Nitrogen monoxide-mediated control of ferritin synthesis: implications for macrophage iron homeostasis

Abstract

Intracellular iron homeostasis is regulated posttranscriptionally by iron regulatory proteins 1 and 2 (IRP1 and IRP2). In the absence of iron in the labile pool, IRPs bind to specific nucleotide sequences called iron responsive elements (IREs), which are located in the 5' untranslated region of ferritin mRNA and the 3' untranslated region of transferrin receptor mRNA. IRP binding to the IREs suppresses ferritin translation and stabilizes transferrin receptor mRNA, whereas the opposite scenario develops in iron-replete cells. Binding of IRPs to the IREs is also affected by nitrogen monoxide (NO), but there are conflicting reports regarding the effect of NO on ferritin synthesis. In this study, we demonstrated that a short exposure of RAW 264.7 cells (a macrophage cell line) to the NO+ donor, sodium nitroprusside (SNP), resulted in a dramatic increase in ferritin synthesis. The SNP-mediated increase of ferritin synthesis could be blocked by MG132, an inhibitor of proteasome-dependent protein degradation, which also prevented the degradation of IRP2 caused by SNP treatment. Moreover, treatment of RAW 264.7 cells with IFN-gamma and lipopolysaccharide caused IRP2 degradation and stimulated ferritin synthesis, changes that could be prevented by specific inhibitors of inducible nitric oxide synthase. Furthermore, the SNP-mediated increase in ferritin synthesis was associated with a significant enhancement of iron incorporation into ferritin. These observations indicate that NO+-mediated modulation of IRP2 plays an important role in controlling ferritin synthesis and iron metabolism in murine macrophages.

Fig 1.

Effects of NO donors and IFN-γ/LPS on RNA-binding activities of IRPs (A and B) and ferritin synthesis (C and D). RAW 264.7 cells were incubated (10 h) with various agents as indicated. (A) Cytosolic protein extracts (10 μg) were subjected to gel retardation assays (see Materials and Methods). CTL, control; L, LPS; I, IFN-γ; W, N-(3-(aminomethyl)-benzyl)acetamide and N, L-N⁶-(1-iminoethyl)-lysine are specific inhibitors of iNOS. (B) Cells were incubated without or with SNP (100 μM) and SNAP (100 μM) for 3 h. (C and D) Ferritin synthesis in RAW 264.7 cells. After 10 h of preincubation with the reagents, cells were pulse-labeled (2 h) with [³⁵S]methionine and harvested. Ferritin was immunoprecipitated by using antiferritin antibodies and analyzed by 12.5% SDS.PAGE followed by autoradiography. Nitrate was assayed by using the Griess reagent as described by Green et al. (35).

Fig 2.

Effects of proteasome inhibitors on RNA-binding activities of IRPs (A), IRP2 protein levels (B), and ferritin synthesis. RAW 264.7 cells were grown in control medium (lane 1, CTL), or were preincubated (lanes 3–6) with MG132 (50 μg/ml) or lactacystin (50 μM) for 30 min, after which the cells were incubated for 3 h with SNP (100 μM), as indicated. (A) Gel retardation assays of protein (10 μg) extracted from RAW 264.7 cells after different treatments. (B) Western blot analysis of protein (50 μg) extracted from RAW 264.7 cells (as in A), by using anti-IRP2 antibodies. (C) After 3 h of preincubation with the reagents, cells were pulse-labeled (2 h) with [³⁵S]methionine, and ³⁵S-ferritin was immunoprecipitated and subjected to SDS/PAGE autoradiography.

Fig 3.

The effect of SNP on ferritin synthesis. RAW 264.7 cells were grown in control medium (lane 1, CTL) or were incubated (3 h) with different concentrations of SNP (A) added for different time intervals (B), as indicated. D.A., densitometric analysis, in arbitrary units.

Fig 4.

Effects of pretreatment with SNP on ferritin synthesis (A) and IRP binding (B). RAW 264.7 cells were grown in control medium (lane 1–4, CTL) or were preincubated with 250 μM SNP (lanes 5–8, SNP 250 μM) for 3 h. The cells were washed with PBS (3×) and incubated in the control medium for indicated time intervals. (A) Autoradiogram of ³⁵S-ferritin; (B) densitometric analysis of ³⁵S-ferritin (n = 3); error bars represent SD; (C) gel retardation assay; (D) densitometric analysis of IRP2-binding activity (n = 3); error bars represent SD.

Fig 5.

Effects of SNP on ⁵⁹Fe uptake by RAW 264.7 cells (A) and ⁵⁹Fe incorporation into ferritin (B and C). RAW 264.7 cells were incubated with 5 μM ⁵⁹Fe₂-Tf in the absence (○) or presence (•) of 500 μM SNP for indicated time intervals, after which the cells were washed with cold PBS (3×). (A) ⁵⁹Fe uptake by control and SNP-treated RAW 264.7 cells. (B) ⁵⁹Fe incorporation into ferritin was determined (see Materials and Methods). (C) ⁵⁹Fe associated with ferritin is expressed as a percentage of total cellular ⁵⁹Fe content. The results are means ± SD of triplicate determinations in a typical experiment performed.

Fig 6.

Effect of SNP and IFN-γ/LPS on ⁵⁹Fe incorporation into ferritin. RAW 264.7 cells were incubated with 5 μM ⁵⁹Fe-Tf for 3 h, after which the cells were washed three times with cold PBS. ⁵⁹Fe-labeled cells were then incubated with either 100 μM SNP or 10 μg/ml of LPS (L) and 100 units/ml of IFN-γ (I), in the presence or absence of iNOS-specific inhibitors (N, W; see Fig. 1) for 18 h. ⁵⁹Fe in ferritin (see Materials and Methods) is expressed as a percentage of total cellular ⁵⁹Fe content. CTL1, percentage of ⁵⁹Fe in ferritin after 3-h incubation with ⁵⁹Fe-Tf, CTL2, percentage of ⁵⁹Fe in ferritin following 18-h reincubation in control medium.

Fig 7.

Effect of cycloheximide on SNP-induced ⁵⁹Fe incorporation into ferritin. RAW 264.7 cells were incubated with 5 μM ⁵⁹Fe₂-Tf for 3 h, after which the cells were washed with cold PBS (3×). After counting ⁵⁹Fe in cell pellets (A), the cells were lysed, and ⁵⁹Fe incorporation into ferritin (B) was measured (see Materials and Methods). ⁵⁹Fe associated with ferritin is expressed as a percentage of total cellular ⁵⁹Fe content. The results are means ± SD of triplicate determinations in a typical experiment performed. Chx, cycloheximide (100 μM).