Dataset Information

Antithrombin regulates matriptase activity involved in plasmin generation, syndecan shedding, and HGF activation in keratinocytes.

ABSTRACT: Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that ?-AT, and not ?-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.

SUBMITTER: Chen YW

PROVIDER: S-EPMC3652837 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Antithrombin regulates matriptase activity involved in plasmin generation, syndecan shedding, and HGF activation in keratinocytes.

Chen Ya-Wen YW Xu Zhenghong Z Baksh Adrienne N H AN Wang Jehng-Kang JK Chen Chiu-Yuan CY Swanson Richard R Olson Steve T ST Kataoka Hiroaki H Johnson Michael D MD Lin Chen-Yong CY

PloS one 20130513 5

Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanis ...[more]

PMID: 23675430

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Project description:The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.

Project description:Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.

Dataset Information

Antithrombin regulates matriptase activity involved in plasmin generation, syndecan shedding, and HGF activation in keratinocytes.

Publications

Antithrombin regulates matriptase activity involved in plasmin generation, syndecan shedding, and HGF activation in keratinocytes.

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