Elsevier

Experimental Eye Research

Volume 90, Issue 1, January 2010, Pages 121-129
Experimental Eye Research

Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: Involvement in thermosensation and wound healing

https://doi.org/10.1016/j.exer.2009.09.020Get rights and content

Abstract

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33° C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

Introduction

The cornea has a highly ordered architecture that allows it to transmit light and function as a barrier between the eye and the external environment. In addition to providing a physical barrier, the cornea has sensory properties that enable it to sustain normal function with special protection against injury. Thus, the cornea possesses a somatic sensory system to detect a variety of stimuli, such as mechanical forces, temperature, and irritant chemicals (Belmonte et al., 2004). Over the last decade, a number of transient receptor potential (TRP) ion channels have been identified as molecular candidates to explain the transduction of the physiological stimuli in the mammalian somatic sensory system (Lee and Caterina, 2005, Caterina, 2007, Schepers and Ringkamp, 2009). However, analysis of the molecules in corneal tissues remains to be done.

The TRP channel family is one of the largest families of calcium-permeable cation channels and is composed of six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPL (mucolipin) and TRPA (ankyrin). It contains thermoreceptors, known as ‘thermoTRPs’: TRP vanilloid 1 (TRPV1), TRPV2, TRPV3, TRPV4, TRP melastatin 8 (TRPM8) and TRP ankyrin 1 (TRPA1), which can be activated by increases or decreases in ambient temperature (Lee and Caterina, 2005, Caterina, 2007, Schepers and Ringkamp, 2009). Moreover, the hallmark of TRP channels is their polymodality; TRPV1, TRPV3, TRPM8 and TRPA1 are also recognized as chemoreceptors, respectively responsive to capsaicin, carvacrol, menthol, mastard and cinnamon oil. Additionally, TRPV4 and TRPA1 act as mechanoreceptors (Levine and Alessandri-Haber, 2007). When these channels were first discovered, attention was primarily focused on their potential contributions to the direct thermal activation of peripheral sensory neurons and the nerve endings. However, recent anatomical, physiological, and behavioral studies have provided evidence for the contribution of epithelial TRPV channels to thermosensation in vitro and in vivo. Among thermoTRPs, TRPV3, which shares 40–50% homology with TRPV1, is expressed at very low levels in sensory neurons, but is prominently expressed in skin keratinocytes (Peier et al., 2002). Nevertheless, TRPV3 knockout (TRPV3−/−) mice have defects in thermal selection and heat-evoked withdrawal behaviors (Moqrich et al., 2005), suggesting that epithelial TRPV3 is indeed involved in cutaneous thermosensation. Moreover, several recent studies have demonstrated that epithelial TRPV channels are also involved in the regulation of intracellular calcium ion ([Ca2+]i) in peripheral migrating cells. TRPV1 is a major physiological Ca2+ influx channel required for the migration of hepatocyte-growth factor (HGF)-pretreated human hepatoblastoma HepG2 cells (Waning et al., 2007), and TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cell migration in prostate cancer PC3 cells (Monet et al., 2009). Since cell migration relies on tight temporal and spatial regulation of the [Ca2+]i concentration (Dreval et al., 2005), calcium-permeable TRPV channels expressed in corneal tissues seem likely to be involved in the regulation of [Ca2+]i in migrating cells. We speculated that TRPV channels are expressed in corneal epithelial cells and play a role in a variety of cellular functions in the cornea. In the present study, therefore, we examined the expression of thermoTRP channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1) in a human corneal cell line (HCE-T cells). This article reports for the first time that functional TRPV3 channels are expressed in corneal epithelial cells of humans and mice, and that TRPV3 activation causes a transient increase of [Ca2+]i. Moreover, appropriate activation by a TRPV3 agonist enhances the cellular viability and increases the wound healing rate of HCE-T cells.

Section snippets

Materials and methods

The Center of Experimental Animal Sciences at Nagoya City University, Japan, approved all of the following experiments.

Identification of TRPV3 mRNA expression

To investigate the expression of thermoTRP channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPA1) in HCE-T cells, we isolated total RNA from the cells and performed RT-PCR with primer sets designed to specifically amplify these TRP channels. As shown in Fig. 1A, products with the predicted sizes and nucleotide sequences of TRPV1, TRPV3 and TRPV4 mRNA were amplified from the cells, whereas TRPV2 transcripts were barely detected and transcripts for TRPM8 and TRPA1 were not observed. The

Discussion

The present study established that functional TRPV3 channels are expressed in HCE-T and mouse corneal epithelial cells. Moreover, we found that both HCE-T cells and mouse corneal epithelial cells have the ability to respond to mild heat stimulation. In these cells, heat stimuli produced an increase of [Ca2+]i with the activation threshold of around 33–34 °C and its activation was mostly inhibited by RuR. Since the TRPV3 channel is RuR-sensitive and only responds to small perturbations at around

Acknowledgements

We thank K. Tanaka and K. Kajita for technical assistance. This investigation was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Sciences (to T.U., S.U., and S.S.).

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