Abstract Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression. Cancer Res; 75(11); 2254–63. ©2015 AACR.

Supplemental Figure Legend. Legends to Supplemental Figures S1-S7.

Supplemental Methods and References. Description of additional methods and procedures used in the study. Also includes Supplemental References.

Supplemental Figures S1-S7. Hepcidin transcript levels in prostate cells under basal conditions and in response to modulation by signaling effectors (S1); Immunohistochemical detection of hepcidin in prostate tissue (S2); Clonegenic survival of DU145 cells treated with DFO (S3); BMPs induce SMAD signaling in prostate cancer cells (S4); Effect of BMP6 and BMP7 on hepcidin in PC3 and LNCaP prostate cancer cells (S5); Effect of Wnt pathway agonists or antagonists in DU145 cells (S6); Immunohistochemical detection of SOSTDC1 in prostate tissue (S7).

Magnetic resonance imaging (MRI) is a powerful diagnostic tool, but its use is restricted to the scanner suite. Here, we demonstrate that a bedside nuclear magnetic resonance (NMR) sensor can assess fluid status changes in individuals at a fraction of the time and cost compared to MRI. Our study recruited patients with end-stage renal disease (ESRD) who were regularly receiving hemodialysis treatments with intradialytic fluid removal as a model of volume overload and healthy controls as a model of euvolemia. Quantitative T 2 measurements of the lower leg of patients with ESRD immediately before and after dialysis were compared to those of euvolemic healthy controls using both a 0.28-T bedside single-voxel NMR sensor and a 1.5-T clinical MRI scanner. In the MRI data, we found that the first sign of fluid overload was an expanded muscle extracellular fluid (ECF) space, a finding undetectable at this stage using physical exam. A decrease in muscle ECF upon fluid removal was similarly detectable with both the bedside sensor and MRI. Bioimpedance measurements performed comparably to the bedside NMR sensor but were generally worse than MRI. These findings suggest that bedside NMR may be a useful method to identify fluid overload early in patients with ESRD and potentially other hypervolemic patient populations.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.

Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin β-subunits. Homodimers or heterodimers of the inhibin β-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHβ subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFβ type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.

Bone morphogenetic proteins (BMPs)/growth and differentiation factors (GDFs) are involved in a wide variety of embryologic, developmental, and physiologic processes. One important area of physiology that requires BMP signaling is the homeostatic regulation of iron in the body. Iron is an essential nutrient that is critical for several fundamental cellular processes including oxygen delivery to tissues and generation of adenosine triphosphate (ATP) in mitochondria. However, excess iron can lead to the generation of reactive oxygen species (ROS) that are highly damaging to cells, and insufficient iron is the major source of anemia worldwide. Therefore, the homeostatic regulation of total body iron content is an important physiologic process that must be exquisitely controlled to prevent the pathologic states of iron excess or iron deficiency. BMP signaling in the liver by the BMP ligands and receptors including the co-receptor hemojuvelin/RGMc regulates the expression of the iron hormone hepcidin to maintain iron homeostasis.

Bone morphogenetic protein (BMP) signaling plays an important role in spermatogenesis and follicle development. Our previous studies have shown that repulsive guidance molecule b (RGMb, also known as Dragon) is a co-receptor that enhances BMP2 and BMP4 signaling in several cell types, and that RGMb is expressed in spermatocytes and spermatids in the testis, and in oocytes of the secondary follicles in the ovary. Here, we demonstrated that specific deletion of Rgmb in germ cells in the testis and ovary did not alter Smad1/5/8 phosphorylation, gonadal structures and fertility. In addition, ovaries from postnatal global Rgmb knockout mice showed similar structures to the wild type ovaries. Our results suggest that RGMb is not essential for normal gonadal function. Summary sentence: RGMb, which is expressed in spermatocytes in the testis and in oocytes in the ovary, is not essential for spermatogenesis and follicle development.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disease that still lacks effective therapy. Repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins (BMPs) and a ligand for neogenin, is expressed in renal tubular epithelial cells. Previous studies showed that RGMb plays negative roles in several types of tumors and prevents the immune system from over activation. The present study was designed to explore the effects of RGMb in ADPKD development. We found that expression of RGMb in kidney was less in PKD mice than wild-type mice. With stimulation of 8-bromo-cAMP, RGMb-null embryonic kidneys had greater cyst index, though their ureteric bud branched less than wild-type mice at E13.5. Postnatal RGMb-null kidneys showed interstitial hyperplasia and decreased tubular structures, especially in the boundary area of renal cortex and medulla. RGMb overexpression dramatically inhibited cyst development and promoted tubulogenesis in MDCK cells grown in 3D collagen gels. Biochemical analysis showed increased p-Smad1/5/8 and decreased p-ERK in RGMb-overexpressing MDCK cells, suggesting modulated BMP signaling. Specific inhibition of p-Smad1/5/8 by LDN193189 reversed the suppression of RGMb on MDCK cyst model. These results reveal RGMb as a novel regulator for ADPKD by promoting renal tubule branching and regulating BMP signaling pathway. Elevating RGMb and enhancing p-Smad1/5/8 are promising new strategies to treat ADPKD.

Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal. In various pathophysiological conditions, however, erythrocyte life span is compromised severely, which threatens the organism with anemia and iron toxicity. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that monocytes that express high levels of lymphocyte antigen 6 complex, locus C1 (LY6C1, also known as Ly-6C) ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate into ferroportin 1 (FPN1, encoded by SLC40A1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1(+)Tim-4(neg) macrophages are transient, reside alongside embryonically derived T cell immunoglobulin and mucin domain containing 4 (Timd4, also known as Tim-4)(high) Kupffer cells (KCs), and depend on the growth factor Csf1 and the transcription factor Nrf2 (encoded by Nfe2l2). The spleen, likewise, recruits iron-loaded Ly-6C(high) monocytes, but these do not differentiate into iron-recycling macrophages, owing to the suppressive action of Csf2. The accumulation of a transient macrophage population in the liver also occurs in mouse models of hemolytic anemia, anemia of inflammation, and sickle cell disease. Inhibition of monocyte recruitment to the liver during stressed erythrocyte delivery leads to kidney and liver damage. These observations identify the liver as the primary organ that supports rapid erythrocyte removal and iron recycling, and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity.

Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling.

Bone morphogenetic protein (BMP) signaling plays an important role in spermatogenesis and follicle development. Our previous studies have shown that repulsive guidance molecule b (RGMb, also known as Dragon) is a coreceptor that enhances BMP2 and BMP4 signaling in several cell types and that RGMb is expressed in spermatocytes and spermatids in the testis and in oocytes of the secondary follicles in the ovary. Here, we demonstrated that specific deletion of Rgmb in germ cells in the testis and ovary did not alter Smad1/5/8 phosphorylation, gonadal structures, and fertility. In addition, ovaries from postnatal global Rgmb knockout mice showed similar structures to the wild-type ovaries. Our results suggest that RGMb is not essential for normal gonadal function.

Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice.

Liver disease due to alpha-1-antitrypsin deficiency (A1ATD) is associated with hepatic iron overload in a subgroup of patients. The underlying cause for this association is unknown. The aim of the present study was to define the genetics of this correlation and the effect of alpha-1-antitrypsin (A1AT) on the expression of the iron hormone hepcidin. Full exome and candidate gene sequencing were carried out in a family with A1ATD and hepatic iron overload. Regulation of hepcidin expression by A1AT was studied in primary murine hepatocytes. Cells co-transfected with hemojuvelin (HJV) and matriptase-2 (MT-2) were used as a model to investigate the molecular mechanism of this regulation. Observed familial clustering of hepatic iron overload with A1ATD suggests a genetic cause, but genotypes known to be associated with hemochromatosis were absent. Individuals homozygous for the A1AT Z-allele with environmental or genetic risk factors such as steatosis or heterozygosity for the HAMP non-sense mutation p.Arg59* presented with severe hepatic siderosis. In hepatocytes, A1AT induced hepcidin mRNA expression in a dose-dependent manner. Experiments in overexpressing cells show that A1AT reduces cleavage of the hepcidin inducing bone morphogenetic protein co-receptor HJV via inhibition of the membrane-bound serine protease MT-2. The acute-phase protein A1AT is an inducer of hepcidin expression. Through this mechanism, A1ATD could be a trigger of hepatic iron overload in genetically predisposed individuals or patients with environmental risk factors for hepatic siderosis.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.