Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Abstract

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by (59)Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.

Figure 1. A. aegypti larval cell ferritin is present in the membrane fraction and secreted into the medium loaded with iron

CCL-125 cells were treated with 33.6 nM ferric citrate and 100 μM FAC (Fe/FAC, control) or 33.6 nM ⁵⁹ferric citrate (0.9 μCi) and 100 μM FAC (⁵⁹Fe/FAC) and incubated for 4 h. Cells were washed and subsequently treated with 100 μM FAC for an additional 14 h. Media and cells were recovered as described in the methods. Protein samples were resolved by a Native-PAGE 4–20% as previously described. (A–C) Cell number, viability and calcein fluorescent quench are similar between Fe/FAC and ⁵⁹Fe/FAC-treated cells. Cell number by ethidium bromide fluorescence, cell viability by Formazan absorbance and calcein fluorescent quench were assayed as described in the methods. *Significantly different relative to control (p<0.001). (D) SYPRO^® Ruby gel stain of proteins from the membrane fraction (Mb, 10 μg protein) and concentrated media samples (3 μg protein). (E) The gel in D was dried and ⁵⁹Fe assessed by autoradiography with exposure to BioMax MR film. (F) Iron-loaded ferritin in culture medium. (Lane 1) Western blot of culture medium with ⁵⁹Fe detected by autoradiography with exposure to BioMax MR film as described in the methods. (Lane 2) Ferritin subsequently detected by Western blot using anti-A. aegypti ferritin-specific rabbit serum as described in the methods. M = molecular mass markers (BIORAD). Graphed data represent means ± SEM of triplicates.

Figure 2. Iron treatment alters iron levels of CCL-125 cells

CCL-125 cells were treated with 0, 100–500 μM FAC, 200 or 500 μM FAC/DES (F/D), and 200 or 500 μM DES (D) for 18 h. (A) Iron is taken up by cells exposed to iron as measured by calcein fluorescent quench. *Significantly different relative to 0 (p<0.0001). ^aSignificantly different relative to 200 μM FAC (p<0.0001). ^bSignificantly different relative to 500 μM FAC (p<0.0001). (B) Cytoplasmic iron levels increase in response to iron exposure as determined by iron ICP-MS. *Significantly different relative to 0 (p<0.04). ^∞Significantly different relative to 100 μM FAC (p=0.054). ^aSignificantly different relative to 200 μM FAC (p<0.005). (C) Iron treatment alters membrane iron levels as determined by iron ICP-MS. *Significantly different relative to 0 (p<0.02). ^∞Significantly different relative to 100 μM FAC (p<0.0002). ^ΦSignificantly different relative to 200 μM FAC (p<0.0009). ^aSignificantly different relative to 200 μM FAC (p<0.0001). ^bSignificantly different relative to 500 μM FAC (p<0.0002). Data represent means ± SEM of triplicates.

Figure 3. Cytoplasmic, membrane and secreted ferritin increases in response to iron dose

CCL-125 cells were treated with 0, 100–500 μM FAC, 200 or 500 μM FAC/DES (F/D), and 200 or 500 μM DES (D) for 18 h. All protein samples were resolved by 17% SDS-PAGE and transferred to nitrocellulose membranes as described in the methods. (A) Ferritin and actin in cytoplasmic extracts (15 μg protein) were detected by Western blot. *Significantly different for relative to 0 (p<0.02). ^aSignificantly different relative to 200 μM FAC (p=0.0557). ^bSignificantly different relative to 500 μM FAC (p<0.04). (B) Ferritin and actin in membrane fractions (15 μg protein) were detected by Western blot. *Significantly different relative to 0 (p<0.05). ^bSignificantly different relative to 500 μM FAC (p=0.0518). (C) The medium was recovered and concentrated. Ferritin and FLAG in media samples (3 μg protein) were detected by Western blot. *Significantly different relative to 0 (p<0.0004). ^aSignificantly different relative to 200 μM FAC (p<0.003). ^bSignificantly different relative to 500 μM FAC (p<0.0006). Ferritin was detected using anti-A. aegypti ferritin-specific rabbit serum, endogenous actin was detected using anti-β-actin-specific mouse serum and exogenous FLAG was detected using anti-FLAG-specific rabbit serum. Purified A. aegypti ferritin (1 μg) was used as a ferritin positive control (+); A549 cell cytoplasmic extract (15 μg protein) served as a positive control for actin (A); purified FLAG-containing bacterial alkaline phosphatase (100 ng, Sigma) served as a positive control for FLAG (F). M = molecular mass markers (BIORAD). A representative Western blot is shown above the graphed data. Graphed data represent means ± SEM of triplicates.

Figure 4. Real-time RT-PCR analysis of mRNA for LCH and HCH

CCL-125 cells were treated with 0, 100-500 μM FAC, 200 or 500 μM FAC/DES (F/D), and 200 or 500 μM DES (D) for 18 h. Ferritin subunit homologue mRNA levels were evaluated using real-time RT-PCR with S7 ribosomal RNA as an endogenous control for total RNA as described in the methods. (A) LCH mRNA increases in a dose dependent manner. *Significantly different relative to 0 (p<0.02). ^∞Significantly different relative to 100 μM FAC (p<0.05). ^aSignificantly different relative to 200 μM FAC (p<0.02). ^bSignificantly different relative to 500 μM FAC (p<0.005). (B) HCH mRNA increases and plateaus at the lowest iron concentrations. *Significantly different relative to 0 (p<0.006). ^aSignificantly different relative to 200 μM FAC (p<0.003). ^bSignificantly different relative to 500 μM FAC (p<0.002). Graphed data represent means ± SEM of triplicates.

Figure 5. Confocal images for localization of ferritin and ceramide-containing membranes in CCL-125 cells

CCL-125 cells were treated with 0 or 100 μM FAC for 18 h. Membranes were identified with BODIPY TR C₅-ceramide (red) and ferritin with anti-A. aegypti ferritin-specific rabbit serum and visualized with Cy 2 TM conjugated goat anti-rabbit IgG (green). (AC) Cells treated with 0 μM FAC for 18 h. (D–F) Cells treated with 100 μM FAC for 18 h. White arrows identify ferritin that is present in ceramide-containing membranes (yellow, upper arrow), cytoplasm (green, middle arrow) and on the margins of the cell (green, lower arrow).

Figure 6. CI-976 reduces viability of CCL-125 cells

CCL-125 cells were concurrently treated with a chemical blocking reagent and 100 μM FAC for 18 h. (A) Cell numbers did not differ among treatment groups as determined by ethidium bromide fluorescence described in the methods. (B) Cell viability was compromised by CI-976 as measured by Formazan absorbance described in the methods. *Significantly different relative to F/E (p<0.0001). **Significantly different relative to F/E (p<0.0006). (C) Calcein fluorescent quench was decreased by CI-976 as described in the methods. *Significantly different relative to F/E (p<0.0002). FAC/EtOH (F/E) was used as control for FAC/BFA (F/B), FAC/CI-976 (F/C) and FAC/CytoD (F/CD) and FAC/HBSS (F/H) served as a control for FAC/PLTX-II (F/P). Graphed data represent means ± SEM of triplicates.

Figure 7. CI-976 blocks ferritin secretion of CCL-125 cells

CCL-125 cells were concurrently treated with a chemical blocking reagent and 100 μM FAC for 18 h. (A) Ferritin does not change in cytoplasmic extracts or the membrane fraction with CI-976 treatment. Cytoplasmic extracts (upper panel, 8 μg protein) or membrane fractions (lower panel, 5 μg protein) for each treatment were resolved by 17% SDS-PAGE and transferred to nitrocellulose membranes. Ferritin was detected by Western blot using anti-A. aegypti ferritin-specific rabbit serum and endogenous actin was detected using anti-β-actin-specific mouse serum. *Significantly different for cytoplasmic ferritin relative to F/H (p<0.05). (B) CI-976 inhibits ferritin secretion. The medium was recovered and concentrated. Media samples for each treatment were resolved either by 18.5% SDS-PAGE and transferred to nitrocellulose membranes (upper panel, 150 μL) or by 4–20% Native-PAGE (lower panel, 250 μL) and Coomassie blue stained for total protein. Ferritin was detected by Western blot using anti-A. aegypti ferritin-specific rabbit serum. *Significantly different relative to F/E (p<0.02). FAC/EtOH (F/E) was used as control for FAC/BFA (F/B), FAC/CI-976 (F/C) and FAC/CytoD (F/CD) and FAC/HBSS (F/H) served as a control for FAC/PLTX-II (F/P). Purified A. aegypti ferritin (1 μg) was used as a ferritin positive control (+); A549 cell cytoplasmic extract (15 μg protein) served as a positive control for actin (A). M = molecular mass markers (BIORAD). Representative Western blots are shown above the graphed data. Graphed data represent means ± SEM of triplicates.