Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine Marguerite S. Buzzaa, Sarah Netzel-Arnetta, Terez Shea-Donohueb, Aiping Zhaob, Chen-Yong Linc, Karin Listd, Roman Szaboe, Alessio Fasanob, Thomas H. Buggee, and Toni M. Antalisa,1 aCenter for Vascular and Inflammatory Diseases and Department of Physiology, bMucosal Biology Research Center, and cDepartment of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201; dDepartment of Pharmacology, Wayne State University and Karmanos Cancer Institute, Detroit, MI 48201; and eProteases and Tissue Remodeling Section, National Institute of Dental and Cranofacial Research, National Institutes of Health, Bethesda, MD 20892 Edited by Masatoshi Takeichi, RIKEN, Kobe, Japan, and approved January 14, 2010 (received for review April 11, 2009) The intestinal epithelium serves as a major protective barrier progress in our knowledge of the components of these structures, between the mammalian host and the external environment. Here their functional regulation remains incompletely understood. we show that the transmembrane serine protease matriptase plays Matriptase [membrane-type serine protease-1 (MT-SP1), a pivotol role in the formation and integrity of the intestinal TADG-15, epithin, SNC19] is an integral membrane trypsin-like epithelial barrier. St14 hypomorphic mice, which have a 100-fold serine protease that is a member of the type II transmembrane reduction in intestinal matriptase mRNA levels, display a 35% reduc- serine protease (TTSP) family (2, 3). Matriptase has a multidomain tion in intestinal transepithelial electrical resistance (TEER). Matrip- structure, consisting of a short cytosolic domain, a transmembrane tase is expressed during intestinal epithelial differentiation and domain, a stem region, and a C-terminal serine protease (catalytic) colocalizes with E-cadherin to apical junctional complexes (AJC) in domain, which is linked to the rest of the molecule by a disulfide differentiated polarized Caco-2 monolayers. Inhibition of matrip- bond (4). Matriptase is widely expressed in virtually all epithelium tase activity using a specific peptide inhibitor or by knockdown of and is specifically found in the epithelial cells lining the esophagus, matriptase by siRNA disrupts the development of TEER in barrier- stomach, jejunum, ileum, and colon of the GI tract (5). The phys- forming Caco-2 monolayers and increases paracellular permeability iological function of matriptase in the GI tract is not known. to macromolecular FITC-dextran. Loss of matriptase was associated Studies of individuals with homozygosity for null and hypo- with enhanced expression and incorporation of the permeability- morphic mutations in the St14 gene encoding matriptase, and associated, “leaky” tight junction protein claudin-2 at intercellular studies of St14 null and hypomorphic mice have revealed a junctions. Knockdown of claudin-2 enhanced the development of critical physiological role for matriptase in skin barrier formation TEER in matriptase-silenced Caco-2 monolayers, suggesting that the and epidermal differentiation (6–8), however the role of reduced barrier integrity was caused, at least in part, by an inability matriptase in other epithelia is less well defined. Using the to regulate claudin-2 expression and incorporation into junctions. matriptase-deficient St14 hypomorphic mouse strain (6) to We find that matriptase enhances the rate of claudin-2 protein turn- investigate matriptase function in intestinal epithelia, we have over, and that this is mediated indirectly through an atypical PKCζ- identified a critical role for matriptase in the formation and dependent signaling pathway. These results support a key role for regulation of the integrity of the intestinal epithelial barrier. Loss matriptase in regulating intestinal epithelial barrier competence, of matriptase, resulting either from genetic depletion in St14 and suggest an intriguing link between pericellular serine protease hypomorphic mice, via siRNA knockdown in the Caco-2 model activity and tight junction assembly in polarized epithelia. of intestinal epithelium, or by chemical inhibition causes a “leaky” barrier, manifested by the impaired ability to develop claudin-2 | intestinal barrier | St14 | type II transmembrane serine transepithelial resistance (TEER) and enhanced paracellular protease | tight junction permeability. This is mechanistically linked to the inappropriate expression of claudin-2, a tight junction protein associated with he intestinal epithelium provides a critical protective barrier increased intestinal permeability and barrier disruption in IBD. Tagainst enteric pathogens, food antigens, and physiochemical stresses caused by digestive and microbial products, and yet must Results be selectively permeable to beneficial nutrients and fluids. Tightly Matriptase Hypomorphic Mutant Mice Have a Leaky Gut. St14 regulated control of barrier function and integrity is critical, as the hypomorphic mice were found consistently to express less than 1% pathogenesis of intestinal diseases such as Crohn's disease, ulcer- of the matriptase mRNA levels detected in intestinal tissues of ative colitis, inflammatory bowel diseases (IBD), and autoimmune littermate control mice (Fig. 1A). The effect of this marked fi diseases are linked to intestinal barrier dysfunction and increased matriptase de ciency on intestinal barrier function was investigated by measurement of transepithelial electrical resistance (TEER) of intestinal permeability (1). The intestinal epithelium is a single fl layer of linked columnar epithelial cells that regulates, through the ex vivo intestinal tissues. TEER re ects paracellular resistance paracellular pathway, the selective passage of ions, fluid, and imparted by tight junctions and the lateral paracellular space, and is macromolecules from the intestinal lumen into the underlying tissues. This paracellular pathway is controlled by intercellular Author contributions: M.S.B., S.N.-A., T.S.-D., A.F., and T.M.A. designed research; M.S.B., apical junctional complexes (AJC) comprising apically located S.N.-A., T.S.-D., and A.Z. performed research; M.S.B., T.S.-D., A.Z., C.-Y.L., K.L., R.S., and tight junctions and lateral adherens junctions and desmosomes T.H.B. contributed new reagents/analytic tools; M.S.B., S.N.-A., T.S.-D., A.Z., A.F., T.H.B., (1). Intercellular AJCs are extremely dynamic structures that and T.M.A. analyzed data; and M.S.B., T.H.B., and T.M.A. wrote the paper. readily adapt to a variety of physiological and pathological stimuli. The authors declare no conflict of interest. They are composed of transmembrane proteins, including occlu- This article is a PNAS Direct Submission. din, claudins, and cadherins, which are linked intracellularly to 1To whom correspondence should be addressed. E-mail: [email protected]. cytoplasmic adaptor proteins, including the family of zonula This article contains supporting information online at www.pnas.org/cgi/content/full/ occludins proteins (e.g., ZO-1) and catenins. Despite significant 0903923107/DCSupplemental. 4200–4205 | PNAS | March 2, 2010 | vol. 107 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.0903923107 Downloaded by guest on September 28, 2021 changed during this period (Fig. S2A), suggesting matriptase protein expression is regulated at a posttranslational level during Caco-2 differentiation. A similar increase in matriptase protein levels (Fig. S2B), in the absence of changes in mRNA (Fig. S2C) was also observed in colonic T84 cells. Matriptase Localizes to AJCs in Polarized Caco-2 Monolayers. Con- focal microscopic examination of matriptase in the polarized fi Fig. 1. Matriptase hypomorph mice possess a leaky gut. (A) Matriptase Caco-2 monolayers revealed speci c localization of matriptase to mRNA levels in intestinal tissue segments of matriptase hypomorph mice sites of intercellular contacts (XY sections, Fig. 3A). Matriptase (Hypo) compared with control littermates (Control) analyzed by quantitative was confined to intercellular apically located junctional complexes, PCR (Q-PCR). Shown is mean from three mice of each genotype. (B) Intestinal whereas polymerized actin was present around the periphery of the permeability measured by TEER in segments of small intestine from cells (XZ sections, Fig. 3A). Negligible matriptase staining was matriptase hypomorph (n = 6) and control littermates (n = 4). Mean ± SEM at detected at either the apical or basal cell surfaces of polarized < 90 min are shown. *P 0.05. Caco-2 monolayers (Fig. 3A, XZ merge panel). Matriptase spe- cifically colocalized with the adherens junction marker E-cadherin a sensitive measure of barrier integrity. Measurement of the mean at the intercellular contacts (Fig. 3B), and was detected below the baseline TEER revealed a 35% reduction in TEER in intestinal apically associated tight junction proteins, ZO-1 and occludin (Fig. tissue segments of St14 hypomorphic mice compared with litter- S3), linking matriptase to the adherens junctions. mate controls (Fig. 1B). Other measures of intestinal function, including smooth muscle contractibility and glucose absorption, Inhibition of Matriptase Activity or siRNA Silencing of Matriptase were not found to be different between St14 hypomorphs and Expression Impairs Caco-2 Epithelial Barrier Formation. Exposure of control littermates. In addition, microscopic evaluation of for- Caco-2 monolayers to the broad-spectrum serine protease inhib- malin-fixed