RGM Family Involved in the Regulation of Hepcidin Expression in Anemia of Chronic Disease

Abstract

The persistent production of inflammatory cytokines causes anemia of chronic disease (ACD). Playing a central role in the pathophysiology of ACD is hepcidin, a key regulator of iron metabolism. The regulation of hepcidin expression is a complex process intricately controlled by multiple pathways. These include the BMP/SMAD, the HFE–TFR2, and the IL-6/STAT3 pathway, each playing a significant role in this regulation. We detail the critical role of the repulsive guidance molecule (RGM) family, especially hemojuvelin (HJV/RGMc), in regulating hepcidin expression in ACD. HJV functions as a co-receptor for BMPs and positively regulates hepcidin expression. RGMa and RGMb may also regulate hepcidin expression and inflammatory responses. RGM family proteins play essential roles in the interplay between inflammation, iron metabolism, and the immune system, and elucidating them could lead to a better understanding of the pathophysiology of ACD and the development of new therapeutic strategies.

1. Introduction

A complex mechanism regulates iron metabolism in the body. Approximately 60–70% of iron is present as a component of heme in hemoglobin, with the remainder in the form of ferritin and hemosiderin as stored iron. Various factors tightly regulate the balance of iron absorption, storage, utilization, and recycling. This precise regulation reflects the vital role iron plays in the body. Indeed, iron is essential in numerous metabolic processes, including electron transfer in mitochondria, neurotransmitter synthesis, protein synthesis, and organ formation [1,2,3,4]. Thus, iron metabolism is closely related to various functions of the body, with particular attention paid to its interaction with the immune system. The homeostasis of iron metabolism and immune system function interact, and inflammatory reactions, notably, cause marked changes in iron dynamics.

Inflammation is the body’s defense response to infection and tissue damage and is characterized by the production of inflammatory cytokines and acute phase proteins, as well as leukocyte activation and infiltration. A moderate inflammatory response is beneficial to the organism, but excessive or chronic inflammation causes tissue damage and is implicated in the pathogenesis of various diseases.

Chronic inflammation often has a major impact on iron metabolism via inflammatory cytokines, leading to anemia of chronic disease (ACD), also known as anemia of inflammation [5]. ACD is associated with a variety of chronic diseases, including infectious diseases, malignancies, autoimmune diseases, chronic kidney disease, and chronic rejection after organ transplantation [6]. Persistent inflammation causes ACD by multiple mechanisms, including the suppression of iron utilization through increased hepcidin, the direct suppression of erythropoiesis by inflammatory cytokines, and a decreased erythropoietin response [6,7]. Because inflammatory and iron deficiency anemia can present similarly, differentiating between the two is clinically important. A useful indicator for this distinction is the serum soluble transferrin receptor (sTfR) level. In iron deficiency anemia, transferrin receptors on erythroid progenitor cells increase to compensate for iron deficiency, resulting in a marked increase in serum sTfR levels. In inflammatory anemia, the increase in sTfR levels is only mild due to the suppression of erythropoiesis by inflammation [8]. Thus, we can effectively differentiate iron deficiency anemia, inflammatory anemia, and a combination of both conditions by using the ratio of serum ferritin to sTfR (ferritin/sTfR ratio) [6].

Hepcidin plays a central role in the pathophysiology of ACD by regulating systemic iron homeostasis [9,10,11]. Hepcidin binds to ferroportin, leading to its internalization and degradation, thereby reducing iron efflux from cells [12]. In ACD, persistent inflammation leads to increased hepcidin production, which may result in reduced iron availability for erythropoiesis and potentially contribute to the development of anemia [13].

The regulation of hepcidin expression is complex and involves multiple signaling pathways, including the BMP/SMAD pathway, the IL-6/STAT3 pathway, and the HFE–TFR2 pathway [10]. Hemojuvelin (HJV), also known as repulsive guidance molecule c (RGMc) or hemochromatosis type 2 protein (HFE2), plays a crucial role in modulating these pathways and consequently, hepcidin expression [14,15]. HJV acts as a co-receptor for BMPs, enhancing BMP signaling and promoting hepcidin transcription [14,16,17].

HJV is a member of the RGM family, which includes repulsive guidance molecule a (RGMa), repulsive guidance molecule b (Dragon, RGMb), and HJV, which have various physiological functions [18,19,20]. While the role of HJV in hepcidin regulation is well established, the potential contributions of RGMa and RGMb to iron homeostasis and their involvement in ACD pathophysiology are emerging areas of research.

This review aims to provide a comprehensive overview of the RGM family’s involvement in hepcidin regulation within the context of ACD, examining the molecular mechanisms by which these proteins modulate hepcidin expression and their potential as therapeutic targets for managing this prevalent form of anemia.

2. History of Hepcidin Discovery

Hepcidin is a master regulator of systemic iron balance [21,22]. Since it was shown that hepcidin, which promotes the suppression of iron absorption, is upregulated in the liver of mice treated with the endotoxin lipopolysaccharide (LPS) and induced inflammation, studies on the relationship between inflammatory anemia and hepcidin have begun [23]. Hepcidin was first reported as a secreted peptide with antimicrobial activity from human urine in 2001 [24]. It was the same peptide with a disulfide bond and antimicrobial activity that was isolated from human blood as LEAP-1 in 2000 [25]. Hepcidin is produced in the liver as an 84-amino acid chain and degraded to 25 amino acids to become its active form [24]. Hepcidin was identified after the responsible gene was cloned in USF-2 KO mice (knockout mice of the transcription factor upstream stimulatory factor 2) who exhibited iron excess [26]. Hepcidin transgenic mice developed severe iron deficiency anemia [27]. These findings reveal a relationship between hepcidin and iron metabolism.

3. Role of Hepcidin in Iron Metabolism

Iron is an essential metallic element for hemoglobin, an enzyme involved in DNA synthesis and mitochondrial respiration. However, when iron is in excess in the body, various free radicals are generated, causing cell, tissue, and organ damage, and other adverse effects on the organism. Therefore, the body’s iron must always be regulated to stay within a specific range [28].

Dietary iron, both heme and non-heme iron, is absorbed via intestinal epithelial cells (Figure 1). Non-heme iron, mainly trivalent, is reduced to divalent by duodenal cytochrome b (Dcyt b) on the intestinal epithelial cell membrane and taken up into the cell by divalent metal transporter 1 (DMT1) [29,30]. After uptake by intestinal epithelial cells, heme iron is transported to the cytoplasm via the heme carrier protein 1 (HCP1), where it is degraded by heme oxygenase and released as iron ions [31,32]. Iron in the cytoplasm is exported to the circulatory system by ferroportin in the basolateral membrane of the intestinal epithelium [33,34,35]. Iron released across the basolateral membrane by ferroportin is oxidized to its ferric (trivalent) form by either membrane-bound hephaestin or soluble ceruloplasmin, then bound to transferrin (TF) in the blood for secure transport and distribution throughout the body [30,36,37].

Hepcidin is a 25-amino acid peptide hormone produced from an 84-amino acid prepropeptide. Hepatocytes mainly secrete hepcidin and circulate in plasma bound to α2-macroglobulin [38]. Hepcidin binds to the cell surface receptor ferroportin, leading to the internalization and degradation of the ferroportin–hepcidin complex in lysosomes, resulting in decreased cellular iron export [12]. Since ferroportin enables iron efflux from intestinal cells, hepatocytes, and macrophages, the internalization of ferroportin into cells by hepcidin binding reduces iron release.

As has been discussed, hepcidin is mainly secreted by hepatocytes but is also expressed and secreted by cardiomyocytes and is found to work mainly only in the heart. In other words, systemic iron metabolism is mainly carried out by hepcidin in the liver, whereas the heart’s own hepcidin finely regulates iron metabolism in the heart [39,40]. This precise regulation of iron metabolism contributes to maintaining proper cardiac function. Lakhal-Littleton et al. performed a functional analysis of cardiac myocyte-specific knockout mice of the hepcidin gene (Hamp) (Hampfl/fl;Myh6.Cre+ mice) and cardiac myocyte-specific knock-in mice of the hepcidin-resistant ferroportin (Slc40a1 C326Yfl/fl;Myh6.Cre+ mice) [41]. The results showed that the iron content of cardiac myocytes was significantly reduced in Hampfl/fl;Myh6.Cre+ mice, causing fatal cardiac dysfunction, including hypertrophy of the left ventricle and a marked reduction in cardiac ejection fraction. In Slc40a1 C326Yfl/fl;Myh6.Cre+ mice, excessive iron efflux from the cardiac myocytes led to iron deficiency in the cardiac myocytes and fatal cardiac dysfunction. These results suggest that the heart’s local hepcidin–ferroportin system is essential for cardiomyocyte iron homeostasis and the protection of cardiac function.

Thus, hepcidin is central in regulating systemic and local iron metabolism. At the systemic level, it maintains iron homeostasis by reducing serum iron, inhibiting iron absorption in the intestinal tract, and regulating iron transport from hepatocytes and macrophages. In contrast, in specific organs, such as the heart, the local hepcidin is responsible for tissue-specific regulation of iron metabolism, essential for maintaining proper organ function.

4. Mechanism of Regulation of Hepcidin Production by Iron Levels in the Body

Hepcidin production is also modulated indirectly by factors other than inflammation that alter the balance of demand and supply of iron in the body, such as anemia and hypoxia, primarily through their effects on erythropoiesis [42]. Hepcidin expression is repressed in the stress erythropoiesis to meet increased erythropoiesis demand [43]. BMP6, a secreted bone morphogenetic protein (BMP) belonging to the TGF-β superfamily, regulates hepcidin production due to body iron [16,17]. BMP6 binds to the BMP receptor complex and induces hepcidin expression via the SMAD system. As discussed below, the BMP receptor complex is regulated complicatedly by the binding of HFE, TFR2 (Transferrin Receptor 2), HJV, and Neogenin [44].

5. Role of Hepcidin in ACD

ACD occurs in various chronic conditions, including infection by microbial pathogens, inflammatory autoimmune diseases such as arthritis and lupus, chronic kidney disease (CKD), and cancer [11,28,45]. This anemia is usually mild to moderate and normocytic but may progress to microcytic in long-standing severe disease [28]. In ACD, iron is sequestered within reticuloendothelial macrophages, and transferrin-bound iron in serum, which is required for hematopoiesis, is reduced [28]. Increased hepcidin expression due to the release of inflammatory cytokines is essential to this phenomenon [28,45]. In chronic inflammation, persistent high hepcidin levels cause a long-term reduction in iron availability, resulting in ACD. Elevated hepcidin expression decreases ferroportin on the surface of intestinal epithelial cells, macrophages, and hepatocytes [12,28,45] (Figure 1). In CKD, hepcidin clearance is impaired, and its accumulation in plasma may facilitate iron sequestration within macrophages, limiting the iron available for hematopoiesis. This, together with increased inflammation and impaired erythropoietin production, contributes to renal anemia [45].

Hepcidin expression is induced during inflammation, mainly via IL-6 [46]. IL-1 and IL-22 also positively regulate hepcidin expression [47,48]. IL-6 binds to the gp130 protein receptor complex and causes phosphorylation of the STAT3 transcription factor via the JAK1/2 tyrosine kinase [49,50,51] (Figure 2). Activated STAT3 translocates into the nucleus, binds to STAT3-responsive elements on the hepcidin promoter, and induces hepcidin transcription [52]. The regulation of hepcidin synthesis by IL-6 also involves the BMP (bone morphogenetic protein) signaling pathway, which acts synergistically with IL-6 to increase hepcidin [53].

Increased hepcidin production during inflammation suppresses the adaptive immune system. Restricted iron utilization leads to suppressed T-lymphocyte proliferation, decreased circulating T-lymphocytes, reduced immune reactivity, and reduced IL-2 production [54,55]. On the other hand, elevated hepcidin expression is essential as a host defense mechanism in infectious diseases [28,45]. Increased hepcidin limits the iron available to bacteria by reducing serum iron, leading to the inhibition of the growth of iron-requiring bacteria [56]. The identification of hepcidin as an antibacterial substance in urine supports this inhibition [24,56]. Hepcidin also plays a role in preventing the formation of non-transferrin-bound iron (NTBI) during infection [57]. To protect a host, hepcidin inhibits the production of NTBI, which promotes the rapid growth of extracellular micro-organisms.

In viral infections, also, the hepcidin regulatory mechanism of macrophages plays an important role in host defense [58]. Through viral infection, macrophages induce hepcidin production in the liver by inflammatory stimuli such as interleukin-6 (IL-6) and lipopolysaccharide (LPS). Increased hepcidin inhibits the extravasation of iron via ferroportin and causes iron to accumulate within macrophages. This iron sequestration inhibits viral replication by limiting the amount of iron required. However, this defense mechanism can be a double-edged sword. Prolonged iron sequestration can lead to anemia and impaired immune function, so hepcidin regulation must be carefully controlled.

On the other hand, viruses can manipulate host hepcidin regulatory mechanisms to create an environment favorable for their replication. For example, in the hepatitis C virus (HCV), a viral core protein has been reported to reduce hepcidin levels [59,60,61]. Iron accumulation within macrophages has been observed in HIV-1 infection, and this has been reported to be positively correlated with mortality [62]. This iron accumulation is caused by the HIV-1 Nef protein, which inhibits HFE function by blocking its trafficking to the cell surface [63]. HFE normally binds to transferrin receptor 1 and attenuates iron uptake into cells. Unfortunately, macrophages are the valuable reservoir for HIV-1, and this iron accumulation can facilitate viral replication [58]. Thus, HIV-1 can create a favorable environment for its survival and replication by manipulating host iron metabolism. Thus, hepcidin is key in the iron scramble between host and pathogen and significantly impacts the progression and prognosis of infection. The host attempts to sequester iron through increased hepcidin and suppress the growth of pathogens, while pathogens also manipulate host iron metabolism for survival.

As mentioned above, hepcidin is also expressed in the heart and regulates local iron metabolism. Chronic heart failure induces a reduction of serum hepcidin levels, which may be an adaptive response to increasing systemic iron availability and a compensatory mechanism for anemia and myocardial iron deficiency associated with heart failure [64]. Cardiac local hepcidin is elevated during cardiac iron deficiency, reducing iron leakage from cardiomyocytes and maintaining intracellular iron stores [41]. Adequate iron supply to cardiomyocytes by cardiac hepcidin contributes to the protection of mitochondrial function during cardiac injury [40,65]. Cardiac hepcidin also has anti-apoptotic effects [66]. These findings suggest that systemic hepcidin and cardiac local hepcidin work in concert to regulate cardiac iron metabolism and function appropriately.

6. Multifaceted Control of Hepcidin Expression through HJV and Associated Molecules

Hepcidin is a peptide hormone produced in the liver that plays a central role in the regulation of iron metabolism. The regulatory mechanism of its expression is complex and involves multiple genes and pathways. The BMP/SMAD pathway, the HFE/TFR2 pathway, and the IL-6/STAT3 pathway are known as major pathways for the regulation of hepcidin expression. Hepcidin expression is regulated by body iron levels via BMP/SMAD and HFE/TFR2 pathways or by inflammation, including ACD, via the IL-6/STAT3 pathway. HJV functions as part of a complex in the BMP/SMAD pathway. Although there is no evidence that HJV is directly involved in the IL-6/STAT3 pathway associated with inflammation, as discussed below, HJV may be deeply involved in ACD.

a

Membrane-bound HJV activates hepcidin transcription through the BMP/Smad pathway

HJV regulates hepcidin expression via the BMP/SMAD pathway. HJV is usually anchored to membranes via the Glycosylphosphatidylinositol (GPI) anchor, called membrane-bound HJV (m-HJV). The m-HJV enhances binding between BMP receptors and their ligands (BMPs) as a co-receptor for BMPs (Figure 2) [14,16,17]. The m-HJV also binds to Neogenin [67]. When the complex formation of m-HJV, BMPs, and BMP receptors is promoted, SMAD1/5/8 is phosphorylated. This phosphorylated SMAD1/5/8 binds to SMAD4 and enters the nucleus. Upon entering the nucleus, the SMAD complex binds to the promoter region of the hepcidin gene and activates hepcidin transcription [14,17]. The binding of m-HJV to Neogenin has been suggested to be crucial for this process (See section “c Regulation of hepcidin expression by Neogenin”) [68]. These findings indicate that m-HJV and Neogenin play an important role in hepcidin transcription via the BMP/SMAD pathway, but the exact mechanism and contribution need further study.

b

Soluble HJV suppresses hepcidin transcription via the BMP/SMAD pathway

HJV is cleaved by two proteases, Matriptase-2 (TMPRSS6) and Furin [69,70,71] (Figure 3).

[TMPRSS6]: TMPRSS6 cleaves membrane-bound HJV and suppresses hepcidin expression. M-HJV promotes hepcidin expression via BMP-2, -4 and -6. TMPRSS6 inhibits this signal by cleaving m-HJV, thereby reducing hepcidin transcription. TMPRSS6 does not cleave soluble HJV, which functions as an antagonist of the BMP pathway. Thus, TMPRSS6 inhibits hepcidin activation by directly suppressing BMP signaling [69,70].

[Furin]: Hypoxia and iron deficiency activate Furin to cleave HJV proteins synthesized in the endoplasmic reticulum of hepatocytes to produce soluble HJVs (s-HJVs) [71]. S-HJVs function as decoy receptors for BMP signaling and prevent BMPs from binding to m-HJVs, leading to the blockage of BMP signaling and hepcidin expression. Thus, Furin also plays a role in inhibiting BMP signaling by cleaving HJV, thereby suppressing hepcidin expression.

c

Regulation of hepcidin expression by Neogenin

Neogenin plays a complex role in regulating hepcidin expression [72]. Neogenin binds to HJV. Studies of Neogenin-deficient mice have shown that Neogenin is essential for appropriate HJV function [73]. In Neogenin-deficient mice, hepcidin expression is completely inhibited, causing severe iron overload, like in the HJV-deficient mice. Contradictory results on the role of Neogenin in iron metabolism were reported at one time; in vitro studies suggested that HJV regulates hepcidin expression independently of Neogenin [74]. However, recent studies using hepatocyte-specific Neogenin knockout mice (Neo1fl/fl; Alb-Cre+) have resolved this discrepancy by confirming that the interaction between Neogenin and HJV is essential for hepcidin expression and iron homeostasis [68]. In hepatocyte-specific Neogenin knockout mice (Neo1fl/fl; Alb-Cre+), a significant decrease in hepcidin mRNA in the liver (approximately 4–14-fold decrease compared to controls) was observed using a qRT-PCR analysis. At the same time, a significant increase in serum iron concentration was followed by a serum iron assay and a substantial increase in liver iron content (significant increase at five weeks of age and even greater increases at eight and fifteen weeks of age) by a liver non-heme iron assay. These results indicate that in Neo1fl/fl; Alb-Cre+ mice, decreased hepcidin expression in the liver and iron excess in the serum and liver are induced. In addition, a mutant of Neogenin (Neo1L1046E), which cannot interact with HJV, failed to induce hepcidin expression. This study emphasizes the importance of in vivo models in hepcidin research and points out the limitations of the studies conducted by Xia et al. using liver tumor cell lines [74]. In addition, Neogenin also interacts with TMPRSS6 to form a three-protein complex containing m-HJV. This complex is thought to facilitate cleavage of m-HJV by TMPRSS6 [75]. TMPRSS6 is a significant repressor of hepcidin expression. TMPRSS6 cleavage of m-HJV results in the downregulation of the BMP/SMAD pathway and hepcidin expression. Thus, Neogenin is involved in positive and negative hepcidin expression regulation by mediating the interaction between HJV and TMPRSS6. These findings suggest that Neogenin contributes to the precise regulation of hepcidin expression in iron metabolism homeostasis.

d

Regulation of hepcidin expression by the HFE–TFR2 pathway

In the HFE–TFR2 pathway, HFE and TFR2 form a complex on the cell membrane [76]. This complex acts as a sensor to sense transferrin-bound iron (Fe-Tf) in blood [77] (Figure 2). As transferrin receptor 1 (TFR1) takes up Fe-Tf, HFE leaves TFR1 and binds to TFR2. The binding of HFE and TFR2 allows cells to sense the presence of circulating iron accurately. When this sensing system is activated, the HFE–TFR2 complex interacts with the HJV and BMP receptor complexes, enhancing hepcidin expression via phosphorylation of SMAD1/5/8 [78]. Mice lacking HFE and TFR2 showed more severe iron excess than those lacking HFE alone [79]. The reduction in hepcidin expression was mild in HFE-deficient mice, more pronounced in TFR2-deficient mice, and most pronounced in HFE/TFR2 double-deficient mice. These results suggest that the coordinated action of HFE and TFR2 is required for iron-responsive hepcidin expression. HJV competitively inhibits the binding of HFE to TFR1, causing HFE to dissociate from TFR1 and bind to TFR2. The deletion mutant of HJV loses its ability to induce hepcidin expression while retaining its ability to bind HFE and TFR2. These findings suggest that HFE–TFR2–HJV complex formation is required for hepcidin expression. HFE has also been found to directly interact with the BMP type I receptor ALK3 (BMPR1A) (Figure 3); HFE inhibits the ubiquitination and proteasomal degradation of ALK3. In addition, HFE increases the expression of ALK3 protein and promotes its translocation to the cell surface [76]. The regulation of hepcidin expression depends on iron oversupply or deficiency. When iron is abundant, hepcidin expression is enhanced via the BMP/SMAD pathway from the HFE–TFR2–HJV complex, inhibiting iron absorption and promoting iron storage. On the other hand, in the case of iron deficiency, suppression of this pathway suppresses hepcidin expression and promotes iron absorption.

e

Regulation of hepcidin expression by IL-6/STAT3 pathway in ACD

The activation of the JAK/STAT3 pathway, mainly through the IL-6 cytokine, is involved in elevated hepcidin expression during inflammation [46]. Inflammatory stimuli, such as bacterial infections, produce IL-6 from macrophages and other immune cells [80]; IL-6 binds to IL-6 receptors on hepatocytes and phosphorylates STAT3 via JAK2. Phosphorylated STAT3 forms dimers and migrates into the nucleus, where it binds to STAT3 response sequences in the promoter region of the hepcidin gene (HAMP) and activates transcription [13,52] (Figure 2).

The BMP/SMAD pathway is essential for hepcidin expression regulation by the IL-6/STAT3 pathway. The STAT3 response sequence on the hepcidin promoter is close to the BMP response sequence, suggesting a close relationship between the two pathways [53]. Interestingly, inactivation of the STAT3 response sequence does not affect hepcidin regulation by BMPs. Still, inactivation of the proximal BMP response sequence significantly reduces hepcidin activation by IL-6, indicating that the complete BMP–SMAD pathway is required for the hepcidin activation by IL-6. Using a BMP–SMAD pathway inhibitor suppressed hepcidin activation by IL-6 and improved anemia in a model of anemia due to inflammation [81]. In liver-specific SMAD4-deficient mice, hepcidin expression levels did not increase after IL-6 treatment, unlike controls, indicating that SMAD4 is essential for hepcidin induction by IL-6 [82]. In experiments using liver-specific Type I BMP receptor Alk3-deficient mice, hepcidin expression was upregulated by IL-6 administration in wild-type mice, whereas it was absent in Alk3-deficient mice [83]. These findings emphasize that the BMP/SMAD pathway is essential for regulating hepcidin expression by the IL-6/STAT3 pathway.

Meanwhile, it was reported that the IL-6/STAT3 pathway also contributes to the activation of hepcidin expression via the BMP/SMAD pathway [84]. In a study using BV2 microglial cells, IL-6 was involved in BMP6 expression during inflammation. LPS and LTA treatment significantly increased BMP6 secretion, suggesting that these inflammatory stimuli activate the BMP/SMAD pathway by increasing BMP6 secretion. IL-6 neutralizing antibody treatment decreased BMP6 secretion and SMAD1/5/9 phosphorylation. By neutralizing IL-6, BMP6 levels in LPS- and LTA-treated cells were significantly reduced, and pSMAD1/5/9 levels decreased in parallel with pSTAT3 levels. This result suggests that, in BV2 microglial cells, IL-6 activates the BMP/SMAD pathway. Furthermore, TFR2 and TMPRSS6 expression was also shown to be altered in an IL-6-dependent manner; neutralizing IL-6 reduced TFR2 levels and significantly decreased TMPRSS6 protein levels in both LPS and LTA treatments. These results indicate that the expression of TFR2 and TMPRSS6 is dependent on IL-6-regulated signaling pathways.

Since these results were obtained in experiments with BV2 microglial cells, these results need to be validated in other hepcidin-synthesizing cells, including hepatocytes, in the future. However, it is suggested that a complex interaction network exists between the IL-6/STAT3 pathway and the BMP/SMAD pathway in the regulation of hepcidin expression under inflammatory conditions.

In summary, regulating hepcidin expression during inflammation involves a complex interplay between the IL-6/STAT3 and BMP/SMAD pathways. These pathways allow for fine-tuning hepcidin levels in response to various physiological and pathological conditions.

7. RGM Family Involved in Hepcidin Synthesis in ACD

HJV, expressed primarily in the liver and skeletal muscle, regulates hepcidin expression in ACD. HJV is a member of the repulsive guidance molecule (RGM) family, which consists of RGMa, RGMb, and HJV (RGMc).

Table 1. Characteristics and functions of RGM family members in hepcidin expression and ACD.

Characteristics	RGMa	RGMb (Dragon)	RGMc (HJV)
Alias	-	Dragon	Hemojuvelin, HFE2
Main expression site	Central nervous system [19,85,86,87,88,89,90]	Lung stroma, lung epithelium, neural tissue, macrophages, bronchial epithelial cells, activated eosinophils [91,92]	Liver and skeletal muscle [14,67,68,74,75,93]
Role in hepcidin synthesis	Promotes hepcidin transcription by acting as a co-receptor for BMPs in HuH7 cells, hepatocarcinoma cell line [78]	May promote hepcidin expression through SMAD1 activation via the BMP signaling pathway, but its direct role in hepcidin regulation remains unclear [94]	Positively regulates hepcidin expression as a co-receptor for BMPs [14,16,17]
Involvement in BMP/SMAD pathway	Acts as a co-receptor for BMPs, promoting BMP/SMAD signaling [15]	Functions as a BMP co-receptor, binding directly to BMP2 and BMP4, and to BMP type I and II receptors. Effects on BMP signaling may be cell-type specific: it inhibits BMP signaling in C2C12 myoblasts, but promotes signaling in others [94,95,96]	Enhances BMP signaling as a co-receptor, crucial for BMP/SMAD pathway activation [14,16,17]
Involvement in IL6/STAT3 pathway	No direct involvement reported in the paper	Negatively regulates IL-6 expression, but no direct involvement in IL6/STAT3 pathway reported [97]	No direct involvement reported, but essential for IL-6-induced hepcidin expression [98]
Association with inflammation	Involved in neuroinflammatory diseases [87,88,90]	Regulates inflammatory responses, negatively regulates IL-6 expression [97]	Essential for hepcidin induction during inflammation [14,98,99]
Associated conditions	Multiple sclerosis [87,88,90], spinal cord injury [19,100,101], cerebral infarction [102,103], blood–brain barrier disruption [93]	Allergic asthma [92], respiratory inflammation [92], promotes respiratory tolerance through binding to PD-L2 [104]	Anemia of chronic disease (ACD) [14,98], hereditary hemochromatosis type 2 [14], inflammatory anemia [83,98,99]
Possible involvement in ACD	RGMa is involved in neuroinflammatory diseases and promotes hepcidin expression through the BMP/SMAD pathway, suggesting a potential contribution to ACD associated with chronic neuroinflammation [78,87,88,90]	RGMb negatively regulates IL-6 expression and influences the BMP/SMAD pathway, potentially indirectly contributing to ACD pathogenesis through modulation of inflammatory responses [94,97]	HJV is essential for hepcidin induction during chronic inflammation and directly involved in the pathophysiology of ACD [14,98]

provides a comprehensive comparison of these three RGM family members, highlighting their key characteristics, expression sites, roles in hepcidin synthesis, involvement in signaling pathways, and associated conditions.

a

Hemojuvelin (HJV/RGMc)

Hemojuvelin (HJV) comes in two forms, membrane-bound (m-HJV) and soluble (s-HJV), each with different functions. m-HJV acts as an auxiliary receptor for several bone morphogenetic factors (BMPs) and positively regulates hepcidin expression via the BMP/SMAD pathway (Figure 2) [14,16,17]. Surface plasmon resonance studies have shown that HJV binds with a high affinity to several BMP ligands, including BMP2, BMP4, BMP5, BMP6, and BMP7 [95]. Of these, HJV shows the highest binding affinity to BMP4 (KD 4.5 nM), followed by BMP6 (KD 8.1 nM) and BMP2 (KD 9.4 nM). Notably, the high affinity of HJV for BMP6 is consistent with the important physiological role of HJV in regulating hepcidin expression and systemic iron homeostasis through mediating BMP6 signaling. In contrast, s-HJV suppresses the BMP/SMAD pathway and negatively regulates hepcidin expression [69,71].

Increased hepcidin expression during inflammation involves activating the JAK/STAT3 pathway, primarily via IL-6 cytokines [46]. IL-6 is a major cytokine in ACD and interacts with the IL-6 receptor to activate the JAK2-STAT3 pathway. Activation of this pathway causes phosphorylated STAT3 to bind to the STAT3 response sequence (STAT3-RE) in the hepcidin gene promoter region, increasing hepcidin expression [53]. Inactivation of the BMP response sequence on the hepcidin gene promoter significantly reduces hepcidin activation by IL-6. Thus, it is well understood that hepcidin gene modification by the BMP–SMAD pathway is essential for the induction of hepcidin expression via the IL-6/STAT3 pathway in ACD [53]. However, in ACD, it is becoming clear that molecules involved in the BMP/SMAD pathway, including HJV, interact with the IL-6/STAT3 pathway and play an important role in inducing hepcidin expression [82,83,99].

Fillebeen et al. showed that HJV is essential for the induction of inflammatory hepcidin by LPS and the concomitant serum iron-lowering response; HJV-deficient mice did not exhibit an adequate serum iron-lowering response to acute inflammation from LPS, FSL1, or E. coli infection [98]. They showed that in HJV-deficient mice, the residual hepcidin induction was insufficient to significantly reduce ferroportin levels in macrophages, particularly in Kupffer cells and splenic macrophages. In HJV-deficient mice, LPS stimulation induces hepcidin expression, but to a markedly lesser extent than in wild-type mice. In vitro experiments have shown that HJV can maintain BMP6-mediated SMAD signaling and promote hepcidin induction during inflammation; SMAD signaling and hepcidin induction in response to BMP6 were severely impaired in HJV-deficient hepatocytes. These results indicate that hepcidin expression in ACD requires a combination of BMP6/HJV/SMAD signaling and activated IL6-dependent STAT3 signaling.

Furthermore, the regulation of hepcidin expression in BV2 microglia is regulated by a complex interplay of multiple pathways, including HJV, depending on the amount of IL-6 [84]. In the baseline IL-6 secreted state, HJV is a co-receptor for the BMP/SMAD pathway and contributes to maintaining basal hepcidin expression.

Neutralizing IL-6 decreased TMPRSS6 expression, which may suppress HJV degradation; TMPRSS6 normally suppresses hepcidin expression by cleaving m-HJV and converting it to s-HJV. Therefore, decreasing TMPRSS6 may increase m-HJV, enhancing hepcidin expression via the BMP/SMAD pathway. It could contribute to the increased hepcidin expression after IL-6 neutralization. In contrast, decreased phosphorylation of STAT3 and SMAD1/5/9, decreased expression of BMP6, and decreased TFR2 levels also occurred after IL-6 neutralization, suggesting a complex regulation of hepcidin expression by IL-6.

In inflammatory conditions with increased IL-6 secretion, both the JAK/STAT pathway and BMP/SMAD pathway are activated; increased IL-6 activates the JAK/STAT pathway, directly promoting hepcidin expression. At the same time, the BMP/SMAD pathway is also activated, suggesting that the HJV-mediated pathway and the JAK/STAT pathway cooperate to promote hepcidin expression. This complex regulatory mechanism is thought to enable the appropriate regulation of hepcidin expression in inflammatory conditions.

These findings suggest that the regulation of hepcidin expression in BV2 microglia is regulated by a complex interaction of multiple pathways, including HJV, in response to changes in IL-6 levels. It is speculated that the regulation is normal at baseline, compensates via a decrease in TMPRSS6 upon IL-6 neutralization, and works in concert with other pathways upon IL-6 increase.

b

RGMa

There are far fewer studies examining the involvement of RGM family molecules other than HJV, namely RGMa and RGMb, in regulating hepcidin expression than in HJV studies. However, RGMa has been the subject of numerous studies, primarily in the central nervous system, and its role in the BMP/SMAD pathway and the regulation of hepcidin expression is becoming more apparent.

RGMa was initially discovered as an axon repulsor in the retinotectal system [18]. Like HJV, RGMa binds to Neogenin receptors and also functions as a co-receptor for BMPs [15].

RGMa is expressed primarily in the central nervous system. During development, RGMa regulates axon guidance, neuronal differentiation, and survival [85]. In particular, RGMa binds to Neogenin, which activates signaling through RhoA and ROCK, inhibiting axon elongation and causing growth cone collapse [86] (Figure 4). The expression level of RGMa is usually low in the adult central nervous system, but its expression is induced during pathological conditions such as cerebral infarction and spinal cord injury [19,103]. Treatment with RGMa-neutralizing antibodies has promoted axonal regeneration and motor function recovery in spinal cord injury models.

RGMa is deeply involved in the pathogenesis of neuroinflammatory diseases [87,88,90]. In experimental autoimmune encephalomyelitis (EAE) in an animal model of multiple sclerosis (MS), RGMa is involved in disease progression. RGMa is expressed on bone marrow-derived dendritic cells, Neogenin on CD4+ T cells, and RGMa–Neogenin interaction promotes T cell activation and inflammatory cytokine production. In particular, RGMa expression is high in Th17 cells, and RGMa causes dephosphorylation of Akt via Neogenin, leading to neuroaxonal degeneration.

Recently, s-HJV was shown to prevent RGMa-induced blood–brain barrier (BBB) disruption by competing with RGMa and binding to Neogenin [93]. s-HJV administration to EAE animal models inhibited RGMa-induced BBB disruption, suppressing immune cell infiltration and improving EAE symptoms. Conversely, the inhibition of s-HJV secretion by liver- or muscle-specific HJV knockout caused BBB disruption, fibrinogen accumulation in the brain, decreased cortical neurons, and behavioral deficits.

In addition, RGMa is also involved in angiogenesis; it is known that in MS and EAE, neovascular vessels form at the site of inflammatory lesions, and RGMa inhibits angiogenesis by suppressing the lumen formation of vascular endothelial cells through Neogenin [89].

Thus, RGMa is a multifunctional protein involved in diverse physiological processes in the nervous, immune, and vascular systems. As our understanding of RGMa’s mechanism of action and its involvement in pathological conditions has increased, the relationship between RGMa and the hepcidin expression system through the BMP/SMAD pathway is becoming more evident. RGMa functions much like HJV in regulating hepcidin expression, as mentioned below.

Like HJV, RGMa has been recognized as one of the factors that regulate hepcidin expression via the BMP/SMAD pathway; RGMa binds to Neogenin and acts as a co-receptor for BMPs, promoting hepcidin transcription by activating the BMP/SMAD pathway (Figure 4). In particular, the N-terminal domain of RGMa (N-RGM) is the primary binding site for BMPs [78,85]. RGMa shows the highest binding affinity for BMP4, followed by BMP2, BMP6, BMP7, and BMP5 [95]. While RGMa generally exhibits the lowest binding affinity for BMP ligands among RGM family members, it maintains a relatively high affinity for the BMP4/BMP2 subfamily.

RGMa has been shown to form complexes with other proteins regulating hepcidin expression. In particular, RGMa has been shown to interact with HFE and TFR2 to form a multiprotein complex. This complex formation has been suggested to regulate hepcidin expression via the BMP/SMAD pathway [78] (Figure 4).

Furthermore, RGMa, like HJV, is known to undergo complex post-translational modifications, which may affect the regulation of hepcidin expression via the BMP/SMAD pathway. In particular, RGMa undergoes autoproteolysis and cleavage by proteases such as Furin to produce soluble fragments [105,106]. Furin also cleaves HJV to generate s-HJV. The soluble fragment of RGMa may have a different function than membrane-bound RGMa, potentially complicating the regulation of hepcidin expression.

Thus, RGMa’s role in regulating hepcidin expression via the BMP/SMAD pathway appears to have much in common with HJV. However, studies of RGMa in ACD are still limited, and the exact physiological role that RGMa plays in regulating hepcidin expression requires further investigation.

c

RGMb

RGMb (also known as Dragon) is the first RGM family member shown to be a BMP co-receptor [94]. Thus, RGMb may be essential in regulating hepcidin expression via the BMP/SMAD pathway. In particular, RGMb has been shown to promote SMAD1 activation via the BMP signaling pathway in HepG2 cells, suggesting that RGMb may be involved in regulating hepcidin expression in the liver [94]. However, whether RGMb is involved in regulating hepcidin expression remains unresolved.

Several molecular mechanisms for activating BMP signaling by RGMb have been reported; RGMb binds strong selectivity for the BMP4/BMP2 subfamily [95]. RGMb’s affinity for these ligands is significantly higher than other RGM family members. It also binds relatively strongly to BMP6 and BMP5 but has the lowest affinity for BMP7. In addition, RGMb also physically binds to BMP type I receptors (ALK2, ALK3, and ALK6) and BMP type II receptors (ActRII and ActRIIB) [94]. RGMb, lacking the C-terminal GPI anchor, failed to increase BMP signaling, indicating that RGMb must bind to the membrane to facilitate BMP signaling [94]. On the other hand, studies using C2C12 myoblasts suggest that RGMb may inhibit BMP signaling; in C2C12 myoblasts, RGMb inhibited BMP signaling induced by constitutively activated BMP receptors and SMAD1 [96]. Thus, the regulation of SMAD1 through the BMP signaling pathway by RGMb may differ between cell types but remains an open question.

RGMb has been reported to be associated with inflammation and immunity more often than other families; RGMb is expressed on macrophages in many tissues, including lung stroma, lung epithelium, and neural tissue, and plays a vital role in regulating inflammatory responses [91]. RGMb is expressed on bronchial epithelial cells, activated eosinophils, and stromal macrophages in the lungs in allergic asthma, and promotes airway irritability and inflammation via IL-25 signaling [92]. RGMb-positive stromal macrophages produce IL-5 and IL-13 and enhance inflammatory responses. RGMb co-localizes with Neogenin, suggesting it may form a BMP–RGMb–Neogenin signaling complex. In the lungs, under inflammatory conditions, Neogenin is upregulated in bronchial epithelial cells and stromal macrophages, suggesting that Neogenin may be involved in the inflammatory response through its interaction with RGMb.

On the other hand, RGMb negatively regulates IL-6 expression. In lung tissue and the livers of RGMb knockout mice, IL-6 expression was upregulated in macrophages and dendritic cells [97]. This BMP-dependent regulation of IL-6 expression by RGMb is mediated through the p38 MAPK and ERK1/2 pathways rather than the SMAD1/5/8 pathway.

RGMb promotes the development of respiratory tolerance by binding to PD-L2, the ligand for programmed death-1 (PD-1) [104]. RGMb, expressed on lung stromal macrophages and alveolar epithelial cells, binds to PD-L2, expressed on lung dendritic cells. PD-L2 is a ligand for programmed death-1 (PD-1), a key player in immune regulation. In contrast, RGMa and RGMc were not shown to bind PD-L2. Xiao et al. proposed that RGMb forms a complex with BMPs, BMP receptors, and Neogenin, and that PD-L2 binding to this complex may regulate BMP and Neogenin signaling in dendritic cells. It is unknown how the RGMb–PD-L2 interaction affects the SMAD1/5/8 pathway and regulation of hepcidin expression in dendritic cells.

Thus, RGMb is deeply involved in the BMP/SMAD and IL-6 pathways, which are upstream of the hepcidin expression regulatory pathway and have diverse regulatory effects on inflammation. RGMb functions as a BMP co-receptor and negatively regulates IL-6 expression. In addition, RGMb promotes immune tolerance and modulates inflammatory responses through binding to PD-L2. Although RGMb’s functions may vary by cell type and situation, it is involved in the complex regulation of inflammatory responses. This suggests that RGMb may be involved in the pathogenesis of ACD, but little is known about this aspect of the disease.

8. Hepcidin Regulation via the RGM Family in Acute Disease

Hepcidin and ferritin are also widely recognized as acute-phase response proteins. Therefore, hepcidin regulation by the RGM family may be important not only in chronic diseases but also in acute diseases such as trauma, acute cardiovascular disease, and acute stroke.

It is becoming clear that abnormalities in iron metabolism homeostasis by HJV and hepcidin play a central role after acute ischemic stroke (AIS); HJV expression in the brain is upregulated after AIS, which in turn induces hepcidin production in brain tissue [107]. The post-ischemic hepcidin elevation was suppressed in HJV knockout mice, reducing infarct volume and inhibiting apoptosis. These results suggest that increased HJV expression after AIS stimulates hepcidin production and exacerbates neuronal injury by promoting iron accumulation in the brain. Results consistent with these findings have been obtained in a previous study by Xiong et al. They showed that activating the IL-6/ STAT3 pathway via the Toll-like receptor 4 (TLR4)/MyD88 signaling pathway after AIS enhanced astrocyte hepcidin production, leading to the downregulation of ferroportin 1 (FPN1) in brain microvascular endothelial cells (BMVECs) and iron accumulation in the brain [108]. Iron accumulation increases oxidative stress and exacerbates neuronal injury and cognitive dysfunction. Thus, the increased HJV expression and consequent increased hepcidin production after AIS may promote brain tissue damage through iron accumulation in the brain.

However, there are seemingly conflicting findings on whether increased hepcidin production after AIS promotes iron accumulation in the brain. You et al. showed that astrocytic hepcidin directly regulates FPN1 in BMVECs, thereby limiting iron influx to the brain and protecting it from excessive iron accumulation [109]. Increasing hepcidin expression in astrocytes improved age-related cognitive decline.

Thus, there appears to be conflicting findings on whether increased astrocyte hepcidin production promotes iron accumulation in the brain. Changes in iron metabolism homeostasis by HJV and hepcidin after AIS may have a two-sided effect on recovery from brain injury.

Few studies have examined the role of RGMa and RGMb in regulating hepcidin after acute disorders. However, RGM families other than HJV may also regulate iron metabolism after acute disorders. Numerous reports have shown that RGMa is upregulated in astrocytes and microglia after head trauma, stroke, and spinal cord injury [19,100,103,110,111,112,113]. Many reports suggest inhibiting RGMa function with neutralizing antibodies or other means improves neuronal function [19,100,101,102]. To date, RGMa has mainly been focused on as an inhibitor of axon regeneration, and its involvement in hepcidin expression has yet to be well investigated. However, given that RGMa functions similarly to HJV in regulating hepcidin expression, as mentioned above, RGMa may play an essential role in the progression of the pathology of acute CNS disease, not only inhibiting neural regeneration but also changing iron metabolism homeostasis.

9. Conclusions

Hepcidin overproduction plays a central role in the pathophysiology of anemia associated with chronic inflammatory diseases (ACD). Multiple pathways, including the BMP/SMAD pathway, the HFE–TFR2 pathway, and the IL-6/STAT3 pathway, interact in complex ways to regulate hepcidin expression. In particular, hemojuvelin (HJV/RGMc), a member of the repulsive guidance molecule (RGM) family, is essential in regulating hepcidin expression. There are two forms of HJV: membrane-bound (m-HJV) and soluble (s-HJV). m-HJV positively regulates hepcidin expression by acting as a co-receptor for BMPs, while s-HJV negatively regulates hepcidin expression by repressing the BMP/SMAD pathway. During inflammation, hepcidin expression is induced by activation of the IL-6/STAT3 pathway, and the BMP/SMAD pathway is also essential for this process. HJV is involved in the interaction of both pathways and plays a vital role in inducing hepcidin expression in ACD. Other members of the RGM family, RGMa and RGMb, may also be involved in regulating hepcidin expression. RGMa, like HJV, functions as a co-receptor for BMPs and has been suggested to promote hepcidin transcription. The link between RGMb and hepcidin transcriptional regulation is unclear, but it has been shown to modulate BMP signaling and negatively regulate IL-6 expression. These findings illustrate the complexity of hepcidin expression regulation in ACD. RGM family proteins play essential roles in the interplay between inflammation, iron metabolism, and the immune system, and may be deeply involved in the pathophysiology of ACD. Future research topics include elucidating the specific roles of RGMa and RGMb in ACD and developing new therapeutic strategies targeting RGM family proteins.

Further elucidation of the interactions of RGM family proteins in regulating hepcidin expression and their role in inflammatory conditions is also needed. Progress in these studies will provide a deeper understanding of the pathophysiology of ACD and may lead to the development of new therapeutic strategies. Therapies targeting RGM family proteins have the potential to overcome the limitations of current ACD therapies and improve patient quality of life.