Elsevier

Cell Calcium

Volume 44, Issue 4, October 2008, Pages 374-385
Cell Calcium

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

https://doi.org/10.1016/j.ceca.2008.01.008Get rights and content

Summary

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4α-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca2+-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca2+-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.

Introduction

Corneal epithelial integrity is required for maintaining tissue transparency and deturgescence. This outermost tear-side facing layer provides a barrier function to protect the cornea from noxious insults. This protective role is dependent on adequate tight junctional resistance [1]. If such resistance is disrupted by corneal injury or infection, corneal swelling and opacification may develop compromising visual performance. These changes result from decreases in transepithelial net osmolyte-coupled fluid flux from the stroma to tears, which is inadequate to overcome the innate tendency for the stroma to imbibe fluid and thicken [2]. On the other hand, epithelial barrier function is maintained despite continuous shedding of terminally differentiated superficial layers provided there is continuous cytokine-modulated corneal epithelial renewal [3], [4], [5]. Therefore, corneal transparency and normal vision are dependent on the ability of the corneal epithelium to undergo continuous renewal and preserve its tight junctional integrity and resistance [6].

Fluctuations in tear film osmolarity can occur in daily living and challenge corneal epithelial barrier function by inducing acute epithelial cell volume changes. In some types of dry eye disease, losses in tear film integrity and changes in its composition can chronically stress this tissue layer [7], [8]. Nevertheless, under physiological conditions, corneal epithelial cells (CEC) withstand such stresses by mounting regulatory volume (RV) responses [9]. During exposure to a hypertonic challenge, these cells initially shrink, which is rapidly compensated for by activation of a RV increase (RVI) response [10]. RVI restores cells to their isotonic cell volume by stimulating the net uptake of osmolytes and subsequent osmotically coupled water influx. Such restoration is in part dependent on activation of Na–K–2Cl 1 co-transporter (NKCC1)-induced osmolyte influx coupled to Na–K pump stimulation [11]. On the other hand, during exposure to a hypotonic challenge, CEC initially swell, and activate a RV decrease (RVD) response to restore their isotonic volume [12]. In human CEC (HCEC), RVD is dependent on increases in K+ and Cl efflux through volume-sensitive ion channels [9], [12] and K–Cl co-transporter 1 (KCC1) [13]. Each of these regulatory volume responses is essential for maintaining corneal transparency, since they restore CEC isotonic volume and prevent compromise of their barrier function [14].

During exposure to an anisosmotic stress, mitogen-activated protein kinase (MAPK) superfamily activation in HCEC induces RV needed for restoration of isotonic cell volume. Exposure to hypertonicity induces protein–protein interaction between p38 and NKCC1. Their association in HCEC is requisite for NKCC1 activation and RVI induction [15]. On the other hand, activation of MAPKs ERK and JNK is required for RVD whereas p38 stimulation occurs subsequent to RVD in rabbit CEC (RCEC) [12].

Ca2+ signaling is critical to induce RVD during exposure to a hypotonic challenge. In RCEC, this response is dependent on ryanodine-sensitive channel stimulation resulting in Ca2+ release from intracellular Ca2+ stores (ICS). ICS depletion is followed by transient increases in plasma membrane Ca2+ influx [16]. This response suggests that in RCEC plasma membrane Ca2+ influx through store operated channels is an important contributor to inducing RVD. In many other tissues, these Ca2+ transients, in turn, stimulate MAPK and volume-sensitive K+ (i.e. Maxi-K+, intermediate K+) channels [17], [18]. However, the identity of plasma membrane associated Ca2+ permeable pathways has not been described in HCEC.

The transient receptor potential (TRP) superfamily forms tetrameric cation permeable channels of varying selectivity. Many members of this superfamily are polymodally activated by a diverse assortment of environmental challenges, including osmotic, mechanical, and thermal stresses and cues [19], [20]. For a variety of tissues, there is emerging evidence that regulatory volume responses are dependent on increases in Ca2+ influx through pathways comprising several different members of the TRP superfamily [21], [22], [23], [24].

The TRP vanilloid subfamily in mammals has 6 members (TRPV 1–6) and they were identified in both excitable and non-excitable cells of multiple tissues [25]. These members were first characterized in invertebrates by a mutagenesis approach: in Drosophila melanogaster, they are encoded by the nan [26] and iav [27] genes, and in Caenorhabditis elegans by the ocr 1-4 [28] and osm-9 genes [29]. The TRPV1-4 members are moderately Ca2+ selective (PCa/PNa = 5–10) whereas the TRPV5-6 members are highly Ca2+ selective (PCa/PNa > 100) [30]. In HCEC, TRPC4 and TRPV1 functional expression was identified [31]. EGF-induced HCEC proliferation is dependent on activation of store operated channels containing TRPC4 whereas TRPV1 stimulation mediates inflammatory cytokine release via MAPK pathways [31], [32].

TRPV4 activation in various studies occurs in either a direct or indirect manner, although definitive evidence of direct activation is still missing [33]. Moreover, different stimuli use distinct pathways to indirectly activate TRPV4 [34]. Cell swelling can activate TRPV4 following cPLA2 stimulation to mobilize plasma membrane polyunsaturated fatty acid release, resulting in the formation of arachidonic acid (AA). Subsequent to AA conversion to epoxyeicosatrienoic acids (i.e., 5′,6′-EET and 8′,9′-EET) by cytochrome P450 epoxygenase, these metabolites activate TRPV4 although it is unclear whether these endogenous ligands can directly bind to the channel [35], [36]. Contrarily, heat and phorbol esters-induced TRPV4 activation is associated with a tyrosine residue (Tyr-556) in the N-terminal portion of TM3 [37].

The activated TRPV4 channel exhibits outwardly rectifying current with a single-channel conductance of 60–310 pS [30], [38], [39]. This wide range may be attributed to differences in measurement conditions in different studies and/or TRPV4 heterologous assembling as well as other factors. Sensitivity and temporality of TRPV4-induced currents to hypotonicity are similar to those of [Ca2+]i transients and to those of cation channels activated by cell swelling in TRPV4-transfected HEK293 cells [40]. A similar role for endogenous TRPV4 has been described in rat and mouse renal ascending thin limb cells where substantial transcellular osmotic gradients occur [41].

Growing evidence indicates that the TRPV4 channel mediates cell volume homeostasis based on the fact that RVD induction requires extracellular Ca2+ ([Ca2+]0). TRPV4-transfected-Chinese hamster ovarian (CHO) cells exhibited RVD during a hypotonic challenge whereas in the non-transfected counterpart there was only persistent swelling [21]. Moreover, TRPV4 expression in epithelial cells appears to be a determinant of membrane fluid permeability since its activation enhances both transcellular and paracellular cytoplasmic membrane permeability. Increases in paracellular fluid permeability are attributable to TRPV4 activation-induced tight junction (TJ) changes, including declines in claudin protein expression, and leakage-linked changes in TJ morphology in mouse mammary gland cells [42]. At the transcellular level, rises in transcellular electrolyte permeability are linked with changes in TRPV4 interaction with aquaporin 5 (AQP5), which is also required for RVD [22], [43]. There is a disparity between how these two proteins interplay in response to hypotonic challenge. In human and mouse salivary gland cells, hypotonicity increased membrane abundance and colocalization of TRPV4 and AQP5 in the apical region of salivary gland cells whereas the absence of either impaired Ca2+ transients and RVD. Additionally, N-terminal deletion of AQP5 suppressed its membrane translocation, TRPV4 activation and RVD, suggesting these two proteins in concert mediate RVD, where hypotonicity-induced TRPV4 stimulation is dependent on AQP5 activity. On the other hand, in mouse lung epithelial cells, hypotonic challenges reduced AQP5 surface abundance. This reduction may be dependent on TRPV4 activation since by inhibition of TRPV4 stimulation or in non-transfected HEK293 cells, such decreases were eliminated. These results indicate that TRPV4 mediates hypotonicity-induced losses of surface-delimited AQP5 content. Apparently, in AQP5+/TRPV4+ epithelia their responses to hypotonic stimulation are cell type specific due to differences in interplay between TRPV4 and AQP5.

The importance of TRPV4 expression for osmotic stress detection has been documented in vivo in both invertebrates and vertebrates. Mechanical- and hypertonicity-induced avoidance, but not chemical-induced avoidance, was (partially) restored by transgenic expression of mammalian TRPV4 in head-sensory neurons of the osm-9 mutated C. elegans [19]. Similarly, TRPV4−/− mice show reduced fluid intake and impaired responses to (hyper-) and hypo-osmotic stimuli, as well as thermal, mechanical sensitivity [44], [45].

In the present study, we describe TRPV4 expression in the intact human corneal epithelium as well as in primary HCEC (pHCEC) and in SV40-immortalized HCEC. TRPV4 is not only localized in the cell periphery, but it is also somewhat evident in the perinuclear domain. Such expression is functional since the either TRPV4 agonist, 4α-PDD, or exposure to a hypotonic medium mediated Ca2+ transients. Furthermore, TRPV4 expression is requisite for RVD since TRPV4 knockdown by effective siRNAs, but not of mismatched siRNAs, markedly suppressed Ca2+ transients and RVD responses. Therefore, TRPV4 activation is essential for mediating a RVD response.

Section snippets

Cell culture

SV40-adenovirus-immortalized HCEC, a generous gift from Dr. Araki-Sasaki, (Kagoshima Miyata Eye Clinic, Kagoshima, Japan), were cultured as previously described [46]. pHCEC were purchased from Cascade Biologics, Inc. (Portland, OR, USA). These cells were grown and maintained in EpiLife medium (Cascade Biologics), supplemented with human corneal growth supplement (HCGS) containing bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin, and mouse epithelial growth factor

TRPV4 expression in human corneal epithelium

To examine whether there is TRPV4 gene and protein expression in HCEC, we performed RT-PCR, immunohistochemistry, FACS, and Western blot analyses, respectively. Fig. 1A shows a micrograph of the intact human cornea. TRPV4 staining is evident throughout the intact corneal epithelial layer (Fig. 1A, right panel). Staining is most intense in the central superficial layer, and diminishes towards the basal and limbal regions. There is minimal TRPV4 staining in the basal limbal zone (Fig. 1A, middle

Discussion

TRPV4 protein expression was detected in HCEC and its primary counterpart as well as in the intact human cornea. Its expression is functional because: (1) 4α-PDD resulted in Ca2+ transients; (2) either ruthenium red, removal of [Ca2+]0, or TRPV4 siRNA knockdown obviated these transients; (3) RVD behavior was selectively inhibited following effective siRNA TRPV4 knockdown whereas this response was not affected by mismatched or nonfunctional siRNA; (4) pre-exposure to CPZ failed to suppress

Acknowledgements

This work was supported by NEI grant 04795 (P.S.R.), William C. Ezell Fellowship from the American Optometric Foundation and Minnie Turner Flaura Fund (Z.P.). The authors thank Yvonne Giesecke and Ines Eichhorn for the technique assistance and Dr. Kathryn Pokorny for critical review and editing of an earlier version of this manuscript.

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