The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Abstract

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.

The ectodomain of matriptase-2 plays an important nonproteolytic role in suppressing hepcidin expression in mice

“…Consistent with our previous studies, 9,24 functional analysis revealed that expression of wild-type Mt2 at both doses was able to fully correct the low serum iron and high hepcidin expression status (Figure 1D-E), and it greatly ameliorated the anemia by significantly increasing the levels of hemoglobin, hematocrit, mean cell volume, and mean corpuscular hemoglobin (Table 2). Thus, the transgenic Mt2 acts similarly to the native Mt2.…”

“…8,9,23 In vivo studies show that a functional Mt2 requires the transmembrane domain and the entire ectodomain. 24 However, whether Mt2 suppresses hepcidin solely by cleaving Hjv as reported by earlier studies 8,23 remains unknown.…”

“…All mouse ORF constructs were the same as described previously (Table 1). 9,24 Most of the constructs were engineered with a FLAG/MYC epitope on the C terminus for the sensitive immunodetection of expressed proteins by a horseradish peroxidase (HRP)-conjugated anti-FLAG antibody. The generation of Mt2 mutants and adeno-associated virus-8 (AAV8) vectors 32 are described in the supplemental Materials and methods (available on the Blood Web site).…”

“…Injection of AAV8-Mt2 mask into Tmprss6 2/2 mice was performed as in our previous studies. 24 The analysis was described in the supplemental Materials and methods. All animals were fed a PicoLab Laboratory Rodent Diet 5L0D containing 240 parts per million iron (LabDiet).…”

Hepcidin and its therapeutic potential in neurodegenerative disorders

“…In hepatocytes, it has been well‐documented that inflammation induces hepcidin gene expression via the interleukin‐6 (IL‐6)/signal transducer and activator of transcription 3 (STAT3) pathway, via iron sensing through the transferrin receptor 2 (TfR2)/human hemochromatosis protein (HFE), and the bone morphogenetic protein (BMP)/hemojuvelin (HJV)/SMAD (a small mothers of decapentaplegic protein) pathways, and downregulated by erythroferrone (ERFE) ‐dependent and ‐independent mechanisms . Recent studies have also demonstrated that hepcidin expression can be upregulated by progesterone and mifepristone, two steroid hormones, through cell surface protein progesterone receptor membrane component‐1, transforming growth factor β1, SMAD1/5/8‐independent signaling by activin B, oxidases such as NADPH‐dependent oxidase 4 or artificially overexpressed urate oxidase (UOX), IL‐1β via inducing CCAAT enhancer‐binding protein δ (C/EBPδ) expression, peroxiredoxin‐2 which is a supporter for STAT3 transcriptional activity, fibroblast growth factor‐encoding gene, and downregulated by the immunophilin FKBP12 (FK506‐binding protein 1A) via binding with BMP type I receptor ALK2, cystathionine β‐synthase, TfR1, and matriptase‐2 …”