TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.

The superficial corneal epithelial layer protects the cornea from losses in tissue transparency and deturgescence resulting from environmental insults. This barrier function maintenance is dependent on the continuous renewal of corneal epithelial cells and the integrity of tight junctions between the superficial epithelial cells in this layer. One environmental stress that can compromise corneal epithelial barrier function is exposure to hyperosmotic tear film, which occurs in dry eye disease.1,2Increases in tear osmolarity promote ocular surface inflammation by activating proinflammatory cytokine release and enhancing inflammatory cell infiltration. These tear gland dysfunction and tear film instability; thus, corneal erosion and opacification may ensue. Although therapeutic approaches such as hypotonic or isotonic artificial tears provide symptomatic relief in dry eye disease patients by lowering their tear osmolarity,3,4development of drugs that can effectively suppress receptor-mediated inflammation is limited. Emerging evidence indicates that the transient receptor potential vanilloid family members mediate responses to osmotic stress. TRPV channels function as a trans-plasma membrane ion entry pathway composed of six transmembrane-spanning subunits in the form of a tetramer. There are seven members (TRPV1-TRPV7) in this subfamily. Only 2 of 7 members have been documented to be activated by osmotic challenges. Our earlier study reveals TRPV4 contributes to hypo-osmosensing mechanism and initiates regulatory volume decrease in HCECs. Similar findings have been made in rat neurons, HaCaT cells, and human airway smooth muscle cells.5–8However, exposure to hyperosmotic challenges does not induce TRPV4 channel activation in HCECs and some other tissues.8–10 Some studies have identified TRPV1 as a hyperosmotic sensor. Liu et al.11 found that hypertonicity sensitized capsaicin induced Ca2+ transients and enhanced TRPV1 translocation to plasma membrane in rat trigeminal neurons. Sharif et al. 12 and Yokoyama et al.13 revealed that an N-terminal variant of the TRPV1 channel is required for hyperosmotic sensing but not for hypertonicity-induced regulatory volume increase in arginine vasopressin (AVP)-releasing neurons in supraoptic nucleus. On the other hand, it remains uncertain whether TRPV1 serves as a hyperosmotic sensor to stimulate fluid intake.14,15 In addition, there is limited information regarding the role of TRPV1 hyperosmosensor in nonneuronal tissues. In HCECs, TRPV1 activation by capsaicin induces increases in IL-6 and IL-8 release through mitogen-activated protein kinase (MAPK) pathway stimulation.16As increases in IL-6 and IL-8 contribute to inflammation occurring in dry eye disease, it is possible that TRPV1 activation by hypertonicity can contribute to these increases. The signaling mechanism through which hypertonic stress increases proinflammatory cytokine release is of great interest. EGF receptor (EGFR) and its linked signaling cascades are not only a key promoter of cell proliferation and migration but also a critical mediator of various pathophysiological events.17EGFR activation has been identified in response to UV light, osmotic stress, membrane depolarization, cytokines, chemokines, and cell adhesion elements. In the corneal epithelium, EGFR transactivation is elicited by lysophosphatidic acid (LPA), adenosine triphosphate (ATP), wounding, and flagellin.18These findings prompted us to determine whether hyperosmotic stimuli–induced increases in proinflammatory cytokine release are dependent on EGFR transactivation and the role of TRPV1 in such processes. MAPK family activation, a downstream event of EGFR stimulation, can also be triggered by osmotic shock. Both hypertonic and hypotonic exposures can activate MAPK.16,19Exposure of the mouse corneal surface to hypertonic stress stimulated ERK, p38, and Jun NH2-terminal kinase (JNK) MAPK signaling, which led to increases in IL-1β, TNFα, and metalloproteinase (MMP)-9 expression levels.20,21Both the duration and the magnitude of MAPK phosphorylation are determinants of types of responses induced by their activation.22In HCECs, the duration and magnitude of ERK and p38 phosphorylation determined EGF-induced proliferation and migration. Prolonged p38 phosphorylation by suppression of ERK signaling pathway promotes EGF-induced migration. On the other hand, proliferation was enhanced when ERK phosphorylation was prolonged by eliminating glycogen synthase kinase (GSK-3)–induced dephosphorylation of ERK.23,24 Such modulation of MAPK-induced signaling by EGF and neural growth factor (NGF) occurs in PC12 cells, a neural precursor cell line. With EGF, ERK MAPK activation peaked at 5 minutes and then rapidly declined. This pattern of ERK activation promoted cell proliferation. In contrast, with NGF, ERK activation remained high for hours, and the cells stopped proliferating and instead differentiated into neurons.25As different responses induced by TRPV1 and EGF activation are both dependent on MAPK signaling, it is convincible that each of the responses is associated with a unique pattern of MAPK stimulation. Another mediator in the process of hypertonicity-induced inflammation is nuclear factor (NF)-κB protein. NF-κB is a latent transcription factor that lies at the center of many inflammatory responses induced by infection and injury.26–28 NF-κB is implicated in mediating dry eye–induced ocular surface inflammation because the inhibition of NF-κB reduces the inflammatory response.1 However, given the complex etiology of dry eye inflammation, including cytokines, chemokines, and MMPs, the importance of NF-κB responsiveness to hypertonic stress is unclear in HCECs. Furthermore, the interaction between MAPK and NF-κB in mediating inflammation depends on types of stimuli and cells.29–32Therefore, investigation is warranted to probe for the role of MAPK and NF-κB in hypertonicity-induced inflammation in corneal epithelial cells. In the present study, we identified that exposure to hyperosmotic stimuli activated the TRPV1 channel. This resulted in EGFR transactivation through metalloproteinase-dependent HB-EGF shedding. TRPV1-EGFR signaling cascades contributed to the phosphorylation of ERK and p38 MAPK and to subsequent activation of NF-κB, leading to increases in IL-6 and IL-8 release.