Membrane-Anchored Serine Protease Matriptase Regulates Epithelial Barrier Formation and Permeability in the Intestine

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 October 1, 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 specifically 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 tissues taken from the ileal and colonic regions of the itor AEBSF during Caco-2 differentiation substantially inhibited hypomorph GI tract showed no significant structural abnormalities the development of TEER (Fig. 4A), suggesting a role for serine in villus architecture, the number of goblet cells, or mucosa and protease enzymatic activities during barrier formation. When crypt features. Together these data suggested a specific intestinal Caco-2 monolayers were exposed to the synthetic inhibitor, CVS- epithelial barrier deficiency in St14 hypomorphic mice. 3983, a selective inhibitor of matriptase proteolytic activity (10), Caco-2 monolayers exhibited a similar inhibition of TEER, with ≈ Matriptase Increases During Differentiation and Formation of Polarized values measuring 50% less on day 8 than in control cultures (Fig. Caco-2 Monolayers. The molecular mechanisms underlying 4A). Discontinuation of inhibitor treatment after day 8 resulted in – matriptase activity in intestinal epithelium were investigated restoration of TEER to the levels of media alone treated cultures using polarized Caco-2 monolayers (9). When grown on transwell (day 12; Fig. 4A), showing that the viability of the cultures was filters, Caco-2 cells spontaneously assemble tight junctions and unaffected. Together, these data implicate matriptase proteolytic display markers of intestinal epithelial cell differentiation, activity in epithelial barrier formation and AJC assembly. including the brush border membrane protease, dipeptidyl pep- Because chemical inhibitors can sometimes display off-target fi tidase IV (DPPIV) (Fig. 2A). Cell-associated matriptase was activities, we developed an alternative approach to speci cally deplete matriptase using siRNA silencing. For these experiments, detected as a 70-kDa band that represents the mature latent fl protein, and a faster-migrating 30-kDa band that represents the Caco-2 cells were transfected with siRNAs at a subcon uent density, and then added to transwell filters at higher density to C-terminal serine protease domain released after reduction of fl the single disulfide bridge that links the two peptide chains fol- accelerate the attainment of con uence and processes of epithelial lowing proteolytic activation of matriptase (4). Caco-2 cells differentiation. Under these conditions, and as reported by others

constitutively expressed low levels of matriptase before reaching (11), intercellular tight junctions are assembled within 4 days. CELL BIOLOGY conﬂuence (day 1; Fig. 2A), which increased dramatically as the Knock-down of matriptase mRNA and protein expression was cells became conﬂuent (by day 4) and developed TEER, with a 5- fold increase in expression detected by day 8 that remained elevated through day 21 (Fig. 2 A and B). These data demonstrate a correlation between matriptase expression and AJC assembly. Matriptase mRNA levels were not correspondingly

Fig. 2. Matriptase expression increases during Caco-2 epithelial cell polar- Fig. 3. Matriptase colocalizes with E-cadherin at apical junctional com- ization and barrier formation. (A) Immunoblot of cell lysates, prepared at the plexes in polarized Caco-2 monolayers. Caco-2 monolayers were grown on indicated times, showing increased levels of DPPIV and matriptase compared transwell ﬁlters for 21 days, immunostained with the indicated antibodies, with GAPDH. Matriptase (∼70 kDa) and the matriptase serine protease and analyzed by confocal analysis. XY images show a single section through domain (∼30 kDa) are indicated. NS indicates a ∼38-kDa band nonspeciﬁcally the monolayer; XZ images show a three-dimensional reconstruction of a recognized by the IM1014 antibody (Fig. S1). (B) TEER measured during for- cross-section of the monolayer. Position of plane of XY sections shown is mation of Caco-2 epithelial barrier (solid line). Graph represents the mean ± indicated by arrow on right of XZ section. (A) Double labeling for matriptase SEM TEER from three separate experiments. Dotted lines show densitometric using the antimatriptase antibody M32 (green) and rhodamine-conjugated quantitation of changes in DPPIV and matriptase from A. Fold increase is phalloidin (red). (B) Double labeling for matriptase using M32 antibody calculated relative to day 1 after normalizing for GAPDH levels. (green) and anti–E-cadherin antibody (red).

Buzza et al. PNAS | March 2, 2010 | vol. 107 | no. 9 | 4201 Downloaded by guest on October 1, 2021 Matriptase Depletion Increases Permeability to Macromolecular FITC- Dextran. TEER is inﬂuenced both by paracellular ion ﬂux and permeability to macromolecules (13). Measurement of the permeability of matriptase-silenced Caco-2 monolayers to the non- ionic macromolecular tracer, FITC-dextran (4kD), which can only traverse the monolayer via the paracellular route, was assessed (Fig. 4D). By day 5, when the development of TEER was substantially delayed in siM1 cultures, monolayers displayed a 3-fold increase in permeability to FITC-dextran compared with control monolayers, linking matriptase to the paracellular pathway.

Matriptase Contributes to AJC Reassembly After Barrier Disruption. The contribution of matriptase to Caco-2 barrier formation and the paracellular pathway, independent of cell proliferation, suggested a functional role during AJC assembly. To test this possibility, assembled tight junction complexes formed in confluent mature Caco-2 monolayers were disrupted by low calcium media and then allowed to reassemble by replacing the media with calcium-containing growth media (calcium switch). In the presence of Fig. 4. Matriptase is required for development of TEER in Caco-2 epithelial the matriptase inhibitor CVS-3983, barrier reassembly was sig- monolayers. (A) Inhibition of matriptase serine protease activity inhibits fi development of TEER in Caco-2 monolayers. Confluent Caco-2 cultures (TEER ni cantly delayed (Fig. 5A). Depletion of matriptase by siRNA in ∼250 ohm·cm2) were treated with the broad-spectrum serine protease Caco-2 monolayers similarly delayed TEER development; within inhibitor AEBSF (25 μM) or the specific matriptase inhibitor CVS-3983 (25 μM) 7 h postcalcium switch, the siM1-treated monolayers developed for 8 days, after which the inhibitors were removed. TEER development only ∼50% of the TEER of the siCtl-treated cultures (Fig. 5B). recovered to that of untreated cultures by day 12 after removal of CVS-3983 The delayed development of TEER was associated with a corre- (right axis). (B) RNAi silencing of matriptase in Caco-2 epithelial monolayers. sponding increase in permeability to FITC-dextran (4kDa) of ∼9- Immunoblot of matriptase protein levels analyzed 24 h after transfection fold relative to control monolayers (Fig. 5C). Together these data with the two matriptase siRNAs (siM1, M2) or control siRNA (siCtl) on the demonstrate the importance of matriptase to AJC assembly and indicated days after plating on transwell filters. Matriptase protein levels are suppressed by >90% by day 1, whereas levels of the related membrane the paracellular pathway. proteases prostasin and DPPIV are unaffected by matriptase siRNAs. (C) RNAi silencing of matriptase inhibits de novo formation of Caco-2 epithelial bar- Known Matriptase Substrates Do Not Account for Matripase riers. Measurement of TEER development on the indicated days from the Regulation of Epithelial Barrier Integrity. The natural substrates of cultures depicted in B.(D) Matriptase-depleted monolayers display increased matriptase that may be proteolytically processed in the lateral paracellular permeability to macromolecular 4 kDa FITC–dextran. Apparent paracellular space of polarized intestinal epithelia are not known. permeabilities (Papp) were measured on day 2 (before barrier formation) and on day 5 postplating onto transwells. Graph represents mean ± SEM from triplicate wells, **P = 0.01. Corresponding average of TEER measured for each culture is shown in parentheses above each bar.

achieved with either of two independent siRNAs (siM1, siM2) by day 1 after addition to transwell ﬁlters, relative to the control siRNA (siCtl) (Fig. S4 and Fig. 4B). Matriptase protein levels remained effectively suppressed through day 7, after which time matriptase levels slowly began to increase, most notably with siM1 (Fig. 4B). The expression of other membrane-associated serine proteases, DPPIV or prostasin, was unaffected by the matriptase targeted siRNAs (Fig. 4B). Over the 7-day period, the matriptase- silenced Caco-2 monolayers (siM1, siM2) exhibited impaired development of TEER, resulting in a ∼70% lower TEER compared with siCtl monolayers at day 7 (Fig. 4C). Extended analysis over a 21-day period showed recovery of TEER as matriptase expression in siM1-silenced monolayers reappeared (Fig. S5). These data show that the depletion of matriptase did not irrever- sibly arrest barrier development, and indicate that matriptase is Fig. 5. Matriptase contributes to AJC reassembly after barrier disruption. (A) Inhibition of matriptase activity delays the development of TEER in Caco- essential for differentiation processes required for normal intes- 2 monolayers disrupted by low calcium. Calcium switch assay performed on tinal epithelial barrier formation. Caco-2 cultures (TEER > 1,000 ohm·cm2). Cells were cultured in the presence or absence of the matriptase inhibitor CVS-3983 (25 μM) during barrier Caco-2 Cell Proliferation in Polarized Monolayers Is Unaffected by reformation after calcium switch. (B) RNAi silencing of matriptase inhibits Matriptase Depletion. Matriptase activity has been associated with Caco-2 barrier reassembly after disruption. Transfected Caco-2 cells (siCtl or cell proliferation via activation of growth factors in some cellular siM1) were grown on transwells for 5 days before calcium switch. (Upper) contexts (12), raising the possibility that the impaired ability to Immunoblot analysis of matriptase protein levels analyzed before calcium − develop TEER in matriptase-silenced Caco-2 monolayers could depletion ( 15 h), after incubation in low-calcium medium (0 h), and after be due to reduced cell proliferation. Cell proliferation assays calcium restoration at 8 and 24 h. (Lower) Development of TEER at the indicated times after calcium restoration. (C) Permeabilities to macro- revealed that there were similar cell numbers in matriptase- molecular FITC–dextran were assessed at 0 and 7 h postcalcium switch. silenced and control Caco-2 monolayers (Fig. S6), showing that Graph shows mean ± SEM from triplicate wells, and data are plotted using a decreased cell proliferation did not account for the inability of log scale. *P < 0.05. Corresponding average of TEER measured for each matriptase depleted monolayers to develop normal TEER. culture is shown in parentheses above each bar.

4202 | www.pnas.org/cgi/doi/10.1073/pnas.0903923107 Buzza et al. Downloaded by guest on October 1, 2021 Known matriptase substrates present in intestinal epithelia, e.g., TEER develops (Fig. S2 and Fig. 6B, siCtl, claudin-2: day 3 vs. day the zymogens of prostasin and urokinase plasminogen activator 6). In contrast, claudin-1 protein levels increase over this same (uPA), or the epidermal growth factor (EGF) receptor, protease- period (Fig. 6B, siCtl, claudin-1: day 3 vs. day 6) as expected. activated receptor–2 (PAR-2), or HGF signaling pathways, were Silencing of matriptase expression prevented the down-regulation not found to be direct targets of matriptase in polarized intestinal of claudin-2, with matriptase-depleted monolayers displaying the epithelial cells (Fig. S7), indicating an alternative molecular elevated claudin-2 levels associated with the less differentiated activity for matriptase during epithelial barrier formation and cells (Fig. 6B, siM1 and siM2, claudin-2: day 3 vs. day 6), whereas AJC assembly. claudin-1 levels were unaffected by matriptase depletion (Fig. 6B, siM1 and siM2, claudin-1: day 3 vs. day 6). Interestingly, loss of Permeability-Associated Tight Junction Protein Claudin-2 Is Selectively claudin-2 is observed predominantly in the insoluble fraction of Deregulated in Matriptase-Silenced Caco-2 Monolayers. Examination extracted lysates (Fig. 6B; day 6 siCtl, claudin-2: IS vs. S), sug- of the expression of AJC proteins involved in barrier assembly in gesting that matriptase facilitates claudin-2 loss from formed tight formed matriptase-silenced Caco-2 monolayers compared with junctional complexes. Indeed, confocal microscopic examination control cultures revealed no significant changes in the levels of the of claudin-2 expression revealed elevated claudin-2 at the intra- tight junction proteins occludin and claudin-1, claudin-3, claudin- cellular junctions of matriptase-silenced Caco-2 monolayers 4, and claudin-8, the adapter protein ZO-1, or the adherens compared with control monolayers (Fig. S8C). junction proteins, E-cadherin, and β-catenin (Fig. 6A and Fig. S9A). The localization of several of these AJC components at Matriptase Hypomorphic Leaky Gut Is Associated with Enhanced intercellular contacts similarly appeared to be unaffected by Claudin-2 Expression in Surface Villous Epithelial Cells. In the small matriptase depletion either during barrier assembly (Fig. S8A)or intestine, claudin-2 expression is normally restricted to the rel- following calcium switch (Fig. S8B). In contrast, there was a sub- atively leaky epithelial cells of the crypts, with its expression stantial 3.5- to 4-fold increase in the levels of the permeability- decreasing substantially in differentiating epithelium along the associated tight junction protein claudin-2 in the matriptase- crypt–villus axis (14). Examination of claudin-2 expression by silenced Caco-2 monolayers (Fig. 6A). The claudins are a family of immunostaining of jejunal tissues from St14 hypomorphic mice tight junctional proteins that form ion specific channels, the compared with control mice showed normal claudin-2 staining in majority of which, including claudin-1, function to enhance the the crypt epithelium of both genotypes (Fig. 6C, arrows). Of “tightness” of epithelial barriers. Some of the claudins however, significance, the surface villous epithelial cells of St14 hypo- such as claudin-2, form ion specific channels that result in the morph small intestines alone displayed substantial claudin-2 “loosening” of epithelial barriers and reduction of epithelial expression that was not evident in the control animals (Fig. 6C, resistance (13). Investigation of the time course of claudin-2 arrowheads). Claudin-2 immunostaining was localized to inter- expression during Caco-2 barrier formation revealed that claudin- cellular regions of the surface epithelium (Fig. 6C, Right). These 2 protein and mRNA expression were highest in Caco-2 cells in vivo findings are consistent with the in vitro findings and before formation of the epithelial barrier, and then were sub- support a role for matriptase in regulating intestinal epithelial stantially down-regulated as Caco-2 monolayers differentiate and barrier integrity through the regulation of claudin-2 protein and its incorporation into intercellular junctional complexes.

Knockdown of Claudin-2 Enhances TEER Development in Matriptase- Silenced Caco-2 Monolayers. Because claudin-2 expression is a determinant of transepithelial resistance and functions to decrease the “tightness” of the epithelial intercellular tight junctions (13), we reasoned that the persistent expression of claudin-2 could contribute to the impaired ability of matriptase-depleted Caco-2 monolayers to develop an effective TEER. Indeed, knockdown of claudin-2 by cotransfection of claudin-2 siRNA in CELL BIOLOGY matriptase-depleted monolayers enhanced TEER development to normal levels (Fig. 7A). Interestingly, knockdown of claudin-2 alone in the Caco-2 monolayers increased TEER development, as has been observed previously (15), suggesting that the low level expression of claudin-2 in control Caco-2 monolayers (Fig. 7A, day 5 siCtl, claudin-2) prevents complete tightening of the Caco-2 epithelial barrier. These ﬁndings support a relationship between claudin-2 levels and the tightness of AJC complex formation, and further implicate the permeability-associated activities of claudin- 2 in the paracellular pathway regulated by matriptase. Fig. 6. Claudin-2 is deregulated in matriptase-depleted intestinal epithelial cells. (A) Immunoblot analysis of junctional proteins present in total cell lysates on day 6 posttransfection. Shown are lysates from two independent trans- Matriptase Does Not Cleave Claudin-2 Directly but Facilitates Claudin- fections (A and B). (B) Immunoblot analysis of claudin-2, claudin-1, and 2 Protein Turnover. Persistent claudin-2 expression was not asso- matriptase protein levels in lysates collected on days 3 and 6 posttransfection. ciated with changes in claudin-2 mRNA levels in matriptase-silenced Triton-soluble fraction (S), and the Triton-insoluble (IS), junction-associated Caco-2 monolayers during barrier formation (Fig. S9B) or in intes- fraction are shown. Average TEERs, day 3: 518 (siCtl), 197 (siM1), and 205 (siM2) tinal epithelial scrapings from St14 hypomorph mice compared with ohm·cm2; day 6: 1,275 (siCtl), 517 (siM1), and 452 (siM2) ohm·cm2.(C) Detection control littermates (Fig. S9C). However, pulse chase analyses com- of claudin-2 on surface of villous epithelium of St14 hypomorphic animals. paring claudin-2 protein stability during barrier formation in Frozen sections of jejunum from control and hypomorph mice immunostained matriptase-silenced Caco-2 monolayers revealed that matriptase with antimouse claudin-2 antibody (green), and counterstained with DAPI for detection of nuclei (blue). Arrows indicate intestinal crypt cells, arrowheads depletion stabilized claudin-2 levels by greater than 3-fold (Fig. 7B), indicate villous epithelium. At high power, claudin-2 staining can be seen to indicating that matriptase facilitates the posttranscriptional turnover localize to intercellular junctions in the hypomorph intestine (Right). Micro- of claudin-2 protein during formation of epithelial barriers. As graphs are representative of four animals of each genotype. Original magni- claudin-2 is not proteolytically processed directly by matriptase (Fig. ﬁcations are 200× (Left and Center) and 1,000× (Right). S10), and as we found no evidence for claudin-2 cleavage peptides in

Buzza et al. PNAS | March 2, 2010 | vol. 107 | no. 9 | 4203 Downloaded by guest on October 1, 2021 Fig. 8. Matriptase activates the PKCζ pathway during epithelial barrier formation. (A) Decreased activation of PKCζ in matriptase-depleted Caco-2 Fig. 7. Enhanced turnover of claudin-2 is functionally associated with monolayers. Cell lysates prepared on day 6 were immunoblotted for phos- ζ ζ matriptase activity. (A) Knockdown of claudin-2 enhances the development phorylated PKC (P-threonine-410), followed by total PKC .(B) Inhibition of ζ of TEER in Caco-2 monolayers. (Left) TEER development in Caco-2 mono- PKC pathway delays TEER development. Caco-2 cells plated on transwells μ ζ ζ layers after silencing of matriptase (siM1), claudin-2 (siCL2), or both together were treated with 50 M PKC -pseudosubstrate inhibitor (PKC -psi) for 48 h. ζ ± (siCL2+ iM1). (Right) Immunoblot analysis of the levels of the indicated Graph shows delay in TEER development in the presence of PKC -psi (mean ζ proteins on day 3 and day 5. Results are representative of three independent SE from triplicate wells). (C) Inhibition of the PKC pathway is associated with experiments using two independent claudin-2 siRNAs. (B) Matriptase medi- sustained expression of claudin-2, whereas claudin-1 is unaffected. Immuno- μ ζ ates enhanced turnover of claudin-2 protein. siCtl- and siM2-transfected cells blot analysis was performed after 48 h treatment with 50 M PKC -psi. were cultured on transwells for 7 days before metabolic labeling and pulse chase. Representative autoradiogram is shown (Upper); plot (Lower) shows Matriptase expression increases during Caco-2 barrier for- average rates of claudin-2 and claudin-1 decay from two independent mation and differentiation, consistent with the higher levels of experiments. Time required to attain 50% of starting claudin-2 levels is greater than 3-fold slower in matriptase-silenced cells. matriptase associated with differentiated epithelia at the intestinal villous tips (19). In polarized Caco-2 monolayers, matriptase localizes to intercellular junctional complexes in association Caco-2 lysates under a variety of conditions, these ﬁndings impli- with E-cadherin along the lateral membrane. Activated PKCζ cated the indirect activation of a signaling pathway by matriptase expression and localization at intercellular junctions is similarly that affects claudin-2 protein turnover. detected in differentiated intestinal epithelia (18, 20). In contrast, claudin-2 expression decreases with Caco-2 differentiation Matriptase Regulates Claudin-2 Turnover via Activation of Atypical (21); this is paralleled in the intestine, where expression de- PKCζ Signaling Pathway. The constitutive turnover of claudin-2 is creases substantially in villous epithelium (14). thought be mediated by an E3 ubiquitin ligase that targets claudins Members of the claudin family that are present at epithelial tight for subsequent lysosomal degradation (16). However, the signal- junctions regulate TEER by a mechanism that does not appear to ing pathways that regulate claudin-2 turnover have not been be related to structural changes in the tight junctions but, rather, established. Atypical protein kinase C (PKC) isoforms are through the formation of pores that selectively control the passage involved in several signal transduction pathways and are essential of cationic or anionic solutes (13). Claudin-2 decreases the tight- for the formation of epithelial tight junctions and establishment of ness of epithelial barriers through the formation of cation- cell polarity (17). We found that matriptase silencing during bar- selective ion channels with particular afﬁnity for sodium ions at the rier formation results in decreased activation of PKCζ (Fig. 8A), tight junctions, but does not contribute to the paracellular per- detected by phosphorylation at Thr-410 which is diagnostic for meability of small noncharged molecules (13, 15). In addition to PKCζ activation (18), indicating that matriptase-mediated barrier decreased TEER, matriptase loss from epithelial monolayers is formation involves activation of a PKCζ signaling pathway. To associated with increased permeability to macromolecules, sug- investigate whether activation of PKCζ regulates the rate of gesting that matriptase may regulate additional pathways asso- claudin-2 turnover, PKCζ activity was functionally inhibited using ciated with tight junction assembly and barrier formation. a cell-permeable PKCζ pseudosubstrate inhibitor (PKCζ-psi). Increased claudin-2 expression and loss of barrier integrity in other TEER development was inhibited in a dose-dependent manner by cell systems is typically in response to cytokines (22) and is charac- PKCζ-psi (Fig. 8B and Fig. S11), which was accompanied by the terized by increased claudin-2 mRNA. The pathways that regulate substantial accumulation of claudin-2 protein (Fig. 8C), in the claudin-2 protein turnover and target it for degradation via lysosomes absence of changes in claudin-2 mRNA levels (Fig. S11B). These (16) remain to be established; however, our data implicate a data support a mechanism by which matriptase regulates claudin-2 matriptase-mediated proteolytic pathway that facilitates the turnover turnover and intestinal epithelial barrier integrity through a PKCζ- of claudin-2 during AJC assembly that is associated with PKCζ dependent signaling pathway. activation. Interestingly, PKCζ signaling has been implicated recently in trypsin and chymotrypsin-enhanced barrier formation (23). Discussion Little is known regarding the regulation of claudin function by The studies presented here provide molecular insight into atypical PKCs, although numerous studies demonstrate their matriptase function in intestinal epithelia and reveal a fundamental involvement in the association of intercellular signaling complexes role for matriptase during epithelial barrier formation and AJC that trigger AJC assembly. Atypical PKC activation induces assembly. Matriptase depletion, either by genetic down-regulation increased phosphorylation of the barrier-forming claudin-4 and its in St14 hypomorphic mice or by RNAi silencing in epithelial localization at cell junctions (24). The present study links PKCζ monolayers, alters the permeability of tight junctions, resulting in activity with increased turnover of the leaky claudin-2 during the decreased epithelial resistance and increased permeability to assembly of tight junctions, further implicating atypical PKCs in macromolecules. The molecular basis of the leaky barrier involves the regulation of this family of tight junction proteins. the stabilization and aberrant incorporation of the permeability- Serine proteases have long been recognized to contribute to associated protein claudin-2 into intercellular junctions mediated protein degradation associated with nutrient digestion in the GI indirectly through an atypical PKC cell-signaling pathway. tract, and increasing evidence suggests that these enzymes also

4204 | www.pnas.org/cgi/doi/10.1073/pnas.0903923107 Buzza et al. Downloaded by guest on October 1, 2021 contribute in a complex way to the regulation of intestinal integrity monolayers was measured using “chopstick: probes. Baseline resistance read- and barrier function. Low levels of trypsin and certain other ings were determined in wells containing membrane inserts only, subtracted endopeptidases are potent inducers of intercellular AJC for- from sample values and expressed in ohm·cm2. Flux of 4-kDa FITC-conjugated mation in intestinal epithelial monolayers (23, 25), whereas higher dextran (Fluka) across Caco-2 monolayers was assayed as described in SI Text. levels of these enzymes disrupt epithelial barriers. Epithelial bar- – rier formation may also be suppressed by serine protease inhibitors Caco-2 Cell Culture and Transfection. Caco-2 cells (ATCC, passage 35 45), cultured under standard cell culture conditions (27), were grown on Trans- (25), further implicating serine protease activities in AJC assem- fi bly. These findings, together with the barrier-protective activity of well lters (Costar) for up to 21 days for formation of polarized epithelial monolayers. For calcium switch experiments, 5-day filter-grown Caco-2 matriptase, implicate pericellular proteolytic processing as an monolayers were exposed to low calcium medium (DMEM + 5 μM CaCl2)for important component of epithelial barrier formation. 15 h, after which medium was replaced with complete DMEM (1.6 mM In summary, we have identified a proteolytic mechanism in the CaCl2) for up to 24 h. When used, the chemical inhibitors AEBSF (Calbio- lateral paracellular space of polarized epithelia important for the chem), CVS-3983 (10) or PKCζ myristolated-psi (Calbiochem), were added to regulation of intestinal epithelial barrier integrity. Compromise culture media and replaced daily. Transfections were performed using of the epithelial barrier is an early event in disorders such as Dharmafect 1 transfection reagent (Dharmacon) using primers listed in the Crohn’s disease and IBD, and the resulting functional change in SI Methods. Cells were plated onto filters 24 h posttransfection at high barrier permeability exacerbates inflammation. Claudin-2 is fre- density (5 × 105 cells/well). Results presented are representative of at least quently up-regulated in active Crohn’s disease and in the three to five independent transfection experiments. inflamed mucosa of patients with ulcerative colitis, where it has been proposed to contribute to increased intestinal permeability Immunofluorescence Microscopy. Caco-2 monolayers grown on transwell fil- and pathogenesis (26). It will be important in future studies to ters for 21 days were methanol fixed at room temperature. Rhodamine- identify the unique substrates of matriptase involved in this conjugated phalloidin was used for detection of actin. XY sections (0.5-μm pathway, and to investigate matriptase dysregulation in the steps) were captured using a Bio-Rad Radiance 2100 confocal microscope pathogenesis of inflammatory diseases of the GI tract. and reconstructed using the Velocity Image Analysis program for XZ images. Intestinal tissues removed from mice were cryopreserved, fixed in methanol, Methods and stained for claudin-2 and DAPI for detection of nuclei. Fluorescence was visualized using a Nikon Eclipse E800 microscope and captured with Axio- Reagent details and additional methods are provided in SI Methods. vision (Zeiss) image capture software. Animals. The St14 (matriptase) hypomorphic mice have been described (6). ’ All experiments were littermate and sibling controlled. Institutional Animal Statistical Analyses. The two-tailed Student s t test was used to compare Care and Use Committee approval was obtained for all experimental pro- averages of normally distributed data with equal variance. A threshold of < fi cedures. For ex vivo analyses, the small intestines were flushed, and the was P 0.05 was considered signi cant. muscle removed and then cut into 1-cm sections. Directly adjacent tissue segments were used for measurements of ex vivo TEER and isolation of RNA. ACKNOWLEDGMENTS. We thank E. Smith, J. Stiltz, and R. Sun for technical Enriched preparations of epithelial cells were obtained by the coverslip assistance. CVS-3983 was provided by Dr. Ed Madison and Corvas Interna- scraping method. tional (San Diego, CA). This work was supported in part by grants from the National Institutes of Health (NIH): DK081376, CA098369, and HL084387 (to T.M.A.); DK48373 (to A.F.); AI/DK49316 (to T.S.D.); HL07698 (to S.N.A.); Measurements of Barrier Integrity. TEER of small intestine segments mounted CA096851 (to C.Y.L.) and the NIH Intramural Program (T.H.B.). M.B. was in microsnapwells apical side up was measured in triplicate using an EVOM supported by Australian National Health and Medical Research Council CJ Voltohmmeter (World Precision Instruments) as described (27). TEER of Caco-2 Martin Fellowship 384359.

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