Biochem Biophys Res Commun. 2017 Jan 29;483(1):245-251. doi: 10.1016/j.bbrc.2016.12.158.

Role of extracellular vesicles in the interaction between epithelial and mesenchymal cells during oviductal ciliogenesis.

Nakano S1, Yamamoto S2, Okada A1, Nakajima T1, Sato M3, Takagi T3, Tomooka Y4.
1 Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.
2 Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Graduate Program in Bioscience, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.
3 Department of Material and Biological Science, Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyou-ku, Tokyo 112-8681, Japan.
4 Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan. Electronic address: tomoylab@rs.noda.tus.ac.jp.

Abstract

Extracellular vesicles (EVs) have been shown to transport miRNA, mRNA and protein, suggesting that they are new communication mediators. Diffusible mesenchymal factors determine the fate of Műllerian epithelial cells into oviductal ciliated cells. In the present study, we investigated whether EVs mediate the communication in the epithelial-mesenchymal interaction during oviductal ciliogenesis. EVs were isolated from cells of oviductal mesenchymal cell line (S1 cells) and characterized by TEM and expression of exosomal marker CD81. CD81 protein was also detected in oviductal mesenchyme, suggesting that CD81-expressing exosomes may be secreted from oviductal mesenchyme, as well as S1 cells. β-actin, Gapdh and Vimentin mRNAs and miRNAs were detected in the exosomes. mRNA in S1 cells was able to be transported into cells of Műllerian epithelial cell line (E1 cells) via the exosomes. The effects of exosomes derived from S1 cells on ciliogenesis of E1 cells were analyzed by in vitro models. Culture with exosomes increased the number of ciliated cells in E1 cells. These results suggest that exosomes derived from mesenchymal cells modulate the oviductal ciliogenesis and open new avenues for developmental study of EVs. Copyright © 2016 Elsevier Inc. All rights reserved.

KEYWORDS: Ciliogenesis; Exosome; Extracellular vesicles; Oviduct

Pubmed

 

Supplement:

Extracellular vesicles (EVs) are secreted by various types of cells and released into the microenvironment. Many scientists have reports the roles of EVs in cancer communications on metastases and angiogenesis, and immunology; however, little is known about the functions of EVs in mammalian organogenesis at developmental stage. EVs including mRNA, miRNA and protein may be involved in phenotype switching in various types of cells, therefore we focused effects of EVs on differentiation of the cells.

We have investigated the development of oviductal epithelium in mice. In adult mice, the oviductal epithelium consists of two major cell populations, secretory cells and ciliated cells. Ciliated cells are dominant in infundibulum and ampulla and secretory cells are dominant in isthmus. In the oviducts, the fate of undetermined epithelial cells was determined by the mesenchyme via secretory factors. In order to molecularly investigate the epithelial-mesenchymal interaction in organogenesis, we developed in vitro culture model. Fate-undetermined epithelial clonal cell line (E1 cells) was differentiated into oviductal ciliated cells by co-culture of oviductal mesenchymal clonal cell line (S1 cells), and Follistatin like-1 (FSTL-1) was found one of the diffusible fate-determining factors derived from S1 cells. These findings led us to consider the possibility that EVs derived from oviductal mesenchymal cells act as diffusible factors and modulate fate determination or ciliogenesis of E1 cells. Therefore, in the present study, we examined the effects of EVs derived from S1 cells on the ciliogenesis of E1 cells.

In vitro analysis revealed that S1 cells secreted CD81-positive EVs that have the exosomes-like size, ca. 100 nm. The CD81 protein was also expressed in oviductal mesenchyme during ciliogenesis in vivo, indicating that CD81-expressing EVs may be secreted from oviductal mesenchyme. Since small amounts of β-actin, Gapdh and Vimentin (they were not signaling molecule) mRNAs were detected, and miRNAs were enriched in the EVs derived from S1 cells, we considered that EVs secreted from oviductal mesenchymal cells may include the miRNAs selectively and small amounts of mRNAs randomly. Thus, we think that included miRNA may be main effectors in the EVs. Co-culture of E1 cells with EGFP-expressing S1 cells revealed that the EVs have capacity for the incorporation into epithelial cells.

Co-culture of E1 cells with the EVs, unlike with the S1 cells, did not induced Foxj1 expression that is essential transcription factor for the fate determination of ciliated cells. However, co-culture with the EVs increased the number of ciliated cells. Taken together, the EVs were not involved in the critical developmental event, fate determination in oviductal epithelium, but modulated ciliogenesis. The finding was consistent with the expectation that miRNAs were main effectors in the EVs, because miRNAs are mainly modulators. Therefore, it is hypothesized that exosomes derived from oviductal mesenchymal cells act as a modulators for ciliogenesis of epithelial cells, and we open new avenues for developmental study of EVs as modulators.