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Hemojuvelin (HJV) regulates iron homeostasis by direct interaction with bone morphogenetic protein (BMP) ligands to induce hepcidin expression through theBMPsignaling pathway in the liver. Crystallography studies indicate that HJV can simultaneously bind to both BMP2 and the ubiquitously expressed cell surface receptor neogenin. However, the role of the neogenin- HJV interaction in the function of HJV is unknown. Here we identify a mutation in HJV that specifically lowers its interaction with neogenin. Expression of this mutant Hjv in the liver of Hjv-/- mice dramatically attenuated its induction of BMP signaling and hepcidin mRNA, suggesting that interaction with neogenin is critical for the iron regulatory function of HJV. Further studies revealed that neogenin co-immunoprecipitated with ALK3, an essential type-I BMP receptor for hepatic hepcidin expression. Neogenin has also been shown to facilitate the cleavage of HJV by furin in transfected cells. Surprisingly, although cleavage of HJV by furin has been implicated in the regulation of HJV function in cell culture models and furincleaved soluble Hjv is detectable in the serum of mice, mutating the furin cleavage site did not alter the stimulation of hepcidin expression by Hjv in mice. In vivo studies validated the important role of HJV-BMP interaction for Hjv stimulation of BMP signaling and hepcidin expression. Together these data support a model in which neogenin acts as a scaffold to facilitate assembly of the HJV.BMP.BMP receptor complex to induce hepcidin expression. [ABSTRACT FROM AUTHOR]

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation. [ABSTRACT FROM AUTHOR]

Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using 54Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased 54Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood–brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity. [ABSTRACT FROM AUTHOR]

ZIP14 (encoded by the solute carrier 39 family member 14 (SLC39A14) gene) is a manganese transporter that is abundantly expressed in the liver and small intestine. Loss-of-function mutations in SLC39A14 cause severe hypermanganesemia. Because the liver is regarded as the main regulatory organ involved in manganese homeostasis, impaired hepatic manganese uptake for subsequent biliary excretion has been proposed as the underlying disease mechanism. However, liver-specific Zip14 KO mice exhibit decreased manganese only in the liver and do not develop manganese accumulation in other tissues under normal conditions. This suggests that impaired hepatobiliary excretion is not the primary cause for manganese overload observed in individuals lacking functional ZIP14. We therefore hypothesized that increased intestinal manganese absorption could induce manganese hyperaccumulation when ZIP14 is inactivated. To elucidate the role of ZIP14 in manganese absorption, here we used CaCo-2 Transwell cultures as a model system for intestinal epithelia. The generation of a ZIP14-deficient CaCo-2 cell line enabled the identification of ZIP14 as the major transporter mediating basolateral manganese uptake in enterocytes. Lack of ZIP14 severely impaired basolateral-to-apical (secretory) manganese transport and strongly enhanced manganese transport in the apical-to-basolateral (absorptive) direction. Mechanistic studies provided evidence that ZIP14 restricts manganese transport in the absorptive direction via direct basolateral reuptake of freshly absorbed manganese. In support of such function of intestinal ZIP14 in vivo, manganese levels in the livers and brains of intestine-specific Zip14 KO mice were significantly elevated. Our findings highlight the importance of intestinal ZIP14 in regulating systemic manganese homeostasis. [ABSTRACT FROM AUTHOR]

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested thatMT2exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenousMT2in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway. [ABSTRACT FROM AUTHOR]