Elsevier

Tissue and Cell

Volume 64, June 2020, 101328
Tissue and Cell

Oxytocin induced epithelium-mesenchimal transition through Rho-ROCK pathway in ARPE-19 cells, a human retinal pigmental cell line

https://doi.org/10.1016/j.tice.2019.101328Get rights and content

Highlights

  • The oxytocin induced the actin fiber formation in the ARPE-19 cells, which is a phenotype mediated by activated Rho GTPase.

  • The actin fiber formation induced by oxytocin was inhibited by ROCK inhibitor, ripasudil, in ARPE-19 cells.

  • Accelerated wound closure and increased cell growth by oxytocin in ARPE-19 cells were inhibited by ripasudil.

  • Collagen gel contraction induced by oxytocin was inhibited by ripasudil in ARPE-19 cells.

Abstract

Previous reports suggest that oxytocin receptors (OXTRs) are expressed in the retinal pigment epithelium in primates. Oxytocinergic signaling activates the Rho-ROCK pathway, which reorganizes the actin cytoskeleton and alters other cellular biophysical characteristics. Such changes could be involved in the epithelial–mesenchymal transition and development of proliferative vitreous retinopathy. Here, we investigated whether oxytocin (OXT) binding to OXTRs in the retinal pigment epithelium can induce Rho-ROCK-mediated cellular activity. We performed four different assays of Rho-ROCK signaling in a human retinal pigment epithelium cell line (ARPE-19) such as induction of actin fibers, wound healing, cell growth, and collagen gel contraction. The assays were performed with or without OXT (100 nM) exposure, as well as with exposure to ripasudil, a specific ROCK inhibitor. The actin stress fiber formation, a phenotype mediated by activated Rho GTPase, was induced by OXT. OXT also accelerated wound closure 19 h after administration, increased cell growth 24 h afterwards, and induced stronger collagen gel contractions. All four cellular responses were inhibited with the addition of 50 μM ripasudil. Taken together, OXT-mediated activation of Rho-ROCK signal transduction could play a role in regulating epithelial–mesenchymal transition in the retinal pigment epithelium, and increase the possibility of subsequent proliferative vitreous retinopathy after vitrectomy.

Introduction

Oxytocin (OXT) and the related neuropeptide arginine vasopressin (AVP) are mainly produced in the cells in the supraoptic nucleus and the paraventricular nucleus of the hypothalamus, which then project to the posterior pituitary to secrete the peptides into the blood. In the periphery, they alter body fluid circulation and smooth muscle (uterine and vascular) contractions; in the central nervous system, they are involved in social recognition, memory, anxiety, drug addiction, and appetite (Donaldson and Young, 2008; Neumann and Landgraf, 2012; Sabatier et al., 2013; Sarnyai, 1998). Recent reports have assessed the physiological roles of OXT and AVP in sensory systems, including the olfactory bulb (Tobin et al., 2010; Tsuji et al., 2017a), retina (Tsuji et al., 2017b), tongue (Sinclair et al., 2010), and spinal cord (Breton et al., 2008).

Radioimmunoassay for OXT and AVP first identified the peptides in human and rodent retina and showed that their expression fluctuated daily (Gauquelin et al., 1983, 1988). Recently, immunohistochemical studies determined that OXT itself was expressed in the photoreceptor layer, and OXT receptor (OXTR) in the retinal pigment epithelium (RPE), of rhesus macaques (Macaca mulatta, Halbach et al., 2015). Furthermore, intracellular calcium was elevated when OXT was added to the cultured human RPE cells (York et al., 2017).

Although the function of OXT in the RPE remains unknown, Obermann et al. suggested that it might be involved in proliferative vitreous retinopathy (PVR) (Obermann et al., 2017). PVR occurs when vitreous humor enters a retinal tear and physically induces retinal detachment; trophic factors in the vitreous then cause RPE cells to undergo epithelial–mesenchymal transition (EMT) and subsequent proliferation and migration, essentially forming a cellular and fibrotic barrier to retinal reattachment. PVR can result from vitrectomy, or from eye diseases such as rhegmatogenous retinal detachment and/or proliferative diabetic retinopathy. Their data indicated that OXTRs bind galectin-3, one of two galectin species (galectin-1 and -3) found in the RPE. Galectins play roles in cell proliferation and migration, and their expression in the RPE would promote PVR; thus, OXT-OXTR signaling might correlate with PVR (Obermann et al., 2017).

In this study, we examined whether oxytocinergic activity affects EMT in the RPE by exposing a human RPE cell line (ARPE-19) to OXT. EMT is characterized by cells with a fibroblast-like morphology (polarized spindle shape, highly-motile, and proliferative) and potent contractile properties. Thus, we assessed morphological change, cell growth, wound healing, and collagen gel contraction induced by OXT. Furthermore, because these morphological changes are associated with the activity of Rho GTPases (Rho), we also performed these assays in the presence of a Rho-associated protein kinase (ROCK; a downstream Rho effector molecule) inhibitor, ripasudil

Section snippets

Materials

OXT was purchased from Peptide Institute Inc. (Osaka, Japan). Texas Red phalloidin and DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ripasudil was purchased from KOWA, Co., Ltd. (Nagoya, Japan). ARPE-19 human RPE cells were a kind gift of Dr. T. Inoue (Department of Ophthalmology, Tokyo University, Tokyo, Japan).

RT-PCR

Primers for human OXTR were designed using Primer-Blast (NCBI). The forward primer was 5′-ttcttcgtgcagatgtggag-3′ and the reverse primer was

OXTR expression and calcium mobilization by OXT

To confirm that OXTRs are expressed in ARPE-19 cells, we used RT-PCR to detect OXTR mRNA, as well as that of the vasopressin receptors 1A and 1B. OXTR, but neither vasopressin receptor mRNA, was amplified in the ARPE-19 cells (Fig. 1A). When cells were incubated in the calcium indicator fura-2/AM, OXT increased the ratio of 340/380 wavelength fluorescence (Fig. 1B), suggesting OXT was mobilizing intracellular calcium in ARPE-19 cells.

Actin fiber formation induced by OXT

We assessed if the Rho-ROCK pathway is downstream of OXT-OXTR

Discussion

OXTR is expressed in RPE cells, and OXT in cone cells (Halbach et al., 2015). Though OXT mobilizes intracellular calcium and produces evoked potentials in RPE cells (York et al., 2017), its functions in RPE cells are unclear. Here we demonstrate that OXT-OXTR signaling can alter cell growth, migration, and contraction in an RPE cell line, and the changes are similar to those seen in EMT. Furthermore, all of the changes induced by OXT could be partially or mostly blocked by Rho-ROCK pathway

CRediT authorship contribution statement

Takahiro Tsuji: Conceptualization, Methodology, Data curation, Writing - original draft, Writing review & editing. Masaru Inatani: Supervision, Writing review & editing. Chiharu Tsuji: Data curation, Writing review & editing. Stanislav M. Cheranov: Data curation. Kazuaki Kadonosono: Supervision, Writing review & editing.

Declaration of Competing Interest

The authors report no conflicts of interest.

Acknowledgement

This study was supported by grants from the Japan Society for the Promotion of Science:19K06939.

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