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. 2015 May 8;290(19):11918-34.
doi: 10.1074/jbc.M115.645903. Epub 2015 Mar 23.

Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium

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Free PMC article

Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium

Yafeng Li et al. J Biol Chem. .
Free PMC article

Abstract

Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation.

Keywords: AMD; C3; CCAAT/Enhancer-binding Protein (C/EBP); Extracellular Signal-regulated Kinase (ERK); Iron; RPE; SMAD3; Signaling; Transforming Growth Factor β (TGF-β).

Figures

FIGURE 1.
FIGURE 1.
Iron up-regulates C3 expression in ARPE-19 cells. A, viability of cells grown as 1-month differentiated monolayers and treated with FAC at increasing doses for 2 days (d). Viability is decreased with 25 μm iron treatment compared with untreated but remains stable for higher doses examined. B and C, TFRC mRNA and C3 mRNA levels, respectively, with increasing FAC treatment doses for 2 days. TFRC mRNA levels decrease with 25 μm iron relative to untreated and are stable at higher doses; similarly, C3 mRNA levels are increased at the lowest iron dose relative to untreated but remain stable at the higher doses. D, C3 protein levels increase, relative to untreated, in the conditioned medium of cells treated with increasing doses of FAC for 2 days. E, C3 mRNA levels are increased relative to control cells at each of the 2-, 4-, and 8-day time points after the initial 250 μm FAC treatment. F, C3 mRNA levels of cells treated with Fe3+, Mn2+, Ni2+, or Cu2+ at 250 μm concentration for 2 days with only Fe3+/FAC showing an increase in C3 mRNA relative to untreated. G and H, C3 mRNA levels of cells treated with 9.75 mg/ml holo-transferrin (holo-Tf) are increased relative to those of apo-transferrin (apo-Tf)-treated cells with a corresponding decrease in TFRC mRNA. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).
FIGURE 2.
FIGURE 2.
Iron-induced C3 expression is not dependent on TGF-β ligand/receptor-mediated canonical signaling. A, C3 mRNA levels in ARPE-19 cells are repressed by exogenous TGF-β1 (10 ng/ml; 2 days (d)), and this inhibition is partially relieved by TGF-β receptor inhibitor SB431542 (100 nm; 2 days). In contrast, PAI-1 mRNA levels are significantly increased by TGF-β1, and this increase is partially inhibited by SB431542. B, TGFB1 mRNA levels show an increase with FAC treatment; TGF-β1 protein levels in the conditioned medium of FAC-treated cells show a significant decrease relative to untreated. C, C3 mRNA levels are increased with addition of neutralizing antibody anti-TGF-β1/2/3 (10 μg/ml; 2 days) when compared with control or IgG treatment conditions. PAI-1 mRNA levels are decreased in the same comparison. D, C3 mRNA levels are not significantly changed with co-treatment with anti-TGF-β1/2/3 antibody and FAC when compared with FAC only. PAI-1 mRNA levels are decreased to similar levels with co-treatment with anti-TGF-β1/2/3 antibody and FAC compared with FAC only. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant).
FIGURE 3.
FIGURE 3.
Non-canonical TGF-β signaling involving ERK1/2 and SMAD3 mediates iron induction of C3. A, C3 mRNA levels in ARPE-19 cells treated with MEK1/2 inhibitor PD98059 (5 μm; 2 days (d)), p38 inhibitor SB202190 (20 μm; 2 days), or JNK1/2/3 inhibitor SP600125 (50 μm; 2 days) alone and plus FAC. Control contains vehicle only (DMSO). Relative to FAC alone, only PD98059 showed a significant decrease in C3 mRNA levels when co-treated with FAC. B, the specific inhibitor of SMAD3, SIS3 (2 μm; 2 days), inhibited FAC-induced C3 up-regulation. SIS3 decreased basal C3 mRNA levels. C, iron-induced increases in C3 protein levels in the conditioned media are diminished by SIS3 and PD98059. D, SIS3 inhibited PAI-1 mRNA levels to the same extent as FAC, whereas PD98059 had no significant effect. Co-treatment with SIS3 and FAC showed no additional suppression of PAI-1 mRNA levels, but co-treatment with PD98059 and FAC restored them to baseline. E, Western blot and densitometry analysis for p-ERK1/2 and ERK1/2 in lysates derived from cells at different time points (0, 1, 3, and 6 h) post-FAC treatment, with or without SIS3 co-treatment. SIS3 showed no effect on the FAC-induced increase of p-ERK1/2 at 1h. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant).
FIGURE 4.
FIGURE 4.
Iron-induced changes in SMAD3 phosphorylation are mediated by non-canonical TGF-β signaling. A, Western blot and densitometry analysis of ARPE-19 cell lysates for p-SMAD3 (Ser-213), p-SMAD3 (Ser-423/425), SMAD3, and TFRC in a FAC treatment time course experiment. The p-SMAD3 (Ser-213) 150-kDa band increased whereas the p-SMAD3 (Ser-423/425) and TFRC bands decreased in intensity in a time-dependent manner. The p-SMAD3 (Ser-213) antibody-detectable 52-kDa band showed no significant change in the same time course. The SMAD3 protein levels also remain unchanged. B, cells transfected with constructs expressing FLAG-tagged SMAD3, SMAD3 EPSM (linker mutations), and SMAD3 EPSM A213S (mutation reverted at residue 213) showed specificity of the antibody used to detect p-SMAD3 (Ser-213) in Western blots. C, Western blot and densitometry analysis of p-SMAD3 (Ser-213) shows that co-treatment with SIS3 or PD98059 and FAC reduced the intensity of this band relative to FAC alone. SIS3 decreased the basal p-SMAD3 (Ser-213) levels. Blotting and analysis of p-SMAD3 (Ser-423/425) demonstrated that SIS3 reduced band intensity to the same extent as FAC alone, whereas PD98059 had no significant effect. Co-treatment with either inhibitor with FAC showed no significant difference from FAC alone. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant).
FIGURE 5.
FIGURE 5.
Iron and TGF-β ligand differentially regulate spatially distinct SMAD3 phosphorylation sites. A, Western blot and densitometry analysis of p-SMAD3 (Ser-213) and p-SMAD3 (Ser-423/425) in cells treated with BSA, IgG, or anti-TGF-β1/2/3 antibody in the presence or absence of FAC show the combined effect of iron and neutralization of canonical TGF-β signaling. SMAD3 protein levels increased after anti-TGF-β1/2/3 antibody treatment relative to control condition, BSA, with no FAC. Only the antibody plus FAC condition is statistically significant. B, Western blot and densitometry analysis following treatment with exogenous TGF-β1 (10 ng/ml; 1 h) show no significant change in p-SMAD3 (Ser-213) and increased p-SMAD3 (Ser-423/425). Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001; ns, not significant).
FIGURE 6.
FIGURE 6.
SMAD3 gene silencing reduces the formation of p-SMAD3 (Ser-213)-containing complexes and suppresses iron-induced C3 up-regulation. A, Western blot and densitometry analysis for SMAD3, p-SMAD3 (Ser-213), and TFRC of ARPE-19 cell lysates derived from untransduced (control), vector, sh-null, and sh-SMAD3 lines, each in the absence or presence of FAC. Efficient knockdown of SMAD3 decreased the formation of p-SMAD3 (Ser-213)-containing complexes at 150 kDa. B, the FAC-induced increase in C3 mRNA levels is observed in the untransduced, vector, and sh-null cell lines but is absent in the sh-SMAD3 cell line. C, Western blot for non-nuclear (Non-Nucl) and nuclear (Nucl) lysates of untreated and FAC-treated cells. The p-SMAD3 (Ser-213)-containing complex at 150 kDa localizes only to the non-nuclear fractions. The p-SMAD3 (Ser-213) antibody-detectable band at 52 kDa is essentially unchanged by FAC treatment in both the non-nuclear and nuclear fractions. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant). d, days.
FIGURE 7.
FIGURE 7.
Iron-induced CEBPD mRNA expression and C/EBP-δ protein nuclear translocation are dependent on SMAD3 activity. A, CEBPD mRNA levels in ARPE-19 cells are decreased by co-treatment with SIS3 and FAC compared with FAC alone, which is increased relative to control. B, CEBPD mRNA levels are increased with addition of neutralizing antibody anti-TGF-β1/2/3 (10 μg/ml; 2 days (d)) when compared with control or IgG treatment conditions. C, CEBPD mRNA levels are elevated with FAC treatment but not significantly changed with co-treatment with anti-TGF-β1/2/3 antibody and FAC. D, Western blot and densitometry analysis for C/EBP-δ in non-nuclear (Non-Nucl) and nuclear (Nucl) lysates of cells treated with FAC in a time course. C/EBP-δ protein levels in the non-nuclear fraction show a time-dependent increase. Nuclear accumulation of C/EBP-δ occurs with the duration of FAC treatment. E, knockdown of SMAD3 in sh-SMAD3 cells impaired FAC-induced increases in nuclear C/EBP-δ when compared with sh-null and untransduced cells. Basal C/EBP-δ levels increased in the sh-SMAD3 cell line relative to the controls. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant).
FIGURE 8.
FIGURE 8.
The bZIP1 domain, a putative C/EBP-δ binding site in the C3 promoter, is critical for iron-induced transcriptional up-regulation of C3. A, ARPE-19 cells transfected with luciferase vectors containing a set of nested but different sized C3 promoter fragments as schematically indicated uniformly displayed responsiveness to FAC treatment. B, cells transfected with the smallest fragment (500 bp) containing a WT sequence, the same fragment with a mutant bZIP1 domain, or the same fragment with a mutant bZIP2 domain are treated with or without FAC. The bZIP1 mutant suppressed baseline and FAC-inducible luciferase activities. In A and B, results were corrected for transfection efficiency as measured by Renilla luciferase activity, which was not significantly changed by FAC. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant). TSS, transcription start site; Luc, luciferase; d, days.
FIGURE 9.
FIGURE 9.
Iron-induced C3 processing and alternative pathway activation can be suppressed by inhibitors of SMAD3 and MEK1/2. A, ELISA of C3a from the conditioned medium of untreated ARPE-19 cells and those treated with different doses of FAC for 2 days (d). Compared with either untreated or 25 μm FAC-treated cells, the 250 μm FAC-treated cells showed a significant increase in C3a production. B, C3a production is significantly decreased by co-treatment with SIS3 or PD98059 and FAC compared with FAC alone at 4 days; co-treatment with both inhibitors and FAC did not further reduce C3a levels compared with co-treatment with a single inhibitor and FAC. C, ELISA using some of the same conditions as in B showed that production of Factor Ba, a marker of alternative complement pathway activation, is decreased by co-treatment with SIS3 or PD98059 and FAC compared with FAC alone. Data are expressed as mean ± S.E. (error bars) (n ≥ 3 with the following statistical notations: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant).
FIGURE 10.
FIGURE 10.
Iron overload in the RPE is associated with C3 activation as demonstrated by localized C3d deposition. A, paraffin-embedded sections of normal and aceruloplasminemia human macular retina with RPE and Bruch's membrane (BrM) labeled are stained with antibodies against C3d (red) and DAPI for nuclei (blue). B, representative images of RPE cells (arrow) and Bruch's membrane (arrowhead) in Optimal Cutting Temperature compound-embedded retina sections of 12-month-old C3−/− and BCre+, Cp−/−, HephF/F mice. Cells with increased L-ferritin levels (green; left panels) have an associated increase in C3d deposition (red; middle panels). The merged images demonstrating co-localization are shown in the right panels. Scale bars are equivalent to 50 μm.
FIGURE 11.
FIGURE 11.
The molecular mechanism of iron-induced RPE C3 production involves ERK1/2, SMAD3, and C/EBP-δ signaling. Increased intracellular iron (Fe3+) in RPE cells treated with FAC stimulates the phosphorylation of ERK1/2 followed by SMAD3 linker (Ser-213) phosphorylation (orange up arrow) in a non-canonical TGF-β pathway leading to up-regulation of C3, the central molecule of the complement cascade (the FAC-induced pathway is delineated by orange arrows). MEK1/2 inhibitor PD98059 and SMAD3 inhibitor SIS3 can block FAC-induced C3 up-regulation. FAC also results in decreased phosphorylation of SMAD3 C-terminal residues (Ser-423/425) at the SSVS motif (orange down arrow). Through SMAD3, FAC increases CEBPD mRNA and C/EBP-δ protein levels possibly by inducing CEBPD expression and relieving SMAD3-mediated inhibition at baseline (orange X over blue inhibitory line). The p-SMAD3 (Ser-213) complex remains extranuclear. In addition, FAC promotes the nuclear accumulation of C/EBP-δ protein. C/EBP-δ likely binds to the bZIP1 domain of the C3 promoter, inducing C3 expression. Once translated into protein, C3 is secreted and cleaved into C3a and C3b. Concurrently, Factor D catalyzes the activation of Factor B to form Bb and the cleavage product Factor Ba. Factors C3b and Bb together form the alternative pathway (AP) C3 convertase to amplify the activation of C3 by forming more C3b and C3a. The iron-induced non-canonical TGF-β pathway (orange) may cross-talk with the basal, canonical TGF-β pathway (blue; see “Discussion”), which is initiated by the binding of TGF-β ligand to the TGF-β receptor complex (types I and II) at the plasma membrane. Endogenous TGF-β binding maintains the phosphorylation levels of SMAD3 at both Ser-213 and Ser-423/425, ultimately acting as a negative regulator of basal C3 expression. Stimulatory arrows and inhibitory lines represent a functional link between the subsequent entities, not necessarily a direct interaction.

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