Page 138 - 2022_02-Haematologica-web

L. Traeger et al.
cycline for 18 hours (h) induced expression of the fusion protein, which was detected at the cell surface (Figure 1A). The FPN-GFP fusion protein was able to export iron, as indirectly indicated by decreased levels of intracellular FTL and FTH1 after FPN-GFP induction (Figure 1B; Online Supplementary Figure S1A). Treatment with hepcidin (50 ng/mL) for 90 minutes (min) caused FPN-GFP to localize to punctate structures in the cytoplasm (Figure 1A), and treat- ment with hepcidin for 18 h caused degradation of the fusion protein (Figure 1C). Because BMP6 induces HepG2 cells to express hepcidin25 (Figure 1D), we were able to investigate the effect of gradual induction of endogenous hepcidin on ferroportin degradation. Treatment with BMP6 (10 ng/mL) for 18 h caused degradation of the FPN-GFP fusion protein as detected by indirect fluorescence and immunoblotting (Figure 1A and C). Pretreatment with chloroquine (100 μM for 2 h), an inhibitor of lysosomal degradation, prevented BMP6-mediated FPN-GFP degrada- tion and caused FPN-GFP to localize to lysosomes in the cytoplasm (Online Supplementary Figure S1B).
BMP6 induces expression of hepcidin through the BMP receptor-SMAD 1/5/8 pathway. After binding to the BMP receptor complex, activated BMP type I receptors phospho- rylate SMAD 1/5/8 proteins, which translocate together with SMAD4 to the nucleus, and induce hepcidin expres- sion.26 The siRNA-mediated inhibition of SMAD4 in HepG2-FPN-GFP cells prevented BMP6-mediated degrada- tion of FPN-GFP (Figure 1E). Taken together, these results show that the HepG2-FPN-GFP cell line expresses inducible, functional FPN-GFP. Both BMP6-induced endogenous hepcidin and exogenous hepcidin cause inter- nalization and degradation of the FPN-GFP fusion protein.
E1 enzyme UBA6 is required for ubiquitination of ferroportin
The human ubiquitin system encodes two E1 enzymes: UBA1 (also known as in UBE1) and UBA6. To identify the E1 enzyme that is involved in ferroportin degradation, HepG2-FPN-GFP cells were transfected with siRNA that targeted each of the E1 enzymes or with a control siRNA (siControl). Twenty-four hours after transfection with siRNA, cells were treated overnight with doxycycline to induce the expression of FPN-GFP and were then incubated with BMP6 for 18 h. Cells that were treated with siControl and BMP6 had decreased cell surface expression of FPN- GFP (Figure 2A). Depletion of UBA1 did not prevent the BMP6-induced localization of FPN-GFP to lysosomes and subsequent degradation. However, depletion of UBA6 pre- vented BMP6-mediated degradation of ferroportin, as indi- cated by the persistence of the FPN-GFP fusion protein at the cell surface (Figure 2A). Immunoblotting confirmed that depletion of UBA6, but not UBA1, impaired degradation of FPN-GFP (Figure 2B; Online Supplementary Figure 2A). The successful depletion of each of the E1 enzymes by the appropriate siRNA was confirmed by quantitative poly- merase chain reaction (qPCR) (Figure 2C). Successful deple- tion of UBA6 by siUBA6 was not affected by the addition of BMP6 (Online Supplementary Figure S2B).
Depletion of UBA6 might block degradation of FPN-GFP or prevent BMP6-induced expression of hepcidin. To con- sider this latter possibility, the ability of UBA6 depletion to inhibit the BMP signal transduction pathway was investi- gated. Depletion of UBA6 had no effect on BMP6-mediated phosphorylation of SMAD1/5/8 (Figure 2B). In addition, siRNA directed against UBA6 did not prevent expression of
endogenous hepcidin in HepG2-FPN-GFP cells (Figure 2D). In contrast, siRNA directed against SMAD4 blunted BMP6- induced expression of hepcidin.
To further demonstrate that depletion of UBA6 blocks degradation of ferroportin, independent of an effect on BMP-induced expression of hepcidin, the effect of exoge- nous hepcidin on the cellular localization of FPN-GFP in HepG2 cells was investigated. To determine the amount of hepcidin produced by HepG2 cells after treatment with BMP6, HepG2 cells were incubated with BMP6 (10 ng/mL) for 18 h and the amount of hepcidin in the tissue culture medium was measured by enzyme-linked immunosorbant assay (ELISA). Under these conditions, BMP6 induced 3.9 ng/mL (± 0.4 ng/mL) of hepcidin, and this concentration of hepcidin (rather than the much higher dose of 30-50 ng/mL used in other studies10,24) was used to treat cells in subse- quent experiments. Cells were transfected with siControl, siUBA1, or siUBA6 and treated overnight with hepcidin (4 ng/mL). In the presence of this low concentration of hep- cidin, FPN-GFP expression at the cell surface persisted in UBA6-depleted cells but not in siControl-treated- or siUBA1-treated cells (Figure 2E). The inability of hepcidin to degrade the FPN-GFP fusion protein in siUBA6 treated cells was confirmed by immunoblot (Online Supplementary Figure S2C). Taken together, these results show that UBA6 is required for hepcidin induced internalization and degrada- tion of FPN-GFP.
The adaptor protein NDFIP1 regulates ferroportin degradation
To identify additional components of the ubiquitin path- way that might be involved in ferroportin degradation, siRNA directed against different E2 and E3 enzymes, as well as other known components of the ubiquitin pathway, were tested for the ability to inhibit BMP6-mediated degra- dation of FPN-GFP (Online Supplementary Table S1, n=77). A commercially available library (Dharmacon, Lafayette, CO, USA), which contains siRNA that were previously verified to silence the corresponding targets and to minimize off-tar- get effects, was used in these studies. HepG2 cells transfect- ed with siRNA directed against SMAD4 were used as pos- itive controls for inhibition of BMP6-mediated degradation of FPN-GFP; siControl was used as a negative control. Eighteen hours after treatment with BMP6, the localization of FPN-GFP was determined by immunofluorescence. In the first screen, we identified 23 siRNA directed against dif- ferent E2 and E3 enzymes that appeared to block FPN-GFP relocalization to the lysosome based on FPN-GFP persist- ence at the cell surface after BMP6 treatment. In second and tertiary screens, all positive candidates were re-evaluated to exclude false positives.
Depletion of each of three E2 enzymes, UBE2R2, UBE2E2 and UBE2J2 partially blocked the internalization of FPN- GFP (Online Supplementary Figure S3A and B), while deple- tion of other, individual E2 enzymes did not impair BMP6- mediated FPN-GFP degradation (data not shown). Treatment with pairwise combinations of UBE2R2, UBE2E2 and UBE2J2 or all three of the E2 enzymes did not further prevent the degradation of ferroportin (data not shown), suggesting that additional E2 enzymes participate in FPN ubiquitination.
In an initial screen, depletion the NEDD family interact- ing protein NDFIP1 and the E3 enzyme ARIH1 impaired BMP6-induced FPN-GFP localization to lysosomes and sub- sequent degradation of the fusion protein (Figure 3A).
480
haematologica | 2022; 107(2)