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CD109 is a component of exosome secreted from cultured cells
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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CD109 is a component of exosome secreted from cultured cells Hiroki Sakakura a, b, Shinji Mii a, Sumitaka Hagiwara b, c, Takuya Kato a, Noriyuki Yamamoto b, Hideharu Hibi b, Masahide Takahashi a, **, Yoshiki Murakumo a, d, * a
Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan c Department of Head and Neck Surgery, Aichi Cancer Center Hospital, Nagoya, Japan d Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2015 Accepted 15 December 2015 Available online xxx
Exosomes are 50e100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-b signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome. © 2015 Elsevier Inc. All rights reserved.
Keywords: CD109 Exosome Mass spectrometry
1. Introduction Exosomes are cup-shaped, lipid-bilayer, 50e100-nm-diameter membrane vesicles, and are released from various types of cells such as epithelial cells, hematopoietic cells, mesenchymal stem cells and some tumor cells [1,2]. Exosomes are released from the intracellular space to the extracellular environment by the fusion of multivesicular bodies with the plasma membrane. During the 1980s, the function of exosomes was thought to be the removal of dispensable proteins and cell debris [3,4]. However, recent studies indicated that exosomes contain proteins, mRNAs and miRNAs,
* Corresponding author. Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. ** Corresponding author. Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail addresses: [email protected] (M. Takahashi), murakumo@ med.kitasato-u.ac.jp (Y. Murakumo).
which can be transported to surrounding cells, suggesting that exosomes may contribute to cellecell communication [5,6]. Exosomes have been identified in most body fluids including blood, saliva, urine and breast milk, and in pathological conditions including malignancies, exosomes released from lesions were detected in these body fluids [1,5,7e10]. Therefore, exosomes in body fluids might be useful biomarkers for pathological conditions [11,12]. CD109, a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, is a member of the a2-macroglobulin/C3, C4, C5 family [13,14]. CD109 was identified as a cell-surface antigen expressed on some types of normal hematopoietic cells and hematopoietic tumor cells [15e18]. CD109 has frequently been detected by immunohistochemical studies with anti-CD109 antibody in several tumor tissues, including squamous cell carcinomas (SCCs) of the oral cavity, esophagus, lung and uterus, basal-like breast carcinoma, malignant melanoma of the skin, soft tissue sarcoma, and urothelial carcinoma of the bladder [19e26]. These
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Please cite this article in press as: H. Sakakura, et al., CD109 is a component of exosome secreted from cultured cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.12.063
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H. Sakakura et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
expression studies suggested that CD109 might be a cancerassociated protein, although the significance of CD109 expression in cancer cells is unclear. However, CD109 is a component of the transforming growth factor (TGF)-b1 receptor system and negatively regulates TGF-b1 signaling in human keratinocytes [27]. CD109 is also involved in epidermal growth factor (EGF) signaling in glioblastoma cells or STAT3 activation in keratinocytes [28e30]. CD109 is cleaved by furinase into two forms, a 180-kDa secreted form and a 25-kDa membrane-attached form. Part of the 180-kDa secreted form is released from the cell surface into culture medium and the rest binds to the 25-kDa fragment on the cell surface in vitro (Fig. 1A) [31]. The processing of CD109 into 180-kDa and 25kDa proteins is necessary for regulating TGF-b1 signaling. Thus, secreted CD109 might play an important role in cell biology, although the mechanism is obscure. In this study, we revealed that CD109 is an exosomal protein and suggest that the signal modulation effects of CD109 might be associated with exosomes in culture medium.
A
Secreted CD109-180kDa
FLAG
CD109-180kDa fragment
CD109/C-9
CD109-25kDa fragment GPI
Furinase cleavage site CD109/11H3 Plasma membrane
2. Materials and methods 2.1. Antibodies and chemicals Anti-CD109/C-9 mouse monoclonal antibody (mAb), which detects the 180-kDa N-terminal fragment of CD109 (CD109180kDa), anti-Alix, anti-HSP70 and anti-Syntenin mAbs and antiCD81 rabbit polyclonal antibody (pAb) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-CD109/11H3 mAb, which detects the 25-kDa C-terminal fragment of CD109 (CD109-25kDa), was provided by Immuno-Biological Laboratories (Fujioka, Gunma, Japan). Anti-FLAG M2 and anti-b-actin mAbs, anti-FLAG M2 Affinity Gel, mouse IgG-Agarose, 3 FLAG peptide, anti-mouse IgG 10-nm Gold antibody and anti-rabbit IgG 5-nm Gold antibody were purchased from Sigma (St Louis, MO, USA). 2.2. Generation of stable transfectants The HEK293 human embryonic kidney cell line was maintained in DMEM with 8% exosome-depleted fetal bovine serum (Exo-FBS) purchased from System Biosciences (Mountain View, CA, USA) at 37 C in 5% CO2. cDNA fragments for FLAG-tagged human CD109 (FLAG-CD109) and FLAG-tagged truncated human CD109 (FLAGsCD109), which does not have a C-terminal (aa 1274-1445) region, were cloned into pcDNA3.1(þ) and used in transfection experiments [21,31]. The stable transfectants were obtained after Geneticin (Invitrogen, Carlsbad, CA, USA) selection (293/FLAGCD109 and 293/FLAG-sCD109, respectively). Fig. 1A and B shows schematic illustrations of FLAG-CD109 and FLAG-sCD109 proteins. Control transfectants were obtained by transfection of empty vector (293/VC).
Lipid raŌ
B
Furinase GPI-anchor cleavage site cleavage site 1270~1273 1420~
Signal sequence ~21
FLAG-CD109 CD109/11H3
FLAG-sCD109 FLAG
C
HEK293 transfectant media
CD109/C-9
Fifty milliliters of filtered media from 293/FLAG-CD109 and 293/ FLAG-sCD109 were loaded into polystyrene columns with 200 ml of FLAG M2 affinity gel. The gel was washed with TBS (50 mM TrisHCl, pH 7.4, 150 mM NaCl), and the bound proteins were eluted by 100 ml 3 FLAG peptide. The IP fractions were used for silver stain, mass spectrometry and western blotting. As a negative control, immunoprecipitation was performed with mouse IgG agarose.
(kDa)
FLAG 150 250
CD109/11H3
150 25
Conditioned medium from HEK293 was centrifuged at 300 g at 4 C for 10 min and then at 15,000 g at 4 C for 20 min. The supernatants were filtered by 0.22-mm nylon filters to remove cells and cell debris. The filtered media were stored at 80 C until analysis. 2.4. Immunoprecipitation (IP)
250
CD109/C-9
2.3. Conditioned medium collection
:180 kDa : 25 kDa
Fig. 1. Detection of secreted CD109 in conditioned medium. (A) Schematic illustration of FLAG-CD109 on the cell surface and secreted CD109-180kDa. Epitope positions for antibodies used in this study (anti-FLAG, -CD109/C-9, and -CD109/11H3 antibodies) are indicated (arrowheads). The furinase cleavage site is also indicated. (B) Protein structures of FLAG-CD109 and FLAG-sCD109 proteins used in this study. FLAG-sCD109 does not have a C-terminal region after the furinase cleavage site. N-terminal signal sequence, furinase cleavage site and C-terminal GPI-anchor cleavage site are indicated. FLAG sequence was inserted after the signal sequence. Epitope positions for anti-FLAG, -CD109/C-9, and -CD109/11H3 antibodies are indicated (arrowheads). (C) Expression of CD109 in 293/FLAG-CD109 and 293/FLAG-sCD109 conditioned medium. Western blotting was performed against conditioned media of 293/FLAG-CD109, 293/FLAGsCD109, and 293/VC by using anti-FLAG, -CD109/C-9, and -CD109/11H3 mAbs. The equal volume of sample was applied in each lane. The dotted and black arrowheads indicate 25-kDa and 180-kDa bands, respectively.
2.5. Mass spectrometry (LC/MS/MS) The samples for LC/MS/MS were prepared as follows [32]. The IP fractions were reduced by incubating with 5 mM dithiothreitol for 30 min and alkylated with 10 mM iodoacetamide for 60 min in the dark. The proteins were demineralized, concentrated by methanol/ chloroform precipitation and digested with trypsin (50 mM NH4HCO3, 1.2 M urea, 0.5 mg trypsin) at room temperature (RT) overnight. LC/MS/MS was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA) connected to an HTC-PAL autosampler and a Paradigm MS4 HPLC (Michrom Bioresources, Auburn, CA, USA). MS and MS/MS data were analyzed by MASCOT searching software in the NCBI database. Ions score is 10log(P), where P is the probability that the observed match is a random event. Individual ions scores >27 indicate the
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identity or extensive homology (p < 0.05).
analyses.
2.6. Isolation of exosomes
2.7. Western blotting
Exosomes of cultured cells were purified as previously described with minor modifications [33,34]. The filtered conditioned media were centrifuged for 70 min at 100,000 g, and the pellets were washed with PBS and centrifuged for 70 min at 100,000 g. The pellets were solubilized in 2 SDS sample buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 25% glycerol, 20 mg/ml bromophenol blue) for western blot analyses or in PBS for electron microscopy
Equal volumes of 2 SDS sample buffer containing 5% 2mercaptoethanol were added to the diluted samples. They were boiled for 2 min, applied to SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking, membranes were incubated with primary antibodies at 4 C overnight, washed with TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20), and incubated with secondary
Fig. 2. Secreted CD109 is present in exosomes from conditioned medium. (A) Silver staining image of CD109 binding proteins in the conditioned medium of 293/FLAG-CD109 cells. FLAG-CD109 in the conditioned medium secreted from 293/FLAG-CD109 cells was immunoprecipitated with anti-FLAG M2 affinity gel and the IP fraction was analyzed by SDSPAGE, followed by silver staining. CD109 was immunoprecipitated (black arrowhead) and some proteins were co-precipitated (open arrowheads). (B) Interaction of CD109180kDa with CD109-25kDa and exosomal proteins. FLAG-CD109 in the conditioned medium secreted from 293/FLAG-CD109 cells was immunoprecipitated with anti-FLAG M2 affinity gel, and the IP fraction was analyzed by western blotting with antibodies for CD109 and exosomal proteins. IP; immunoprecipitation. (C) Detection of CD109 in the exosome fraction. Using differential centrifugation, exosomes in the 293/FLAG-CD109 and 293/VC conditioned media were isolated and analyzed by western blotting with the indicated antibodies. (D) Electron micrographs of 293/FLAG-CD109 in the exosome. The upper panel shows cup-shaped vesicles (open arrowheads) of approximately 50e100 nm diameter. The exosomes were immunostained with anti-CD109-11H3 (10-nm gold: gray arrowheads) and anti-CD81 (5-nm gold: black arrowheads) (lower panel) antibodies. Scale bars: 100 nm.
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antibodies conjugated to horseradish peroxidase for 60 min at RT. After washing with TBS-T, antigeneantibody reactions were detected by LAS4000 (GE Healthcare, Tokyo, Japan) using the ECL Detection Kit (GE Healthcare). 2.8. Electron microscopy (EM) The exosome fraction was prepared for EM examination by negative stain and immunostain. For negative stain, the exosome pellet was resuspended in PBS and loaded onto formvar/carboncoated EM grids (EM Japan, Tokyo, Japan), fixed with 2% paraformaldehyde (PFA) and negatively stained with 1% aqueous uranyl acetate, followed by drying at RT. For immunogold staining, the exosome pellet was loaded onto EM grids and fixed with 2% PFA. The grids were placed on liquid drops containing the diluted antibody for overnight at RT. After washing with PBS, the grids were treated with 5-nm or 10-nm gold-labeled secondary antibody. The exosome samples were stained with 1% aqueous uranyl acetate, fixed with 0.13% methylcellulose and dried at RT. The samples were examined using a JEM-1400EX transmission electron microscope (JEOL, Tokyo, Japan). 3. Results 3.1. CD109-180kDa and CD109-25kDa fragments are present in conditioned medium in association with exosomal proteins To elucidate the function of secreted CD109-180kDa in the medium, we first tried to isolate proteins that interacted with CD109-180kDa using FLAG-CD109 overexpressing cells. Western blot analysis confirmed the expression of CD109 in conditioned
media of 293/FLAG-CD109 and 293/FLAG-sCD109 cells. A 180-kDa band was detected in the conditioned media of both cell lines. Notably, a weak 25-kDa band was also detected in the 293/FLAGCD109 conditioned medium (Fig. 1C). Secreted FLAG-CD109180kDa in the conditioned medium of 293/FLAG-CD109 was immunoprecipitated using an anti-FLAG M2 affinity gel column, and precipitated proteins were analyzed by SDS-PAGE and silver staining. A major band of about 180 kDa, probably FLAG-CD109180kDa, and several minor bands were observed in the immunoprecipitates compared to control immunoprecipitates, suggesting that secreted CD109 might interact with several proteins in the conditioned medium (Fig. 2A). The IP fractions were analyzed by LC/MS/MS. CD109 was detected by the highest score and the other 32 proteins were detected as co-precipitated proteins (Table 1). Because some proteins such as CD81 and CD9 were exosomal proteins, the ExoCarta exosomal proteins and RNA database (http:// www.exocarta.org/) was searched for the 32 proteins, and 20 out of 32 proteins were revealed to be exosomal proteins (Table 1, asterisks). Next, the IP fractions were analyzed by western blotting with anti-CD109 antibodies and antibodies against exosomal proteins, Alix, HSP70, Syntenin and CD81. FLAG-CD109-180kDa was detected in the IP fraction of 293/FLAG-CD109 medium. Interestingly, CD109-25kDa was co-precipitated with FLAG-CD109-180kDa from 293/FLAG-CD109 conditioned medium. Alix, HSP70, Syntenin and CD81 were also detected in the IP fraction. CD109 and exosomal proteins were undetected in control samples (Fig. 2B). These findings indicated that CD109-180kDa is associated with exosomal proteins, and CD109-25kDa is also present in conditioned medium in association with CD109-180kDa.
Table 1 Results of LC/MS/MS analysis of FLAG-CD109 associated proteins in the conditioned medium of 293/FLAG-CD109. No.
Protein name
Score
1** 2* 3* 4 5* 6* 7* 8* 9 10* 11* 12 13* 14* 15 16 17 18* 19* 20* 21 22 23* 24 25 26 27* 28 29* 30* 31* 32* 33*
Activated T-cell marker CD109 [Homo sapiens] Syntenin [Homo sapiens] Prostaglandin F2 receptor negative regulator [Homo sapiens] Lactadherin [Homo sapiens] Heat shock cognate 71 kDa protein isoform 1 [Homo sapiens] Epidermal cytokeratin 2 [Homo sapiens] EGF-like repeats and discoidin I-like domains 3 [Homo sapiens] Apolipoprotein J precursor [Homo sapiens] Mutant beta-actin [Homo sapiens] CD81 antigen [Homo sapiens] Neutral amino acid transporter B [Homo sapiens] Chain H, structure of the Hirulog 3 [Homo sapiens] ALG-2 interacting protein 1 [Homo sapiens] Keratin 10 [Homo sapiens] Putative [Homo sapiens] Cytokeratin 9 [Homo sapiens] Chain A, crystal structure of the 9-10 8 glycine insertion mutant of ubiquitin [Homo sapiens] Serum albumin [Homo sapiens] Alpha-enolase isoform 1 [Homo sapiens] CD9 antigen [Homo sapiens] Unnamed protein product [Homo sapiens] Unnamed protein product [Homo sapiens] Copine-8 [Homo sapiens] Chain A, human Rap1a, residues 1-167 [Homo sapiens] Transducin beta-1 subunit [Homo sapiens] Unnamed protein product [Homo sapiens] Proapolipoprotein [Homo sapiens] Epithelial cell marker protein 1 [Homo sapiens] Immunoglobulin superfamily member 8 [Homo sapiens] Alpha-2-HS-glycoprotein [Homo sapiens] Histone 1, H2bn, isoform CRA_b [Homo sapiens] Tetraspanin-6 [Homo sapiens] Integrin beta-1 isoform 1A precursor [Homo sapiens]
3712 680 483 448 289 262 236 176 171 153 133 119 111 111 91 80 79 74 57 53 52 49 48 42 42 42 39 37 37 33 36 35 33
CD109 is indicated by **, and exosomal proteins are indicated by *.
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3.2. CD109 is present in the exosome Exosomes of 293/FLAG-CD109 were collected from conditioned medium by a differential centrifugation method and analyzed by western blotting. Both CD109-180kDa and CD109-25kDa fragments were found in the exosome fraction of 293/FLAG-CD109 (Fig. 2C). It should be noted that CD109-25kDa was undetectable in the exosome-free fraction of conditioned medium, whereas a considerable amount of CD109-180kDa was present in the exosome-free fraction. Then, localization of CD109 in the exosome was analyzed by EM. We validated the typical exosomes in the exosome fraction of 293/FLAG-CD109 by negative stain (Fig. 2D, upper panel). Immunogold stain using anti-CD109/11H3 and anti-CD81 antibodies indicated CD109-25kDa and CD81 were localized in the exosome (Fig. 2D, lower panel). These results indicated that the CD109-25kDa fragment in conditioned medium is present in the exosome, whereas the CD109-180kDa fragment is present both in the exosome and in exosome-free medium. 3.3. CD109-25kDa fragment is necessary for localization of CD109 in the exosome We next evaluated the region of CD109 necessary for its localization in the exosome. We generated HEK293 overexpressing FLAG-sCD109, which does not contain the C-terminal region of CD109 after the furinase cleavage site (293/FLAG-sCD109, Fig. 1A, B). FLAG-sCD109 in the conditioned medium was immunoprecipitated by anti-FLAG M2 affinity gel column and the IP fraction was analyzed by western blotting. FLAG-CD109-180kDa was detected in the IP fraction of 293/FLAG-sCD109 as well as 293/FLAG-CD109; however, Syntenin and CD81 were not co-precipitated with FLAGsCD109 (Fig. 3A). The same results were obtained by the batch method of immunoprecipitation using anti-FLAG M2 affinity gel (Supplementary Fig. S1). Then, exosomes of 293/FLAG-sCD109 were collected from the conditioned medium by differential centrifugation and analyzed by western blotting. In contrast to 293/FLAGCD109, only exosomal proteins were detected, and CD109-180kDa and CD109-25kDa were not detected in the exosome fraction of 293/FLAG-sCD109 (Fig. 3B). These results indicated that CD10925kDa is necessary for the localization of CD109 in the exosome. 4. Discussion Recent studies have suggested the importance of the exosome in cell biology, which is involved in cellecell communication by transmitting proteins, mRNAs and miRNAs. Exosomes are also involved in cancer development, progression, metastasis and drug resistance [35]. Thus, association with an exosome in the extracellular space is an important factor to investigate the function of proteins secreted from cells. Previously, we reported that CD109 was cleaved by furinase and the N-terminal 180-kDa fragment of CD109 was released from the cell surface into the culture medium in vitro [31]. In the present study, we investigated the significance of secreted CD109-180kDa in the culture medium. We reveal that part of secreted CD109180kDa is present in the exosome and the rest is in the exosomefree fraction in the conditioned medium. We also found that CD109-25kDa, which is anchored to GPI, is secreted into the conditioned medium as an exosomal protein, forming a complex with CD109-180kDa. It was previously reported that lipid-rafts are taken up into exosomes and several GPI-anchored proteins are present in the exosomes [36,37]; thus, CD109 should exist in the exosome as a GPI-anchored protein similar to that which exists on the cell membrane. Although various types of CD109 are present on the cell surface
Fig. 3. The CD109-25kDa fragment is required for the presence of CD109 in the exosome. (A) CD109-180kDa does not interact with exosomal proteins in the absence of CD109-25kDa. FLAG-CD109 in the conditioned media of 293/FLAG-CD109 and 293/ FLAG-sCD109 were immunoprecipitated with anti-FLAG M2 affinity gel and subjected to western blotting using the indicated antibodies. IP; immunoprecipitation. (B) CD109-180kDa is not present in the exosome in the absence of CD109-25kDa. Using differential centrifugation, exosomes of 293/FLAG-CD109, 293/FLAG-sCD109 and 293/ VC were isolated and analyzed by western blotting using the indicated antibodies. (C) A schematic illustration of various types of CD109 in the culture medium. The complex of CD109-180kDa and CD109-25kDa is present on the cell surface. Part of CD109180kDa is released from the cell surface into the extracellular space. Some of the complexes of CD109-180kDa and CD109-25kDa are taken up in exosomes and are released into the extracellular space as exosomal proteins.
and in the extracellular space, the biological significance of each CD109 fragment is obscure (Fig. 3C). Especially, the difference in function between the CD109-180kDa fragments in the exosome and the exosome-free fraction is unknown at present. We previously reported that secreted CD109 in the conditioned medium is important for the attenuation of TGF-b1 signaling, and that a furinase-resistant CD109 mutant is also secreted into the conditioned medium but does not have a suppressive effect on TGF-b1
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signaling [28,31]. The furinase-resistant CD109 mutant is thought to be present in the exosomes because it is anchored to GPI, and should not be present in the exosome-free medium. Therefore, the CD109-180kDa fragment secreted in the exosome-free medium may be important for the negative effect on TGF-b1 signaling. It is interesting to note that epidermal growth factor receptor (EGFR), which interacts with CD109 on the cell surface, is an exosomal protein [38,39]. It has been reported that EGFR is taken up into exosomes and then released into the extracellular space. The Nterminal ectodomain of EGFR is shed and released from the cell surface or exosome surface, and the C-terminal fragment remains in the exosome. The released N-terminal fragment can bind to the ligand in the medium [39]. Thus, the CD109-180kDa fragment in the exosome-free medium might be biologically more active than that in the exosomes. Although we did not evaluate the function of CD109 in the exosome, it is known that CD109 is involved in cell biology of the adjacent cells. The addition of recombinant CD109 to the culture medium downregulated TGF-b1 signaling in keratinocytes in vitro, and conditioned medium of SK-MG-1 cells overexpressing CD109 attenuated TGF-b1 signaling in wild-type SK-MG-1 cells [28,29]. It has been reported that TGF-b1 is a cargo molecule of exosomes in cancer and is transmitted to adjacent cells to stimulate myofibroblast differentiation or tumor growth [40,41]. Because CD109 is highly expressed in some types of human cancers, exosomal CD109 might be transmitted to adjacent cancer cells or mesenchymal cells and regulate TGF-b1 signaling in these cells. The relationship between CD109 in the exosome and TGF-b1 signaling requires further study. Exosomes are released from various types of tumor cells, and thus, they could be useful biomarkers [1,42]. Tumor-derived exosomes are often detected in body fluids in high concentrations. By isolating exosomes, numerous non-specific proteins in the body fluids outside exosomes can be removed so that tumor-associated biomarker proteins or miRNAs in exosomes can be detected easily and accurately. CD109 is highly expressed in some tumor tissues, especially in SCCs [19e26]. We recently reported that CD109180kDa released from tumor tissue was detected in blood in a tumor-bearing mouse model in vivo [43]. If CD109 in exosomes from tumors can be detected in body fluids from, it might be a useful tumor marker. CD109 is overexpressed in carcinomas in situ as well as in SCCs in the oral cavity [23]. Therefore, CD109 in exosomes can be used as a tumor marker not only in blood but also in saliva. Taken together, we revealed that CD109 is a novel exosomal protein and may have biological functions associated with exosomes. Further investigations of CD109 in the exosome are needed to verify the function of CD109 and the potential use of CD109 as a biomarker.
Conflict of interest The authors have no conflict of interest.
Acknowledgments We thank Mr. K. Imaizumi, Mr. K. Uchiyama and Mrs. K. Ushida for their technical assistance. This work was supported by Grantsin-Aid for Global Center of Excellence (GCOE) research commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to MT) and for Scientific Research (C) commissioned by MEXT of Japan (21590435 to YM).
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H. Sakakura et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7 Pathol. Int. 60 (2010) 735e743. [27] K.W. Finnson, B.Y. Tam, K. Liu, et al., Identification of CD109 as part of the TGFbeta receptor system in human keratinocytes, FASEB J. 20 (2006) 1525e1527. [28] J.M. Zhang, Y. Murakumo, S. Hagiwara, et al., CD109 attenuates TGF-b1 signaling and enhances EGF signaling in SK-MG-1 human glioblastoma cells, Biochem. Biophys. Res. Commun. 459 (2015) 252e258. [29] I.V. Litvinov, A.A. Bizet, Y. Binamer, et al., CD109 release from the cell surface in human keratinocytes regulates TGF-b receptor expression, TGF-b signalling and STAT3 activation: relevance to psoriasis, Exp. Dermatol. 20 (2011) 627e632. [30] S. Mii, Y. Murakumo, N. Asai, M. Jijiwa, et al., Epidermal hyperplasia and appendage abnormalities in mice lacking CD109, Am. J. Pathol. 181 (2012) 1180e1189. [31] S. Hagiwara, Y. Murakumo, S. Mii, et al., Processing of CD109 by furin and its role in the regulation of TGF-beta signaling, Oncogene 29 (2010) 2181e2191. [32] T. Nishioka, M. Nakayama, M. Amano, et al., Proteomic screening for Rhokinase substrates by combining kinase and phosphatase inhibitors with 143-3z affinity chromatography, Cell Struct. Funct. 37 (2012) 39e48. [33] C. Thery, S. Amigorena, G. Raposo, et al., Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Curr. Protoc. Cell Biol. (2006). Chapter 3:Unit 3.22. [34] Y. Yoshioka, Y. Konishi, N. Kosaka, et al., Comparative marker analysis of extracellular vesicles in different human cancer types, J. Extracell. Vesicles 2 (2013) 20424. [35] X. Zhang, X. Yuan, H. Shi, et al., Exosomes in cancer: small particle, big player,
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CD109 is a component of exosome secreted from cultured cells Hiroki Sakakura a, b, Shinji Mii a, Sumitaka Hagiwara b, c, Takuya Kato a, Noriyuki Yamamoto b, Hideharu Hibi b, Masahide Takahashi a, **, Yoshiki Murakumo a, d, * a
Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan c Department of Head and Neck Surgery, Aichi Cancer Center Hospital, Nagoya, Japan d Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2015 Accepted 15 December 2015 Available online xxx
Exosomes are 50e100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-b signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome. © 2015 Elsevier Inc. All rights reserved.
Keywords: CD109 Exosome Mass spectrometry
1. Introduction Exosomes are cup-shaped, lipid-bilayer, 50e100-nm-diameter membrane vesicles, and are released from various types of cells such as epithelial cells, hematopoietic cells, mesenchymal stem cells and some tumor cells [1,2]. Exosomes are released from the intracellular space to the extracellular environment by the fusion of multivesicular bodies with the plasma membrane. During the 1980s, the function of exosomes was thought to be the removal of dispensable proteins and cell debris [3,4]. However, recent studies indicated that exosomes contain proteins, mRNAs and miRNAs,
* Corresponding author. Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. ** Corresponding author. Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail addresses: [email protected] (M. Takahashi), murakumo@ med.kitasato-u.ac.jp (Y. Murakumo).
which can be transported to surrounding cells, suggesting that exosomes may contribute to cellecell communication [5,6]. Exosomes have been identified in most body fluids including blood, saliva, urine and breast milk, and in pathological conditions including malignancies, exosomes released from lesions were detected in these body fluids [1,5,7e10]. Therefore, exosomes in body fluids might be useful biomarkers for pathological conditions [11,12]. CD109, a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, is a member of the a2-macroglobulin/C3, C4, C5 family [13,14]. CD109 was identified as a cell-surface antigen expressed on some types of normal hematopoietic cells and hematopoietic tumor cells [15e18]. CD109 has frequently been detected by immunohistochemical studies with anti-CD109 antibody in several tumor tissues, including squamous cell carcinomas (SCCs) of the oral cavity, esophagus, lung and uterus, basal-like breast carcinoma, malignant melanoma of the skin, soft tissue sarcoma, and urothelial carcinoma of the bladder [19e26]. These
http://dx.doi.org/10.1016/j.bbrc.2015.12.063 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: H. Sakakura, et al., CD109 is a component of exosome secreted from cultured cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.12.063
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H. Sakakura et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
expression studies suggested that CD109 might be a cancerassociated protein, although the significance of CD109 expression in cancer cells is unclear. However, CD109 is a component of the transforming growth factor (TGF)-b1 receptor system and negatively regulates TGF-b1 signaling in human keratinocytes [27]. CD109 is also involved in epidermal growth factor (EGF) signaling in glioblastoma cells or STAT3 activation in keratinocytes [28e30]. CD109 is cleaved by furinase into two forms, a 180-kDa secreted form and a 25-kDa membrane-attached form. Part of the 180-kDa secreted form is released from the cell surface into culture medium and the rest binds to the 25-kDa fragment on the cell surface in vitro (Fig. 1A) [31]. The processing of CD109 into 180-kDa and 25kDa proteins is necessary for regulating TGF-b1 signaling. Thus, secreted CD109 might play an important role in cell biology, although the mechanism is obscure. In this study, we revealed that CD109 is an exosomal protein and suggest that the signal modulation effects of CD109 might be associated with exosomes in culture medium.
A
Secreted CD109-180kDa
FLAG
CD109-180kDa fragment
CD109/C-9
CD109-25kDa fragment GPI
Furinase cleavage site CD109/11H3 Plasma membrane
2. Materials and methods 2.1. Antibodies and chemicals Anti-CD109/C-9 mouse monoclonal antibody (mAb), which detects the 180-kDa N-terminal fragment of CD109 (CD109180kDa), anti-Alix, anti-HSP70 and anti-Syntenin mAbs and antiCD81 rabbit polyclonal antibody (pAb) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-CD109/11H3 mAb, which detects the 25-kDa C-terminal fragment of CD109 (CD109-25kDa), was provided by Immuno-Biological Laboratories (Fujioka, Gunma, Japan). Anti-FLAG M2 and anti-b-actin mAbs, anti-FLAG M2 Affinity Gel, mouse IgG-Agarose, 3 FLAG peptide, anti-mouse IgG 10-nm Gold antibody and anti-rabbit IgG 5-nm Gold antibody were purchased from Sigma (St Louis, MO, USA). 2.2. Generation of stable transfectants The HEK293 human embryonic kidney cell line was maintained in DMEM with 8% exosome-depleted fetal bovine serum (Exo-FBS) purchased from System Biosciences (Mountain View, CA, USA) at 37 C in 5% CO2. cDNA fragments for FLAG-tagged human CD109 (FLAG-CD109) and FLAG-tagged truncated human CD109 (FLAGsCD109), which does not have a C-terminal (aa 1274-1445) region, were cloned into pcDNA3.1(þ) and used in transfection experiments [21,31]. The stable transfectants were obtained after Geneticin (Invitrogen, Carlsbad, CA, USA) selection (293/FLAGCD109 and 293/FLAG-sCD109, respectively). Fig. 1A and B shows schematic illustrations of FLAG-CD109 and FLAG-sCD109 proteins. Control transfectants were obtained by transfection of empty vector (293/VC).
Lipid raŌ
B
Furinase GPI-anchor cleavage site cleavage site 1270~1273 1420~
Signal sequence ~21
FLAG-CD109 CD109/11H3
FLAG-sCD109 FLAG
C
HEK293 transfectant media
CD109/C-9
Fifty milliliters of filtered media from 293/FLAG-CD109 and 293/ FLAG-sCD109 were loaded into polystyrene columns with 200 ml of FLAG M2 affinity gel. The gel was washed with TBS (50 mM TrisHCl, pH 7.4, 150 mM NaCl), and the bound proteins were eluted by 100 ml 3 FLAG peptide. The IP fractions were used for silver stain, mass spectrometry and western blotting. As a negative control, immunoprecipitation was performed with mouse IgG agarose.
(kDa)
FLAG 150 250
CD109/11H3
150 25
Conditioned medium from HEK293 was centrifuged at 300 g at 4 C for 10 min and then at 15,000 g at 4 C for 20 min. The supernatants were filtered by 0.22-mm nylon filters to remove cells and cell debris. The filtered media were stored at 80 C until analysis. 2.4. Immunoprecipitation (IP)
250
CD109/C-9
2.3. Conditioned medium collection
:180 kDa : 25 kDa
Fig. 1. Detection of secreted CD109 in conditioned medium. (A) Schematic illustration of FLAG-CD109 on the cell surface and secreted CD109-180kDa. Epitope positions for antibodies used in this study (anti-FLAG, -CD109/C-9, and -CD109/11H3 antibodies) are indicated (arrowheads). The furinase cleavage site is also indicated. (B) Protein structures of FLAG-CD109 and FLAG-sCD109 proteins used in this study. FLAG-sCD109 does not have a C-terminal region after the furinase cleavage site. N-terminal signal sequence, furinase cleavage site and C-terminal GPI-anchor cleavage site are indicated. FLAG sequence was inserted after the signal sequence. Epitope positions for anti-FLAG, -CD109/C-9, and -CD109/11H3 antibodies are indicated (arrowheads). (C) Expression of CD109 in 293/FLAG-CD109 and 293/FLAG-sCD109 conditioned medium. Western blotting was performed against conditioned media of 293/FLAG-CD109, 293/FLAGsCD109, and 293/VC by using anti-FLAG, -CD109/C-9, and -CD109/11H3 mAbs. The equal volume of sample was applied in each lane. The dotted and black arrowheads indicate 25-kDa and 180-kDa bands, respectively.
2.5. Mass spectrometry (LC/MS/MS) The samples for LC/MS/MS were prepared as follows [32]. The IP fractions were reduced by incubating with 5 mM dithiothreitol for 30 min and alkylated with 10 mM iodoacetamide for 60 min in the dark. The proteins were demineralized, concentrated by methanol/ chloroform precipitation and digested with trypsin (50 mM NH4HCO3, 1.2 M urea, 0.5 mg trypsin) at room temperature (RT) overnight. LC/MS/MS was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA) connected to an HTC-PAL autosampler and a Paradigm MS4 HPLC (Michrom Bioresources, Auburn, CA, USA). MS and MS/MS data were analyzed by MASCOT searching software in the NCBI database. Ions score is 10log(P), where P is the probability that the observed match is a random event. Individual ions scores >27 indicate the
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identity or extensive homology (p < 0.05).
analyses.
2.6. Isolation of exosomes
2.7. Western blotting
Exosomes of cultured cells were purified as previously described with minor modifications [33,34]. The filtered conditioned media were centrifuged for 70 min at 100,000 g, and the pellets were washed with PBS and centrifuged for 70 min at 100,000 g. The pellets were solubilized in 2 SDS sample buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 25% glycerol, 20 mg/ml bromophenol blue) for western blot analyses or in PBS for electron microscopy
Equal volumes of 2 SDS sample buffer containing 5% 2mercaptoethanol were added to the diluted samples. They were boiled for 2 min, applied to SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking, membranes were incubated with primary antibodies at 4 C overnight, washed with TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20), and incubated with secondary
Fig. 2. Secreted CD109 is present in exosomes from conditioned medium. (A) Silver staining image of CD109 binding proteins in the conditioned medium of 293/FLAG-CD109 cells. FLAG-CD109 in the conditioned medium secreted from 293/FLAG-CD109 cells was immunoprecipitated with anti-FLAG M2 affinity gel and the IP fraction was analyzed by SDSPAGE, followed by silver staining. CD109 was immunoprecipitated (black arrowhead) and some proteins were co-precipitated (open arrowheads). (B) Interaction of CD109180kDa with CD109-25kDa and exosomal proteins. FLAG-CD109 in the conditioned medium secreted from 293/FLAG-CD109 cells was immunoprecipitated with anti-FLAG M2 affinity gel, and the IP fraction was analyzed by western blotting with antibodies for CD109 and exosomal proteins. IP; immunoprecipitation. (C) Detection of CD109 in the exosome fraction. Using differential centrifugation, exosomes in the 293/FLAG-CD109 and 293/VC conditioned media were isolated and analyzed by western blotting with the indicated antibodies. (D) Electron micrographs of 293/FLAG-CD109 in the exosome. The upper panel shows cup-shaped vesicles (open arrowheads) of approximately 50e100 nm diameter. The exosomes were immunostained with anti-CD109-11H3 (10-nm gold: gray arrowheads) and anti-CD81 (5-nm gold: black arrowheads) (lower panel) antibodies. Scale bars: 100 nm.
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antibodies conjugated to horseradish peroxidase for 60 min at RT. After washing with TBS-T, antigeneantibody reactions were detected by LAS4000 (GE Healthcare, Tokyo, Japan) using the ECL Detection Kit (GE Healthcare). 2.8. Electron microscopy (EM) The exosome fraction was prepared for EM examination by negative stain and immunostain. For negative stain, the exosome pellet was resuspended in PBS and loaded onto formvar/carboncoated EM grids (EM Japan, Tokyo, Japan), fixed with 2% paraformaldehyde (PFA) and negatively stained with 1% aqueous uranyl acetate, followed by drying at RT. For immunogold staining, the exosome pellet was loaded onto EM grids and fixed with 2% PFA. The grids were placed on liquid drops containing the diluted antibody for overnight at RT. After washing with PBS, the grids were treated with 5-nm or 10-nm gold-labeled secondary antibody. The exosome samples were stained with 1% aqueous uranyl acetate, fixed with 0.13% methylcellulose and dried at RT. The samples were examined using a JEM-1400EX transmission electron microscope (JEOL, Tokyo, Japan). 3. Results 3.1. CD109-180kDa and CD109-25kDa fragments are present in conditioned medium in association with exosomal proteins To elucidate the function of secreted CD109-180kDa in the medium, we first tried to isolate proteins that interacted with CD109-180kDa using FLAG-CD109 overexpressing cells. Western blot analysis confirmed the expression of CD109 in conditioned
media of 293/FLAG-CD109 and 293/FLAG-sCD109 cells. A 180-kDa band was detected in the conditioned media of both cell lines. Notably, a weak 25-kDa band was also detected in the 293/FLAGCD109 conditioned medium (Fig. 1C). Secreted FLAG-CD109180kDa in the conditioned medium of 293/FLAG-CD109 was immunoprecipitated using an anti-FLAG M2 affinity gel column, and precipitated proteins were analyzed by SDS-PAGE and silver staining. A major band of about 180 kDa, probably FLAG-CD109180kDa, and several minor bands were observed in the immunoprecipitates compared to control immunoprecipitates, suggesting that secreted CD109 might interact with several proteins in the conditioned medium (Fig. 2A). The IP fractions were analyzed by LC/MS/MS. CD109 was detected by the highest score and the other 32 proteins were detected as co-precipitated proteins (Table 1). Because some proteins such as CD81 and CD9 were exosomal proteins, the ExoCarta exosomal proteins and RNA database (http:// www.exocarta.org/) was searched for the 32 proteins, and 20 out of 32 proteins were revealed to be exosomal proteins (Table 1, asterisks). Next, the IP fractions were analyzed by western blotting with anti-CD109 antibodies and antibodies against exosomal proteins, Alix, HSP70, Syntenin and CD81. FLAG-CD109-180kDa was detected in the IP fraction of 293/FLAG-CD109 medium. Interestingly, CD109-25kDa was co-precipitated with FLAG-CD109-180kDa from 293/FLAG-CD109 conditioned medium. Alix, HSP70, Syntenin and CD81 were also detected in the IP fraction. CD109 and exosomal proteins were undetected in control samples (Fig. 2B). These findings indicated that CD109-180kDa is associated with exosomal proteins, and CD109-25kDa is also present in conditioned medium in association with CD109-180kDa.
Table 1 Results of LC/MS/MS analysis of FLAG-CD109 associated proteins in the conditioned medium of 293/FLAG-CD109. No.
Protein name
Score
1** 2* 3* 4 5* 6* 7* 8* 9 10* 11* 12 13* 14* 15 16 17 18* 19* 20* 21 22 23* 24 25 26 27* 28 29* 30* 31* 32* 33*
Activated T-cell marker CD109 [Homo sapiens] Syntenin [Homo sapiens] Prostaglandin F2 receptor negative regulator [Homo sapiens] Lactadherin [Homo sapiens] Heat shock cognate 71 kDa protein isoform 1 [Homo sapiens] Epidermal cytokeratin 2 [Homo sapiens] EGF-like repeats and discoidin I-like domains 3 [Homo sapiens] Apolipoprotein J precursor [Homo sapiens] Mutant beta-actin [Homo sapiens] CD81 antigen [Homo sapiens] Neutral amino acid transporter B [Homo sapiens] Chain H, structure of the Hirulog 3 [Homo sapiens] ALG-2 interacting protein 1 [Homo sapiens] Keratin 10 [Homo sapiens] Putative [Homo sapiens] Cytokeratin 9 [Homo sapiens] Chain A, crystal structure of the 9-10 8 glycine insertion mutant of ubiquitin [Homo sapiens] Serum albumin [Homo sapiens] Alpha-enolase isoform 1 [Homo sapiens] CD9 antigen [Homo sapiens] Unnamed protein product [Homo sapiens] Unnamed protein product [Homo sapiens] Copine-8 [Homo sapiens] Chain A, human Rap1a, residues 1-167 [Homo sapiens] Transducin beta-1 subunit [Homo sapiens] Unnamed protein product [Homo sapiens] Proapolipoprotein [Homo sapiens] Epithelial cell marker protein 1 [Homo sapiens] Immunoglobulin superfamily member 8 [Homo sapiens] Alpha-2-HS-glycoprotein [Homo sapiens] Histone 1, H2bn, isoform CRA_b [Homo sapiens] Tetraspanin-6 [Homo sapiens] Integrin beta-1 isoform 1A precursor [Homo sapiens]
3712 680 483 448 289 262 236 176 171 153 133 119 111 111 91 80 79 74 57 53 52 49 48 42 42 42 39 37 37 33 36 35 33
CD109 is indicated by **, and exosomal proteins are indicated by *.
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3.2. CD109 is present in the exosome Exosomes of 293/FLAG-CD109 were collected from conditioned medium by a differential centrifugation method and analyzed by western blotting. Both CD109-180kDa and CD109-25kDa fragments were found in the exosome fraction of 293/FLAG-CD109 (Fig. 2C). It should be noted that CD109-25kDa was undetectable in the exosome-free fraction of conditioned medium, whereas a considerable amount of CD109-180kDa was present in the exosome-free fraction. Then, localization of CD109 in the exosome was analyzed by EM. We validated the typical exosomes in the exosome fraction of 293/FLAG-CD109 by negative stain (Fig. 2D, upper panel). Immunogold stain using anti-CD109/11H3 and anti-CD81 antibodies indicated CD109-25kDa and CD81 were localized in the exosome (Fig. 2D, lower panel). These results indicated that the CD109-25kDa fragment in conditioned medium is present in the exosome, whereas the CD109-180kDa fragment is present both in the exosome and in exosome-free medium. 3.3. CD109-25kDa fragment is necessary for localization of CD109 in the exosome We next evaluated the region of CD109 necessary for its localization in the exosome. We generated HEK293 overexpressing FLAG-sCD109, which does not contain the C-terminal region of CD109 after the furinase cleavage site (293/FLAG-sCD109, Fig. 1A, B). FLAG-sCD109 in the conditioned medium was immunoprecipitated by anti-FLAG M2 affinity gel column and the IP fraction was analyzed by western blotting. FLAG-CD109-180kDa was detected in the IP fraction of 293/FLAG-sCD109 as well as 293/FLAG-CD109; however, Syntenin and CD81 were not co-precipitated with FLAGsCD109 (Fig. 3A). The same results were obtained by the batch method of immunoprecipitation using anti-FLAG M2 affinity gel (Supplementary Fig. S1). Then, exosomes of 293/FLAG-sCD109 were collected from the conditioned medium by differential centrifugation and analyzed by western blotting. In contrast to 293/FLAGCD109, only exosomal proteins were detected, and CD109-180kDa and CD109-25kDa were not detected in the exosome fraction of 293/FLAG-sCD109 (Fig. 3B). These results indicated that CD10925kDa is necessary for the localization of CD109 in the exosome. 4. Discussion Recent studies have suggested the importance of the exosome in cell biology, which is involved in cellecell communication by transmitting proteins, mRNAs and miRNAs. Exosomes are also involved in cancer development, progression, metastasis and drug resistance [35]. Thus, association with an exosome in the extracellular space is an important factor to investigate the function of proteins secreted from cells. Previously, we reported that CD109 was cleaved by furinase and the N-terminal 180-kDa fragment of CD109 was released from the cell surface into the culture medium in vitro [31]. In the present study, we investigated the significance of secreted CD109-180kDa in the culture medium. We reveal that part of secreted CD109180kDa is present in the exosome and the rest is in the exosomefree fraction in the conditioned medium. We also found that CD109-25kDa, which is anchored to GPI, is secreted into the conditioned medium as an exosomal protein, forming a complex with CD109-180kDa. It was previously reported that lipid-rafts are taken up into exosomes and several GPI-anchored proteins are present in the exosomes [36,37]; thus, CD109 should exist in the exosome as a GPI-anchored protein similar to that which exists on the cell membrane. Although various types of CD109 are present on the cell surface
Fig. 3. The CD109-25kDa fragment is required for the presence of CD109 in the exosome. (A) CD109-180kDa does not interact with exosomal proteins in the absence of CD109-25kDa. FLAG-CD109 in the conditioned media of 293/FLAG-CD109 and 293/ FLAG-sCD109 were immunoprecipitated with anti-FLAG M2 affinity gel and subjected to western blotting using the indicated antibodies. IP; immunoprecipitation. (B) CD109-180kDa is not present in the exosome in the absence of CD109-25kDa. Using differential centrifugation, exosomes of 293/FLAG-CD109, 293/FLAG-sCD109 and 293/ VC were isolated and analyzed by western blotting using the indicated antibodies. (C) A schematic illustration of various types of CD109 in the culture medium. The complex of CD109-180kDa and CD109-25kDa is present on the cell surface. Part of CD109180kDa is released from the cell surface into the extracellular space. Some of the complexes of CD109-180kDa and CD109-25kDa are taken up in exosomes and are released into the extracellular space as exosomal proteins.
and in the extracellular space, the biological significance of each CD109 fragment is obscure (Fig. 3C). Especially, the difference in function between the CD109-180kDa fragments in the exosome and the exosome-free fraction is unknown at present. We previously reported that secreted CD109 in the conditioned medium is important for the attenuation of TGF-b1 signaling, and that a furinase-resistant CD109 mutant is also secreted into the conditioned medium but does not have a suppressive effect on TGF-b1
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signaling [28,31]. The furinase-resistant CD109 mutant is thought to be present in the exosomes because it is anchored to GPI, and should not be present in the exosome-free medium. Therefore, the CD109-180kDa fragment secreted in the exosome-free medium may be important for the negative effect on TGF-b1 signaling. It is interesting to note that epidermal growth factor receptor (EGFR), which interacts with CD109 on the cell surface, is an exosomal protein [38,39]. It has been reported that EGFR is taken up into exosomes and then released into the extracellular space. The Nterminal ectodomain of EGFR is shed and released from the cell surface or exosome surface, and the C-terminal fragment remains in the exosome. The released N-terminal fragment can bind to the ligand in the medium [39]. Thus, the CD109-180kDa fragment in the exosome-free medium might be biologically more active than that in the exosomes. Although we did not evaluate the function of CD109 in the exosome, it is known that CD109 is involved in cell biology of the adjacent cells. The addition of recombinant CD109 to the culture medium downregulated TGF-b1 signaling in keratinocytes in vitro, and conditioned medium of SK-MG-1 cells overexpressing CD109 attenuated TGF-b1 signaling in wild-type SK-MG-1 cells [28,29]. It has been reported that TGF-b1 is a cargo molecule of exosomes in cancer and is transmitted to adjacent cells to stimulate myofibroblast differentiation or tumor growth [40,41]. Because CD109 is highly expressed in some types of human cancers, exosomal CD109 might be transmitted to adjacent cancer cells or mesenchymal cells and regulate TGF-b1 signaling in these cells. The relationship between CD109 in the exosome and TGF-b1 signaling requires further study. Exosomes are released from various types of tumor cells, and thus, they could be useful biomarkers [1,42]. Tumor-derived exosomes are often detected in body fluids in high concentrations. By isolating exosomes, numerous non-specific proteins in the body fluids outside exosomes can be removed so that tumor-associated biomarker proteins or miRNAs in exosomes can be detected easily and accurately. CD109 is highly expressed in some tumor tissues, especially in SCCs [19e26]. We recently reported that CD109180kDa released from tumor tissue was detected in blood in a tumor-bearing mouse model in vivo [43]. If CD109 in exosomes from tumors can be detected in body fluids from, it might be a useful tumor marker. CD109 is overexpressed in carcinomas in situ as well as in SCCs in the oral cavity [23]. Therefore, CD109 in exosomes can be used as a tumor marker not only in blood but also in saliva. Taken together, we revealed that CD109 is a novel exosomal protein and may have biological functions associated with exosomes. Further investigations of CD109 in the exosome are needed to verify the function of CD109 and the potential use of CD109 as a biomarker.
Conflict of interest The authors have no conflict of interest.
Acknowledgments We thank Mr. K. Imaizumi, Mr. K. Uchiyama and Mrs. K. Ushida for their technical assistance. This work was supported by Grantsin-Aid for Global Center of Excellence (GCOE) research commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to MT) and for Scientific Research (C) commissioned by MEXT of Japan (21590435 to YM).
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