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, 284 (34), 22580-9

Hemojuvelin-neogenin Interaction Is Required for Bone Morphogenic protein-4-induced Hepcidin Expression

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Hemojuvelin-neogenin Interaction Is Required for Bone Morphogenic protein-4-induced Hepcidin Expression

An-Sheng Zhang et al. J Biol Chem.

Abstract

Hemojuvelin (HJV) is a glycosylphosphatidylinositol-linked protein and binds both bone morphogenic proteins (BMPs) and neogenin. Cellular HJV acts as a BMP co-receptor to enhance the transcription of hepcidin, a key iron regulatory hormone secreted predominantly by liver hepatocytes. In this study we characterized the role of neogenin in HJV-regulated hepcidin expression. Both HJV and neogenin were expressed in liver hepatocytes. Knockdown of neogenin decreased BMP4-induced hepcidin mRNA levels by 16-fold in HJV-expressing HepG2 cells but only by about 2-fold in cells transfected with either empty vector or G99V mutant HJV that does not bind BMPs. Further studies indicated that disruption of the HJV-neogenin interaction is responsible for a marked suppression of hepcidin expression. Moreover, in vivo studies showed that hepatic hepcidin mRNA could be significantly suppressed by blocking the interaction of HJV with full-length neogenin with a soluble fragment of neogenin in mice. Together, these results suggest that the HJV-neogenin interaction is required for the BMP-mediated induction of hepcidin expression when HJV is expressed. Combined with our previous studies, our results support that hepatic neogenin possesses two functions, mediation of cellular HJV release, and stimulation of HJV-enhanced hepcidin expression.

Figures

FIGURE 1.
FIGURE 1.
HFE2 and neogenin expression profiles in isolated rat liver cells. A, qRT-PCR analysis of HFE2 mRNA in isolated rat liver hepatocytes (Hep, n = 5), Kupffer cells (KC, n = 7), sinusoidal endothelial cells (SEC, n = 4), and hepatic stellate cells (HSC, n = 6). Results are expressed as the amounts relative to GAPDH. The mean values and the S.D. for each cell population are presented. B, qRT-PCR analysis of neogenin mRNA in isolated rat liver cell populations. The analysis was performed as described in A. C, Western blot analysis of HJV and Neo proteins in isolated rat liver cells. Cell extract protein (250 μg) was separated in SDS-PAGE under reducing conditions. Equal protein loading was confirmed by Ponceau S staining of the membrane (not shown). Membranes were probed with antibodies against HJV and neogenin. Cell lysate from HEK293 cells stably expressing both HJV and neogenin (HEK) was included as a positive control. Experiments were performed using two different sets of isolated cells and showed consistent results.
FIGURE 2.
FIGURE 2.
Titration of the response of hepcidin mRNA expression to BMP4. tTA-HepG2 cells were subcultured into 12-well plates. After 48 h of incubation, cells at about 60% confluence were first incubated with MEM, 1 mm pyruvate, 1× NEAA, 1% FCS for 6 h to serum starve the cells. Cells were then incubated in the same medium with BMP4 at 0, 0.1, 0.5, 1, 5, 10, 25, and 50 ng/ml for 18 h followed by total RNA isolation, cDNA preparation, and qRT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels are expressed as the amount relative to that of GAPDH in each specific sample. The results are from three separate experiments, and the mean values and the S.D. are presented.
FIGURE 3.
FIGURE 3.
Knockdown of neogenin suppresses BMP4-induced hepcidin expression. A, knockdown of neogenin decreases pSmad1/5/8. Control-, HJV-, and G99V HJV-HepG2 cells in 12-well plates were transfected with either control (Ctrl) or neogenin (Neo) siRNA using RNAiMAX reagent. After about 48 h, cells were serum-starved for 6 h followed by incubation with the same medium (1 ml/well) supplemented with 5 ng/ml BMP4 for 16 h. CM was then collected, and cell lysate was prepared. About 15% of CM and the total lysate were subjected to SDS-PAGE followed by immunodetection of neogenin (Neo), pSmad1/5/8 (pSmad), HJV, and β-actin in the lysate (L) and HJV in CM. B, knockdown of neogenin decreases the BMP4-induced hepcidin mRNA. Neogenin knockdown and BMP4 treatment in control-, HJV-, and G99V HJV-HepG2 cells were performed in essentially the same manner as described in A. Total RNA was isolated followed by cDNA preparation and qRT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels are expressed as the amount relative to that of GAPDH in each specific sample. C, knockdown of neogenin decreases hepcidin mRNA in the absence of exogenous BMP4. All the experimental procedures were performed as described in B except that control- and HJV-HepG2 cells were incubated with different concentrations of BMP4 (0, 0.5, 1, and 5 ng/ml). D, neogenin rescue analysis. HJV-HepG2 cells were first transfected with either control (Ctrl) or neogenin (Neo) siRNA on day 1. About 24 h later, cells were introduced with either human neogenin cDNA (hNeo) or pcDNA3 empty vector (pcDNA3). On day 3, cells were serum-starved with MEM, 1 mm pyruvate, 1× NEAA, 1% FCS for 6 h followed by incubation with the same medium (1 ml/well) supplemented with 5 ng/ml BMP4 for 16 h. At about 72 h after the siRNA transfection, CM was collected, and cell lysate was prepared for immunodetection (left panel) as well as for qRT-PCR analysis of hepcidin mRNA (right panel) as described in A and B, respectively. All the experiments were repeated at least three times with consistent results. The qRT-PCR results in B and D were assessed by one-way analysis of variance, and the statistical significant differences relative to the corresponding controls were determined by Tukey's post-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 4.
FIGURE 4.
Inhibition of HJV release by a furin convertase inhibitor does not suppress BMP4-induced hepcidin expression. A, Western blot analysis. tTA-HepG2 cells stably expressing HJV or transfected with empty vector (Ctrl) were maintained in the absence of dox. At about 48 h after subculturing into 12-well plates, cells were first incubated with MEM, 1 mm pyruvate, 1× NEAA, 1% FCS plus 2 μg/ml dox for 6 h to serum starve the cells and to induce HJV expression. Cells were then incubated in the same medium containing 5 ng/ml BMP4 with or without 5 μm decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, a FCI, for 18 h. CM was collected, and cell lysates were prepared. About 20% of CM and the total lysate were subjected to SDS-PAGE followed by immunodetection of pSmad1/5/8 (pSmad), HJV, and β-actin in the lysate (L) and HJV in CM. This experiment was repeated three times with consistent results. B, qRT-PCR analysis of hepcidin mRNA. Incubation of tTA-HepG2-control and -HJV cells with FCI and BMP4 was performed in essentially the same manner as described in A. Total RNA was isolated followed by cDNA preparation and qRT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels are expressed as the amount relative to that of GAPDH in each specific sample. The results are from four separate experiments, and the mean values and the S.D. are presented.
FIGURE 5.
FIGURE 5.
Soluble neogenin suppresses BMP4-induced hepcidin expression only when HJV is expressed. A, qRT-PCR analysis of hepcidin mRNA. tTA-HepG2 cells stably transfected with empty vector (control), HJV, or G99V HJV were maintained in the absence of dox. At about 48 h after subculturing into 12-well plates, cells were first incubated with MEM, 1 mm pyruvate, 1× NEAA, 1% FCS plus 2 μg/ml dox for 6 h to serum starve the cells and to induce HJV expression. Cells were then incubated in the same medium containing BMP4 at 0 or 5 ng/ml with or without 40 nm Neo FNIII 5–6 (Neo) for 18 h. Total RNA was isolated followed by cDNA preparation and qRT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels are expressed as the amount relative to that of GAPDH in each specific sample. The results are from four separate experiments and were assessed by one-way analysis of variance. The statistical significant differences relative to the corresponding controls were determined by Tukey's post test. *, p < 0.05; ***, p < 0.001. B, Western blot analysis. Incubation of tTA-HepG2-control, -HJV, and -G99V HJV cells with Neo FNIII 5–6 (Neo) and BMP4 was performed essentially the same as described in A. Total cell lysates (L) were subjected to SDS-PAGE followed by immunodetection of pSmad1/5/8 (pSmad), HJV, and β-actin. This experiment was repeated three times with consistent results. C, flow cytometry analysis of cell surface HJV after incubation with Neo FNIII 5–6. Control- and HJV-HepG2 cells were incubated with or without 40 nm Neo FNIII 5–6 for 18 h. Cells were then detached from flasks with cell dissociation buffer and incubated with affinity-purified rabbit anti-HJV antibody (4 μg/ml) in Hanks' buffer supplemented with 3% fetal bovine serum for 30 min at 4 °C followed by incubation with phycoerythrin-conjugated goat anti-rabbit IgG (1:500 dilution) in the same buffer for 30 min at 4 °C. Flow cytometry analysis was performed on a BD Biosciences FACSCalibur flow cytometer. Rabbit IgG and control-HepG2 cells were used as negative controls. The levels of cell surface HJV are expressed as arbitrary units. The results are from three separate experiments and the mean values and the S.D. are presented. p values are calculated using two-tailed Student's t test to compare the difference between the HepG2-HJV cells incubated with (HJV/Neo FNIII 5–6) and without (HJV) Neo FNIII 5–6.
FIGURE 6.
FIGURE 6.
Soluble neogenin suppresses hepatic hepcidin expression in mice. Eight-week-old male 129EvSv mice were injected intraperitoneally with purified neogenin FNIII 5–6 (Neo FNIII 5–6) at 3 mg/kg body weight or the carrier buffer (Ctrl) twice at 8:30 am and 3:30 pm on the same day. Injected mice had free access to the regular rodent diet. At 24 h after the first injection, animals were euthanized while under anesthesia. Liver tissues were collected for qRT-PCR analysis of hepcidin and GAPDH mRNA. The hepcidin mRNA levels were calculated as the amount relative to that of GAPDH in each specific sample and are expressed as the amount relative to the carrier buffer-injected mice. Each group consisted of three mice. The mean values and the S.D. are presented. p values are calculated using two-tailed Student's t test to compare the difference between the two groups.
FIGURE 7.
FIGURE 7.
A model of neogenin in HJV-induced hepcidin expression in hepatocytes. A, HJV-neogenin is required for the proper assembly of HJV-BMP ligand-BMPR I/BMPR II complex to initiate the BMP signaling and to induce hepcidin expression. B, disruption of HJV-neogenin interaction either by neogenin (Neo) knockdown or by soluble neogenin (sNeo) leads to the formation of the aberrant and non-functional HJV-BMP ligand-BMPR I complex. FNIII 5, neogenin fibronectin type III 5 domain. FNIII 6, neogenin fibronectin type III 6 domain. BMPRI, type I BMP receptor. BMPRII, type II BMP receptor.

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