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Regulation of ferroportin degradation
of transporters of small molecules, which includes more than 300 membrane-bound proteins.11,12 Ferroportin is pre- dominantly expressed in tissues associated with iron trans- port including enterocytes, hepatocytes, macrophages and erythrocytes.10,11,13 The protein has 12 membrane-spanning domains, which create a channel through which iron is transported. Binding of hepcidin to the main extracellular cavity of ferroportin causes ligation of ubiquitin molecules to multiple intracellular lysine residues.14 Polyubiquitination of ferroportin induces the internalization of the hepcidin- ferroportin complex followed by degradation within lyso- somes.10,14,15 Degradation of ferroportin results in decreased serum iron, because enterocytes, hepatocytes and macrophages are no longer able to transfer intracellular iron to the circulation.16 In addition to the ability of hepcidin to induce degradation of ferroportin, the hormone is also able to inhibit iron export by directly occluding the iron channel.17 Occlusion of the channel may be especially important for cells, such as mature red blood cells, which lack the endocytic machinery required to degrade ferro- portin.17
Ubiquitin is a 76 amino acid polypeptide that can be attached to lysine residues in proteins. The attachment of ubiquitin to a protein regulates the protein’s localization, stability and/or activity.18 The process of ubiquitination involves the activation and transient conjugation of ubiqui- tin to a carrier protein, with subsequent final ligation of the ubiquitin molecule to a substrate. In general, ubiquitination requires three different kinds of enzymes: a ubiquitin acti- vating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3). The human ubiquitin system encodes two different E1 enzymes (UBA1 and UBA6), approximately 50 different E2 enzymes, and more than 600 E3 enzymes.19–21 The ubiquitin E3 ligases are important for substrate recognition and are divided into three different classes.22 Depending on the class of ligase, the E3 enzyme either directly transfers ubiquitin to a substrate (“HECT” and “RBR” ligases) or acts as an adaptor to facilitate the transfer of ubiquitin from an E2 enzyme directly to the sub- strate (“RING” E3 ligases).22,23 Binding of the E3 enzyme to the substrate may also require an adaptor protein that acts as a scaffold between the E3 enzyme and the target protein.
In this study, an in vitro small interfering RNA (siRNA) screen was performed to determine which proteins in the ubiquitin pathway are involved in ferroportin degradation. A previous study used a modified HEK293 cell line, in which expression of ferroportin was induced by the addi- tion of ponasterone.24 Exogenous hepcidin and putative inhibitors of ferroportin degradation were added to this cell line and the level of ferroportin was then measured. To per- mit screening for specific enzymes involved in ferroportin ubiquitination without using exogenous hepcidin, we established a HepG2 cell line that expresses the ferroportin- green fluorescent protein (FPN-GFP) fusion protein in response to doxycycline. In this cell line, BMP6 can be used to gradually induce the expression of endogenous hepcidin. The HepG2-FPN-GFP cell line was used to show that the alternative E1 enzyme UBA6 as well as the NEDD4 family binding protein NDFIP1 are able regulate the degradation of ferroportin in response to BMP6, as well as exogenous hep- cidin. Depletion of the E3 ligase ARIH1 indirectly inhibited ferroportin degradation by impairing BMP6-mediated hep- cidin induction. In vivo, depletion of Ndfip1 in the murine liver increased the level of hepatic ferroportin and increased circulating iron.
Methods
HepG2-FPN-GFP cell line
A plasmid encoding human ferroportin (NM_016917) fused to GFP was a gift from Tomas Ganz (David Geffen School of Medicine, UCLA).14 DNA encoding the fusion protein was ligated into the NheI and NotI sites of plasmid pTRE2hyg (Clontech cat#631014). The plasmid was transfected into the HepG2 “tet-on advanced” cell line (Clonetech cat#630932) using Effectene trans- fection reagents (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions and successfully transfected cells were selected using hygromycin (0.4 mg/mL). Individual cell lines were established and were confirmed to express FPN-GFP in the presence of doxycycline (2 μg/mL). One colony, which expressed a high level of FPN-GFP after treatment with doxycycline, was selected for further experiments. HepG2-FPN-GFP cells were maintained in Eagle Minimum Essential Medium, 10% FBS, L-glu- tamine (2 mM), G418 (100 ng/mL), hygromycin (0.4 mg/mL), penicillin (100 units/mL), and streptomycin (100 μg) at 37°C in 5% CO2 and 95% humidity.
Adeno-associated virus administration
All experiments using mice were approved by the Partners Subcommittee on Research Animal Care at Massachusetts General Hospital, Boston, MA, USA (Protocol # 2007N000052). Wild-type mice on a C57BL/6J background were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Animals were fed a standard diet (380 ppm iron). Adeno-associated virus (AAV) short hairpin RNA (shRNA) AAV2/8-GFP-U6-m-Ndfip1-shRNA (AAV2/8-shNdfip1) and AAV2/8-GFP-U6-scrmb-shRNA (AAV2/8- shControl) were obtained from Vector BioLabs (Malvern, PA, USA). Eight-week-old male mice were injected intravenously with 1x1011 particles of AAV2/8-shNdfip1 or AAV2/8-shControl via the tail vein. Six weeks later, mice were anesthetized with 4% isoflu- rane and whole blood was collected by cardiac puncture. Liver and spleen were harvested for further analysis.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.3.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± standard deviation (SD). The Shapiro-Wilk test was performed to test for normality. Correlation analysis were performed using Pearson correlation. Comparison of two groups was performed using the Student’s t-test for parametric data and the Mann-Whitney-U test for non-parametric data. Comparison of more than two groups was performed using one-way ANOVA with Tukey post hoc test (for parametric data) or the Kruskal-Wallis test with Dunn’s post hoc test (for non-parametric data). After adjusting for multiple comparisons, a P value <0.05 was consid- ered statistically significant.
A detailed description of other methods can be found in the Online Supplementary Appendix.
Results
Preparation and characterization of the HepG2-FPN- GFP cell line
Binding of hepcidin to ferroportin induces the polyubiq- uitination, internalization and lysosomal degradation of the ligand-channel complex.10 To identify the specific enzymes that mediate ubiquitination of ferroportin, we established a stable HepG2 cell line that inducibly expresses FPN-GFP (HepG2-FPN-GFP) in the presence of doxycycline. Treatment of HepG2-FPN-GFP cells with 2 μg/mL of doxy-
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