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Mast Cell Tissue Inhibitor of Metalloproteinase-1 Is Cleaved and Inactivated Extracellularly by -Chymase¹

Brendon T. Frank^*, J. Caleb Rossall^*, George H. Caughey^*, and Kenneth C. Fang^2,*,

^* Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, CA 94143

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously reported that mast cell -chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. -Chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1’s -chymase-mediated cleavage products reveals hydrolysis at Phe¹²-Cys¹³ and Phe²³-Val²⁴ in loop 1 and Phe¹⁰¹-Val¹⁰² and Trp¹⁰⁵-Asn¹⁰⁶ in loop 3 of the NH₂-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to -chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, -chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with -chymase increases gel B activity 2- to 5-fold, suggesting that -chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of -chymase blocks degranulation-induced TIMP-1 processing (absent in -chymase-deficient HMC-1 cells). Purified -chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell -chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

	Introduction

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Activated mast cells respond to allergen, tumor invasion, and fibrogenic injury by releasing tryptase and -chymase, which are granule-associated tryptic and chymotryptic serine proteases, respectively. Upon degranulation, both proteases participate in proteolytic and nonproteolytic pathways regulating matrix protein degradation, receptor activation, peptide inactivation, and fibroblast, smooth muscle, or epithelial cell mitogenesis (1). Tryptase and -chymase may also act in a broader range of homeostatic and pathologic tissue remodeling processes via interactions with a family of Ca²⁺- and Zn²⁺-dependent matrix metalloproteinases (MMPs)³ (2, 3, 4, 5, 6, 7). Secreted or cell surface-associated membrane-type MMPs participate in physiologic pathways such as embryonic development, organ morphogenesis, angiogenesis, and wound healing, and also contribute to the pathogenesis of arthritis, cancer, cardiovascular disease, and lung fibrosis (8). Posttranslational regulation of MMP activity depends upon conversion of proenzymes to mature active forms and their inhibition by tissue inhibitors of metalloproteinases (TIMPs). Proteolytic activation of pro-MMPs involves one or more activator proteases which remove the propeptide domain containing a critical Cys residue, thus disrupting the cysteine switch which confers active site latency (9). TIMPs inhibit activity of active MMP species and block activation of pro-MMPs by forming TIMP-MMP complexes in a 1:1 molar ratio. NH₂-terminal TIMP domains occupy the MMP active site and COOH-terminal domains confer binding specificity through interactions with COOH-terminal MMP domains (10).

Mast cells may regulate local MMP activity by contributing zymogen-activating serine proteases and by secreting pro-MMPs. Our previous work demonstrates that mast cells secrete progelatinase B (progel B; MMP-9), which is activated extracellularly by degranulated -chymase upon cleavage of the catalytic domain at two sites (2, 3, 4). -Chymase-dependent activation of progel B in vivo is hypothesized to switch nonangiogeneic tissues to an angiogenic phenotype in premalignant lesions in a murine model of epithelial carcinogenesis (11). In addition to progel B, -chymase also directly cleaves and activates procollagenase (MMP-1) and prostromelysin (MMP-3) (6, 7). Mast cells also express these two MMPs plus progelatinase A (MMP-2), which shares in vitro substrate specificity with gelatinase B (gel B) (4, 12, 13).

Since cells secrete pro-MMPs bound to TIMPs in a complex, zymogen activation likely requires prior TIMP processing which precedes proteolytic activation of the MMP moiety. TIMP-1 binds to progel B, which is secreted as a monomer or dimer, or in disulfide-mediated linkage with neutrophil gelatinase-associated lipocalin (NGAL) to form a ternary complex of progel B monomer, TIMP-1 and NGAL (progel B-TIMP-1-NGAL) (14, 15). Unbound TIMP-1 may be inactivated in vitro by cleavage, degradation, or chemical modification via proteolytic and nonproteolytic mechanisms involving serine or thiol proteases and reactive oxygen species, respectively (16, 17, 18, 19). However, mechanisms which regulate processing of MMP-bound TIMP-1 remain unclear. In addition to its ability to block pro-MMP activation and inhibit activity of mature MMPs, TIMP-1 also demonstrates erythroid-potentiating activity, stimulates steroidogenesis, regulates mitogenesis, and controls apoptosis via mechanisms which are independent of its MMP inhibitory activity (10). Thus, processing of TIMP-1 in the inflammatory milieu may not only alter the protease:antiprotease balance to favor proteolysis, but also attenuate or abolish its non-MMP inhibitory effects on specific cell populations. We report here that mast cell -chymase cleaves free TIMP-1 and processes the progel B-TIMP-1 complex by inactivating bound TIMP-1 and activating progel B, without involvement of other secreted proteases, and that degranulation of -chymase regulates processing of TIMP-1 secreted by mast cells.

	Materials and Methods

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Cell culture

BR dog mastocytoma cells or HMC-1 cells (obtained from J. Butterfield, Mayo Clinic, Rochester, MN) were maintained in continuous suspension culture in DME-H16 medium supplemented with 2% bovine calf serum (2) or IMDM with 2% FCS (20), respectively, as previously described. Murine bone marrow-derived mast cells (MBMMC) obtained from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were cultured in RPMI 1640 medium supplemented with 10% bovine calf serum and 50% medium conditioned by WEHI-3B cells, as previously described (21). Cells were maintained at a final concentration of 1 x 10⁶ cells/ml and incubated at 37°C in humidified 5% CO₂ and 95% air. Cells were harvested by centrifuging at 500 x g for 5 min, washing three times in Ca²⁺-and Mg²⁺-free PBS, and resuspending in serum-free culture medium to a final concentration of 10–15 x 10⁶ cells/ml.

RNA blotting

Poly(A)⁺ RNA was isolated from BR mastocytoma cells incubated alone or in the presence of 100 ng/ml recombinant canine KL (kindly provided by Keith Langley, Amgen, Thousand Oaks, CA) using the Poly(A)⁺ RNA MicroFast Track extraction kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA blotting and hybridization of random primed [-³²P]ATP-labeled 0.79-kb dog TIMP-1 cDNA (gift from Philip Davies, Merck Research Laboratories, Rahway, NJ) or 2.3-kb dog gel B (3) cDNA to nylon membranes, posthybridization washings, and autoradiography were performed as previously described (4). Densitometric data were obtained by analysis of autoradiographic signals generated by hybridizing the blot with labeled probe. To account for possible variations in signal intensity due to differing concentrations of mRNA present in each lane, the blot was also hybridized with a labeled probe for -actin. Densitometric data were then compared with control values obtained with the -actin probe.

Immunoblotting

Aliquots of medium conditioned by mast cells were concentrated by Microcon-10 filters (Amicon, Beverly, MA), subjected to electrophoresis, and blotted onto polyvinylidene difluoride membrane (Polyscreen; NEN Life Science Products, Boston, MA). Membranes were washed in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.3% Tween 20 and incubated with polyclonal rabbit anti-TIMP-1 (Chemicon, Temecula, CA; rabbit polyclonal Ab is specific for TIMP-1 and does not cross-react with other TIMPs in the manufacturer’s quality control assays) at 22°C for 1 h. After incubation of membranes with HRP-linked anti-rabbit Ig for 1 h and luminol and peroxidase reagents (Phototope-HRP Western Blot Detection kit; New England Biolabs, Beverly, MA) according to the manufacturer’s protocols, signals for immunoreactive proteins were visualized on Hyperfilm ECL (Amersham Pharmacia, Piscataway, NJ).

Substrate cleavage

Purified recombinant human (rh) TIMP-1 (Chemicon) or the progel B-TIMP-1-NGAL complex (Calbiochem, La Jolla, CA) was incubated either alone or in combination with different concentrations of activator proteases including: purified rh-chymase (22), purified rh-tryptase (Promega, Madison, WI), human neutrophil elastase (HNE; Sigma, St. Louis, MO), L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma), and purified rh stromelysin (MMP-3) catalytic domain (22 kDa; Calbiochem). Reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 10 mM CaCl₂, and 0.02% NaN₃ (reaction buffer), incubated at 37°C for various time periods, and stored at 0°C before further analysis. To determine the electrophoretic profile of reaction products, aliquots were subjected to SDS-PAGE using 16% or 4–20% gradient Tris-glycine gels (Invitrogen) under reducing conditions, with detection of proteins by Coomassie Blue R250 (Fisher Scientific, Tustin, CA) or immunoblotting using polyclonal rabbit anti-TIMP-1 or polyclonal rabbit anti-MMP-9 Abs (Triple Point Biologics, Portland, OR).

TIMP-1 activity

MMP inhibitory activity of rhTIMP-1 was determined by incubating intact or -chymase-processed inhibitor with active gel B monomer (Calbiochem). rhTIMP-1 was incubated alone or in the presence of various concentrations of -chymase in reaction buffer at 37°C for different time periods. Reactions were stopped by incubation with Ala-Ala-Pro-Phe-chloromethyl ketone (AAPF-CMK; Enzyme Systems Products, Livermore, CA) at 22°C for 10 min at a final concentration of 25 µM. Active gel B monomer was incubated alone or with aliquots of each reaction mix in a 1:1 molar ratio at 22°C for 10 min. The gel B activity in solution was determined in a final volume of 200 µl of reaction buffer containing 25 µM AAPF-CMK in 96-well plates (Becton Dickinson, Franklin Lakes, NJ) with 10 µM DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH₂ (Cha = b-cyclohexylalanyl; Abz = 2-aminobenzoyl(anthraniloyl)] (DNP-PGCHAK; Calbiochem) (23). Fluorescent products released by cleavage of substrate were detected at different times at excitation and emission wavelengths of 360 nm and 460 nm, respectively, using a CytoFluor 2350 fluorometer (Millipore, Danbury, CT) and a sensitivity parameter of 6.

NH₂-terminal sequencing

Amino acid sequence was determined by the Biomolecular Resource Center at the University of California San Francisco. The reaction mixture containing the TIMP-1 products of -chymase cleavage was electrophoresed onto a 4–20% Tris-glycine polyacrylamide gel and blotted onto sequencing grade polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid buffer containing 10% methanol. Protein bands identified by Coomassie blue staining of the membrane were excised and subjected to Edman degradation using a Procise 491 Protein Sequencer (PE Biosystems, Foster City, CA). Protein sequence alignments were performed using MacVector software (Oxford Molecular Group, Hunt Valley, MD).

Gelatin zymography

The gel B activity resulting from -chymase cleavage of the progel B-TIMP-1-NGAL complex was detected by gelatin zymography as previously described (2). Briefly, reaction products were electrophoresed through 10% polyacrylamide gels copolymerized with gelatin (1 mg/ml). After electrophoresis, gels were washed twice for 30 min in 2.5% Triton X-100 and incubated at 37°C for 18 h in 40 mM Tris buffer (pH 7.5) containing 200 mM NaCl and 10 mM CaCl₂. Gels were stained with Coomassie blue for 10 min and destained in 10% acetic acid containing 50% methanol. Clear zones of lysis against a blue background indicated gelatinase activity.

Enzyme assays

Soluble MMP activity was determined by detection of products released upon cleavage of fluorescently labeled proteins or synthetic peptides by active proteases. The gel B monomer or the progel B-TIMP-1-NGAL complex was incubated alone or with activator proteases at 37°C for different time periods in 200 µl of reaction buffer in 96-well plates with various concentrations of DNP-PGCHAK (23) or DQ gelatin (fluorescein conjugate from pig skin) (Molecular Probes, Eugene, OR) to measure gel B activity or FITC-conjugated casein (Sigma) to assay stromelysin activity. Specific activity of active gel B monomer or active stromelysin catalytic domain using DQ gelatin or FITC casein was 186 fluorescent units/µg/min or 280 fluorescent units/µg/min, respectively. Background levels of substrate cleavage by -chymase, HNE, or trypsin were subtracted from individual determinations of soluble activity.

Degranulation studies

Harvested cells were resuspended in serum-free culture medium to a final concentration of 10–15 x 10⁶ cells/ml. Cells were incubated at 37°C for different time periods either alone, with 2 µM calcium ionophore A23187 (Sigma), or with ionophore in combination with individual inhibitors (Sigma) to identify protease classes involved in the processing of endogenous TIMP-1. Various concentrations of PMSF, aprotinin, or AAPF-CMK were used to block serine protease activity, while E64 or phosphoramidon and BB94 (gift from Marc Navre) were used to inhibit activity of cysteine proteases or metalloproteases (Affymay Research Institute, Santa Clara, CA), respectively. Aliquots removed at specified intervals were centrifuged to pellet cells. To identify the electrophoretic profile of products resulting from processing of native dog TIMP-1, various concentrations of purified dog -chymase (24) were incubated with control BR cell supernatants for different periods of time. Cell supernatants and cleavage reactions were stored at -20°C before analysis by immunoblotting.

Statistical analysis

Differences with a p < 0.05 using Student’s two-tailed t test were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mastocytoma TIMP-1 expression

Our previous studies established that mast cells express progel B, which could be purified in zymogen form free of TIMP-1 from dog BR mastocytoma cells, which phenotypically resemble human mast cells in their protease complement (2, 4, 25). Since cells usually secrete pro-MMPs complexed to TIMPs in a 1:1 stoichiometric ratio, expression of progel B by mastocytoma cells predicted coexpression of TIMP-1, which binds the zymogen and active forms of progel B (10, 26). As seen in Fig. 1, A and B, autoradiography reveals a single TIMP-1 mRNA signal whose steady-state level remains unchanged following KL stimulation of BR cells; by contrast, KL increases gel B mRNA levels by 5-fold, as previously described (4). As seen in Fig. 1C, analysis of media conditioned by cells incubated for 18 h reveals immunoreactive bands at 12 and 14 kDa (bands b and c), an unexpected electrophoretic profile since native dog TIMP-1 migrates at 35 kDa, a size which differs from that of human TIMP-1 (28.5 kDa) due to the presence of an additional N-linked glycosylation site (27, 28, 29). Since serine proteases process TIMP-1 in vitro (30), cells were incubated in the presence of increasing concentrations of PMSF to determine whether proteolysis obscures the identification of native TIMP-1 bands. Incubation with PMSF yields additional 8-, 20-, and 27-kDa immunoreactive bands, while the maximum concentration yields an 35-kDa band (band a). Thus, serine protease inhibition reveals the presence of native dog TIMP-1 protein and its intermediate cleavage products. These data demonstrate that mast cells express TIMP-1 mRNA and protein, which may be processed extracellularly by serine proteases released from secretory granules.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Mast cell expression of TIMP-1. A, Poly(A)+ RNA isolated from BR cells incubated in medium alone (C) or in the presence of 100 ng/ml KL was separated on a 1% agarose gel containing 6.1% formaldehyde and transferred to nylon membrane which was hybridized with an [-32P]-labeled probe for dog TIMP-1 or gel B. B, Densitometric data were obtained in duplicate experiments by analysis of autoradiographic signals after 96 h and normalized to values obtained with the -actin probe. Values are expressed as a percentage of the control signal obtained from unstimulated cells. C, BR cells were incubated in serum-free medium alone (C) or in the presence of 2 mM or 10 mM PMSF. After 18 h, cells were pelleted and supernatants of conditioned medium were subjected to SDS-PAGE on 16% Tris-glycine gels under reducing conditions and transferred to polyvinylidene difluoride membrane with detection of immunoreactive bands by polyclonal anti-TIMP-1 Ab. Note that serine protease inhibition reveals the presence of native 35-kDa dog TIMP-1.

-Chymase cleaves and inactivates TIMP-1

As shown in Fig. 2A, incubation of rhTIMP-1 with rh-chymase at a 1:1 molar ratio for 2 h generates two principle products with molecular sizes of 18 and 10 kDa. By contrast, the tryptic mast cell serine protease, tryptase, has no effect in agreement with previously published data (5). The electrophoretic profile of -chymase-processed TIMP-1 products differs from that of HNE which yields bands at 17 and 16 kDa (30, 31) and additional products of 8–10 kDa. Thus, these data suggest that -chymase cleavage of unbound TIMP-1 involves limited hydrolysis to generate products that differ from those generated by HNE. As seen in Fig. 2B, incubation of -chymase with TIMP-1 at a 0.5:1 molar ratio yields a faint 18-kDa band. Increasing the molar ratio yields a more intense 18-kDa band and an additional band at 10 kDa. The time course shown in Fig. 2C illustrates that at a ratio of 1:1, -chymase generates the 18-kDa band by 5 min with the 10-kDa band visible by 15 min. The later appearance of the 10-kDa band suggests that 1) it derives from a separate cleavage site on the TIMP-1 parent protein that is less avidly cleaved or 2) that it derives from the 18-kDa band via cleavage at a site that is accessible only after generation of the 18-kDa band. The effect of -chymase processing on the MMP inhibitory activity of TIMP-1 was explored by determining its effect on the ability of TIMP-1 to inhibit soluble activity of active gel B. As shown in Fig. 2D, -chymase processing of TIMP-1 decreases its ability to inhibit active gel B monomer by >80% compared with that of unprocessed TIMP-1. Thus, cleavage by -chymase attenuates the ability of TIMP-1 to inhibit gel B activity in solution.

View larger version (65K): [in this window] [in a new window]   FIGURE 2. -Chymase cleaves and inactivates TIMP-1. A, Purified human TIMP-1 (25 µg/ml) was incubated alone or with purified human -chymase, a molar equivalent of active tryptase monomer (which exists in tetrameric form in solution) or HNE (25 µg/ml) at 37°C for 1 h in reaction buffer. Reaction products were subjected to SDS-PAGE on 4–20% gradient gels under reducing conditions and detected by immunoblotting using polyclonal anti-TIMP-1 Ab. B and C, Concentration- and time-dependent -chymase processing of TIMP-1. B, TIMP-1(25 ng) was incubated alone (C) or with increasing (0.5, 1, 2.5, or 5) molar equivalents of -chymase at 37°C for 1 h in reaction buffer. Reaction products were detected by immunoblotting using anti-TIMP-1 Ab. An 18-kDa cleavage product resulting from incubation with 0.5 molar equivalents of -chymase becomes more intense in the presence of higher concentrations of the enzyme. An 10-kDa product is visible upon incubation with concentrations of -chymase at molar equivalence or greater. C, Aliquots of TIMP-1 (25 µg/ml) incubated with -chymase (25 µg/ml) were removed at the indicated time intervals and placed at 0°C in the presence of 2 µM AAPF-CMK and reducing buffer. Note the appearance of the 18- and 10-kDa bands by 5 and 15 min, respectively. D, -Chymase inactivation of TIMP-1. -Chymase was incubated alone or with TIMP-1 at a 2.5:1 molar ratio at 37°C for 0 h (uncleaved) or 12 h (cleaved) with the reactions stopped by the addition of 25 µM AAPF-CMK to inhibit -chymase activity. Active gel B monomer was incubated with aliquots of each reaction mix at a 1:1 molar ratio to TIMP-1 for 10 min before determinations of gelatinolytic activity in solution using the fluorogenic substrate DNP-PGCHAK. Values are expressed as a percentage of inhibition of active gel B monomer relative to that by uncleaved TIMP-1 and represent the mean ± SE of inhibition analyzed in triplicate (*, p < 0.005 compared with uncleaved).

-Chymase cleavage sites of TIMP-1

To clarify the mechanism of -chymase processing of TIMP-1, we characterized the electrophoretic profile of the cleavage products and identified sites of hydrolysis. As seen in Fig. 3A, analysis of cleavage products by SDS-PAGE yields two bands at 18 and 10 kDa under reducing conditions. NH₂-terminal sequencing of the 18-kDa product yields two sequences (X₁NX₂DLVI and VGTPEV) in a molar ratio of 1:3, while that of the 10-kDa product yields two sequences (VAPWN and NSLSLAQ) in a molar ratio of 1:4, as shown in Fig. 3B. Alignment of these sequences with the primary sequence of human TIMP-1 (29) reveals that -chymase cleaves the Phe¹²-Cys¹³ and Phe²³-Val²⁴ bonds in loop 1 and the Phe¹⁰¹-Val¹⁰² and Trp¹⁰⁵-Asn¹⁰⁶ bonds in loop 3 of the NH₂-terminal domain (residues 1–124 (32)). Whereas residue X₁ is indeterminate due to the inability of Edman degradation to detect Cys residues, no residue was assigned to position X₂ due to insufficient discrimination of chromatographic peak amplitudes in successive cycles. Thus, the P1 residue at each of the identified cleavage sites is aromatic (Phe or Trp) as expected of hydrolysis by a chymotryptic enzyme. These aromatic residues are completely conserved in all mammalian TIMP-1. By contrast, the residue on the other (P1') side of the scissile bond varies in mammalian TIMPs. Met or Leu substitutes for Val²⁴ or Val¹⁰² in rodent sequences, respectively, while His, Arg, and Ser substitute for Asn¹⁰⁶ in the rat, mouse, and canine sequences, respectively (Ref. 33 and GenBank Accession no. AAD10632). As seen in Fig. 4, HNE cleaves TIMP-1 at a single site at the Val⁶⁹-Cys⁷⁰ bond which is situated in an area of intermolecular contact with stromelysin-1 (MMP-3) (32, 34). By contrast, -chymase inactivates free TIMP-1 by hydrolyzing at four sites in loops 1 and 3, which are distant from the active site and located in the five-stranded -barrel of the NH₂-terminal domain, yielding two identifiable cleavage products.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. N-terminal sequences of -chymase-processed TIMP-1 cleavage products. A, Purified human -chymase and TIMP-1 were incubated alone or together at a 0.5:1 molar ratio at 37°C for 30 min. Samples were analyzed by SDS-PAGE under reducing conditions with proteins detected by Coomassie blue (A). Cleavage generates two products 18 (band a) and 10 kDa (band b) in size which are not visualized in the purified preparations of either -chymase or TIMP-1. B, Bands a and b were transferred to polyvinylidene difluoride membrane, identified by Coomassie staining, and excised for automated peptide sequencing. Sequences a1 and a2 (picomoles indicated in parentheses) identify band a as a doublet resulting from two cleavages at scissile bonds containing Phe12 and Phe23 as the P1 residues. Sequences b1 and b2 also identify band b as a doublet with two overlapping sequences resulting from cleavage at scissile bonds containing Phe101 and Trp105. Peptide sequences were aligned with the cDNA predicted amino acid sequence of human TIMP-1 (29 ) using MacVector software.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. Sites of -chymase cleavage in the NH2-terminal domain of TIMP-1. A, Loop-specific -chymase processing. Purified human -chymase cleaves the NH2-terminal domain (residues 1–124) at four sites (indicated by arrowheads), including Phe12-Cys13 and Phe23-Val24 in loop I and Phe101-Val102 and Trp105-Asn106 in loop III, separating it from the COOH-terminal domain (residues 127–184). Cleavage product containing COOH-terminal domain and portions of NH2-terminal domain with intact disulfide linkages (yellow) is indicated in blue. By contrast, HNE cleaves at a single site at the Val69-Cys70 bond (31 ). B, Ribbon diagram of bound TIMP-1 showing locations of scissile bonds cleaved by -chymase or HNE. Four scissile bonds containing aromatic P1 residues favored by -chymase are located away from the long edge of TIMP-1 (red), which contains the Val69-Cys70 bond cleaved by HNE and interacts with the stromelysin (MMP-3) (blue) active site cleft (32 34 ). C, Surface localization of aromatic P1 residues. Scissile bonds containing Phe (green) or Trp (purple) residues at the P1 position are exposed on the surface of TIMP-1. Positions of the P1 residues are shown in relation to those of Val69 (gray) and Cys70 (yellow) in the bond cleaved by HNE. Figures drawn and rendered with SwissPdbViewer v.3.5 based on the crystal structure of the complex formed between TIMP-1 and the catalytic domain of stromelysin-1 (MMDB Id: 9217, PDB Id: 1UEA) (34 ).

-Chymase processing of the progel B-TIMP-1-NGAL complex

To determine whether -chymase also cleaves bound TIMP-1, increasing concentrations of the enzyme were incubated with a ternary molecular complex comprised of progel B, TIMP-1, and NGAL (progel B-TIMP-1-NGAL). As shown in Fig. 5A, incubation of -chymase with the progel B-TIMP-1-NGAL complex at a 0.5:1 molar ratio of -chymase to the TIMP-1 component results in the appearance of an 18-kDa band. Increasing the molar concentration of -chymase yields an additional band at 10 kDa, while a 10-fold molar excess decreases the 28.5-kDa band of intact TIMP-1. Thus, the electrophoretic profile of cleavage products resulting from -chymase processing of bound TIMP-1 is similar to that generated upon cleavage of free TIMP-1. These data suggest that bound TIMP-1 is susceptible to hydrolysis by -chymase.

View larger version (85K): [in this window] [in a new window]   FIGURE 5. -Chymase cleavage of the progel B-TIMP-1-NGAL complex. Purified human progel B-TIMP-1-NGAL complex (145 ng) was incubated alone (C) or in the presence of the indicated molar equivalents of purified human -chymase at 37°C for 1 h in reaction buffer. Reaction products were subjected either to immunoblotting with antisera to TIMP-1 (A) or gel B (B) after SDS-PAGE under reducing conditions or to gelatin zymography performed under nonreducing conditions (C).

-Chymase activation of complexed progel B

To determine the effect of -chymase processing of TIMP-1-bound progel B, the gel B activity of the progel B-TIMP-1-NGAL complex in the presence of activator serine proteases was assayed in solution over 2 h using the fluorogenic substrate DNP-PGCHAK. As seen in Fig. 6, incubation of -chymase with the complex at a 0.1:1 molar ratio increases activity 2-fold. Activity of the complex alone likely occurs due to contaminating amounts of gel B monomer (35). Coincubation of the complex with -chymase at ratios of 0.5:1 or 1:1 increases activity by 3.5- or 4.5-fold, respectively. Incubation of molar excesses of -chymase with the complex does not further increase activity (data not shown). Similar molar ratios of trypsin, an activator of progel B monomer (35) (and additional log ratios between 10^-10 and 10^-1; data not shown), do not increase activity to the magnitude induced by -chymase processing. These data demonstrate that the observed increases in the complex’s soluble activity following -chymase processing do not result from its activation of contaminating progel B monomer. Incubation of the complex with a molar equivalent or less (data not shown) of HNE does not increase gelatinolytic activity of the complex. Therefore, -chymase processing of the progel B-TIMP-1-NGAL complex activates the zymogen, increasing the gelatinase activity of the complex in solution.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. -Chymase processing increases soluble activity of the progel B-TIMP-1-NGAL complex. The progel B-TIMP-1-NGAL complex (145 ng) was incubated in the absence or presence of purified human -chymase, HNE, or trypsin at 37°C for 2 h in reaction buffer containing excess fluorogenic substrate, DNP-PGCHAK. Activator proteases were incubated with the complex in ratios relative to the TIMP-1 moiety at 0, 0.1, 0.5, and 1 molar equivalents. Soluble gel B activity was determined in the presence of an excess of the fluorogenic substrate DNP-PGCHAK. Values represent the mean ± SE of gelatinolytic activity analyzed in triplicate (*, p < 0.002; **, p < 0.0001; ***, p = 0.0001 compared with complex alone).

As seen in Fig. 7, the activity of progel B-TIMP-1-NGAL in solution after processing by activator proteases was compared with that of a molar equivalent of active gel B monomer. Whereas -chymase increases the activity of the progel B-TIMP-1-NGAL complex, neither HNE alone nor a 4-h pretreatment of the complex with HNE before addition of 0.2 or 0.5 molar equivalents of active MMP-3 (a mechanism of sequential proteolytic processing previously shown to activate the progel B-TIMP-1 complex (30)) has any effect. Maximal soluble activity of -chymase-processed complex represents 80% of the maximal activity of a molar equivalent of an active 82-kDa TIMP-free gel B monomer (relative to the 92-kDa progel B moiety in the complex). Thus, these data suggest that processing of the progel B-TIMP-1-NGAL complex by -chymase overcomes TIMP-1 inhibition and results in recovery of the soluble activity of the gel B moiety following -chymase-dependent zymogen activation.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Relative gel B activity of -chymase-processed progel B-TIMP-1-NGAL complex. Seventy-three nanograms of the progel B-TIMP-1-NGAL complex (46 ng of 92-kDa progel B moiety) were incubated alone or in the presence of either purified human -chymase or HNE without or with active stromelysin (MMP-3). -Chymase or HNE was incubated alone with the complex at ratios of molar equivalence with the TIMP-1 moiety at 37°C for 8 h, while 0.2 or 0.5 molar equivalents of active stromelysin were added after prior treatment of the complex with HNE for 4 h. Reactions were performed in the presence of an excess of the fluorogenic substrate DNP-PGCHAK. Soluble gel B activity of the complex alone or in the presence of activator proteases was compared with that of 41 ng of active 82-kDa gel B monomer. Values represent the mean ± SE of gelatinolytic activity analyzed in triplicate (*, p < 0.001 compared with the complex alone or with HNE with or without stromelysin).

To determine the effect of degranulation on mast cell TIMP-1 processing, cells were incubated alone or in the presence of 2 µM calcium ionophore A23187 for 5 min. As seen in Fig. 8A, supernatant of control dog BR mastocytoma cells contains an immunoreactive band at 35 kDa (band a), suggesting constitutive secretion of native TIMP-1; an additional band at 14 kDa (band b) suggests synchronous release of a related TIMP-1 product. Supernatant of either HMC-1 cells, a human mast cell leukemia line (20), or MBMMC reveals a similar pattern with bands migrating at 30 or 31 kDa, the expected sizes of human (with glycosylation-dependent variation from 28.5 to 34 kDa) or murine TIMP-1, respectively (29, 36, 37)). Supernatant of HMC-1 or MBMMC cells also contains the 14-kDa immunoreactive TIMP-1 band. These data suggest that mast cells constitutively release native TIMP-1 and that TIMP-1 secretion occurs in cells with either a normal or malignant phenotype. Upon degranulation, the electrophoretic profile of the supernatants of BR or MBMMC cells changes with the appearance of more bands between 17 and 29 kDa, in addition to the native and 14-kDa TIMP-1 bands present in control supernatants. By contrast, the profile of TIMP-1 bands present in HMC-1 degranulation supernatants remains identical to that of the control. HMC-1 cells are similar to BR or MBMMC cells in their expression of tryptase, which does not cleave TIMP-1 (as seen in Fig. 1), but differ in their lack of expression of -chymase (38). Therefore, these data suggest that degranulated mast cell -chymase cleaves TIMP-1 extracellularly.

View larger version (46K): [in this window] [in a new window]   FIGURE 8. Effects of degranulation and purified -chymase on endogenous mast cell TIMP-1. A, BR dog mastocytoma cells, HMC-1 cells, or MBMMC were incubated alone or degranulated using 2 µM calcium ionophore A23187 for 5 min at 37°C. Supernatants were subjected to SDS-PAGE on 16% gels under reducing conditions and immunoreactive bands were detected using polyclonal anti-TIMP-1 Ab. Note the absence of TIMP-1 processing in -chymase-deficient HMC-1 cells and the constitutive expression of both native TIMP-1 and the 14-kDa form in these mast cells. B, To identify proteases involved in degranulation-dependent processing of endogenous TIMP-1, BR cells were incubated in the absence (-) or presence of ionophore, both alone (+) and in combination with molar excesses of class-specific inhibitors: 10 mM PMSF, 10 µM aprotinin, 250 µM E64, 250 µg/ml phosphoramidon, or 500 nM BB94. Note attenuation of TIMP-1 processing by PMSF with the absence of band c, suggesting inhibition of serine protease-dependent processing of endogenous TIMP-1. C, To identify the specific mast cell serine protease involved in degranulation-dependent TIMP-1 processing, BR cells were incubated alone (-), with ionophore alone (+), and in combination with increasing concentrations (200 nM, 2 µM, 20 µM, 200 µM, 400 µM, and 2 mM) of AAPF-CMK, a specific inhibitor of -chymase, for 15 min at 37°C before analysis of supernatants by immunoblotting with polyclonal anti-TIMP-1 Ab. Note the diminution in the intensity of band c and that of intermediate TIMP-1 products in the presence of increasing concentrations of AAPF. D, To demonstrate that -chymase cleaves endogenous TIMP-1, 50 ng of purified dog -chymase was incubated for 30 or 60 min with cell-free supernatants of medium conditioned by BR cells incubated alone for 5 min at 37°C. Exogenous -chymase generates additional 8-, 12-, 17-, and 27-kDa bands, which are also seen in conditioned medium (CM) of BR cells incubated for 18 h at 37°C in the presence of 2 mM PMSF (as shown in Fig. 1C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
TIMPs act in diverse biological processes via mechanisms which may be either dependent on or independent of their ability to inhibit MMPs. In part, their myriad effects may be due to interactions involving structurally distinct NH₂- and COOH-terminal domains, which block MMP-active sites and bind to MMP COOH domains, respectively (8). However, regulation of these TIMP domains is unclear. A unique feature of the gelatinase class of MMPs is binding of the zymogens, progelatinase A (MMP-2) and progel B, to TIMP-2 and TIMP-1, respectively (39, 40). Thus, TIMP-1 not only inhibits activity of mature gel B, but also blocks activation of the proenzyme. We previously reported that -chymase, a chymotryptic serine protease unique to mast cells, activates progel B by cleaving the catalytic domain (3). The work here demonstrates that -chymase also cleaves and inactivates TIMP-1 whether it exists free or bound in a complex with progel B. By cleaving TIMP-1 at exposed surface sites, -chymase overcomes its inhibitory effects and converts complexed progel B to mature species with gelatinolytic activity in solution.

Whereas previous work demonstrated that mast cells express MMPs (2, 4, 12, 13, 25), the current data provide evidence that HMC-1 cells, dog BR mastocytoma cells, and MBMMC also secrete TIMP-1. Our previous data showed that ligation of mast cell kit receptor by KL up-regulates progel B mRNA expression 5-fold. By contrast, TIMP-1 mRNA levels remain unchanged following KL stimulation, possibly due to distinct mechanisms which regulate TIMP-1 expression via enhancement of mRNA stability (41). Thus, KL-kit interactions may alter local progel B to TIMP-1 ratios, favoring release of TIMP-1-free progel B, which would be more susceptible to activation. Mast cells not only release the native form of TIMP-1, but also an 14-kDa form whose presence in conditioned medium is explained neither by extracellular proteolytic processing nor by translation of alternatively spliced mRNA. Whether posttranslational intracellular processing or nonproteolytic extracellular mechanisms generate the 14-kDa form remains a subject of investigation. Both the native and 14-kDa forms of TIMP-1 also appear to be products of normal MBMMC, thus suggesting that the secreted TIMP-1 products of cultured canine and human mast cells do not only represent a malignant phenotype.

-Chymase inactivates TIMP-1 by hydrolyzing bonds in the NH₂-terminal domain. Whereas cleavage by HNE, trypsin, or chymotrypsin destroys the inhibitory activity of unbound TIMP-1 (31), only HNE has previously been shown to degrade TIMP-1 in the progel B-TIMP-1 complex (30). Like HNE, -chymase cleaves TIMP-1, whether the inhibitor exists free or in a complex with progel B. However, -chymase generates products that differ in size from those generated by HNE, suggesting that hydrolysis by -chymase occurs at different sites. The four scissile bonds identified all contain aromatic P1 residues (Phe or Trp), which are favored by -chymase and highly conserved in mammalian species, as seen in Fig. 4; peptide sequencing did not identify cleavage at sites containing other -chymase-preferred P1 residues (including Tyr and a hydrophobic residue, Leu), which localize to the surface of TIMP-1 (34, 42). This suggests that cleavage at these sites may not be favorable due to steric hindrance or that the cleavage products may either be transient or insufficient in quantity for detection and sequencing. Therefore, although -chymase and HNE are similar in their inactivation of free or bound TIMP-1 via NH₂-terminal domain processing, they differ in the number as well as general location of TIMP-1 bonds cleaved.

Conversion of complexed progel B to active forms requires processing which overcomes zymogen latency conferred by the cysteine switch (9) and inhibition of active species by bound TIMP-1. Cells secrete pro-MMPs noncovalently bound to TIMPs with 1:1 stoichiometry, suggesting that physiologic regulation of MMP activity depends on one or more activators, acting alone or in concert, to activate secreted proenzymes. Proteases which hydrolyze either free TIMP-1 or uncomplexed progel B monomer in vitro cannot cleave both components when complexed to each other (17, 30, 31). By contrast, at catalytic or stoichiometric molar ratios, -chymase processing of the progel B-TIMP-1-NGAL complex results not only in cleavage of both TIMP-1 and progel B, but also in conversion of the zymogen to mature forms with up to a 5-fold increase in soluble activity. Activation of the zymogen in the progel B-TIMP-1-NGAL complex also suggests that NGAL (bound via disulfide linkage to progel B without altering the protease’s activity (15, 43, 44)) does not hinder access of -chymase to either TIMP-1 or progel B. Processing by catalytic amounts of -chymase alone compares favorably with other proteolytic mechanisms which activate the complex via 1) molar excesses of either active stromelysin or matrilysin (MMP-7), which increase gelatinolytic activity by competitively binding TIMP-1 (without cleavage) and activating progel B (45, 46); or 2) sequential involvement of HNE, which cleaves and inactivates TIMP-1, and mature stromelysin, which activates progel B (30). In the present work, combined HNE and stromelysin processing of the complex did not increase its gel B activity, possibly due to differences in substrates and reaction conditions. Thus, -chymase processing of the progel B-TIMP-1-NGAL complex not only cleaves and inactivates bound TIMP-1, whose hydrolysis appears to be rate limiting, but also converts progel B from zymogen to active forms.

Access to the NH₂-terminal domain of complexed TIMP-1 is critical for cleavage and attenuation of its inhibition of gel B. Inactivation of TIMP-1 by HNE depends on cleavage of the Val⁶⁹-Cys⁷⁰ bond in the "C-connector loop" which forms the long edge that occupies the MMP-active site cleft (34). This region remains exposed and accessible to HNE when TIMP-1 binds to progel B. Other serine proteases such as trypsin and chymotrypsin can cleave free TIMP-1, but cannot process bound TIMP-1, suggesting that their preferred scissile bonds are obscured in the complex (30). By contrast, TIMP-1 regions cleaved by -chymase are distant from the long edge and are thus likely to be accessible whether the inhibitor is free or bound, as seen in Fig. 4. -Chymase also cleaves TIMP-1 at multiple sites in loops 1 and 3, which may separate the NH₂- and COOH-terminal domains. This contrasts with the single cleavage of TIMP-1 by HNE in loop 1, which leaves the domains linked via the disulfide bonds. Perhaps the liberated, inactivated NH₂-terminal domain arising from -chymase cleavage dissociates from the complex, facilitating subsequent activation of progel B. Whether the TIMP-1 COOH domain retains its non-MMP inhibitory functions following -chymase processing remains unknown.

Hyperplasia and degranulation are key features of the mast cell response in inflamed tissues. Degranulating mast cells release -chymase in active form from secretory granules where it is stored in high concentrations, second only in abundance to tryptase (1, 47). The current data demonstrate that secreted mast cell TIMP-1 remains in its native form until ionophore-induced degranulation releases -chymase, which cleaves the inhibitor to generate products similar to those following incubation of purified -chymase with mast cell supernatants. Lack of TIMP-1 processing by degranulating HMC-1 cells, which do not express -chymase, further substantiates the dependence of mast cell TIMP-1 processing on exocytosed -chymase. Whether nonproteolytic mechanisms may also process secreted TIMP-1 remains unknown. Mast cells express -chymase at levels of 5 pg/cell (48), yielding a cellular concentration of 300 µM (which underestimates the concentration in secretory granules due to differences in granule and cytosolic volumes) that predicts even higher local tissue concentrations upon degranulation. In human tissues, -chymase is found at a concentration of as much as 45 µg/g of tissue (48, 49), compared with a TIMP-1 concentration of 12.4 µg/g (50). Immunolocalization studies in a murine model of squamous epithelial carcinogenesis demonstrate limited diffusability of chymase; thus, it remains in the vicinity of degranulating mast cells situated near basement membranes, which are sites of extracellular matrix remodeling (11). The abundance of stored -chymase and its limited diffusability suggest that in vivo mast cell degranulation may generate transient and localized molar excesses of -chymase relative to its substrates such as TIMP-1. We speculate that such a quantum proteolytic event (51) may be an important mechanism whereby exocytosed mast cell -chymase regulates both the MMP inhibitory and noninhibitory functions of TIMP-1 in remodeling tissues. Evidence of ongoing and gradual (or piecemeal) degranulation of mast cells in fibrotic tissues (52) suggests that such a mechanism may not only occur transiently, but also persist indefinitely. Therefore, the quiescence or activation of mast cells in lung, skin, heart, or gut tissues may determine not only the protease to antiprotease balance of MMPs to TIMPs which is critical in matrix remodeling and fibrogenesis (8, 52, 53), but also the ability of TIMP-1 to regulate apoptosis and mitogenesis via pathways which are independent of its MMP inhibitory functions (11, 54).

The current data suggest a role for mast cell -chymase in physiologic pathways which regulate MMP activity. Unlike other proteases which require cascading activation of multiple proenzymes before activation of the target pro-MMP (2, 3, 5, 8, 17, 35, 55, 56, 57), -chymase activates zymogen monomers of progel B, procollagenase, and stromelysin, without a requirement for other proteases or cofactors (2, 3, 6, 7). Data also show that infiltration of premalignant lesions by mast cells activates progel B via -chymase processing, coincident with the activation of angiogenesis, thus demonstrating an in vivo role for -chymase-dependent pro-MMP activation in tissue remodeling (11). In summary, our data show that cultured mast cells, such as HMC-1 or BR cells and MBMMC, secrete TIMP-1, which is processed by exocytosed -chymase. Uniquely, -chymase processes the progel B-TIMP-1-NGAL ternary complex by inactivating bound TIMP-1 and converting progel B to mature active forms. -Chymase hydrolysis of TIMP-1 occurs at four scissile bonds containing exposed aromatic residues distant from regions which interact with the MMP active site. Cleavage attenuates the inhibitory activity of TIMP-1 and may separate its NH₂- and COOH-terminal domains. Therefore, release of -chymase may provide a novel mechanism for recovering and augmenting activity of progel B complexed to TIMP-1 during extracellular matrix remodeling in mast cell-rich environments.

	Acknowledgments

 
We thank Ralph Reid and Wilfred W. Raymond for helpful discussions and Paul J. Wolters for providing murine mast cells. We also acknowledge the excellent technical assistance of Afshin Bidgol, John Blount, and Diego Muilenburg.

	Footnotes

 
¹ This work was supported by Grants HL-03345 and HL-24136 from the National Institutes of Health. K.C.F. is the recipient of a Mentored Clinical Scientist Development Award from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Kenneth C. Fang, Box 0911, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0911.

³ Abbreviations used in this paper: MMP, matrix metalloproteinase; AAPF-CMK, Ala-Ala-Pro-Phe chloromethyl ketone; DNP-PGCHAK, DNP-Pro-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH₂; gel B, gelatinase B; HNE, human neutrophil elastase; KL, recombinant canine kit ligand; MBMMC, murine bone marrow-derived mast cells; NGAL, neutrophil gelatinase-associated lipocalin; progel B, progelatinase B; TIMP, tissue inhibitor of metalloproteinase; rh, recombinant human.

Received for publication October 4, 2000. Accepted for publication December 7, 2000.

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