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