The neogenin-hemojuvelin axis in murine liver is crucial for hepcidin expression

cDNA constructs.

Murine Neo1 ORF (NM_008684) and murine deleted in colorectal cancer (Dcc) ORF (NM_007831), each containing a C-terminal FLAG/MYC epitope (fNeo1 and fDcc, respectively), were purchased in pCMV6 vectors (#MR226235 and #MR226661; OriGene Technologies). Sequence integrity was validated by DNA sequencing. The fDcc construct was subcloned into an AAV8 construct containing a strong liver-specific promoter, as described previously (54). This promoter consists of two copies of the human α1-microglobulin/bikunin enhancer combined with the promoter from the human thyroid hormone-binding globulin gene. AAV8 constructs containing N-terminal 3xFLAG-tagged Hjv (fHjv) or the fHjv^A183R mutant were generated previously (31). All AAV8 viral vectors were produced at the Molecular Virology Support Core, OHSU.

Animal studies.

All animal procedures were approved by OHSU/Institutional Animal Care and Use Committee. Homozygous hepatocyte-specific Neo1^−/− mice (Neo1^fl/fl;Alb-Cre⁺) on a C57BL/6J background were generated in our previous studies (24). Global Hjv^−/− mice on a 129/SvEvTac (129/S) background were obtained from Dr. Nancy Andrews (Duke University), and Hjv^−/− mice on a C57BL/6J background were generated via backcrossing for ten generations. To obtain experimental groups, Neo1-Hjv double-mutant (Neo1^fl/fl;Alb-Cre⁺;Hjv^−/−), Neo1 single-mutant (Neo1^fl/fl;Alb-Cre⁺;Hjv^+/+), Hjv single-mutant (Neo1^fl/fl;Alb-Cre⁻;Hjv^−/−), and wild-type (Neo1^fl/fl;Alb-Cre⁻;Hjv^+/+) littermates were generated by crossing Neo1^fl/fl;Alb-Cre^−/−;Hjv^−/− male with Neo1^fl/fl;Alb-Cre^+/−;Hjv^+/+ female on a uniform C57BL/6J background. All mice were fed a PicoLab Laboratory Rodent Diet-5L0D containing 240-ppm iron (LabDiet). Mice of both genders were euthanized for analysis at the ages indicated in the figure legends.

To evaluate the function of the fHjv^A183R mutant, eight-week-old Hjv^−/− male mice (129/S background) were intraperitoneally injected with sterile PBS, AAV8-fHjv, or fHjv^A183R at doses of ~5× 10¹¹ and/or ~10¹³ genome-particles per mouse. To study the function of Dcc, eight-week-old Neo1^−/− male mice (C57BL/6J background) were intraperitoneally injected with sterile PBS or AAV8-fDcc at ~0.29× 10¹² or ~1.35×10¹² genome-particles per mouse. All mice were euthanized for analysis three weeks post-administration. Blood was collected via cardiac puncture for serum iron assays. Livers were rapidly removed, snap-frozen in liquid nitrogen, and stored at −80°C for qRT-PCR and western blot analysis. Age- and gender-matched wild-type, Hjv^−/−, and Neo1^−/− mice were included as additional controls.

Blood parameters and serum iron assays.

Blood parameters were analyzed using a Hemavet 950 automated analyzer (Drew Scientific). Serum iron concentrations were measured using the Pointe Iron/TIBC Reagent Set (Pointe Scientific).

Tissue nonheme iron assays.

Tissue nonheme iron levels were determined as previously described (49) with the following modifications: 50–150 mg of wet tissue was digested in 250–750 μL of acid buffer at 65°C for 72 hrs. The supernatant was collected by centrifugation at 10,000 x g for 5 min, followed by the addition of chromogen (1.86 mM bathophenanthroline sulfonate and 143 mM thioglycolic acid in water). Absorbance was measured at 535 nm using an xMark Microplate Spectrophotometer (Bio-Rad). Each sample was analyzed in triplicate. Iron concentrations are expressed as micrograms of iron per gram (μg/g) of wet tissue.

qRT-PCR.

Total RNA was extracted from mouse liver tissues using a NucleoSpin RNA kit (Macherey-Nagel). cDNA was synthesized using Oligo deoxythymidine primers (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). qRT-PCR analysis of Neo1, Hjv, Hamp (hepcidin), Id1, Dcc, IL-6, and β-actin transcripts was performed in triplicates for each sample using Power SYBR Green PCR master mix (Applied Biosystems) on a QuantStudio 7 Flex qPCR instrument (ThermoFisher) at the OHSU Integrated Genomics Laboratory. All primer sets (Supplemental Tables S1) were validated against the reference gene (β-actin) to ensure comparable amplification efficiencies. Results are expressed as mRNA levels relative to β-actin for each sample.

Cell line and transfection.

Hep3B cells were obtained from ATCC (#HB-8064). Cells were transiently transfected with pCMV6-fDcc, pCMV6-fNeo1, or an empty vector using Lipofectamine-3000 (Invitrogen) to assess the cell surface localization of fDcc via biotinylation and α/γ-secretase cleavage using specific inhibitors. The inhibitors used in the studies include α-secretase inhibitor, Aderbasib (INCB007839; #HY-10293; MedChemExpress); γ-secretase inhibitors, DAPT (D5942; Sigma-Aldrich) and LY450139 (SML1938; Sigma-Aldrich); and proteasome inhibitor, MG132 (#47490; EMO Millipore Corp). Biotinylation of cell surface proteins was performed on ice. Approximately 64 h post-transfection, Hep3B cells were washed three times with ice-cold PBS+ buffer (PBS supplemented with 0.5 mm CaCl₂ and 1 mm MgCl₂) and incubated with 0.25 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in PBS+ buffer for 30 min. The reaction was quenched by washing the cells five-times with ice-cold PBS+ buffer containing 0.1% glycine (pH 7.6). Cells were solubilized, and the resulting lysate was cleared by centrifugation at 16,000 x g for 5 min. Biotinylated proteins were isolated using streptavidin resin (G Biosciences), eluted with 1x Laemmli buffer, and subjected to SDS-PAGE and immunodetection using antibodies against NEO1, Dcc, FLAG, Na⁺K⁺ ATPase, and β-actin.

To detect α-secretase-cleaved fDcc or fNeo1 ectodomains in the conditioned medium, the complete medium (MEM/10% FBS) was replaced with Opti-MEM/1% FBS at 46 h post-transfection. Cells were then incubated in the presence or absence of 25 μM Aderbasib for an additional 18 h prior to collection. Proteins in ~600 μL of conditioned medium were precipitated using 6% TCA, and the resulting protein pellets were resuspended in 1x Laemmli buffer for SDS-PAGE and immunodetection. To detect γ-secretase-cleaved fDcc-intracellular domain (fDcc-ICD), Hep3B cells were transiently transfected with the pCMV6-fDcc construct. At approximately 63 h post-transfection, cells were incubated with 10 μM MG-132 and 10 μM DAPT or 10 μM LY450139 for 7 h. Approximately 90% of the cell lysate was subjected to pull-down using anti-FLAG affinity gel (Sigma #A2220), followed by elution using the 3xFLAG peptide at ~200 μg/ml (Sigma F4799). The eluted proteins and ~10% of the input lysate were then subjected to SDS-PAGE and immunodetection using specific antibodies.

Immunodetection.

Liver membrane fractions were prepared as previously described (55). Protein extracts (~200 μg) from the liver membrane preparations were separated via SDS-PAGE (11%) under reducing conditions. Endogenous Neo1 and β-actin were detected by using rabbit anti-human NEO1-ectodomain (40), mouse anti-β-actin (Sigma), and the corresponding secondary antibodies. Transduced fHjv and fHjv^A183R were detected using an HRP-coupled mouse anti-FLAG M2 IgG (Sigma). Transduced fDcc was detected using a goat anti-mouse Dcc antibody (#AF844; R&D Systems) and donkey anti-goat IgG Alexa Fluor Plus 680 (Invitrogen). All images were captured by using the c600 western blot imaging system (Azure Biosystems, Inc). The intensities of specific bands were quantified by using the ImageJ software. Protein levels are expressed relative to β-actin.

Statistical analysis.

Statistical analysis was performed using GraphPad Prism 11. For multiple comparisons, one-way ANOVA followed by Tukey’s post hoc test was used. A two-tailed t-test was used to compare two data sets. All data are presented as mean ± SD. A P value <0.05 was considered statistically significant.

Hjv is a more potent inducer of hepcidin expression than hepatic Neo1.

The genetic background of mice significantly impacts systemic iron load (56, 57). To determine the relative contributions of hepatic Neo1 and Hjv to hepcidin expression, we utilized hepatocyte-specific Neo1^−/− (Neo1^fl/fl;Alb-Cre⁺;Hjv^+/+) and global Hjv^−/− (Neo1^fl/fl;Alb-Cre⁻;Hjv^−/−) mice on a uniform C57BL/6J background to avoid confounding genetic background effects. Hepatic hepcidin (Hamp) mRNA levels in eight-week-old mice of both genders were analyzed via qRT-PCR analysis and compared to age-matched wild-type (Neo1^fl/fl;Alb-Cre⁻;Hjv^+/+) controls. Earlier studies showed no sex-related differences in hepatic Hamp mRNA levels in mice on a C57BL/6J background (58). As expected, significant decreases in Neo1 and Hjv mRNA levels confirmed successful knockout in the respective models (Figure 1A/B). In the liver, Hjv is predominantly expressed in hepatocytes, whereas Neo1 is found in both hepatocytes and non-parenchymal cells (47, 48). The modest Neo1 mRNA levels detected in the liver of Neo1^−/− mice (Figure 1A) likely originate from these nonparenchymal cells. Consistent with previous studies (24, 48, 49), qRT-PCR analysis revealed that Hamp mRNA levels were significantly reduced in both Neo1^−/− and Hjv^−/− mice compared with wild-type controls (Figure 1C). Notably, Hjv^−/− mice exhibited a more pronounced reduction than Neo1^−/− mice, retaining approximately 3.53% and 15.71% of the Hamp mRNA levels observed in wild-type controls, respectively (Figure 1C). These results suggest that Hjv is a more potent inducer of hepcidin expression than hepatic Neo1.

As expected, reduced hepcidin expression in Neo1^−/− and Hjv^−/− mice resulted from impaired Bmp-signaling, as evidenced by a parallel decrease in hepatic Inhibitor of DNA binding-1 (Id1) mRNA (Figure 1D). Id1 is a direct downstream target and a sensitive indicator of Bmp signaling status. Because hepatic Hamp mRNA levels correlate closely with serum hepcidin concentrations (59–61), this reduction caused severe systemic iron overload in both knockout models (Figure 1E/F). This manifests as marked increases in serum iron concentrations and liver nonheme iron levels, the latter of which serves as a reliable indicator of total body iron stores. Mechanistically, low hepcidin promotes iron efflux into the circulation through ferroportin in the duodenum. Although the difference in liver nonheme iron accumulation between Neo1^−/− and Hjv^−/− mice was not statistically significant, the latter showed a greater accumulation relative to wild-type controls (8.7-fold vs 9.98-fold increase; Figure 1F). These findings align with the more pronounced reduction in Hamp mRNA observed in Hjv^−/− mice (Figure 1C).

Given that hepatic hepcidin expression relies heavily on the Neo1-Hjv interaction (24, 31) and that Neo1^−/− and Hjv^−/− mice displayed respective decreases of approximately 84.29% and 96.47% of hepatic Hamp mRNA levels, respectively (Figure 1C), our results suggest that this Neo1-Hjv association accounts for approximately 84% of total expression. The remaining ~12% of Hjv-mediated hepcidin induction (reflecting the difference between 84.29% and 96.47%) likely proceeds through a Neo1-independent pathway.

Neogenin and hemojuvelin in murine liver constitute a crucial axis to induce hepcidin expression, acting primarily through their interaction.

To investigate the extent to which Hjv and Neo1 induce hepcidin independently, we generated hepatic Neo1-Hjv double-mutant mice (Neo1^fl/fl;Alb-Cre⁺;Hjv^−/−; hereafter Neo1^−/−;Hjv^−/−) on a C57BL/6J background. At eight weeks of age, these mice showed no visible phenotypic abnormalities or changes in body weight compared with their wild-type counterparts (Supplemental Figure S2A). We reasoned that if combined hepatic Neo1 and Hjv ablation further reduced hepatic hepcidin expression compared to Hjv^−/− mice, it would indicate that both Hjv and hepatic Neo1 possess distinct mechanisms for inducing hepcidin that are independently of their physical interaction.

Hepatic Hamp mRNA levels and body iron load were analyzed in eight-week-old mice of both sexes and compared with age, sex, and background-matched wild-type, Neo1^−/−, and Hjv^−/− animals. Simultaneous ablation of both hepatic Neo1 and Hjv resulted in a further reduction in Hamp mRNA compared to Hjv^−/− mice (Figure 2A–C). Notably, the dual deficiency of hepatic Neo1 and Hjv resulted in a ~99.70% reduction in hepatic Hamp mRNA levels, representing a nearly complete abolition of expression. These results suggest that hepatic Neo1 retains a Hjv-independent mechanism to induce hepcidin expression, albeit to a limited extent (~3.23%). This is calculated from the difference between ~3.53% in Hjv^−/− mice and ~0.30% in Neo1^−/−;Hjv^−/− mice (Figure 2C). Additionally, a slight but non-significant reduction in hepatic Id1 mRNA levels was observed in Neo1^−/−;Hjv^−/− mice relative to Hjv^−/− mice (Figure 2D), raising the possibility that this Neo1-induced hepcidin expression may also be mediated through the Bmp signaling pathway.

Elevated body iron load typically causes progressive iron deposition in the pancreas and heart, in addition to the liver (48, 49). Consistent with the lower Hamp mRNA levels in Neo1^−/−;Hjv^−/− mice, we detected trending increases in liver nonheme iron levels and significant elevations in iron accumulation in the pancreas and heart compared to single-mutant counterparts (Figure 2F–H). Since the majority of the body iron is found in the hemoglobin of red blood cells (2), we measured hematological parameters and detected significant increases in hemoglobin, hematocrit, and mean corpuscular volume in Neo1^−/−;Hjv^−/− mice (Supplemental Figure S2B-E). These results rule out the possibility that the elevated tissue iron load in Neo1^−/−;Hjv^−/− mice is due to impaired iron utilization for erythropoiesis; rather, the elevated erythroid parameters are likely derived from an increased systemic iron supply.

Taken together, the above data suggest that the hepatic Neo1/Hjv axis accounts for approximately 99% of hepcidin expression. Within this axis, ~84% of hepcidin expression is specifically attributable to the Neo1-Hjv interaction, while approximately 12% and 3% of hepcidin expression are likely derived from Hjv- and Neo1-specific pathways, respectively, acting independently of their interaction.

Overexpression of Hjv^A183R, which does not bind Neo1, indicates that Hjv can induce hepcidin independently of Neo1 association.

To gain insight into the mechanism by which Hjv induces hepcidin independently of Neo1, we compared the effects of Hjv and its mutant, Hjv^A183R, on hepcidin expression and Neo1 levels in Hjv^−/− mice. The alanine-to-arginine substitution at residue-183 in murine Hjv disrupts the Hjv-Neo1 interaction without affecting Hjv trafficking to the plasma membrane in transfected cells (Figure 3A) (31, 33). Because no commercial antibody can sensitively detect endogenous Hjv in murine liver, a FLAG epitope was engineered onto the N-terminus of Hjv and Hjv^A183R constructs (fHjv and fHjv^A183R; Figure 3B) for immunodetection. The addition of the N-terminal FLAG tag does not interfere with Hjv-mediated induction of hepcidin (24, 31). Both fHjv and fHjv^A183R were expressed in the livers of Hjv^−/− mice via an AAV8 viral vector under a hepatocyte-specific promoter. Previous studies demonstrated that AAV8-mediated transduction results in hepatocyte-specific expression with a homogeneous distribution throughout the liver, similar to native Neo1 and Hjv mRNA (47, 62, 63). Administration of the AAV8 vector alone had no effect on iron homeostasis (64, 65).

Consistent with our earlier observations (31), expressing fHjv mRNA at levels comparable to those of endogenous Hjv mRNA in wild-type mice (Figure 3C) fully corrected low Hamp mRNA levels in Hjv^−/− mice (Figure 3D). In contrast, expression of similar levels of fHjv^A183R mRNA resulted in only a modest increase (~15%) of Hamp mRNA relative to fHjv (Figure 3C/D). However, a full correction of hepcidin expression was achieved when fHjv^A183R was overexpressed at ~30.62-fold higher mRNA levels than those in wild-type mice and ~24.15-fold higher protein levels than fHjv (Figure 3C–F). The relatively faster migration of fHjv^A183R in immunoblots (Figure 3E; lanes 7–9 versus 10–15) is likely due to changes in charge caused by the amino acid substitution, as previously reported for TfR1^T104D mutant (66). The lack of changes in IL-6 mRNA levels ruled out inflammation as a cause for this increase (Figure 3G). Instead, parallel increases in Id1 mRNA levels suggest that hepcidin induction occurred via the Bmp-signaling pathway, consistent with fHjv (Figure 3H). As predicted, the increased hepcidin expression following fHjv^A183R overexpression reduced serum iron concentrations (Figure 3I). Together, these observations suggest that in the absence of Neo1 association, Hjv can still induce hepcidin expression via the Bmp-signaling pathway, though with significantly reduced efficiency.

We next tested whether hepcidin induction by fHjv^A183R overexpression correlates with increased levels of membrane-associated Neo1-ECD/TMD in the liver, similar to wild-type fHjv. In agreement with earlier studies (24), two distinct Neo1 bands were detected in liver membrane preparations from wild-type mice using an antibody against the human NEO1 ectodomain (Figure 3E; top panel, lanes 1–3). The upper band represents full-length Neo1 (Neo1), while the lower band corresponds to Neo1-ECD/TMD, likely derived from γ-secretase cleavage (Figure 3J) (37). Ablation of Hjv led to a marked decrease in hepatic Hamp mRNA and Neo1-ECD/TMD levels (Figure 3C/D; Figure 3E, top panel, lanes 4–6; Figure 3K). Lack of Hjv does not alter hepatic Neo1 mRNA levels (24). However, in contrast to the full restoration of Hamp mRNA levels and the substantial normalization of Neo1-ECD/TMD levels in Hjv^−/− mice via fHjv expression, overexpression of fHjv^A183R rescued Hamp mRNA expression but failed to alter Neo1-ECD/TMD levels (Figure 3C/D; Figure 3E, top panel, lanes 7–9 vs 13–15; Figure 3K). This could be due to the disrupted interaction between fHjv^A183R and Neo1. The relatively faint β-actin bands for the high fHjv^A183R group (Figure 3E; lowest panel; lanes 13–15) were likely due to the interference from strong fHjv^A183R signals, which were detected by an HRP-conjugated anti-FLAG antibody prior to β-actin detection. Taken together, these data suggest that Hjv can induce hepcidin expression through both Neo1-dependent and Neo1-independent mechanisms, both of which facilitate the Bmp-signaling pathway. However, the latter is significantly less efficient.

Studies of Hjv heterozygous mice indicate that Hjv induces hepcidin expression with low efficacy in the absence of hepatic Neo1.

Although the Neo1-independent pathway is less efficient, its low efficacy underscores the importance of the Neo1-Hjv interaction for optimal hepcidin expression. Previous studies have shown that Hjv^+/− mice are indistinguishable from wild-type mice regarding hepcidin expression and body iron levels (48, 49), suggesting that Hjv is not a limiting factor when hepatic Neo1 is present. We next compared hepatic Hamp mRNA levels between Neo1^−/−;Hjv^+/− and Neo1^−/−;Hjv^+/+ mice, both of which lack hepatic Neo1. We reasoned that if Hjv heterozygosity further reduces the already low hepcidin expression in these mice, it would support a critical role for hepatic Neo1 in facilitating Hjv function.

Eight-week-old mice of both genders on a C57BL/6J background were analyzed. Consistent with earlier observations (48, 49), deletion of a single copy of the Hjv allele in Neo1^+/+;Hjv^+/− mice had no significant impact on Hamp mRNA, Id1 mRNA, serum and tissue iron levels, or red blood cell parameters (Figure 4A–L). However, in the absence of hepatic Neo1, the heterozygous mutation of Hjv (Neo1^−/−;Hjv^+/−) resulted in a ~4.6-fold reduction in hepatic Hamp mRNA levels compared to Neo1^−/−;Hjv^+/+ counterparts (Figure 4A–C; Supplemental Figure S3). The approximate 10.8% of decrease in Id1 mRNA in Neo1^−/−;Hjv^+/− mice versus Neo1^−/−;Hjv^+/+ mice (reflecting the difference between 42.4% and 53.2%; Figure 4D) suggests that this reduction in Hamp mRNA resulted from impaired Bmp signaling. The lack of a statistically significant difference in Id1 mRNA levels between Neo1^−/−;Hjv^+/− and Neo1^−/−;Hjv^+/+ mice (Figure 4D) could be attributed to the sensitivity limit of qRT-PCR. In line with the modest decrease in Hamp mRNA, we detected a trend toward increased liver nonheme iron, alongside significant increases in pancreatic and cardiac iron in Neo1^−/−;Hjv^+/− mice (Figure 4F–H). The lack of change in hemoglobin, hematocrit, and mean corpuscular volume in these mice (Figure 4J–L) indicates no impairment of iron utilization for erythropoiesis. Additionally, comparable decreases in hepatic Hamp mRNA were detected in 5-week-old Neo1^−/−;Hjv^+/− mice compared to their corresponding Neo1^−/−;Hjv^+/+ controls, but not between 5-week-old Neo1^+/+;Hjv^+/− and Neo1^+/+;Hjv^+/+ mice (Supplemental Figure S4). Together, these data indicate that in the absence of hepatic Neo1, Hjv becomes a limiting factor for hepcidin induction, suggesting that hepatic Neo1 deficiency markedly impairs the efficacy of Hjv-mediated hepcidin expression.

Overexpression studies of the Neo1 homologue, Dcc, imply that hepatic Neo1 retains residual hepcidin-inducing activity independent of Hjv.

To determine whether hepatic Neo1 possesses any residual ability to induce hepcidin expression independent of Hjv, we initially attempted to overexpress the Neo1^L1046E mutant, which disrupts the Hjv-binding site (33), in Neo1^−/− mice. However, the Neo1 coding sequence approached the packaging limit of the AAV8 vector, preventing the generation of sufficient AAV8 viral titers. To circumvent this, we utilized murine Dcc (Figure 5A). Dcc is a Neo1 homologue with ~1000-fold lower binding affinity for Hjv (33); it is smaller (1447 versus 1492 amino acids) and shares ~69% amino acid sequence similarity with Neo1. While endogenous Dcc expression is predominantly restricted to the developing central nervous system (67, 68), it shares similar domain structures and post-translational processing with Neo1 (69, 70).

A FLAG/MYC epitope was engineered onto the C-terminus of the Dcc construct (fDcc) due to the lack of a suitable antibody for the intracellular portion of Dcc. Adding a similar epitope to the C-terminus of Neo1 (fNeo1) has previously been shown not to affect its induction of hepcidin (24). When expressed in Hep3B cells (a human hepatoma cell line), fDcc trafficked to the plasma membrane, as confirmed by cell-surface biotinylation on ice and the subsequent pulldown of biotinylated proteins using streptavidin beads (Figure 5B; panels 1–3; lane 13–18). The western blot images were obtained by sequential probing with anti-mouse Dcc ectodomain, anti-human NEO1 ectodomain, and anti-FLAG epitope antibodies. The enrichment of Na⁺K⁺ ATPase and the absence of detectable β-actin in biotinylated fractions confirmed that only plasma membrane proteins were labeled (Figure 5B; panel 4/5). Consistent with earlier studies (70) and similar to Neo1 (37), the ectodomain of Dcc underwent active shedding, which was largely inhibited by the α-secretase inhibitor Aderbasib (Figure 5A; 5B; panel 6; lane 4–7). Dcc is also subjected to γ-secretase cleavage within its transmembrane domain, releasing the Dcc-intracellular domain (Dcc-ICD) into the cytosol (70). In the presense of the proteasomal inhibitor MG-132, released fDcc-ICDs were detectable in cell extracts via anti-FLAG antibody; this signal was largely abolished by the γ-secretase inhibitors LY450139 and DAPT (Figure 5C; panel 2; lane 8–11). These data indicate that Dcc undergoes post-translational processing in hepatoma cells in a manner similar to that of fNeo1 (37), suggesting that Dcc could serve as a model to study Neo1 function.

To investigate its biological function in vivo, we expressed fDcc at two different levels in the livers of Neo1^−/− mice via intraperitoneal administration of an AAV8-fDcc viral vector. qRT-PCR analysis revealed that hepatic fDcc mRNA levels in the low- and high-expression groups were approximately 6.8- and 60.6-fold higher, respectively, than endogenous Neo1 mRNA in wild-type mice (Figure 6A/B). Western blot analysis indicated that the expressed fDcc protein migrated at the predicted molecular weight on SDS-PAGE (Figure 6C), with protein levels proportional to mRNA levels (Figure 6D). Consistent with earlier studies (67, 68), negligible levels of endogenous Dcc mRNA and protein were detected in either wild-type or Neo1^−/− mice (Figure 6B; 6C, top panel, lanes 1–4). However, unlike Neo1 (Figure 3E; top panel, lanes 1–3), only one fDcc band was detected using an antibody against mouse Dcc ectodomain (Figure 6C, top panel, lanes 5–10). Functional analysis revealed that expression of lower fDcc levels in the liver partially rescued low Hamp mRNA, leading to corresponding reductions in serum iron (Figure 6E/F; Supplemental Figure S5). High fDcc levels resulted in a complete correction of low Hamp mRNA and high serum iron to levels comparable to wild-type counterparts (Figure 6E/F). The parallel increases in hepatic Hamp and Id1 mRNA suggest that fDcc-mediated Hamp induction occurs via enhanced Bmp signaling (Figure 6E/G). Furthermore, the absence of changes in IL-6 mRNA levels (Figure 6H) ruled out inflammation as a driver of increased Hamp expression. Together, these overexpression studies suggest that Dcc retains residual hepcidin-inducing activity by enhancing Bmp signaling, implying that its homologue, Neo1, possesses a similar Hjv-independent signaling capacity.