Iron Together with Lipid Downregulates Protein Levels of Ceruloplasmin in Macrophages Associated with Rapid Foam Cell Formation

Abstract

Aim: Iron accumulation in foam cells was previously shown to be involved in atherogenesis. However, the mechanism for iron accumulation was not clarified. Ceruloplasmin (Cp) is an important factor in cellular iron efflux and was found to be downregulated in atherosclerotic plaques in our previous study. The current study is to investigate the role of Cp in atherosclerosis.

Methods: We used RAW264.7 cells, a well-accepted cell model of atherosclerosis, which were treated with lipopolysaccharides (LPS), ferric ammonium citrate (FAC) or deferoxamine, and oxidized low density lipoprotein (ox-LDL) to detect the regulation of Cp and its influence in iron efflux and lipid accumulation using biochemical and histological assays.

Results: Our results showed that the Cp protein level increased after 200-μM FAC treatment in LPS-activated RAW264.7 cells. Ox-LDL treatment (50 μg/ml) moderately reduced both mRNA and protein levels and ferroxidase activity of Cp (p＜0.05). No significant difference was observed in the expression of ferritin and ferroportin, two important iron-related proteins for iron storage and efflux, respectively, after ox-LDL treatment. However, co-treatment with ox-LDL and FAC drastically reduced the expression of Cp. Accordingly, the ferroxidase activities simultaneously decreased, whereas the protein levels of Ft and Fpn1 significantly increased, indicating further iron accumulation. Moreover, co-treatment with FAC and ox-LDL enhanced the accumulation of cholesterol compared with ox-LDL-only treatment to trigger apoptosis.

Conclusion: Our findings suggest that physiological interaction of iron and lipid obstructs iron efflux and accelerates the lipid accumulation in macrophages during foam cell formation, which implicates the role of iron in the pathology of atherosclerosis.

Fig. 1.

Both iron and LPS promote the expression and activities of ceruloplasmin (A) Cellular iron Perl's stain. The relative values are shown (inset). (B) Western blot for iron related proteins, Ft, Fpn1, and TfR. (C) The expression and ferroxidase activity of Cp, revealed by real-time quantitative PCR, western blotting and ferroxidase enzyme activity assays. FAC: ferric ammonium citrate; DFO: deferoxamine; LPS: lipopolysaccharides; Ft: ferritin; Fpn1: Ferroportin 1; TfR: Transferrin receptor; Cp: ceruloplasmin; PCR: polymerase chain reaction. * p < 0.05 compared with PBS-treated group, ^# p < 0.05 compared with LPS-treated group.

Fig. 2.

Uptake of the oxidized-LDL by activated RAW264.7 cells significantly reduces the expression of Cp (A) The content of CE, FC, TC, and CE/TC(%) in LPS-activated RAW264.7 cells after the treatment of ox-LDL. (B) Representative western blots to show the effect of ox-LDL on Cp, Ft, and Fpn1 expression. ox-LDL: oxidized low density lipoprotein; TC: total cholesterol; FC: free cholesterol; CE: cholesteryl ester. * p < 0.05 compared with LPS-treated group.

Fig. 3.

Physiological interaction of iron and ox-LDL accelerates the decrease of ceruloplasmin expression in LPS-treated RAW264.7 cells (A) Co-effect of iron and ox-LDL on mRNA and protein levels and activity of Cp, (B) the protein levels of Ft, and (C) the protein levels of Fpn1. (D) Effects of ox-LDL on the expression of Cp in an iron dependent manner. Delayed-200 in the last lane means 2-h delayed treatment of FAC after ox-LDL addition. The treatment for the rest lanes is simultaneous addition of FAC and ox-LDL. Hp: hephaestin; REL: Relative intensity. * p < 0.05 vs. ox-LDL treated group, ^# p < 0.05 vs. FAC group in (A–C); * p < 0.05 vs. non-treated group, ^# p < 0.05 vs. ox-LDL-only group in (D).

Fig. 4.

Cellular localization of Cp is determined in RAW264.7 cells Immuno-fluorescence assays showed upregulated cellular expression of Cp by iron is reversed by the additon of ox-LDL in RAW264.7 cells. Most Cp is localized in the cytoplasm as shown by dots, which are suggested to be lipid rafts²⁶⁾. DAPI: 4′,6-diamidino-2-phenylindole.

Fig. 5.

Iron promotes the lipid deposition in ox-LDL-treated RAW264.7 cells (A) Iron enhancement of lipid uptake in RAW264.7 cells. (B) More lipid in the deformed RAW264.7 cells when co-treated with iron and ox-LDL, revealed by oil red stain. (C) A severe imbalance between lipid influx and efflux after the co-treatment of FAC with ox-LDL. CD36: a scavenger receptor of cholesterol influx; ABCA1 (ATP-binding cassette transporter A1): an important transporter for cholesterol efflux. *p < 0.05 vs. ox-LDL treated group in (A); *p < 0.05 vs. non-treated group, ^# p < 0.05 vs. ox-LDL group in (C).

Fig. 6.

Physiological interaction of iron with ox-LDL increases apoptosis of RAW264.7 cells (A–B) Flow cytometry assays demonstrated the enhancement of apoptotic rates of RAW264.7 by co-treatment of FAC with ox-LDL versus FAC or ox-LDL treatment alone. (C) Cell survival rate was significantly decreased after the co-treatment of FAC with ox-LDL versus FAC or ox-LDL treatment alone by counting the living cells. (D) Apoptotic index (Bcl2/Bax ratio) confirmed the occurrence of apoptosis. PI: propidium iodide; Bcl2 (B-cell lymphoma-2): an anti-apoptotic regulator; Bax: a proapoptotic regulator; *p < 0.05, **p < 0.01, ***p < 0.001.