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Regulation of ferroportin degradation
absence of exogenous hepcidin for 20 min, and protein lysates were immunoprecipitated with an antibody direct- ed against NDFIP1. In the absence of hepcidin, a small amount of FPN-GFP was detected in the immunoprecipitat- ed protein lysate. Treatment with hepcidin caused an increase in the amount of FPN-GFP that co-immunoprecip- itated with NDFIP1 (Figure 3E). Taken together, the results suggest that NDFIP1 interacts with FPN and is involved in hepcidin-induced FPN internalization and degradation.
A second adaptor protein (NDFIP2), which like NDFIP1 facilitates ubiquitination by HECT E3 enzymes, shares 79% similarity with NDFIP127. To investigate the potential role of NDFIP2 in the regulation of FPN, the effect of NDFIP2 depletion on BMP6-induced FPN-GFP degradation was assessed. While siNDFIP1 treatment prevented degra- dation of FPN-GFP, depletion of NDFIP2 had no effect on BMP6-mediated degradation of the FPN-GFP (Figure 3F; Online Supplementary Figure S3D).
NDFIP1 recruits members of the NEDD4 family of E3 lig- ases to target proteins.28 To investigate whether NEDD4 family members (NEDD4, NEDD4L, ITCH, WWP1, WWP2, SMURF1, SMURF2, HECW1, HECW229) regulate FPN levels, the localization of FPN-GFP in cells treated with siRNA directed against each of these enzymes was exam- ined. None of the siRNA directed against members of the NEDD4 family, either alone or in pair-wise combinations, prevented BMP6 mediated FPN-GFP degradation (Online Supplementary Figure S4A and B). These results indicate that either more than two of these enzymes are involved in BMP6-induced FPN degradation or additional, as yet unidentified, enzymes are able to interact with NDFIP1 and mediate FPN degradation.
ARIH1 indirectly regulates ferroportin by inhibiting BMP6-mediated induction of hepcidin
ARIH1 is a member of the Ariadne family of RBR E3 lig- ases. Treatment of HepG2-FPN-GFP cells with siRNA directed against ARIH1 inhibited BMP6-mediated degrada- tion of FPN-GFP (Figure 3A). ARIH1 was successfully depleted by transfection of siARIH1 in both the absence (Figure 4A) and the presence of BMP6 (Online Supplementary Figure S4C), as determined by qPCR. The addition of low dose exogenous hepcidin to HepG2-FPN-GFP cells, howev- er, reduced the level of FPN-GFP on the surface of ARIH1- depleted cells (Figure 4B). The ability of exogenous hepcidin to degrade FPN-GFP in siARIH1 treated cells was confirmed by immunoblot (Online Supplementary Figure S2C). We con- sidered the possibility that depletion of ARIH1 inhibits FPN degradation by interfering with the ability of BMP signaling to induce hepcidin gene expression. In the absence of BMP6, the depletion of ARIH1 reduced basal hepcidin mRNA levels (Figure 4C). Depletion of siARIH1 impaired BMP6-stimulated induction of hepcidin mRNA by 80% (Figure 4D). ARIH1 depletion also inhibited BMP6-mediat- ed induction of ID1, another target of the BMP signaling pathway (Figure 4E). Interestingly, BMP6-induced phos- phorylation of SMAD1/5/8 proteins was not affected by ARIH1 depletion (Figure 4F). These results suggest that ARIH1 has an indirect effect on the stability of FPN by alter- ing BMP6- mediated hepcidin induction through a non- canonical pathway.
The Ariadne RBR E3 ligase ARIH2 (also known as TRIAD1) is the closest relative to ARIH1 with 54% similar- ity.30 To consider the possibility that this second member of the Ariadne family is involved in the indirect regulation of
FPN, the effect of ARIH2 depletion on BMP6-induced FPN- GFP degradation was assessed. In contrast to ARIH1, deple- tion of ARIH2 had no effect on BMP6-mediated degrada- tion of the FPN-GFP protein expression (Figure 4F; Online Supplementary Figure S4D).
Silencing of Ndfip1 stabilizes hepatic ferroportin
in vivo
The adaptor protein NDFIP1 was identified as a protein that is involved in FPN degradation in vitro. To address whether NDFIP1 is important for FPN degradation in vivo, mice were injected with an AAV2/8 encoding a shRNA directed against Ndfip1, under the control of a U6 promoter. The AAV serotype 8 was used in these studies because it has a high efficiency of transduction in hepatocytes.31 In both AAV2/8-shNdfip1 and AAV2/8-shControl injected ani- mals, GFP expression was detected in the liver, indicating successful systemic administration of the virus (Figure 5A). In animals injected with AAV2/8-shNdfip1, hepatic Ndfip1 mRNA levels were significantly reduced compared to con- trol animals (Figure 5B). Mice injected with AAV2/8- shNdfip1 had a 3-fold increase in FPN protein level in the liver compared to control mice (Figure 5C and D). Hamp mRNA and serum hepcidin levels were similar in both groups, suggesting that higher FPN levels were not caused by induction of hepcidin (Figure 5E and F). Increased hepat- ic FPN was associated with a 28% increase in serum iron levels in AAV2/8-shNdfip1, compared to AAV2/8-shControl, mice (Figure 5G) and there was a correlation between serum iron and FPN levels (Online Supplementary Figure S5A). Hepatic FTL levels were increased and TfR1 mRNA was decreased in AAV2/8-shNdfip1-treated mice (Online Supplementary Figure S5B and D). As expected because of the targeting of AAV8 to the liver,32 splenic Ndfip1 mRNA levels were not decreased in AAV2/8-shNdfip1 mice (Online Supplementary Figure S5E). The results show that the AAV2/8-mediated depletion of Ndfip1 increases the level of hepatic FPN and that Ndfip1 is required for FPN degrada- tion in the liver.
Discussion
This study identified components of the ubiquitin system that are important for FPN degradation. A HepG2 cell line that inducibly expresses functional FPN-GFP fusion protein was established. BMP6-induced expression of hepcidin, which caused the internalization and degradation of the fusion protein and permitted analysis of FPN degradation under conditions in which the level of hepcidin increases gradually. In vitro, the alternative E1 enzyme UBA6, as well as the adaptor protein NDFIP1, were critical for hepcidin- induced FPN degradation. Depletion of either UBA6 or NDFIP1 inhibited hepcidin-induced internalization and degradation of FPN-GFP. The E3 ligase ARIH1 indirectly regulated FPN stability by altering BMP6-mediated hep- cidin induction through a non-canonical pathway. In vivo, the depletion of Ndfip1 in the murine liver increased the level of hepatic FPN and increased circulating iron.
In 2007, UBA6 was identified as a second ubiquitin acti- vating E1 enzyme. UBA1 and UBA6 have non-redundant functions and each enzyme is essential for biological processes.33,34 UBA6 is widely expressed in different tissues but contributes to only approximately 1% of overall cellular ubiquitination.33,35 In addition to activating ubiquitin for
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