Pro-EMAP II is not primarily cleaved by caspase-3 and -7

F. R. Zhang1 and M. A. Schwarz1,2

1 Departments of Pediatrics, Cardiothoracic Surgical Research, and Surgery, Childrens Hospital Research Institute, Los Angeles 90027; Keck School of Medicine, University of Southern California, Los Angeles, California 90089; and 2 Departments of Surgery and Pediatrics, University of Medicine and Dentistry of New Jersey- Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Endothelial monocyte-activating polypeptide (EMAP) II is a unique cytokine, also known as p43, the active mature form of which exhibits antiangiogenic properties in vivo and in vitro. The proteolytic enzymes associated with the cleavage and release of the active mature form, however, remain unclear. Here we show that, in contrast to prior observations, purified pro-EMAP II is not cleaved by either caspase-3 or -7 in vivo or in vitro. Thus other proteolytic processes, which allow it to induce apoptosis via caspase-3 activation in migrating and dividing endothelium, may be involved in the release of the active mature EMAP II.

neovascularization; endothelial monocyte-activating polypeptide II; antiangiogenesis; p43; caspase-7; endothelial monocyte activating polypeptide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIAL MONOCYTE-ACTIVATING POLYPEPTIDE (EMAP) II [now identified as p43 (11)] is a unique cytokine-like protein that was first described by its mild proinflammatory properties, its ability to induce leukocyte and mononuclear phagocyte migration, and the induction of tissue factor in endothelial cells (7, 8). Subsequently, three different hypotheses have emerged regarding the role of the mature approx 22-kDa form of EMAP II. One theory assumes that EMAP II is a product of apoptosis that induces the release of mature EMAP IIs via caspase-7, where it then functions as an inflammatory mediator (1, 9). In contrast, there is a growing body of literature that supports the role of the mature form of EMAP II as an antiangiogenic protein that is capable of inhibiting the growth of primary tumors and distal metastasis through its ability to induce apoptosis in the actively dividing and migrating endothelium by caspase-3 activation (16). The initial observations that the mature form of EMAP II had antiangiogenic properties were confirmed in independent laboratories, which found that not only does EMAP II inhibit tumor growth (2) but it also has the capability to increase the sensitivity of melanomas to tissue necrosis factor (10, 20) while diminishing tumor recurrence after phototherapy (4). Lastly, the mature portion of EMAP II has been shown to have significant homology with tRNA synthetase complexes (19).

Further evidence supporting a functional role for EMAP II comes from its temporal-spatial distribution during vertebrate development (21). During fetal development, EMAP II is strongly expressed at a region critical for vessel differentiation and formation, the epithelial-mesenchymal junction. Conversely, in the adult, EMAP II is predominately localized to perivascular regions, implying that it is a static modulator of vessel formation and angiogenesis. Furthermore, detailed studies of lung development indicate that EMAP II's expression is at a region of active fetal angiogenesis and that as EMAP II expression declines in this region, there is a sharp increase in the angiogenic growth factor vascular endothelial growth factor (6) and its receptor flk-1 (5), giving rise to the vasculature. The implication that EMAP II plays an important regulatory role in lung neovascularization was further supported by studies in an allograft model of fetal lung neovascularization and morphogenesis, where delivery of the mature form of EMAP II not only inhibited fetal lung neovascularization but also arrested distal alveolar epithelial cell differentiation. Conversely, delivery of a blocking EMAP II antibody generated to the COOH-terminal region of EMAP II induced an increase in vessel formation within the lung allograft (17).

This spectrum of roles attributed to mature EMAP II, as an active inducer of apoptosis (2, 16) in endothelial cells versus its being a product of apoptosis (1), was confusing. This lead us to explore whether mature EMAP II is a byproduct of apoptosis or an active inducer of cellular apoptosis. Therefore, we set forth to determine whether purified pro-EMAP II (p43) was cleaved in vitro by caspase-3 and -7 or in vivo using standard, commercially available cells that had apoptosis chemically induced. After purification of pro-EMAP II using a PET28a his tag system, we were unable to detect the mature form of EMAP II after incubation with either activated caspase-3 or -7. Furthermore, use of a standard commercial in vivo preparation of Jurkat and HL-60 cells of either stable noninduced cell lysate or cell lysate from etoposide (a chemotherapeutic that activates caspase-3, -7, and -9) induced apoptosis, revealed that there was no cleavage of the mature form of EMAP II. Lastly, in a fetal lung coculture system where pro-EMAP II is highly expressed at the region of mesenchymal-epithelial contact, there was minimal cellular apoptosis. In contrast, in the presence of exogenous mature EMAP II, there was a marked increase in cellular apoptosis in the region of mesenchymal-epithelial contact. These studies led us to believe that mature EMAP II is an active inducer of apoptosis and that the mature EMAP II is not a byproduct of apoptosis primarily cleaved by caspase-3 or -7 from pro-EMAP II.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Synthesis of recombinant pro-EMAP II from Escherichia coli. The cDNA of human pro-EMAP II was cloned from RT-PCR products of U937 cells' total RNA based on TA vector (Invitrogen). Primers were 5'-primer 5'-CG GAA TTC ATG GCA AAC AAT GAT GCT GTT CTG AAG-3' and 3'-primer 5'-GTA TGT GGC CAC ACA CTC AGC ATT-3'. Confirmation of the clone was provided by sequence analysis, after which the cDNA was inserted into PET28a 6X his tag-containing plasmid. E. coli (DE3) underwent transformation with the pro-EMAP II/PET28a plasmid and was induced with 1-4 mM of isopropylthiogalactoside (IPTG). After 3-4 h of induction, the cells were pelleted and lysed, and the EMAP II protein was purified through the use of a nickel column under native conditions as per protocol (Qiagen) with all procedures performed at 4°C. Briefly, pelleted cells were lysed with 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 10 mM imidazole in the presence of 1 mg/ml of lysozyme. After sonication, cellular debris was removed by centrifugation before being loaded on the Ni-NTA slurry. At this point pro-EMAP II was noted to be partially soluble. After washing the column, we eluted pro-EMAP II off with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole. Importantly, only the soluble purified pro-EMAP II that came off the column was used for the experiments. Pro-EMAP II was then dialyzed at 4°C against PBS containing 5% glycerol three times before being aliquoted and frozen at -80°C.

In vitro cleavage assays. Two micrograms of purified pro-EMAP II per reaction were placed at 30°C either in the presence of vehicle (PBS) or 60 units of commercially prepared human recombinant caspase-3 (14,000 U/µg) or caspase-7 (15,600 U/µg; BIOMOL, Plymouth Meeting, PA) according to the manufacturer's protocol. The recombinant caspase enzymes used were cleaved and activated from the proenzyme, with the caspase-3 isoform containing the 17- plus 12-kDa heterodimer and the caspase-7 isoform containing the 20- plus 12-kDa heterodimer. A sample was obtained at the beginning of incubation (0 min) and at 5, 15, 30, 45, 60, and 120 min and overnight. Equal amounts of the incubated reaction were loaded on a 12% Tris-glycine gel. After electrophoresis, the gel was stained with Bio-safe Coomassie dye (Bio-Rad, Hercules, CA) for 1 h before destaining at room temperature in distilled H2O for 2 h. Pictures were taken, and the gel was allowed to air-dry in a gel frame. In addition, equal amounts of protein were electrophoresed on a 12% SDS-PAGE gel, transferred to Immobilon-P, blocked overnight in a casein-based blocking solution (Boehringer-Mannheim, Indianapolis, IN), and probed with a rabbit anti-EMAP II antibody (generated from the COOH terminus and recognizing the pro- and mature forms of EMAP II). Specific binding was detected using a chemiluminescence substrate (Pierce, Rockford, IL) and XAR-5 film (Eastman Kodak, Rochester, NY). Activity of caspase-3 and -7 was assessed by analyzing cleavage of poly(ADP-ribose)polymerase (PARP). PARP, purified from thymus by DNA-cellulose affinity chromatography (BIOMOL) in 50 mM HEPES, 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM dithiothreitol, 1 mM EDTA, and 10% glycerol, was incubated with control, caspase-3, or caspase-7. Samples were analyzed at the beginning of incubation (0 min), 30 min, 1 h, and overnight. Equal amounts of the incubated reaction were loaded on a 6% Tris-glycine gel. After electrophoresis, the protein was transferred to Immobilon-P, blocked overnight, and probed with an anti-PARP antibody (BIOMOL). Specific binding was detected using a chemiluminescence substrate and XAR-5 film.

In vivo cleavage assays. Jurkat cell (human Jurkat macrophage cell) and HL-60 cell line (human HL-60 leukemia cells) extracts were purchased from BIOMOL in either an noninduced (catalog nos. SW-115 and SW-101, respectively) or apoptosis-induced state (catalog nos. SW-116 and SW-102, respectively). Apoptosis was induced in both cell lines with the chemotherapeutic agent etoposide. Induction of apoptosis pathways was confirmed by Western blot, which indicated the activation of caspase-3, caspase-7, caspase-9, and PARP (3, 22). Equal amounts of noninduced and induced cell extracts were loaded side by side and electrophoresed on a 12% SDS-PAGE gel, transferred to Immobilon-P, blocked overnight in a casein-based blocking solution (Boehringer-Mannheim), and probed with a rabbit anti-EMAP II antibody generated from the COOH-terminal end of EMAP II. Specific binding was detected using a chemiluminescence substrate (Pierce) and XAR-5 film (Eastman Kodak). Quantitative analysis was accomplished using a Molecular Dynamics personal densitometer. Noninduced and induced cell extracts were analyzed by Western blot for etoposide activation of caspase-3 and -7. Equal amounts of protein were loaded, electrophoresed on a 12% SDS-PAGE gel, transferred to Immobilon-P, blocked overnight, and probed for caspase-3 (PharMingen, San Diego, CA) and caspase-7 (BIOMOL).

Coculture. Organotypic murine lung cultures were performed following the protocol of Schuger et al. (13-15). Timed-gestation, 15-day-old embryos underwent dissection from Swiss-Webster mice (Simonsen, Morgan Hill, CA). Lungs were isolated and underwent digestion in PBS containing 0.3% trypsin and 0.1% EDTA for 10 min at 37°C before being filtered through a 100-µm-pore mesh. The mixed epithelial-mesenchymal cells were then resuspended in MEM (GIBCO-BRL) with nonessential amino acids and plated at a concentration of 2-2.5 × 106 cells/ml in eight-well chamber slides. Experiments were performed in the presence of vehicle or recombinant mature EMAP II (mature 3.2 µg/ml) (17).

Immunohistochemistry analysis. As previously described (21), cells in the eight-well dish were fixed in 4% paraformaldehyde and dehydrated. We utilized a peptide-generated polyclonal antibody of EMAP II (1 µg/ml). With the use of a histostain kit from Zymed (San Francisco, CA), after being blocked, cells were exposed to the primary antibody overnight at 4°C. Cells were then incubated with secondary biotinylated antibody as per the manufacturer's protocol. A brief incubation with the streptavidin-horseradish peroxidase conjugate system (Zymed) was followed by development using the chromogen substrate aminoethylcarbazole.

Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling analysis of cocultures. Cocultured cells were exposed to vehicle or EMAP II (3.2 µg/ml). Cells were evaluated on day 3 for apoptosis. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and exposed to the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction (containing terminal deoxynucleotidyltransferase and a nucleotide mixture in a reaction buffer), after which the cells were exposed to a fluorescein (FITC) antibody, counterstained with propidium iodine (0.05 µg/ml), mounted with PBS-glycerol, and observed under a fluorescent microscope (Olympus).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purification of recombinant pro-EMAP II. To determine whether cleavage of mature EMAP II (approx 22 kDa) was through caspase-3 or -7, it was important to develop an easy and reproducible production system for soluble recombinant pro-EMAP II. We used a PET28a 6X his tag system to quickly and efficiently isolate pro-EMAP II under native conditions. Recombinant pro-EMAP II was expressed in E. coli (Fig. 1), induced with 1-4 mM IPTG, and the E. coli was pelleted after 3-4 h of induction (Fig. 1). The purified, recombinant proform of EMAP II (Fig. 1) had a MR of approx 43 kDa on both reduced and nonreduced SDS-PAGE gels.


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Fig. 1.   Purification of recombinant proendothelial monocyte-activating polypeptide (pro-EMAP) II. Pro-EMAP II in a PET28a 6X his tag system was isolated after isopropylthiogalactoside (IPTG) induction from Escherichia coli. Lane 1 on this Coomassie blue gel is the initial expression of pro-EMAP II. Lane 2 represents pro-EMAP II induction with IPTG, and lane 3 represents purified pro-EMAP II. N, noninduced; I, induced; P, purified.

Pro-EMAP II is not cleaved in vitro or in vivo by activated caspase-3 or -7. To clarify whether mature EMAP II was a cleavage product of activated caspase-3 or -7, we took purified recombinant pro-EMAP II and incubated it with vehicle, activated caspase-3 (Fig. 2, A and C), or caspase-7 (Fig. 2, B and C) before evaluating cleavage by Coomassie blue stain on an SDS-PAGE gel. Despite incubating for extended periods of time (starting at 5 min up to overnight), we were unable to detect cleavage of the mature form of EMAP II (Fig. 2, A-C) from pro-EMAP II at any of these time points by either Coomassie blue staining (Fig. 2, A-C) or enhanced chemiluminescence (Fig. 2F). Simultaneously, activity of the caspase-7 and -3 enzyme was assessed by analysis of their ability to cleave PARP. Caspase-7 (Fig. 2D) and caspase-3 (Fig. 2E) were found to cleave PARP as early as 1 h after incubation. These results were confirmed using a standardized, commercially produced, in vivo induction of apoptosis using the chemotherapeutic agent etoposide. Etoposide has been shown to induce cellular apoptosis through the activation of caspase-3, -7, and -9 (3, 22, and manufacturer). Consistent with our in vitro results, despite known activation of caspase-3 (Fig. 3C) and caspase-7 (Fig. 3B) in vivo, the mature form of EMAP II was not cleaved (Fig. 3A).


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Fig. 2.   Pro-EMAP II is not cleaved by activated caspase-3 or -7. Purified pro-EMAP II was incubated with or without caspase-3 or -7 at 30°C, and samples were obtained at 0, 5, 15, 30, 45, 60, 90, and 120 min or after overnight incubation and run on an SDS-PAGE gel followed by Coomassie blue stain (C) or enhanced chemiluminescence detection after Western analysis (F). Mature EMAP II was not generated by cleavage of pro-EMAP II by either caspase-3 (A, C) or caspase-7 (B, C) at either the early time points (A, B) or after overnight incubation (C, F). Concomitant assessment of caspase-7 (D) and caspase-3 (E) activity was determined by their ability to cleave poly(ADP-ribose)polymerase (arrows in D and E) as early as 1 h after incubation. ON, overnight.



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Fig. 3.   In vivo activation of caspase-3 and -7 by etoposide does not generate cleavage of mature EMAP II. Commercially available Jurkat and HL-60 whole cell extract from cells that were either not induced or chemically induced (etoposide) to undergo apoptosis were purchased, and whole cell lysate was run on an SDS-PAGE gel. Western analysis for EMAP II indicated that in both the noninduced and induced cell lines only pro-EMAP II was present without the mature form, despite the presence of documented activated caspase-7 (B, arrow) and caspase-3 (C, arrow) in the induced cells, thus indicating that in vivo in the presence of activated caspase-3 and -7, mature EMAP II is not cleaved from pro-EMAP II.

Cellular presence of pro-EMAP II does not induce apoptosis, whereas exogenous, mature EMAP II induces apoptosis in culture. Lastly, to confirm that mature EMAP II is an inducer of apoptosis and not a product of apoptosis, we looked at the distribution of pro-EMAP II in fetal lung coculture, where it is highly expressed, and compared it to cellular apoptosis. Immunohistochemistry (Fig. 4A) indicated that pro-EMAP II (Western analysis, Fig. 4B) is highly expressed at the junction (Fig. 4A, arrows) of fetal lung mesenchymal and epithelial (Fig. 4A, M and EC, respectively) cells in coculture. Furthermore, fluorescent TUNEL analysis (counterstained with rhodamine to outline the cells) determined that although there was minimal to no apoptosis at baseline in this region (Fig. 4C), the addition of exogenous mature EMAP II induced a marked increase in cellular apoptosis (indicated by cells positive for FITC) that localized to the mesenchymal-epithelial cell contact region (Fig. 4D, arrows).


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Fig. 4.   In contrast to exogenous mature EMAP II inducing apoptosis in vivo, pro-EMAP II expression is not associated with cellular apoptosis. After a standardized protocol, fetal murine lungs were dissociated and placed in culture, where they formed epithelial cysts. Immunohistochemistry for determination of EMAP II spatial expression in the lung coculture showed that EMAP II was highly expressed at the region of epithelial-mesenchymal contact (A). Western analysis indicated that it is pro-EMAP II protein that was being generated in the coculture (B). With the use of a TUNEL assay to analyze for apoptosis (FITC), propidium (red)-counterstained cells in coculture indicated that, although there was a minimal basal amount of apoptosis in cocultures at the region where pro-EMAP II protein is highly expressed (C, EC), in the presence of exogenously added mature EMAP II there is a marked increase in cellular apoptosis that is localized to the region (D, EC as indicated by the arrows) where pro-EMAP II is normally expressed. These results again substantiate the hypothesis that mature EMAP II induces apoptosis, whereas pro-EMAP II is not associated with the induction of apoptosis in these regions. EC, epithelial cells forming an epithelial cyst; M, mesenchymal cells. Magnification: A, ×400; B and C, ×200.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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We have shown that mature-EMAP II is not primarily cleaved from pro-EMAP II (p43) by caspase-3 or -7 in vitro or in vivo. Furthermore, although pro-EMAP II is highly expressed at the region of contact coculture of mesenchymal-epithelial cells, there is a minimal amount of baseline apoptosis present in this region. In contrast, addition of exogenous mature EMAP II induced a marked increase in apoptosis at the region where pro-EMAP II is expressed. Therefore, our findings suggest that mature EMAP II is an active protein that is capable of inducing apoptosis and not an apoptotic byproduct generated by cleavage of activated caspase-3 or -7.

Although caspase-3 and -7 have been implicated in a significant amount of proteolytic processes associated with cellular apoptosis, we were confused by conflicting studies showing that mature EMAP II is either 1) an active inducer of cellular apoptosis via caspase-3 induction (16) and of upregulation of the Fas-associated death domain with concomitant downregulation of Bcl-2 (2) in endothelial cells, or, according to contradictory findings, 2) a byproduct, in its mature state, of pro-EMAP II cleavage by caspase-7 during activation of the apoptosis cascade (1). Thus we set forth to determine whether, in a pure system, mature EMAP II is an apoptosis byproduct. Our in vitro studies using purified pro-EMAP II and in vivo studies using standardized etoposide-induced apoptosis revealed that pro-EMAP II is not primarily cleaved by caspase-3 or -7. These findings are in direct contrast to those findings of Behrensdorf et al. (1), who were able to directly cleave a reticulocyte lysate-generated pro-EMAP II with caspase-3 and -7. There are several possible explanations for these findings: 1) instead of using purified EMAP II protein, Behrensdorf et al. (1) used the lysate from a reticulocyte system that contained other cellular products in addition to pro-EMAP II, which may have altered their findings, or 2) the murine caspase-3 and -7 generated in E. coli homogenates used by Behrensdorf et al. (1) did not contain the two heterodimers (12 plus 17 kDa for caspase-3 and 20 plus 12 kDa for caspase-7) that form the active tetramer that has the two independently functioning catalytic sites necessary for the residues to bind the substrate and catalyze it (18).

The presence of the aspartic acid and the ASTD motif at the NH2 terminus of mature EMAP II suggests that this is a likely cleavage site for interleukin-converting enzyme; however, this does not ensure that this region is a caspase cleavage site. Although the preferred tetrapeptide recognition motif differs significantly among the caspases, it has been shown that one must have recognition of at least four amino acids NH2 terminal to the cleavage site as a necessary requirement for efficient catalysis. Furthermore, not all proteins that contain the optimal tetrapeptide sequence are cleaved, implying that other tertiary structural elements (the necessary interaction with other proteins) may influence substrate recognition (18). Further supporting our position is the finding by Behrensdorf et al. (1) that in Meth A fibrosarcoma cells (the cell line from which EMAP II was first identified) "the appearance of active caspase-7 in the lysates of apoptotic Meth A fibrosarcoma cells precedes the appearance of mature EMAP II by 4-6 hours" (1), yet they were able to generate caspase-7 cleavage of EMAP II in 90 min. These findings are in direct agreement with our in vivo findings that, in the presence of activated caspase-3 and -7, mature EMAP II is not present, suggesting that mature EMAP II is not generated by this proteolytic pathway.

In addition, we show that the presence of the pro-EMAP II protein generated de novo in cell culture does not generate local apoptosis. In contrast, the addition of mature EMAP II not only generates apoptosis but does so in the exact location where pro-EMAP II is so highly expressed. These findings again suggest that mature EMAP II has an active and independent function from pro-EMAP II and is not an apoptotic byproduct. Although our observations have clarified how EMAP II is not cleaved, they have not elaborated on how pro-EMAP II is cleaved. We believe that, as suggested by Quevillon et al. (11), the presence of an upstream KEKE motif is a likely candidate for the cleavage site of pro-EMAP II (p43). The KEKE motif has been shown to be a facilitator of protein-protein interactions. Based on the location of the KEKE motif, just upstream of the cleavage site of mature EMAP II, we hypothesize that it facilitates the interaction between pro-EMAP II and an as-yet unidentified protein such as the multicatalytic protease (12). An interaction with a protein at this site could facilitate the active cleavage of mature EMAP II.

In summary, we have taken the first steps to expand our understanding of the function of EMAP II by clarifying the in vivo cleavage of pro-EMAP II to its mature form. We have shown that pro-EMAP II (p43) is not cleaved by caspase-3 or -7 in vitro or in vivo. Furthermore, while the basal expression of pro-EMAP II in cell culture does not generate apoptosis, addition of mature EMAP II does induce apoptosis in the exact region of pro-EMAP II production. Although these studies do not determine the systems that metabolize pro-EMAP II, importantly they have begun to clarify the notion that cleavage of pro-EMAP II leading to the generation of mature EMAP II is less likely to be a byproduct of apoptosis. The identification of the protease involved in the cleavage of pro-EMAP II (p43) is the focus of our ongoing research.


    ACKNOWLEDGEMENTS

We give special thanks to Susan Smith for excellent technical skills.


    FOOTNOTES

This research was supported in part by American Lung Association Grants RG-084-N and CI-001N; the Webb-Berger Foundation; and National Heart, Lung, and Blood Institute Grants HL-60061 and HL-03981.

Present address of M. A. Schwarz: UMDNJ-Robert Wood Johnson Medical School, 125 Paterson St., CAB 7036, New Brunswick, NJ 08903.

Address for reprint requests and other correspondence: M. A. Schwarz, UMDNJ-Robert Wood Johnson Medical School, 125 Paterson St., CAB 7036, New Brunswick, NJ 08903 (E-mail: m.schwarz{at}umdnj.edu).

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

First published January 18, 2002;10.1152/ajplung.00141.2001

Received 24 April 2001; accepted in final form 8 January 2002.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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