Biochemical and Biophysical Research Communications
CD109 is a component of exosome secreted from cultured cells
Introduction
Exosomes are cup-shaped, lipid-bilayer, 50–100-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, which can be transported to surrounding cells, suggesting that exosomes may contribute to cell–cell 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], [7], [8], [9], [10]. 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 α2-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 [15], [16], [17], [18]. 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 [19], [20], [21], [22], [23], [24], [25], [26]. These expression studies suggested that CD109 might be a cancer-associated protein, although the significance of CD109 expression in cancer cells is unclear. However, CD109 is a component of the transforming growth factor (TGF)-β1 receptor system and negatively regulates TGF-β1 signaling in human keratinocytes [27]. CD109 is also involved in epidermal growth factor (EGF) signaling in glioblastoma cells or STAT3 activation in keratinocytes [28], [29], [30]. 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 25-kDa proteins is necessary for regulating TGF-β1 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.
Section snippets
Antibodies and chemicals
Anti-CD109/C-9 mouse monoclonal antibody (mAb), which detects the 180-kDa N-terminal fragment of CD109 (CD109-180kDa), anti-Alix, anti-HSP70 and anti-Syntenin mAbs and anti-CD81 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-β-actin mAbs, anti-FLAG M2 Affinity
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/FLAG-CD109 conditioned medium (Fig. 1C). Secreted FLAG-CD109-180kDa in
Discussion
Recent studies have suggested the importance of the exosome in cell biology, which is involved in cell–cell 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
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 Grants-in-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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