⁺-specific chelator TPEN (lanes 3 and 4). Total cellular RNA was isolated, and MT-1-, MT-2-, and GAPDH-specific RT-PCR were performed. DETA/NO increases MT-1 and MT-2 mRNA expression, which is inhibited by TPEN, whereas TPEN alone has no effect. Values are mean ± SD of three independent experiments. *, P < 0.01; **, P < 0.005 as compared with lane 2.