Neogenin inhibits HJV secretion and regulates BMP-induced hepcidin expression and iron homeostasis

Abstract

Neogenin, a deleted in colorectal cancer (DCC) family member, has been identified as a receptor for the neuronal axon guidance cues netrins and repulsive guidance molecules repulsive guidance molecules (RGM). RGMc, also called hemojuvelin (HJV), is essential for iron homeostasis. Here we provide evidence that neogenin plays a critical role in iron homeostasis by regulation of HJV secretion and bone morphogenetic protein (BMP) signaling.Livers of neogenin mutant mice exhibit iron overload, low levels of hepcidin, and reduced BMP signaling. Mutant hepatocytes in vitro show impaired BMP2 induction of Smad1/5/8 phosphorylation and hepcidin expression. Neogenin is expressed in liver cells in a reciprocal pattern to that of hepcidin, suggesting that neogenin functions in a cell nonautonomous manner. Further studies demonstrate that neogenin may stabilize HJV, a glycosylphosphatidylinositol-anchored protein that interacts with neogenin and suppresses its secretion. Taken together, our results lead the hypothesis that neogenin regulates iron homeostasis via inhibiting secretion of HJV, an inhibitor of BMP signaling, to enhance BMP signaling and hepcidin expression. These results reveal a novel mechanism underlying neogenin regulation of HJV-BMP signaling. IntroductionIron, a component of many metalloproteins, plays a crucial role in various physiologic processes, such as oxygen sensing and transport, mitochondrial electron transfer in the respiratory chain, and catalytic activity of diverse enzymes. Dysregulation of iron homeostasis leads to either iron deficiency, such as anemia, or iron overload characteristic of hereditary hemochromatosis, a relatively common inherited disorder associated with progressive organ dysfunction. 1 Cellular iron overload is toxic and causes cell death, at least in part due to free radical formation and lipid peroxidation.Hemojuvelin (HJV) is a member of a family of glycosylphosphatidylinositol (GPI)-linked cell surface proteins. It has an essential role in iron homeostasis. 2 HJV contains a putative N-terminal signal peptide, a tri-amino acid motif (the RGD site), a partial von Willebrand factor type D domain, and a C-terminal GPI anchor consensus sequence. 2,3 It is predominately expressed in the skeletal muscle, heart, and liver. 3,4 Mutations in the HJV gene is a leading cause of juvenile hemochromatosis, [5][6][7][8] and patients have low levels of hepcidin, 5,8 a crucial hormone secreted by the liver for iron homeostasis. HJV mutant mice exhibit reduced hepcidin expression and iron overload, 5,6 resembling patients with HJV mutations. Consistently, expression of HJV increases, while suppression of HJV decreases, hepcidin expression in cultured cells. 9 The mechanisms underlying HJV regulation of hepcidin expression and iron homeostasis are beginning to be elucidated. HJV was shown to function as a coreceptor for bone morphogenetic proteins (BMP). It binds to BMP2/4/6 and forms a complex with BMP receptors (type I and II). 10-12 HJV deficient mice/cells exhibit impaired BMP signaling ...

Neogenin Promotes BMP2 Activation of YAP and Smad1 and Enhances Astrocytic Differentiation in Developing Mouse Neocortex

“…Neogenin mutant mice (neogenin m/m ), kindly provided by Dr. Sue Ackerman (The Jackson Laboratory), were maintained in C57BL/6 strain background as described previously (Mitchell et al, 2001;Lee et al, 2010;. Neogenin f/f (neo f/f ) mice were generated by Ozgene as illustrated in Figure 6.…”

“…In addition, neogenin is important for endochondral bone formation , neural tube formation (Mawdsley et al, 2004;Kee et al, 2008), digit patterning (Hong et al, 2012), ion metabolism (Zhang et al, 2005;Kuns-Hashimoto et al, 2008;Lee et al, 2010), and muscle differentiation (Kang et al, 2004). Neogenin is highly expressed in the embryonic and adult neural stem cells (NSCs) (Gad et al, 1997;Fitzgerald et al, 2007;van den Heuvel et al, 2013).…”

Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

“…There were no hematologic abnormalities suggestive of the presence of a secondary, anemia-induced iron overload phenotype in mice with liverspecific Alk2 or Alk3 deficiency (supplemental Figure 3). Moreover, hepatic expression of hereditary hemochromatosis diseasecausing genes, including transferrin receptor-2 (Tfr2), 30 Hfe, 31,32 hemojuvelin (Hjv), 9,13,14,33 and ferroportin 1 (Fpn1), 34 did not differ between genotypes (supplemental Figure 4). These results demonstrate a mild iron overload phenotype in Alk2 fl/fl ; Alb-Cre mice and a severe iron overload phenotype in Alk3 fl/fl ; Alb-Cre mice and indicate that both Alk2 and Alk3 have roles in the regulation of systemic iron homeostasis.…”

“…5,6 Recent studies have demonstrated a critical role for bone morphogenetic protein (BMP) signaling in the regulation of hepcidin expression by iron. [7][8][9][10][11] Binding of BMP ligands to type I and type II BMP receptors induces the type II receptor to phosphorylate and activate the type I receptor. The activated type I receptor, in turn, phosphorylates intracellular signaling molecules, including SMADs 1, 5, and 8.…”

RGMs: Structural Insights, Molecular Regulation, and Downstream Signaling

“…The most compelling evidence is the iron overload observed in the livers of mice in which the gene encoding Neogenin (NEO) has been knocked out [28]. Structural experiments showing a direct physical interaction with RGMs acting as a link between these two signaling pathways led to the crystal structure of a ternary complex comprising the BMP ligand BMP2, the full-length extracellular domain of RGMb, and the two membrane proximal FNIII domains of Neogenin [17].…”