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 that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In mice, increased expression of exogenous MT2 in 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 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 MT2 cleaves 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.
Negative regulation of the NF-κB transcription factor is essential for tissue homeostasis in response to stress and inflammation. NF-κB activity is regulated by a variety of biochemical mechanisms including phosphorylation, acetylation, and ubiquitination. In this study, we provide the first experimental evidence that NF-κB is regulated by SUMOylation, where the RelA subunit of NF-κB is SUMOylated by PIAS3, a member of the PIAS (protein inhibitor of activated STAT) protein family with E3 SUMO ligase activity. PIAS3-mediated NF-κB repression was compromised by either RelA mutant resistant to SUMOylation or PIAS3 mutant defective in SUMOylation. PIAS3-mediated SUMOylation of endogenous RelA was induced by NF-κB activation thus forming a negative regulatory loop. The SUMOylation of endogenous RelA was enhanced in IκBα null as compared with wild type fibroblasts. The RelA SUMOylation was induced by TNFα but not leptomycin B mediated RelA nuclear translocation. Furthermore, RelA mutants defective in DNA binding were not SUMOylated by PIAS3, suggesting that RelA DNA binding is a signal for PIAS3-mediated SUMOylation. These results support a novel negative feedback mechanism for NF-κB regulation by PIAS3-mediated RelA SUMOylation.
Altered innate immunity is a feature of certain skin inflammatory diseases such as psoriasis and atopic dermatitis (AD). In this study, we provide evidence that deficiency in Trim32 (a tripartite motif (TRIM) protein with innate antiviral activity) contributes to a Th2 biased response and predisposes to features of AD in mice. Upon treatment with the TLR7 agonist imquimod (IMQ), Trim32 knockout (KO) mice displayed compromised psoriasiform phenotypes and defective Th17 response. Instead, IMQ treatment of Trim32 KO mice induced AD-like phenotypes with enhanced skin infiltration of eosinophils and mast cells, elevation of Th2 cytokines/chemokines expression, and reduced expression of filaggrin protein expression. Furthermore, while the induction of phosphorylated Stat3 and RelA were compromised following IMQ treatment in the KO mice, phosphorylated Stat6 was elevated. CCL20 induction by TNFα and IL-17A was reduced in Trim32 deficient keratinocytes whereas CCL5 induction by TNFα and IL-4 was enhanced. In addition, Trim32 protein levels were elevated in mice treated with IMQ. Unlike Trim32 overexpression in psoriasis, TRIM32 levels were low in AD patients. Based on Trim32 induction by IMQ, the lower levels of TRIM32 in AD skin compared to healthy control and psoriatic skin suggest a defective TRIM32 pathway in AD pathogenesis.
Edited by F. Peter Guengerich Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an ironregulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2 ؊/؊ mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.
Loss of p53’s proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14’s mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells.
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