Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity
2008; Elsevier BV; Volume: 283; Issue: 43 Linguagem: Inglês
10.1074/jbc.m801970200
ISSN1083-351X
AutoresRoman Szabo, John P. Hobson, Karin List, Alfredo Molinolo, Chen‐Yong Lin, Thomas Bugge,
Tópico(s)Liver physiology and pathology
ResumoHepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless β-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase. Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless β-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase. Recent mining of vertebrate genomes uncovered an unexpectedly large number of new membrane-associated trypsin-like serine proteases. The biochemical and physiological functions of most of these new serine proteases are undefined and the subject of active investigation. Trypsin-like serine proteases are typically synthesized as inactive zymogens that are irreversibly activated by a single endoproteolytic cleavage within a highly conserved activation site. They are subsequently inactivated by specific serine protease inhibitors that bind directly to the active site (1Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (742) Google Scholar, 2Rawlings N.D. Barrett A.J. Methods Enzymol. 1994; 244: 19-61Crossref PubMed Scopus (521) Google Scholar, 3Rau J.C. Beaulieu L.M. Huntington J.A. Church F.C. J. Thromb. Haemost. 2007; 5: 102-115Crossref PubMed Scopus (254) Google Scholar). Three functionally distinct classes of serine protease inhibitors, termed serpin-, Kazal-, and Kunitz-type inhibitors, have been identified in vertebrates. Whereas the serpin-type inhibitors have been extensively studied due to their preeminent role in regulating coagulation and fibrinolysis (3Rau J.C. Beaulieu L.M. Huntington J.A. Church F.C. J. Thromb. Haemost. 2007; 5: 102-115Crossref PubMed Scopus (254) Google Scholar), the Kazal-and Kunitz-type serine protease inhibitors in vertebrates are comparatively less explored.Hepatocyte growth factor activator inhibitor (HAI 2The abbreviations used are: HAI, hepatocyte growth factor activator inhibitor; β-geo, fusion of β-galactosidase and neomycin phosphotransferase; IC50, half-maximal (50%) inhibitory concentration; PAI, plasminogen activator inhibitor; uPA, urokinase plasminogen activator. 2The abbreviations used are: HAI, hepatocyte growth factor activator inhibitor; β-geo, fusion of β-galactosidase and neomycin phosphotransferase; IC50, half-maximal (50%) inhibitory concentration; PAI, plasminogen activator inhibitor; uPA, urokinase plasminogen activator.)-1 and HAI-2 (also known as placental bikunin), encoded by Spint1 and Spint2 genes, respectively, are two recently discovered and closely related membrane-associated Kunitz-type serine protease inhibitors. These unusual serine protease inhibitors are type I transmembrane glycoproteins that contain two extracellular Kunitz-type inhibitory domains (4Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Itoh H. Kataoka H. Hamasuna R. Kitamura N. Koono M. Biochem. Biophys. Res. Commun. 1999; 255: 740-748Crossref PubMed Scopus (26) Google Scholar, 6Muller-Pillasch F. Wallrapp C. Bartels K. Varga G. Friess H. Buchler M. Adler G. Gress T.M. Biochim. Biophys. Acta. 1998; 1395: 88-95Crossref PubMed Scopus (50) Google Scholar, 7Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 8Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). HAI-1 was originally described as an endogenous inhibitor of hepatocyte growth factor activator (8Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). However, studies published shortly after the identification of HAI-1 show that the shed extracellular domain of HAI-1 can be isolated from tissue fluids and cell culture supernatants in a complex with the extracellular domain of the transmembrane serine protease matriptase (encoded by the ST14 gene) (9Lin C.Y. Anders J. Johnson M. Dickson R.B. J. Biol. Chem. 1999; 274: 18237-18242Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), strongly suggesting a physiological role of HAI-1 in matriptase inhibition. This was confirmed in several recent genetic studies in mice and zebrafish that revealed an essential role of matriptase inhibition by HAI-1 during vertebrate embryonic development (10Tanaka H. Nagaike K. Takeda N. Itoh H. Kohama K. Fukushima T. Miyata S. Uchiyama S. Uchinokura S. Shimomura T. Miyazawa K. Kitamura N. Yamada G. Kataoka H. Mol. Cell Biol. 2005; 25: 5687-5698Crossref PubMed Scopus (79) Google Scholar, 11Fan B. Brennan J. Grant D. Peale F. Rangell L. Kirchhofer D. Dev. Biol. 2007; 303: 222-230Crossref PubMed Scopus (55) Google Scholar, 12Szabo R. Molinolo A. List K. Bugge T.H. Oncogene. 2007; 26: 1546-1556Crossref PubMed Scopus (109) Google Scholar, 13Mathias J.R. Dodd M.E. Walters K.B. Rhodes J. Kanki J.P. Look A.T. Huttenlocher A. J. Cell Sci. 2007; 120: 3372-3383Crossref PubMed Scopus (97) Google Scholar, 14Carney T.J. von der Hardt S. Sonntag C. Amsterdam A. Topczewski J. Hopkins N. Hammerschmidt M. Development. 2007; 134: 3461-3471Crossref PubMed Scopus (83) Google Scholar).The analysis of the functions of HAI-2 in vertebrate physiology has been complicated by the reported early embryonic lethality of Spint2 null mice (15Mitchell K.J. Pinson K.I. Kelly O.G. Brennan J. Zupicich J. Scherz P. Leighton P.A. Goodrich L.V. Lu X. Avery B.J. Tate P. Dill K. Pangilinan E. Wakenight P. Tessier-Lavigne M. Skarnes W.C. Nat. Genet. 2001; 28: 241-249Crossref PubMed Scopus (347) Google Scholar). However, HAI-1 and HAI-2 are both type I transmembrane proteins and display 39-56% amino acid identity in their two Kunitz-type inhibitor domains, suggesting that HAI-2 could be a second physiological inhibitor of matriptase. Indeed, we show here that the kinetics of matriptase inhibition by HAI-2 is equipotent to that of HAI-1, and that matriptase and HAI-2 form SDS-stable complexes. Furthermore, HAI-2 efficiently blocks matriptase-mediated activation of two physiological candidate substrates, the prostasin zymogen and cell surface-bound pro-urokinase plasminogen activator (uPA). By generating a mouse strain with a promoterless β-galactosidase marker gene inserted into the endogenous Spint2 locus, we show that HAI-2 co-localizes with matriptase and HAI-1 in the epithelia of all major organ systems. Collectively, these new data strongly implicate HAI-2 as a physiologically relevant inhibitor of matriptase, possibly acting in a redundant or partially redundant manner with HAI-1 to regulate epithelial cell surface proteolysis in adult tissues.EXPERIMENTAL PROCEDURESChromogenic Peptide Hydrolysis Assay—The chromogenic peptide Glu-Gly-Arg-p-nitroanilide was purchased from Bachem Bioscience (King of Prussia, PA), the soluble recombinant human HAI-1 and HAI-2 extracellular domains were from R&D Systems (Minneapolis, MN). To study the inhibition of matriptase proteolytic activity, 1 nm recombinant human matriptase serine protease domain (16Friedrich R. Fuentes-Prior P. Ong E. Coombs G. Hunter M. Oehler R. Pierson D. Gonzalez R. Huber R. Bode W. Madison E.L. J. Biol. Chem. 2002; 277: 2160-2168Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) was incubated with 100 μm chromogenic peptide substrate in 50 mm Tris-HCl, pH 8.0, 1 mm CaCl2, 0.1% Tween 20 for 20 min at 37 °C in the presence of 0-500 nm HAI-1 or HAI-2 proteins. Changes in absorbance at 405 nm were monitored over time on a SAFIRE2™ Microplate Reader (Tecan, Durham, NC). All measurements were performed in triplicate.Formation of HAI/Matriptase Inhibitory Complexes—100 ng of the recombinant active human matriptase serine protease domain in 50 mm Tris/HCl, pH 8.0, 100 mm NaCl buffer was incubated with 300 ng of human recombinant HAI-1 or HAI-2 for 30 min at room temperature. Protein complexes were analyzed by 4-12% reducing SDS-PAGE and Western blotting using a polyclonal anti-mouse HAI-2 primary antibody (R&D Systems) and anti-goat secondary antibody conjugated with alkaline phosphatase (EMD Chemicals-Calbiochem, La Jolla, CA) and a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium detection system (Roche Applied Science). For non-immunological detection of HAI-matriptase complex formation, the assay was performed using 20 ng of active human matriptase serine protease domain and 50 ng of human recombinant HAI-1 or HAI-2, followed by 4-12% reducing SDS-PAGE and silver staining using a SilverQuest kit (Invitrogen) according to the manufacturer's instructions.Prostasin Zymogen Activation Assay—0.1 μm human soluble prostasin zymogen prepared as described (17Netzel-Arnett S. Currie B.M. Szabo R. Lin C.Y. Chen L.M. Chai K.X. Antalis T.M. Bugge T.H. List K. J. Biol. Chem. 2006; 281: 32941-32945Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) was incubated with 5 nm recombinant active human matriptase serine protease domain for 1 h at 37 °C in 50 mm Tris/HCl, pH 8.0, 100 mm NaCl buffer. To evaluate the effect of HAI-1 and HAI-2 on matriptase-mediated activation of pro-prostasin, matriptase was preincubated with 100 nm human recombinant HAI-1 or HAI-2 proteins for 30 min at 37 °C prior to the pro-prostasin activation assay. Proteins were boiled and analyzed by reduced SDS-PAGE followed by Western blot as described above using a monoclonal anti-prostasin antibody (BD Biosciences, San Jose, CA), goat anti-mouse secondary antibody conjugated with alkaline phosphatase (DakoCytomation, Glostrup, Denmark).Activation of Pro-uPA on the Surface of THP-1 Cells—Matriptase-dependent cell surface activation of pro-uPA was determined as described previously (18Kilpatrick L.M. Harris R.L. Owen K.A. Bass R. Ghorayeb C. Bar-Or A. Ellis V. Blood. 2006; 108: 2616-2623Crossref PubMed Scopus (89) Google Scholar). Briefly, THP-1 acute monocytic leukemia cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, l-glutamine, and antibiotics (all from Invitrogen). Before the experiment, the cells were washed twice in serum-free RPMI 1640 buffered with 25 mm HEPES, pH 7.4, and incubated for 5 min at room temperature in 100 mm NaCl, 50 mm glycine/HCl, pH 3.0, buffer to dissociate cell surface-associated uPA. Cells were washed twice in serum-free RPMI 1640, 25 mm HEPES, pH 7.4, resuspended in the same medium at 107 cells/ml, and incubated with 300 nm HAI-2, 250 nm plasminogen activator inhibitor-1 (R&D Systems), or in the absence of inhibitors for 15 min at room temperature. 100 nm pro-uPA (a kind gift of Dr. Stephen Leppla, NIAID, National Institutes of Health) or 100 nm high molecular weight two-chain uPA (Molecular Innovations, Novi, MI) were then added, and the mixture was incubated for 30 min at 37 °C. Cells were washed twice with serum-free RPMI 1640, 25 mm HEPES, pH 7.4, resuspended at 106 cells/ml in 50 mm Tris/HCl, pH 7.4, 100 mm NaCl, 0.01% Tween 20 with 0.5 mm SpectrozymeUK (American Diagnostica, Stamford, CT), and incubated at 37 °C for 4 h. uPA substrate hydrolysis was measured by the increase in absorbance at 405 nm using a Victor3V spectrophotometer (PerkinElmer Life Sciences).Tissue Acquisition—All procedures involving live animals were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium following Institutional Guidelines and standard operating procedures. Spint2 knock-in mice containing a promoterless β-galactosidase gene trap inserted into intron 1 of the mouse Spint2 gene were generated from the embryonic stem cell line KST272 obtained from Bay Genomics (San Francisco, CA) (15Mitchell K.J. Pinson K.I. Kelly O.G. Brennan J. Zupicich J. Scherz P. Leighton P.A. Goodrich L.V. Lu X. Avery B.J. Tate P. Dill K. Pangilinan E. Wakenight P. Tessier-Lavigne M. Skarnes W.C. Nat. Genet. 2001; 28: 241-249Crossref PubMed Scopus (347) Google Scholar). The generation of the Spint1 null mice and β-galactosidase-tagged ST14 knock-in mice have previously been described (12Szabo R. Molinolo A. List K. Bugge T.H. Oncogene. 2007; 26: 1546-1556Crossref PubMed Scopus (109) Google Scholar, 19List K. Haudenschild C.C. Szabo R. Chen W. Wahl S.M. Swaim W. Engelholm L.H. Behrendt N. Bugge T.H. Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (281) Google Scholar, 20List K. Szabo R. Molinolo A. Nielsen B.S. Bugge T.H. Am. J. Pathol. 2006; 168: 1513-1525Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The Spint2 knock-in mice were genotyped for the presence of the β-galactosidase gene trap by PCR using HAI-2-geo51 (5′-ATCTGCAACCTCAAGCTAGC-3′) and HAI-2-geo31 (5′-CAGAACCAGCAAACTGAAGG-3′) primers. The Spint1 null mice and ST14 knock-in mice were genotyped by PCR as described previously (12Szabo R. Molinolo A. List K. Bugge T.H. Oncogene. 2007; 26: 1546-1556Crossref PubMed Scopus (109) Google Scholar, 19List K. Haudenschild C.C. Szabo R. Chen W. Wahl S.M. Swaim W. Engelholm L.H. Behrendt N. Bugge T.H. Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (281) Google Scholar, 20List K. Szabo R. Molinolo A. Nielsen B.S. Bugge T.H. Am. J. Pathol. 2006; 168: 1513-1525Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar).Whole Mount X-gal Staining—Six-month-old wild-type or heterozygous β-galactosidase-tagged Spint2 knock-in mice or β-galactosidase-tagged ST14 knock-in mice were euthanized by CO2 inhalation. Organs were excised, and slices of each tissue were placed in 4% paraformaldehyde in phosphate-buffered saline for 30 min, rinsed in phosphate-buffered saline, and stained overnight at 37 °C with a β-galactosidase staining kit (Roche Applied Science). The tissues were post-fixed for 16 h in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were counterstained with nuclear fast red and subsequently examined for HAI-2 or matriptase expression. All microscopic images were acquired on a Zeiss AxioImager Z1 light microscope using an AxioCam HRc digital camera system (both Carl Zeiss AG, Gottingen, Germany).Immunohistochemistry—Six-month-old wild-type or Spint1 null mice were euthanized by CO2 inhalation. Tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and cut into sections 5 μm thick. Antigens were retrieved by incubation for 10 min at 37 °C with 10 μg/ml proteinase K (Fermentas, Hanover, MD). The sections were blocked for 1 h at room temperature with 2% bovine serum albumin in phosphate-buffered saline and incubated overnight at 4 °C with the goat anti-mouse HAI-1 primary antibody (R&D Systems). Bound antibodies were visualized using a biotin-conjugated anti-goat secondary antibody (Vector Laboratories, Burlingame, CA) and a Vectastain ABC kit (Vector Laboratories) using 3,3′-diaminobenzidine substrate (Sigma).In Situ Hybridization—In situ hybridization of digoxigenin-labeled probes to paraformaldehyde-fixed, paraffin-embedded sections was carried out using the Link-Label ISH Core Kit II (BioGenex, San Ramon, CA) following the manufacturer's instructions. The Spint2-specific digoxigenin-labeled RNA probes were prepared by in vitro transcription of the +40- to +347-bp fragment of the mouse Spint2 open reading frame cloned into a pCRII-TOPO vector using the DIG RNA Labeling Kit (Roche Applied Science) according to the manufacturer's instructions. 100 ng of sense or antisense RNA probe was applied on parallel tissue sections and hybridized overnight at 37 °C. After the hybridization, signals were detected using the Link-Label ISH Core Kit II (BioGenex) as described in the instruction manual, and the sections were counterstained with nuclear fast red.RESULTSHAI-2 Is a Potent Inhibitor of Matriptase That Blocks the Proteolytic Activation of Its Candidate Physiological Targets the Prostasin Zymogen, and Cell Surface Pro-uPA—HAI-2 consists of two Kunitz-type inhibitor domains followed by a single span transmembrane domain and lacks the additional N-terminal region of unknown function found in HAI-1 (7Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Although this accounts for the significant difference in the overall molecular weight between the two inhibitors (28.2 and 56.9 kDa for nascent unmodified proteins, respectively), the two proteins nevertheless exhibit a high degree of homology in their inhibitory domains. Comparing the two Kunitz-type inhibitor domains of the human and murine forms of HAI-1 and HAI-2 revealed a 39-56% overall amino acid identity and a 57-67% amino acid homology (Fig. 1A). The most highly conserved motifs included the reactive site loop and the secondary binding segment of the Kunitz domain (Fig. 1A), which were previously shown to be involved in the binding of the Kunitz domain to target proteases, including matriptase (16Friedrich R. Fuentes-Prior P. Ong E. Coombs G. Hunter M. Oehler R. Pierson D. Gonzalez R. Huber R. Bode W. Madison E.L. J. Biol. Chem. 2002; 277: 2160-2168Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Because HAI-1 is an essential inhibitor of matriptase in both mice and zebrafish (12Szabo R. Molinolo A. List K. Bugge T.H. Oncogene. 2007; 26: 1546-1556Crossref PubMed Scopus (109) Google Scholar, 14Carney T.J. von der Hardt S. Sonntag C. Amsterdam A. Topczewski J. Hopkins N. Hammerschmidt M. Development. 2007; 134: 3461-3471Crossref PubMed Scopus (83) Google Scholar), this suggested that HAI-2 could also be a relevant inhibitor of this widely expressed membrane serine protease. HAI-1 has been shown previously to form SDS-stable complexes with matriptase that can be visualized by SDS-PAGE under non-boiling conditions (9Lin C.Y. Anders J. Johnson M. Dickson R.B. J. Biol. Chem. 1999; 274: 18237-18242Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Therefore, to investigate the possible interaction between matriptase and HAI-2, we incubated the recombinant matriptase serine protease domain with recombinant soluble extracellular domain of either HAI-2 or HAI-1. Protease-inhibitor complex formation was analyzed by reducing SDS-PAGE followed by either silver staining or Western blotting using an HAI-2 antibody that cross-reacts with HAI-1. As reported previously, a novel protein species appeared with a molecular weight predicted for the matriptase-HAI-1 complex when matriptase was incubated with HAI-1 (Fig. 1, B and C, lane 3). This was associated with a decrease in the amount of non-complexed HAI-1 (Fig. 1, B and C, compare lanes 2 and 3). Interestingly, the formation of similar matriptase-HAI-2 complexes was readily detected when matriptase was incubated with HAI-2 (Fig. 1, B and C, lane 5), also associated with a decrease in non-complexed HAI-2 (Fig. 1, B and C, compare lanes 4 and 5). Next, we determined if HAI-2 displayed inhibitory activity against matriptase by determining the IC50 of the soluble HAI-2 toward the matriptase serine protease domain. In agreement with the high amino acid identity of the Kunitz-type inhibitor domains of the two transmembrane serine protease inhibitors, HAI-2 potently inhibited matriptase, displaying an IC50 that was indistinguishable from that of HAI-1 (0.5 nm and 0.63 nm, respectively) (Fig. 1D).HAI-2 Blocks Matriptase-mediated Activation of Its Candidates Substrates Pro-prostasin and Cell Surface-bound Pro-uPA—Matriptase likely promotes oral epithelial and epidermal differentiation by proteolytically activating the prostasin (PRSS8/CAP1) zymogen, because ST14- and PRSS8-ablated epidermis are phenocopies of each other and active prostasin is absent in ST14 null mice (17Netzel-Arnett S. Currie B.M. Szabo R. Lin C.Y. Chen L.M. Chai K.X. Antalis T.M. Bugge T.H. List K. J. Biol. Chem. 2006; 281: 32941-32945Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 21Leyvraz C. Charles R.P. Rubera I. Guitard M. Rotman S. Breiden B. Sandhoff K. Hummler E. J. Cell Biol. 2005; 170: 487-496Crossref PubMed Scopus (230) Google Scholar). To determine if HAI-2 can block prostasin activation by matriptase, recombinant prostasin zymogen was expressed in HEK293T cells and released from the cell surface by phosphatidylinositol-specific phospholipase C. As reported previously (17Netzel-Arnett S. Currie B.M. Szabo R. Lin C.Y. Chen L.M. Chai K.X. Antalis T.M. Bugge T.H. List K. J. Biol. Chem. 2006; 281: 32941-32945Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), incubation of soluble prostasin zymogen with low amounts of matriptase lead to the formation of active two-chain prostasin, which displayed a slightly increased electrophoretic mobility in high percentage SDS-PAGE after reduction of the single disulfide bridge that links the two chains (Fig. 1E, compare lanes 2 and 3). This activation of prostasin by matriptase was completely blocked when matriptase was preincubated with either HAI-1 (Fig. 1E, compare lanes 3 and 4) or HAI-2 (Fig. 1E, compare lanes 3 and 5).We next determined if HAI-2 was capable of inhibiting matriptase activity in a physiologically relevant setting. Recent studies have shown that matriptase is required for the conversion of pro-uPA to active two-chain uPA on the surface of the acute monocytic leukemia cell line THP-1, ultimately leading to plasminogen activation on the cell surface (18Kilpatrick L.M. Harris R.L. Owen K.A. Bass R. Ghorayeb C. Bar-Or A. Ellis V. Blood. 2006; 108: 2616-2623Crossref PubMed Scopus (89) Google Scholar). To investigate if HAI-2 could regulate matriptase activity under physiologically relevant conditions, we therefore measured uPA activity on the surface of THP-1 cells that were preincubated with either pro-uPA or active high molecular weight uPA in the presence or absence of soluble HAI-2 protein. HAI-2 displayed no inhibitory activity toward active two-chain uPA when assayed in vitro (data not shown). In agreement with this, the presence of HAI-2 had no effect on cell surface uPA activity when THP-1 cells were preincubated with active uPA (Fig. 1F). However, HAI-2 almost completely abolished uPA activity on the surface of cells that were preincubated with pro-uPA (Fig. 1F). In contrast, when the known uPA inhibitor plasminogen activator inhibitor-1 was added to THP-1 cells instead of HAI-2, a strong reduction of cell surface uPA activity was evident irrespectively of whether the cells were incubated with pro-uPA or active uPA (Fig. 1F). These data indicate that, unlike plasminogen activator inhibitor-1, HAI-2 does not target uPA protease activity per se, but rather the activation of the uPA zymogen by endogenous matriptase.Generation of β-Galactosidase-tagged Spint2 Mice for Delineation of HAI-2 Matriptase Co-localization Studies in Vivo—The data presented above potentially implicated HAI-2 in the regulation of matriptase proteolytic activity. However, for a protease inhibitor to serve as a physiological inhibitor of a cognate protease requires the physical proximity of the two proteins in tissues. HAI-2 and matriptase are both membrane-anchored proteins, making it likely that HAI-2 must be expressed by the same cells that express matriptase to inhibit the protease. Therefore, we next set out to delineate the potential co-localization of HAI-2 and matriptase in cell lineages that form adult murine tissues. Furthermore, we mapped the expression of HAI-2 relative to HAI-1 to determine the possible functional overlap between the two membrane serine protease inhibitors.An initial test of several commercially available anti-mouse HAI-2 antibodies revealed prohibitively high nonspecific immunohistochemical background using a variety of staining conditions, as well as cross reactivity with HAI-1 (data not shown). A similar lack of specificity was observed with all commercially available and in-house-generated matriptase antibodies. To analyze the expression of HAI-2 in mouse tissues, we therefore employed the technique of enzymatic gene trapping. A search of ES cell clones available through the International Gene Trap Consortium (www.genetrap.org) revealed one ES cell clone, KST272, with a promoterless β-galactosidase-neomycin phosphotransferase fusion gene (β-geo) inserted into intron 1 of the mouse Spint2 gene, which encodes HAI-2 (15Mitchell K.J. Pinson K.I. Kelly O.G. Brennan J. Zupicich J. Scherz P. Leighton P.A. Goodrich L.V. Lu X. Avery B.J. Tate P. Dill K. Pangilinan E. Wakenight P. Tessier-Lavigne M. Skarnes W.C. Nat. Genet. 2001; 28: 241-249Crossref PubMed Scopus (347) Google Scholar) (HAI-2/β-geo allele, Fig. 2A). The insertion results in the expression of a β-geo reporter protein from the endogenous promoter of the Spint2 gene, thus allowing identification of HAI-2-expressing cells in situ by X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. This technique has previously been shown to be a reliable alternative to immunohistochemistry in the absence of suitable antibodies or when proteins are expressed below the level of immunological detection (20List K. Szabo R. Molinolo A. Nielsen B.S. Bugge T.H. Am. J. Pathol. 2006; 168: 1513-1525Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Voss A.K. Thomas T. Gruss P. Dev. Dyn. 1998; 212: 171-180Crossref PubMed Scopus (64) Google Scholar, 23Wurst W. Gossler A. Joyner A. Gene Targeting: A Practical Approach. 2nd Ed. Oxford University Press, New York2000: 207-254Google Scholar).FIGURE 2HAI-2 generation of a β-galactosidase-tagged spint2 mouse strain. A, schematic representation of the mouse HAI-2 gene locus targeted with a β-geo gene trap vector. ES cell clone KST272 has a gene trap consisting of the engrailed-2 (En2) splice acceptor site/CD4 transmembrane domain/β-galactosidase-neomycin phosphotransferase fusion gene (β-geo), and a bovine growth hormone polyadenylation site (pA) inserted between exon 1 and 2 (E1 and E2) of mouse Spint2 gene. Clone KST272 gives rise to a fusion protein that consists of an N-terminal signal peptide (SP) from the endogenous mouse HAI-2 protein fused to the β-galactosidase-neomycin phosphotransferase (β-geo) fusion protein (HAI-2/β-geo). B-D, representative histological sections showing high expression of HAI-2-β-galactosidase protein (blue) in epithelium of distal tubules in kidney (B, arrowhead), surface epithelium (C, arrowhead), and goblet cells (C, open arrowhead) in colon, epithelium in trachea (D, arrowhead), and low expression in proximal tubules in kidney (B, arrows). B′-D, expression of endogenous HAI-2 mRNA detected by in situ hybridization in distal and proximal tubules in kidney (B′, arrowhead and arrow, respectively), surface epithelium and goblet cells (C′, arrowhead and open arrowhead, respectively) in colon, and in tracheal epithelium (D′, arrowhead) in adult wild-type mouse. Insets: In situ hybridization of mouse HAI-2 sense probe in wild-type kidney (B′), colon (C′), and trachea (D′). Scale bars:25 μm (B, B′, D, and D
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