TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium

Abstract

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc.

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

“…34 Consistent with the latter possibility, barrier permeability in CE monolayers is regulated by GSK101 but not the TRPV1 agonist capsaicin. 61 Our finding that mouse CECs do not respond to hypertonic stimuli may reflect the rareness of TRPV1-expressing cells, low TRPV1 expression in adult tissue, and/or presence of full-length protein variants that are not activated by cell shrinking. 48 We detected TRPV1 reporter signals in a subset of nerve ending-like processes, an observation that accords with histological and functional studies showing heat-, hypertonicity-and mechano-dependent firing in a capsaicin-and hypertonicity-responding population of polymodal nociceptor afferents.…”

“…Consistent with prominent transcript expression, immunohistochemistry showed robust TRPV4 expression in central and limbal regions and a well-defined basal-to-squamous gradient of TRPV4-ir and TRPV4 eGFP , a pattern that resembles the basal-apical TRPV4 expression patterns reported in urothelial, skin, and mammary epithelia, 17,18,60 but differs from the rabbit cornea in which TRPV4 was localized to apical cells. 61 Basal expression implicates TRPV4 in differentiation/stemness and/or barrier permeability functions, 15,21,33 whereas plasma membrane TRPV4-ir reflects the well-known roles of the channel in ion transport, Ca 2+ homeostasis, and volume regulation. 45,62,63 Although the function of cytosolic TRPV4-ir puncta is less clear, total internal reflection microscopy studies have linked such puncta to stimulus-and development-dependent trafficking of endosomes, membrane translocation, insertion, and/or internalization.…”

Transient receptor vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function

“…Recent publications indicate an epithelial function of the channel opposed to that in endothelium i.e. stabilization of the epithelial barrier in the skin ( Akazawa et al, 2013 ), the urogenital tract ( Janssen et al, 2016 ) and the corneal epithelium ( Martinez-Rendon et al, 2017 ). We demonstrated TRPV4 expression in alveolar epithelial type I (ATI) and type II (ATII) cells ( Figure 2B-C ).…”

“…In other tissues however, the channel maintains physiological cell barriers, e.g. in skin ( Akazawa et al, 2013 ), the urogenital tract ( Janssen et al, 2016 ) and the corneal epithelium ( Martinez-Rendon et al, 2017 ). In tracheal epithelial cells TRPV4 channels regulate ciliar beat frequency ( Lorenzo et al, 2008 ) and in alveolar epithelial cells TRPV4 activation by 4α-phorbol esters produced blebs and breaks in lung septa ( Alvarez et al, 2006 ) by unknown molecular mechanisms.…”

TRPV4 regulates migration and tube formation of human retinal capillary endothelial cells

“…Activation of TRPV4 modulates the extracellular melatonin in human non-pigmented ciliary ECs [ 25 ]. Activation of TRPV4 is necessary for the correct establishment of tight junctions in corneal epithelia as well as the regulation of both the barrier function of tight junctions and their ability to respond to epidermal growth factor [ 26 ]. However, no studies had focused on TRPV4 function in HRCECs.…”