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The Generation of Endostatin Is Mediated by Elastase¹

Laboratory for Surgical Research, Department of Surgery, The Children’s Hospital, Boston, Massachusetts 02115 [W. W., M. A. M., D. W., J. F.]; Departments of Surgery [M. A. M., J. F.] and Cellular Biology [J. F.], Harvard Medical School, Boston, Massachusetts 02115; and Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322 [J. L. A.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
Endostatin, a potent inhibitor of angiogenesis and tumor growth, is a COOH-terminal fragment of collagen XVIII derived through cleavage of an Ala-His linkage by an as yet unidentified endostatin-processing enzyme. Endostatin was originally isolated from the conditioned medium of hemangioendothelioma (EOMA) cells. By investigating the processing of collagen XVIII to endostatin by EOMA cells, we show here that the generation of endostatin can be mediated by an elastase activity. We also show that several members of the elastase family can act as an endostatin-processing enzyme by specifically cleaving the Ala-His linkage and releasing endostatin from a precursor molecule. We further suggest that the generation of endostatin from collagen XVIII is at least a two-step process, involving a metal-dependent early step and an elastase activity-dependent final step.

	Introduction

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
Angiogenesis inhibitors, which inhibit angiogenesis specifically and selectively, have been shown to suppress tumor growth in mice without toxicity or drug resistance. Many of the known angiogenesis inhibitors are fragments of larger molecules. For instance, endostatin, a potent angiogenesis inhibitor, is a fragment of collagen XVIII (1) , and angiostatin is a fragment of plasminogen (2) . Elucidation of the mechanisms that control the release of angiogenesis inhibitors from their parental molecules will have a profound influence on our understanding of angiogenesis and may also lead to potential therapeutic applications.

The generation of angiostatin has been shown to involve a variety of pathways that appear to be different in different model systems (3, 4, 5, 6, 7, 8, 9) . However, the mechanism by which endostatin is generated from collagen XVIII has not been elucidated. Collagen XVIII consists of an NH₂-terminal noncollagenous domain, a series of collagen-like domains, and a COOH-terminal NC1³ domain (Fig. 1A ; Ref. 1 ). Endostatin, originally isolated from the medium of hemangioendothelioma (EOMA) cells, is generated from collagen XVIII through the cleavage of an Ala-His linkage by an unidentified EPE. The present study was undertaken to identify the enzymes involved in the generation of endostatin. In this study, we report that members of the elastase family of enzymes can function as an EPE by specifically cleaving recombinant NC1 at the Ala-His linkage and releasing endostatin. In addition, we show that at least two steps, a metal-dependent early step and an elastase-dependent final step, were involved in the generation of endostatin from collagen XVIII.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Generation of endostatin by EOMA cells involves an elastase activity. A, Schematic representation of collagen XVIII and endostatin. Open squares, collagen-like domains. Arrow, cleavage site of endostatin. Es, endostatin. B and C, generation of endostatin by EOMA cells. Cells were plated, grown to near confluency, and refed with serum-free medium with (C) or without (B) various protease inhibitors. CM (3 ml) of EOMA (B and C) and other cells (B) were collected 24 h later. Endostatin was enriched by incubation with heparin beads and subsequently detected by Western blotting with an antibody against endostatin. BAE, bovine aortic endothelial cells; BCE, bovine capillary endothelial cells; BSMC, bovine smooth muscle cells; CCL188, human colon CCL 188 carcinoma cell line; MS1, mouse capillary endothelial cells immortalized with SV40 large T; SVR, MS1 transformed with H-ras. D and E, conversion of endostatin from rNC1 by CM of EOMA cells. Purified rNC1 (100 ng) was incubated with CM (30 µl, collected 3 days after cell were plated) for the indicated time (16 h in E) in the absence (D) or presence (E) of protease inhibitors. Cleavage products were detected by Western blotting with an antibody against endostatin.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

Cell Culture.
EOMA cells were cultured as described previously (1) . Cells were maintained in DMEM supplemented with 10% heat-inactivated calf serum and 1% glutamine-penicillin-streptomycin. Cells were passaged by trypsinization (Trypsin/EDTA; Life Technologies, Inc.), followed by dilution (100–200-fold) in DMEM medium containing 10% calf serum.

Expression and Purification of rNC1 Protein.
The cDNA encoding NC1 was amplified from a mouse 1 (XVIII) cDNA clone mc3b (kindly provided by N. Fukai and B. Olsen, Harvard Medical School, Boston, MA) by PCR and was cloned into the EcoRI and XbaI sites of the expression vector pSecTagA (Invitrogen). The vector contains a secretion signal at the NH₂-terminus and myc and His-tags at the COOH-terminus to facilitate purification and detection.

The rNC1 expression vector was introduced into 293T human epithelial kidney cells using LipofectAMINE (Life Technologies, Inc.). Transfected cells were selected with Zeocin (400 µg/ml) in DMEM with 10% calf serum, expanded, and thereafter maintained in medium containing Zeocin. After reaching confluence, NC1-expressing cells were washed with PBS and changed into serum-free medium (Opti-MEM, supplemented with insulin-transferrin-selenium). After a 48 h incubation, the medium was collected and filtered through a 0.45 µm filter. His-tagged NC1 protein was purified using a TALON affinity column (Clontech) according to the manufacturer’s instructions. The purified protein was dialyzed against PBS and stored at -80°C in aliquots of 50 µl.

	Western Blot Analysis.

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
Proteins were separated on a 12.5% SDS-PAGE, electrotransferred onto a PVDF membrane, and probed with a rabbit polyclonal antibody against endostatin, followed by an horseradish peroxidase-conjugated sheep antirabbit antibody (Amersham). Three different rabbit antibodies were used for these experiments. The antibody raised against recombinant mouse endostatin was used routinely, whereas two other antibodies raised either against recombinant human endostatin or a peptide (GLSGTFRAFLSSRLQDLYSIVRRADRGSVC) derived from mouse endostatin were used to verify the results. After washing the PVDF membrane extensively, endostatin-related proteins were visualized using ECL Western blotting detection reagents (Amersham).

	Generation of Endostatin from rNC1.

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
rNC1 (100 ng) was incubated with CM from EOMA cells (30 µl, collected 3 days after cells were plated) at 37°C for the indicated time either in the absence or presence of protease inhibitors. Cleavage products were examined by Western analysis.

	Detection of Endostatin in Conditioned Medium of EOMA Cells.

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
CM (3 ml) was incubated with 30 µl of heparin Sepharose beads (Pharmacia Biotech, Inc.) on a rotating shaker for 1 h at 4°C to concentrate endostatin. After washing three times with 10 mM Tris (pH 7.0), the beads were boiled in 20 µl of SDS sample buffer. Endostatin was then detected by Western blotting.

	Purification of an EPE.

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
An extract of porcine pancreas (Life Technologies, Inc.) was chosen as the source of purification, because it was found to contain an EPE activity and was readily available in larger quantities than EOMA CM. The extract was first dialyzed against 10 mM Tris (pH 7.0) and then applied to a Hitrap Q Sepharose column (5 ml) (Pharmacia Biotech, Inc.) preequilibrated with the same buffer. Proteins were eluted through a stepwise profile with 0.2, 0.3, 0.4, and 2 M NaCl at a flow rate of 1 ml/min using fast protein liquid chromatography (Pharmacia). Each fraction was screened for its ability to cleave rNC1 to endostatin. Fractions with cleavage activity, which eluted at 0.2 M NaCl, were pooled and applied to a Superose 12 gel filtration column (Pharmacia) using fast protein liquid chromatography. Active fractions were then fractionated on a SynChropak RP-4 column using high performance liquid chromatography. The activity eluted at a concentration of 50–53% acetonitrile in 0.1% trifluoroacetic acid.

	Protein Sequencing.

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
After SDS-PAGE, proteins were blotted to a PVDF membrane. Coomassie blue-stained proteins were excised from the membrane and applied directly to a Perkin Elmer/Applied Biosystems Division model 477A protein sequencer, with an on-line model 120A PHP-amino acid analyzer. Protein sequencing was conducted by the HHMI/Harvard Medical School Biopolymers Facilities.

	Results

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
Generation of Endostatin by EOMA Cells May Involve Several Proteolytic Activities.
To investigate the processing of endostatin, CM from EOMA cells, from which endostatin was first identified, was used for study. For controls, CM from several other collagen XVIII-producing cell lines was also examined. The presence of endostatin was determined by Western blot analysis using antibodies specific for endostatin as described in "Materials and Methods." As expected, endostatin was detected in the CM of EOMA cells (Fig. 1B) . It was not, however, present in the medium of a cell line that does not produce collagen XVIII (CCL188), nor was it detected in the media of several cell lines that produce collagen XVIII (BAE, BCE, and BASMC). Endostatin was also found, albeit at a lower level than in the CM of EOMA cells, in the medium of a mouse capillary endothelial cell line SVR (transformed with SV40 large T and H-ras) but was absent in the medium of its parental cell line MS1 (expressing only SV40 large T; Fig. 1B ; Ref. 10 ).

In addition to endostatin, several other endostatin-related peptides were detected in the CM of EOMA cells. One of the peptides migrates at M_r 32,000 and therefore is likely to be NC1. The presence of these intermediate-sized fragments indicates that the generation of endostatin from collagen XVIII by EOMA cells might involve several proteolytic activities or several steps.

Generation of Endostatin by EOMA Cells Is Inhibited by an Elastase Inhibitor and by a Matrix Metalloprotease Inhibitor.
To examine the proteolytic activity involved in the generation of endostatin by EOMA cells, a panel of class-specific protease inhibitors was tested. Generation of endostatin in the CM of EOMA cells was significantly impaired in the presence of elastatinal, a specific inhibitor of elastases, but not by several other classes of protease inhibitors (Fig. 1C and Table 1 ). The elastase inhibitor, however, did not inhibit the generation of the cleavage product that corresponds to NC1 (Fig. 1C) . A metal chelator, 1,10-phenanthroline, on the other hand, blocked the generation of both NC1 and endostatin, implying that a metal-dependent activity was required for the generation of a precursor of endostatin, e.g., NC1. A MMP might be responsible for such a metal-dependent activity, because 1,10-phenanthroline can specifically inhibit MMP activity. These results suggest that the proteolytic activities mediating the generation of endostatin might involve both an elastase activity and a MMP activity.

To determine whether an elastase was involved in the cleavage of rNC1 to endostatin by the CM of EOMA cells, the effect of elastatinal was tested (Fig. 1E and Table 1) . As expected, this inhibitor significantly blocked the cleavage of rNC1. AEBSF, a general serine protease inhibitor, also blocked the cleavage of rNC1, but some other serine protease inhibitors that are more specific to trypsin or chymotrypsin had little effect on the cleavage. These results suggest that an elastase-like serine protease is involved in the cleavage of rNC1 to endostatin.

Elastases Can Directly Cleave rNC1 to Endostatin.
We next asked whether an elastase-like protease directly cleaves NC1 to endostatin or whether such a protease triggers a cascade of proteolytic processes, leading to the release of endostatin. To address this question, a protease that is capable of directly cleaving rNC1 to endostatin at the site of Ala-His (EPE) was isolated as described in "Materials and Methods" and identified to be a member of the elastase family (Fig. 2A) . We chose an extract of porcine pancreas for the purification, because we found that the presence of residual amounts (0.5% v/v) of TC trypsin (an extract of porcine pancreas) greatly enhanced the accumulation of endostatin in the CM of EOMA cells, and that this extract was capable of cleaving rNC1 to endostatin. The purified enzyme was able to cleave rNC1 to a M_r 20,000 peptide, the size of endostatin. NH₂-terminal sequencing of this cleavage product showed that it begins with amino acids HTHQDFQP, identical to that of endostatin (1) .

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. An elastase-like protease can directly release endostatin from rNC1. A, SDS-PAGE gel of a purified EPE. Fractions eluted from an RP-4 column were resolved on an SDS-PAGE gel and stained with Coomassie blue 250. Fraction 23 contains the EPE activity. B, the NH2-terminal sequence of the purified EPE is aligned with several members of the elastase family. Nonconserved amino acids are underlined; amino acids that were determined with low confidence are shown in lower case; X, an undetermined amino acid. C, rNC1 was incubated with the purified EPE in the presence of inhibitors for elastase or other proteases. D, rNC1 was incubated with PPE and HNE in the presence or absence of elastase inhibitor, elastatinal, for 16 h. Cleavage products were analyzed for the presence of endostatin by Western blotting and were also NH2-terminal sequenced.

We also tested the cleavage of rNC1 by representatives of two major classes of elastase, porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE) (Fig. 2D) . Both elastases were capable of directly converting rNC1 to a peptide with a size similar to that of endostatin. The cleavage activities were also blocked by elastatinal. NH₂-terminal sequencing of the cleavage products showed that the main cleavage site is the Ala-His linkage. Cleavage after alanine is consistent with the substrate preference of elastase (13) .

	Discussion

Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 
In this report, we describe for the first time a mechanism for the processing of the angiogenesis inhibitor endostatin from collagen XVIII. We show that the generation of endostatin by EOMA cells can be prevented by either a metal chelator or an elastase inhibitor and suggest that the processing of collagen XVIII to endostatin involves at least two steps, a metal-dependent early step and an elastase activity-dependent final step (Fig. 3) . In addition, we identify an elastase-like protease as an EPE, which can specifically cleave rNC1 at the Ala-His site to release a peptide with an identical size and NH₂-terminal sequence (HTH) to that of the endostatin protein isolated originally (1) .

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A model for the generation of endostatin from collagen XVIII. At least two steps are involved in the generation of endostatin (Es) from collagen XVIII, a metal-dependent early step and an elastase activity-dependent final step. 1,10-Phenanthroline, a metal chelator, inhibits the generation of endostatin by preventing the generation of a precursor of endostatin. Elastases (hypocalcemic factor, PPE, and HNE) are capable of cleaving a precursor at the Ala-His site and releasing endostatin. Elastatinal, a specific elastase inhibitor, blocks the generation of endostatin at this final step.

(a) Several other collagen XVIII-producing cells could not generate endostatin, even when large amounts of the purified EPE were supplemented into the cultures (not shown). One possibility is that native collagen XVIII is not a suitable substrate for elastase and that the generation of endostatin may involve more than one step. For instance, native collagen XVIII may need to be initially modified or processed, allowing it to be readily cleaved by an elastase in a subsequent step. EOMA cells, as well as SVR cells, appear to be capable of this initial modification or processing.

(b) Elastase inhibitors blocked the generation of endostatin in the culture of EOMA cells with a concomitant accumulation of NC1 (Fig. 1C) , indicating that NC1 is likely an intermediate product of collagen XVIII processing.

(c) A metal chelator, although not inhibiting the cleavage of rNC1 to endostatin (Fig. 1E) , impaired the production of both NC1 and endostatin by EOMA cells (Fig. 1C) , suggesting that a metal-dependent activity (e.g., MMP activity) is required for generating a precursor of endostatin (e.g., NC1). Finally, another cell line SVR that also produces endostatin was shown to have an elevated level of MMP ⁽¹⁰⁾ , consistent with a potential role of MMP in the generation of an endostatin precursor.

Elastases are a group of proteases that can cleave elastin, an important connective tissue protein. They have also been demonstrated to process a number of molecules from their precursors, including tumor necrosis factor (14) and interleukin-1 (15) . Here we show that an elastase is also involved in the generation of a potent angiogenesis inhibitor, endostatin. Our observation that several members of the elastase family can convert NC1 to endostatin implies that there may not be a unique elastase for the processing of endostatin and that different elastases may be involved in its generation in different tissues.

Endostatin-like molecules ranging from M_r 22,000–38,000 with distinct NH₂-terminal sequences have been found in the circulation and in many tissues (16 , 17) , but the physiological roles of these endostatin-related proteins have not yet been established. Whether functional endostatin can be produced in vivo and whether elastases are involved in its generation remain to be determined. It is very plausible, however, that endostatin can be generated by elastases in vivo, given that elastases are produced in a highly regulated manner by a variety of vascular-related cells, including neuotrophil leukocytes (13) , platelets (18) , and smooth muscle cells (19) .

It is also possible that collagen XVIII is processed into endostatin in vivo by a mechanism different from the in vitro processing described here. In a series of preliminary experiments, we searched for enzymes other than elastase that could cleave rNC1 to endostatin and found that although many proteases including thrombin, plasmin, chymotrypsin, and corneal extracts containing MMPs did not generate endostatin, cathepsin B and cathepsin L could convert rNC1 into a fragment with a size similar to that of endostatin. We did not further characterize the cleavage of rNC1 by cathepsins. However, at the time of this writing, Bjorn Olsen informed us about the finding of he and his colleagues that cathepsin L generates endostatin in EOMA cells.⁴ Pure trypsin can also cleave rNC1, but the size of its cleavage product is larger than endostatin. It remains to be determined whether cathepsins B and L cleave rNC1 at the Ala-His site. Because it was shown that a number of different proteases were involved in the generation of angiostatin in different systems, the presence of many forms of endostatin-like molecules in various tissues suggests that multiple pathways may also be involved in the generation of endostatin-like molecules. Identification of the enzymes involved in each case and the factors that regulate their activities will be important for a better understanding of angiogenesis in vivo.

The regulation of angiogenesis is thought to be controlled by a balance of stimulators and inhibitors (20) . Many of the angiogenesis inhibitors identified to date are fragments of larger molecules. How these inhibitors are generated remains largely unknown. Our study of the processing of endostatin from collagen XVIII in cell culture has shown that the generation of endostatin from its parental molecule involves at least two steps and that the final step can be mediated by an elastase activity. Elucidation of the regulatory machinery that controls the release of angiogenesis inhibitors from their precursors will provide a better understanding of the process of angiogenesis and may also lead to potential therapeutic applications.

	ACKNOWLEDGMENTS

 
We thank Dr. Michael O’Reilly for EOMA cells; Drs. Thomas Boehm and Kashi Javaherian for generously providing antibodies against endostatin; Dr. Naomi Fukai and Björn Olsen for mc3b cDNA clones; Dr. Yuen Shing for helpful advice on protein purification; Catherine Butterfield for help with tissue culture; and Drs. Deborah Freedman, David Resnick, Thomas Boehm, and Steven Pirie-Shepherd for very thoughtful discussions and critical reading of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by Grant R01 CA64481 from the NIH (to J. F.), a grant to Children’s Hospital from EntreMed, Inc. (Rockville, MD), Grant RPG-97-013 from the American Cancer Society (to M. A. M.), and by Grants KO8 AR 02096-01 and RO3 AR 44947-03 from NIAMS (to J. L. A.). W. W. is supported by the Susan G. Komen Breast Cancer Foundation.

² To whom requests for reprints should be addressed, at Children’s Hospital, Hunnewell 103, 300 Longwood Avenue, Boston, MA 02115.

³ The abbreviations used are: NC1, noncollageneous domain 1 of collagen XVIII; rNC1, recombinant NC1; CM, conditioned medium; EPE, endostatin-processing enzyme; HNE, human neutrophil elastase; PPE, porcine pancreatic elastase; MMP, matrix metalloprotease; PVDF, polyvinylidene difluoride; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; TLCK, N--p-tosyl-L-lysine chloromethyl ketone; pAPMSF, (4-amidinophenyl)-methanesulfonyl fluoride.

⁴ B. Olsen, personal communication.

Received 10/ 4/99. Accepted 10/29/99.

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Top ABSTRACT Introduction Materials and Methods Western Blot Analysis. Generation of Endostatin from... Detection of Endostatin in... Purification of an EPE. Protein Sequencing. Results Discussion REFERENCES

 

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