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ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2

2006; Springer Nature; Volume: 25; Issue: 22 Linguagem: Inglês

10.1038/sj.emboj.7601400

1460-2075

Nathan V. Lee, Makoto Sato, Douglas S. Annis, Joseph A. Loo, Lily Wu, Deane F. Mosher, M. Luisa Iruela‐Arispe,

Galectins and Cancer Biology

Article2 November 2006free access ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2 Nathan V Lee Nathan V Lee Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Search for more papers by this author Makoto Sato Makoto Sato Department of Urology, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Douglas S Annis Douglas S Annis Department of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Joseph A Loo Joseph A Loo Department of Biological Chemistry and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA Search for more papers by this author Lily Wu Lily Wu Department of Urology, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Deane F Mosher Deane F Mosher Department of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author M Luisa Iruela-Arispe Corresponding Author M Luisa Iruela-Arispe Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Search for more papers by this author Nathan V Lee Nathan V Lee Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Search for more papers by this author Makoto Sato Makoto Sato Department of Urology, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Douglas S Annis Douglas S Annis Department of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Joseph A Loo Joseph A Loo Department of Biological Chemistry and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA Search for more papers by this author Lily Wu Lily Wu Department of Urology, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Deane F Mosher Deane F Mosher Department of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author M Luisa Iruela-Arispe Corresponding Author M Luisa Iruela-Arispe Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Search for more papers by this author Author Information Nathan V Lee1, Makoto Sato2, Douglas S Annis3, Joseph A Loo4, Lily Wu2, Deane F Mosher3 and M Luisa Iruela-Arispe 1 1Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA 2Department of Urology, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA 3Department of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA 4Department of Biological Chemistry and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA *Corresponding author. Department of Molecular, Cell and Developmental Biology, Molecular Biology Institute, UCLA, 611 Charles E. Young Drive East Boyer Hall 559, Los Angeles, CA 90095, USA. Tel.: +1 310 794 5763; Fax: +1 310 794 5766; E-mail: [email protected] The EMBO Journal (2006)25:5270-5283https://doi.org/10.1038/sj.emboj.7601400 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Matrix metalloproteases regulate both physiological and pathological events by processing matrix proteins and growth factors. ADAMTS1 in particular is required for normal ovulation and renal function and has been shown to modulate angiogenesis. Here we report that TSP1 and 2 are substrates of ADAMTS1. Using a combination of mass spectrometry and Edman degradation, we mapped the cleavage sites and characterized the biological relevance of these processing events. ADAMTS1 cleavage mediates the release of polypeptides from the trimeric structure of both TSP1 and 2 generating a pool of antiangiogenic fragments from matrix-bound thrombospondin. Using neo-epitope antibodies we confirmed that processing occurs during wound healing of wild-type mice. However, TSP1 proteolysis is decreased or absent in ADAMTS1 null mice; this is associated with delayed wound closure and increased angiogenic response. Finally, TSP1−/− endothelial cells revealed that the antiangiogenic response mediated by ADAMTS1 is greatly dependent on TSP1. These findings have unraveled a mechanistic explanation for the angiostatic functions attributed to ADAMTS1 and demonstrated in vivo processing of TSP1 under situations of tissue repair. Introduction Remodeling of the extracellular matrix is an essential requirement for development, repair and homeostasis of normal tissues (Mott and Werb, 2004). Among the molecules responsible for these events are the matrix metalloproteases. These constitute a major group of extracellular and membrane-bound enzymes involved in the selective digestion and processing of proteins, glycoproteins and growth factors located outside the cell (Mott and Werb, 2004; Lee et al, 2005b). Like many other metalloproteases, ADAMTS1 (A Disintegrin And Metalloprotease with ThrombosSpondin) is a secreted, zinc-binding enzyme broadly expressed during development and in several adult tissues (Thai and Iruela-Arispe, 2002; Lee et al, 2005a). However, unlike many other metalloproteases in which loss-of-function showed a minimal phenotype, genetic inactivation of ADAMTS1 results in either embryonic lethality (in about 40% of null mice) or postnatal lethality due to severe kidney dysfunction (Shindo et al, 2000; Mittaz et al, 2004; Lee et al, 2005a). Mice homozygous for the null allele also display multiple defects in the female reproductive tract including poor fertility and anomalies in uterine structure (Russell et al, 2003). In addition, mice suffer from stunted growth and showed adrenal abnormalities (Shindo et al, 2000). Together, these outcomes stress the relevance of ADAMTS1 during development and homeostasis of several adult organs. Mechanistic understanding of the phenotype associated with ADAMTS1 inactivation requires a concrete knowledge of its catalytic profile, that is, biological substrates, and information of other noncatalytic functions. Towards this goal, several groups have identified: (1) substrates for ADAMTS1 that, to date, include aggrecan, versican and nidogen (Kuno et al, 2000; Sandy et al, 2001; Rodriguez-Manzaneque et al, 2002; Canals et al, 2006); (2) catalytic modifiers, such as fibulin1 (Lee et al, 2005a) and (3) noncatalytic function such as sequestration of VEGF to reduce signaling via this ligand (Luque et al, 2003). Some of the substrates identified for ADAMTS1 have been linked to the pathologies displayed by the null mouse such as inability to ovulate due to lack of versican cleavage (Russell et al, 2003). Nonetheless, many of the other defects remain to be explained at a molecular level. To further expand our knowledge of substrates for ADAMTS1, we tested several potential candidates. Our strategy focused on extracellular proteins located in basement membranes, as the expression profile of ADAMTS1 includes several epithelia (kidney, lung and epidermis) and blood vessels (Thai and Iruela-Arispe, 2002; Gunther et al, 2005). Furthermore, basement membrane components have been shown to regulate differentiation and migration of endothelial cells during angiogenesis (Kalluri, 2003). Here we showed that TSP1, a constitutive component of epithelial and endothelial basement membrane, is cleaved by ADAMTS1. Thrombospondins (TSPs) are a family of secreted glycoproteins broadly and highly expressed during development (Iruela-Arispe et al, 1993). In addition, TSPs have been associated with the regulation of several processes in the adult including angiogenesis, wound healing and collagen fibril assembly (Bornstein et al, 2000; Lawler, 2000, 2002; Lawler and Detmar, 2004). From all five members of the TSP family, only TSP1 and 2 inhibit angiogenesis in vitro and in vivo (Lawler and Detmar, 2004). The antiangiogenic domain has been mapped to the type I (or TSR) repeats present in TSP1 and 2, a motif that is absent in TSPs 3, 4 and 5. Here we show that processing of TSP1 by ADAMTS1 releases bioactive polypeptides with antiangiogenic properties, demonstrate that this cleavage event occurs in vivo, and explore the biological consequences of TSP processing in mice that lack ADAMTS1. Results TSP1 is cleaved by ADAMTS1 To test the hypothesis that basement membrane proteins are substrates for ADAMTS1, we exposed both TSP1 and laminin to the enzyme in vitro. Analysis of the digestion by electrophoretic mobility under reducing conditions revealed two smaller polypeptides of 110 and 36 kDa in the TSP1 sample visible by Coomassie. In contrast, under the same conditions ADAMTS1 did not cleave laminin (Figure 1A). An assortment of several matrix proteins, including TSP1, has been previously used in ADAMTS1 enzymatic assays and indicated no cleavage (Rodriguez-Manzaneque et al, 2002). However, those analyses were performed using antibodies and nonreduced conditions and this prevented visualization of released fragments. Figure 1.TSP1 and 2 are cleaved by ADAMTS1 at unique sites. (A) Coomassie stained gel of full-length TSP1 and Laminin incubated with ADAMTS1. Arrows indicate the 110 and 36 kDa cleavage fragments. (B) Western immunoblot of TSP1 incubated with ADAMTS1 or thrombin. (C) Western immunoblots of TSP1 and 2 incubated with ADAMTS1. (D) Western immunoblot of TSP1 incubated with ADAMTS1, catalytically inactive ADAMTS1 (E385A) or a truncated ADAMTS1 form that only harbors type I repeats (TSRs) for indicated times. (E) Western immunoblot of TSP2 incubated with ADAMTS1, catalytically inactive ADAMTS1 (E385A) or a truncated ADAMTS1 form that only harbors type I repeats (TSRs) for indicated times. Open arrow, fragment already present in preparation that is not susceptible to ADAMTS1 cleavage. Download figure Download PowerPoint Purification of TSP1 entails thrombin-induced platelet degranulation and because thrombin is a recognized protease for TSP1 (Lawler et al, 1986a, 1986b), we asked if TSP1 fragments could potentially be the result of contaminating thrombin activity. To test this, TSP1 was incubated with 2 or 5 U of thrombin. Analysis by Western immunoblot with a polyclonal anti-TSP1 antibody (GPC) confirmed that the fragment derived from ADAMTS1 cleavage, 36 kDa, was distinct from the fragment released by thrombin, 25 kDa (Figure 1B). TSP2 is also a substrate for ADAMTS1 TSP1 and 2 share identical structural features and are 32–82% conserved in amino-acid sequence depending on the specific domain (Bornstein, 1992). Thus, we were interested in determining whether TSP2 might also be a substrate for ADAMTS1. In vitro digestion assays revealed that ADAMTS1 released two fragments of 42 and 30 kDa (Figure 1C). To ensure that cleavage of both TSP1 and 2 by ADAMTS1 did not result from possible contaminating proteases, a catalytically inactive ADAMTS1 (E385A) and the ADAMTS1 C-terminal fragment (TSRs) lacking the catalytic domain were incubated with TSP1 and 2 in parallel. The inactive ADAMTS1 (E385A) and the C-terminal fragment were purified from the same cell expression system following a similar protocol. Consequently, any contaminating protease would also be present in these preparations. Both TSP1 and 2 were cleaved only by active ADAMTS1 (Figure 1D and E). These experiments confirmed that TSP1 and 2 cleavage resulted specifically from the catalytic activity of ADAMTS1. In addition, at an enzyme:substrate (E:S) ratio of 1:2.5, TSP1 was cleaved by ADAMTS1 in 5 min (Figure 1D). At the same ratio, TSP2 was cleaved by ADAMTS1 releasing a 42 kDa fragment in 15 min and into a second 30 kDa fragment in 1 h (Figure 1E). In certain TSP1 protein preparations, a 60 kDa fragment was already present in the starting material, but were not susceptible to ADAMTS1 (Figure 1A and D, open arrow). To assess the cleavage efficiency of TSP1 and 2 by ADAMTS1, the proteins were incubated with varying ratios of ADAMTS1 for 1 h at 37°C. E:S ranged from 1:1 to 1:40. Within 1 h, half of the starting full-length TSP1 was processed at an E:S of 1:40 (Figure 2A). Cleavage of TSP2 by ADAMTS1 was not as effective; an E:S of 1:5 was required to cleave 30% of the starting full-length TSP2 (Figure 2B). However, proteolysis of both TSP1 and 2 was dose-dependent as more ADAMTS1 yielded increasingly more cleavage products (Figure 2A and B, arrows). Figure 2.ADAMTS1 cleavage of TSP1 and 2 occurs in a dosage-dependent manner. (A, B) Western immunoblots of TSP1 and 2 incubated with ADAMTS1 for 1 h at 37°C at E:S ranging from 1:1 to 1:40. (C, D) Western immunoblots of TSP1 and 2 incubated with ADAMTS1 in pH ranging from 5.0 to 8.5. Arrowheads, intact TSP1 and 2; arrows, TSP1 and 2 cleavage fragments. Tables under each blot indicate densitometric quantification of the bands. Numbers are in percentile of relative intensity in relation to the darkest band in the blot. Download figure Download PowerPoint To determine if proteolysis occurs at physiological pH, TSP1 and 2 were incubated with ADAMTS1 at a pH range of 5.0 to 8.5 at an E:S of 1:20. Maximum efficiency for cleavage of TSP1 occurred at pH 6.5 to 8.5 and for TSP2 at pH 7.0 to 8.5. TSP2 was cleaved by ADAMTS1 at two distinct sites to generate a 42 and a 30 kDa polypeptide. Kinetics experiments revealed a sequential release of these fragments (Figure 1E). In addition, increasing molar ratio of ADAMTS1 favors the generation of the 30 kDa fragment (Figure 2B). These data suggest that the initial cleavage releases the 42 kDa fragment and a second event releases the 30 kDa fragment. Cleavage of TSP1 and 2 by ADAMTS1 is not shared by ADAMTS4 ADAMTS1 and ADAMTS4 display high sequence homology and share the substrates, aggrecan and versican (reviewed by Apte, 2004). Thus, we sought to determine whether TSP1 and 2 are also cleaved by ADAMTS4. Both TSP1 and 2 were not cleaved by ADAMTS4 compared to ADAMTS1 at the same molar concentration (Figure 3A and B). To verify that ADAMTS4 was active, aggrecan was digested with both ADAMTS1 and ADAMTS4. As expected, both were able to cleave aggrecan, although ADAMTS4 was a more effective enzyme for aggrecan than ADAMTS1 (Figure 3C). Aggrecan is cleaved by both ADAMTS1 and ADAMTS4 to 200 kDa as well as 65 kDa (Sandy et al, 2000; Rodriguez-Manzaneque et al, 2002). ADAMTS4 is more efficient at cleaving aggrecan hence majority of the product was 65 kDa in mass. In contrast, ADAMTS1 is less efficient at cleaving aggrecan yielding mainly 200 kDa fragments. Figure 3.TSP1 and 2 are not substrates of ADAMTS4. (A, B), Western immunoblots of TSP1 and 2 incubated with ADAMTS1, ADAMTS4 and vehicle. (C) Western immunoblots of aggrecan incubated with ADAMTS1, ADAMTS4 or vehicle. (D, E) Western immunoblots of TSP1 and 2 incubated with varying amounts of 87 kDa ADAMTS1 and 65 kDa ADAMTS1, as indicated. Arrowheads, intact TSP1 and 2; arrows, TSP1 and 2 cleavage fragments. Download figure Download PowerPoint Heparin binding domains of TSP1, TSP2 and ADAMTS1 are necessary for efficient cleavage The N-terminal domain of TSP1 and 2, as well as TSR repeats of ADAMTS1 have been shown to interact with heparin (Murphy-Ullrich et al, 1993; Kuno and Matsushima, 1998; Rodriguez-Manzaneque et al, 2000). We considered that these domains might facilitate docking and subsequent cleavage. To test this possibility, TSP1 was incubated with the full-length active ADAMTS1 (87 kDa) and with truncated active ADAMTS1 (65 kDa), which lacks part of the spacer region and the last two TSRs, and displays lower affinity for heparin (Rodriguez-Manzaneque et al, 2000). The truncated form of ADAMTS1 required a higher E:S (2:1) to achieve near complete processing of intact TSP1 within 2 h in comparison to 87 kDa ADAMTS1, which completely processed intact TSP1 at an E:S of 1:10 (Figure 3D). TSP2 was also incubated with 65 and 87 kDa forms of ADAMTS1. The 65 kDa form of ADAMTS1 was rather inefficient at processing intact TSP2, as an E:S of 1:1 was required to release the 42 kDa TSP2 N-terminal fragment visible by Western blot (Figure 3E). The 87 kDa ADAMTS1 was more efficient at cleaving intact TSP2 (Figure 3E). The reciprocal experiment was carried out by incubating truncated mutants of TSP1 and 2 lacking the heparin binding domain (delN-1 and delN-2) with ADAMTS1. In comparison to intact TSP1, which was completely processed, delN-1 was not cleaved as efficiently, as only half was processed (Figure 4B (i)). In turn, delN-2 was not cleaved by ADAMTS1 (Figure 4B (ii)). To discard concerns related to structural changes of deletion mutants, we tested another deletion mutant, NoC, which has the N-module necessary for heparin binding. Both NoC-1 and NoC-2 were cleaved by ADAMTS1 (Figure 4C (i and ii)). Figure 4.Heparin binding domains are important for cleavage. (A) schematic diagram of full-length TSP1 and 2 and deletion mutants. (B (i, ii), Western immunoblots and Coomassie staining of full-length TSP1 and delN-1 or TSP2 and delN-2 digested with ADAMTS1. (C (i, ii)), Western immunoblots and Coomassie staining of full-length TSP1 and truncated mutant NoC-1truncated or TSP2 and NoC-2 digested with ADAMTS1. Open arrows, ADAMTS1 protein. (D (i, ii)) TSP1 and 2 digested with ADAMTS1 in presence of increasing amounts of heparin. Arrowhead, full-length protein; arrows, cleaved fragments. Download figure Download PowerPoint To test whether heparin affects TSP1 and 2 processing, we incubated both TSP proteins with ADAMTS1 at an E:S of 1:40 in the presence of increasing amounts of heparin. TSP1 cleavage was not affected by heparin (Figure 4D (i)), similarly, the more carboxy terminal cleavage site in TSP2, which yield the 42 kDa fragment was not altered (Figure 4D (ii)). However, generation of the 30 kDa fragment was suppressed by heparin (Figure 4D (ii)). This would indicate that rather than altering enzymatic activity, heparin binds to one of the sites in TSP2 and hampers the ability of ADAMTS1 to dock and cleave within this site. Nonetheless, based on the previous findings, the heparin-binding region in TSP1 and 2 appears to function independent from heparin to facilitate processing by ADAMTS1. Identification of cleavage sites Analysis of with MALDI-TOF MS determined that the 36 kDa fragment corresponds to the N-terminus of TSP1 and the 110 kDa fragment corresponds to the C-terminus. Analysis repeated with LC MS yield the same result (data not shown). Because the 110 kDa fragment exposes the cleavage site at the N-terminal region, we transferred it onto PVDF membrane and performed N-terminal Edman degradation sequencing. One of the resulting peptides contained the LRRPPL sequence indicating that cleavage occurred between residues: glutamic acid 311 and leucine 312 (Figures 5A and 6A). This site is consistent with the classification of glutamyl endopeptidase attributed to ADAMTS1 based on cleavage of other substrates such as aggrecan and versican (Rodriguez-Manzaneque et al, 2002; Westling et al, 2002). Similarly, higher-molecular weight TSP2 doublets released by ADAMTS1 cleavage were also analyzed by MALDI-TOF MS and determined to correspond to the C-terminal fragment lacking the N-terminal domain (data not shown). Both fragments were transferred to PVDF, Edman sequencing resolved only the lower doublet fragment and revealed the sequence LIGGPP (Figure 5B). This indicates that one of the cleavage sites in TSP2 lies between glutamic acid 306 and leucine 307 (Figure 6A). Figure 5.TSP1 and 2 cleavage sites and schematic representation of resulting fragments. (A) TSP1 is cleaved at one site C1 by ADAMTS1 adjacent to amino-acid sequence LRRPPL (determined by Edman degradation sequencing). (B) TSP2 is cleaved at two sites C1 and C2. Site C1 is adjacent to amino acids LIGGPP. Proteins were visualized with Coomassie. Asterisk in (A) represents ADAMTS1. Download figure Download PowerPoint Figure 6.N-terminal fragments remain trimerized releasing the monomeric C-terminal fragments. (A) Sequence alignment of TSP1 and 2 including region in proximity of cleavage sites. Boxed region represents pro-collagen domain. Underlined sequence corresponds to the coiled-coil region. Asterisks denote cysteines involved in interchain disulfide bonds. Inset, schematic representation of N-terminal and C-terminal fragments in reducing (R) and nonreducing (NR) conditions. (B, C) Western immunoblots of TSP1 and 2 incubated with ADAMTS1 or vehicle under nonreducing and reducing conditions. Download figure Download PowerPoint The second cleavage site in TSP2 remains to be determined; however, the monoclonal antibody used to detect TSP2 (3C5.3) recognizes the N-module, indicating that this cleavage is likely occur towards the N-terminal region. Proteolytic activity of ADAMTS1 releases monomeric C-terminal peptides from TSP1 and 2 Native TSP1 and 2 exist as homotrimers linked by intermolecular disulfide bonds and the coiled-coil oligomerization domain (Engel, 2004). Additional intramolecular disulfide bonds exist within the linker region between the coiled-coil domain and the procollagen homology domain (Misenheimer et al, 2000). Based on the mapped cleavage site, ADAMTS1 proteolysis could have two possible outcomes: (1) release the C-terminus fragment from the N-terminus fragment; or (2) cleavage could target the disulfide bond region resulting in a nicked protein where the N-terminus remains attached to the C-terminus by disulfide bonds. To distinguish between these two possibilities, digested proteins were separated under reducing and nonreducing conditions (Figure 6B and C). The 36 kDa N-terminus fragment of TSP1 (reducing condition) was detected as a 108 kDa fragment (nonreducing) (Figure 6B), indicating that the N-terminus remained trimerized after ADAMTS1 cleavage. Since the N-terminus is the only known region required to mediate trimer formation, the 110 kDa C-terminus fragment is released in a monomeric form. Analysis of the TSP2 digestion products revealed that ADAMTS1 cleavage of this molecule also releases the C-terminus fragment as a monomer leaving the N-terminus in a trimeric form. Under nonreducing conditions, the 42 kDa N-terminus fragment was shifted to a 145-kDa band (Figure 6C). Trimerization of the 42 kDa would yield a complex of approximately 126 kDa in size. The discrepancy between the 142 kDa band and the expected 126 kDa band is likely due to glycosylation. In addition, the 30 kDa N-terminus fragment (reducing condition) shifted to a 90-kDa band (nonreducing condition) corresponding to a trimerized 30-kDa fragment (Figure 6C). ADAMTS1 cleaves murine TSP1 Since murine TSP1 shares high sequence identity to the human orthologue, we assessed whether mTSP1 is also cleaved by ADAMTS1 (Figure 7B). Adenoviral constructs expressing active ADAMTS1, inactive ADAMTS1 or GFP were used to infect 293T cells. Conditioned medium (CM) was collected 24 h postinfection and was subsequently incubated with either purified hTSP1 or mTSP1 from mouse LE II cells at 37°C for 2 h. Under these conditions, mTSP1 was also cleaved by ADAMTS1 releasing a 36 kDa fragment indicating that the cleavage site was likely to be in the same region in both species (Figure 7A). Figure 7.Characterization of TSP1 neo-epitope antibodies. (A) Western immunoblot of murine and human TSP1 incubated with CM from adenovirus infected cells expressing GFP (cmvGFP), inactive ADAMTS1 (cmvE385A) or full-length active ADAMTS1 (cmvATS1). (B (i)) Sequence alignment of human and murine TSP1 residues flanking cleavage site. (ii) Schematic diagram of TSP1 and fragments recognized by neo-epitope antibodies (78 and 79) and antibody to spanning region (80). (C) Western immunoblot of intact TSP1 and TSP1 fragments resulting from processing with either thrombin or ADAMTS1 under reducing and nonreducing conditions. Arrowhead, full-length TSP1; arrow, TSP1 fragment. Download figure Download PowerPoint Characterization of neo-epitope antibodies To further explore the significance of the processing events, we developed neo-epitope antibodies against the cleaved sites and the spanning region using flanking peptides, NRELVSE (#78) and LKRPPLC (#79), and the spanning TEENRELVSELKRPPL peptide (#80). All three antibodies were affinity-purified with their corresponding immunizing peptides and cross-absorbed to eliminate unwanted reactivities. We then tested their specificity against TSP1 cleaved by either thrombin or ADAMTS1. Antibody #78 specifically recognized the 36 kDa TSP1 fragment released by ADAMTS1 cleavage. Similarly, antibody #79 specifically recognized the 110 kDa TSP1 fragment. Antibody #80 recognized intact TSP1 as well as thrombin-cleaved TSP1 as the polypeptide retained the spanning region. All antibodies worked in reduced and nonreduced conditions (Figure 7C). TSP1 is cleaved by ADAMTS1 during wound healing It has been previously shown that ADAMTS1 is upregulated in inflammatory situations, a feature also shared by TSP1 (Agah et al, 2002). Thus, we investigated whether TSP1 fragments could be detected in excisional skin wound healing assays at 2-day postinjury. We made full-thickness wounds on the dorsal skin of ADAMTS1 null mice and wild-type siblings. Intact TSP1 expression was found at the leading wound edge epithelium, fibrin clot and hair follicles in the ADAMTS1 null animals (Figure 8A (d), arrow denotes the leading edge). In comparison, less intense staining of intact TSP1 was found in the leading wound edge of the wild-type siblings (Figure 8A (a)). However, comparable levels of TSP1 protein were localized to the fibrin clot and hair follicles. In contrast, both N- and C-terminal fragments of TSP1 were present on the leading edge in the wound epithelium and hair follicles in wild-type animals (Figure 8A (b and c)). ADAMTS1 null animals displayed less intense staining of both N- and C-terminal TSP1 fragments (Figure 8A (e and f)). Figure 8.TSP1 cleavage by ADAMTS1 occurs during excisional wound healing. (A) (a–f) Immunohistochemical staining of intact TSP1 and TSP1 fragments in 2-day excisional wound serial sections using the spanning region antibody (#80) or neo-epitope antibodies (#78 and #79). (Arrow in a–f, invading front of epidermal cells; C: fibrin clot; E: epidermis; F: hair follicles) (A). (g–l), Immunohistochemical staining of intact TSP1 and fragments within fibrin clot. Arrows in (g and j), intact TSP1; arrows in (h), N-terminal TSP1 fragment. (B) Western immunoblot of TSP1 (intact and fragments) immunoprecipitated with anti-TSP1 antibodies (#78, 79 or 80) and anti-occludin (occ). Western immunoblot was performed using either #78, 79 or 80 (arrowhead: C-terminal TSP1 fragment; arrow: N- terminal TSP1 fragment; asterisk: intact TSP1). Download figure Download PowerPoint ADAMTS1 has been shown to be expressed by CD11b positive cells within the clot by 1-day postinjury (Krampert et al, 2005). We analyzed the localization of TSP1 fragments within the clot of 2-day wounds. As expected, intact TSP1 was found in both wild-type and ADAMTS1 null clots (Figure 8A (g and j), arrows). However, the N-terminal TSP1 fragment was only found in the clot of wild-type animals (Figure 8A (h), arrows). The C-terminal fragment was not visible in either wild type or ADAMTS1 null animals. A finding that suggests that this fragment is likely soluble and short lived (Figure 8A (i and l)). Immunoprecipitation of wound lysates using the antiamino fragment antibody (#78) pulled down a 40 kDa species in wild-type sample, this fragment was not detected on the ADAMTS1 null sample (Figure 8B, arrow). Similarly, immunoprecipitation with the anticarboxy fragment antibody (#79) pulled down a 110-kDa fragment in the wild-type sample and to a lower degree in the ADAMTS1 null sample (Figure 8B, arrowhead). Immunoprecipitation with the antispanning peptide antibody (#80) was able to precipitate a 145 kDa fragment that corresponds to the intact TSP1 (Figure 8B, asterisk). At 5-day postinjury, wounds were closed in the wild-type animals and the new epithelial layer covering the wound was thicker than the surrounding epithelia (Figure 9A (a)). In contrast, ADAMTS1 null wounds remained open with poor epithelial migration (Figure 9A (b), arrows). Evaluation of capillary density revealed more vessels in the dermis of ADAMTS1 null mice than in control littermates (Figure 9C and D). Figure 9.ADAMTS1 null animals exhibits delayed wound healing and increased angiogenesis. (A) Hematoxylin and eosin stained 5-day wound cross-sections of wild-type and ADAMTS1 null animals. (B) Excisional wound opening area quantified over 5 days in wild-type and adamts1 null animals. Difference in open wound area between wild-type and adamts1 null from day 1 onward has a P-value <0.005. (C) Hematoxylin and eosin (a, d) anti-PECAM stained (b, c, e, f) blood vess