Mast Cell Tissue Inhibitor of Metalloproteinase-1 Is Cleaved and Inactivated Extracellularly by α-Chymase This information is current as Brendon T. Frank, J. Caleb Rossall, George H. Caughey and of September 24, 2021. Kenneth C. Fang J Immunol 2001; 166:2783-2792; ; doi: 10.4049/jimmunol.166.4.2783 http://www.jimmunol.org/content/166/4/2783 Downloaded from References This article cites 56 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/166/4/2783.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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. Fang2*† 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 Downloaded from 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 http://www.jimmunol.org/ progel B without involvement of other proteases. The Journal of Immunology, 2001, 166: 2783–2792. ctivated mast cells respond to allergen, tumor invasion, inhibitors of metalloproteinases (TIMPs). Proteolytic activation of and fibrogenic injury by releasing tryptase and ␣-chy- pro-MMPs involves one or more activator proteases which remove A mase, which are granule-associated tryptic and chymo- the propeptide domain containing a critical Cys residue, thus dis- tryptic serine proteases, respectively. Upon degranulation, both rupting the cysteine switch which confers active site latency (9). proteases participate in proteolytic and nonproteolytic pathways TIMPs inhibit activity of active MMP species and block activation regulating matrix protein degradation, receptor activation, peptide of pro-MMPs by forming TIMP-MMP complexes in a 1:1 molar inactivation, and fibroblast, smooth muscle, or epithelial cell mi- by guest on September 24, 2021 ratio. NH2-terminal TIMP domains occupy the MMP active site togenesis (1). Tryptase and ␣-chymase may also act in a broader and COOH-terminal domains confer binding specificity through range of homeostatic and pathologic tissue remodeling processes interactions with COOH-terminal MMP domains (10). 2ϩ 2ϩ via interactions with a family of Ca - and Zn -dependent matrix Mast cells may regulate local MMP activity by contributing zy- 3 metalloproteinases (MMPs) (2–7). Secreted or cell surface-asso- mogen-activating serine proteases and by secreting pro-MMPs. ciated membrane-type MMPs participate in physiologic pathways Our previous work demonstrates that mast cells secrete progelati- such as embryonic development, organ morphogenesis, angiogen- nase B (progel B; MMP-9), which is activated extracellularly by esis, and wound healing, and also contribute to the pathogenesis of degranulated ␣-chymase upon cleavage of the catalytic domain at arthritis, cancer, cardiovascular disease, and lung fibrosis (8). Post- two sites (2–4). ␣-Chymase-dependent activation of progel B in translational regulation of MMP activity depends upon conversion vivo is hypothesized to switch nonangiogeneic tissues to an an- of proenzymes to mature active forms and their inhibition by tissue giogenic 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 *Cardiovascular Research Institute and †Department of Medicine, University of Cal- ifornia, San Francisco, CA 94143 prostromelysin (MMP-3) (6, 7). Mast cells also express these two Received for publication October 4, 2000. Accepted for publication December MMPs plus progelatinase A (MMP-2), which shares in vitro sub- 7, 2000. strate specificity with gelatinase B (gel B) (4, 12, 13). The costs of publication of this article were defrayed in part by the payment of page Since cells secrete pro-MMPs bound to TIMPs in a complex, charges. This article must therefore be hereby marked advertisement in accordance zymogen activation likely requires prior TIMP processing which with 18 U.S.C. Section 1734 solely to indicate this fact. precedes proteolytic activation of the MMP moiety. TIMP-1 binds 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 Devel- to progel B, which is secreted as a monomer or dimer, or in di- opment Award from the National Institutes of Health. sulfide-mediated linkage with neutrophil gelatinase-associated li- 2 Address correspondence and reprint requests to Dr. Kenneth C. Fang, Box 0911, pocalin (NGAL) to form a ternary complex of progel B monomer, Cardiovascular Research Institute, University of California, San Francisco, CA TIMP-1 and NGAL (progel B-TIMP-1-NGAL) (14, 15). Unbound 94143-0911. E-mail address: [email protected] TIMP-1 may be inactivated in vitro by cleavage, degradation, or 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- chemical modification via proteolytic and nonproteolytic mecha- Ala-Lys(N-Me-Abz)-NH2; gel B, gelatinase B; HNE, human neutrophil elastase; KL, nisms involving serine or thiol proteases and reactive oxygen spe- recombinant canine kit ligand; MBMMC, murine bone marrow-derived mast cells; NGAL, neutrophil gelatinase-associated lipocalin; progel B, progelatinase B; TIMP, cies, respectively (16–19). However, mechanisms which regulate tissue inhibitor of metalloproteinase; rh, recombinant human. processing of MMP-bound TIMP-1 remain unclear. In addition to Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2784 ␣-CHYMASE CLEAVAGE OF TIMP-1 its ability to block pro-MMP activation and inhibit activity of ma- fore further analysis. To determine the electrophoretic profile of reaction ture MMPs, TIMP-1 also demonstrates erythroid-potentiating ac- products, aliquots were subjected to SDS-PAGE using 16% or 4–20% tivity, stimulates steroidogenesis, regulates mitogenesis, and con- gradient Tris-glycine gels (Invitrogen) under reducing conditions, with de- tection of proteins by Coomassie Blue R250 (Fisher Scientific, Tustin, CA) trols apoptosis via mechanisms which are independent of its MMP or immunoblotting using polyclonal rabbit anti-TIMP-1 or polyclonal rab- inhibitory activity (10). Thus, processing of TIMP-1 in the inflam- bit anti-MMP-9 Abs (Triple Point Biologics, Portland, OR). matory milieu may not only alter the protease:antiprotease balance TIMP-1 activity to favor proteolysis, but also attenuate or abolish its non-MMP inhibitory effects on specific cell populations. We report here that MMP inhibitory activity of rhTIMP-1 was determined by incubating intact ␣ mast cell ␣-chymase cleaves free TIMP-1 and processes the progel or -chymase-processed inhibitor with active gel B monomer (Calbio- chem). rhTIMP-1 was incubated alone or in the presence of various con- B-TIMP-1 complex by inactivating bound TIMP-1 and activating centrations of ␣-chymase in reaction buffer at 37°C for different time pe- progel B, without involvement of other