Essential Role of Endocytosis of the Type II Transmembrane Serine Protease TMPRSS6 in Regulating Its Functionality

Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2011; 286(33):29035-43. DOI: 10.1074/jbc.M111.223461
Source: PubMed


The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.

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    ABSTRACT: Hemojuvelin (HJV) and matriptase-2 (MT2) are co-expressed in hepatocytes, and both are essential for systemic iron homeostasis. HJV is a glycosylphosphatidylinositol-linked membrane protein that acts as a co-receptor for bone morphogenetic proteins to induce hepcidin expression. MT2 regulates the levels of membrane-bound HJV in hepatocytes by binding to and cleaving HJV into an inactive soluble form that is released from cells. HJV also interacts with neogenin, a ubiquitously expressed transmembrane protein with multiple functions. In this study, we showed that neogenin interacted with MT2 as well as with HJV and facilitated the cleavage of HJV by MT2. In contrast, neogenin was not cleaved by MT2, indicating some degree of specificity by MT2. Down-regulation of neogenin with siRNA increased the amount of MT2 and HJV on the plasma membrane, suggesting a lack of neogenin involvement in their trafficking to the cell surface. The increase in MT2 and HJV upon neogenin knockdown was likely due to the inhibition of cell surface MT2 and HJV internalization. Analysis of the Asn-linked oligosaccharides showed that MT2 cleavage of cell surface HJV was coupled to a transition from high mannose oligosaccharides to complex oligosaccharides on HJV. These results suggest that neogenin forms a ternary complex with both MT2 and HJV at the plasma membrane. The complex facilitates HJV cleavage by MT2, and release of the cleaved HJV from the cell occurs after a retrograde trafficking through the TGN/Golgi compartments.
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    ABSTRACT: Iron is a crucial factor for life. However it also has the potential to cause the formation of noxious free radicals. These "double-edged sword"characteristics demand a tight regulation of cellular iron metabolism. In this review we discuss the various pathways of cellular iron uptake, cellular iron storage and transport. Recent advances in understanding the reduction and uptake of non-transferrin-bound iron are discussed. We also discuss recent progress in the understanding of transcriptional and translational regulation by iron. Furthermore we discuss recent advances in the understanding of the regulation of cellular and systemic iron homeostasis and several key diseases resulting from iron deficiency and overload. We discuss the knockout mice available for studying iron metabolism and the related human conditions.
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    ABSTRACT: Matriptase-2, a recently identified cell surface protease is the key enzyme of iron homeostasis modulating the expression of the liver peptide hormone hepcidin. Hepatocyte growth factor activator inhibitors type 1 and 2 (HAI-1 and HAI-2) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned media. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned media of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound hemojuvelin (HJV). Altogether, our study characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homeostasis, with clinical importance for a treatment of iron overload diseases.
    Preview · Article · Jan 2013 · Biochemical Journal
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