Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium-chloride cotransporter (KCC) in mediating (1) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (2) increases in cell cycle progression; and (3) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G(0)/G(1)-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G(2)/M-phase and accelerated the transition from G(0)/G(1)-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (1) activation of the p44/42MAPK signaling pathway, (2) cell cycle progression, and (3) increases in cell proliferation.
The aim of this study was to examine cell-to-cell metabolite transfer and connexin distribution in the rabbit corneal epithelium, in the stationary state, and during wound healing.Rabbit corneas were wounded with a surgical tool, producing a 3-mm-wide elongated debridement. Corneas were allowed to heal in vivo for up to 45 hours. Monoclonal antibodies against connexins Cx 26, Cx 32, Cx 43, and Cx 50 were used to stain cryostat sections. Cell-to-cell metabolite transfer capacity was assessed by a modification of the scrape-loading technique using lucifer yellow as the organic ion tracer.The rabbit corneal epithelium contains Cx 43 and Cx 50, localized in the cell's plasma membrane, as shown previously for other species. Cx 26 and Cx 32 are not detectable. Tracer transfer occurred in both basal and suprabasal cell layers. After wounding, the migrating epithelial monolayer lacked Cx 43 and Cx 50. This change was apparent 6 hours after injury and persisted until complete wound closure (approximately 24 hours). The Cx 50 membrane stain was increased elsewhere, in particular in the transition zone between monolayered and multilayered epithelium. Consistent with the expression changes, migrating cells displayed no or minimal cell-to-cell tracer transfer, whereas in the periphery of the wound, tracer transfer was enhanced in comparison to the control specimen.Corneal epithelial healing involves biphasic changes in the expression of connexins and cell-to-cell communications. These alterations may be critical for the optimization of the healing response.
The corneal wound healing response to an alkali burn results in dysregulated inflammation and opacity. Transient receptor potential vanilloid type1 (TRPV1) ion channel activation by such a stress contributes to this unfavorable outcome. Accordingly, we sought to identify potential drug targets for mitigating this response, in human corneal epithelial cells (HCEC).SV40-immmortalized HCEC were transduced with lentiviral vectors to establish stable c-Jun N-terminal kinase1 (JNK1), nuclear factor-κB1 (NF-κB1), and dual specificity phsophatase1 (DUSP1) shRNAmir sublines. Immunoblotting evaluated the expression of NF-κB1, DUSP1, protein kinase Cδ (PKCδ), and the phosphorylation status of cell signaling mediators. Enzyme-linked immunosorbent assay (ELISA) evaluated interleukin-6 (IL-6) and interleukin-8 (IL-8) release.Capsaicin (CAP; a selective TRPV1 agonist), induced time-dependent activation of transforming growth factor-activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) cascades temporally followed by increased nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation, rises in both PKCδ protein levels and IL-6 and IL-8 release. All of these responses were blocked by the TAK1 inhibitor 5z-7-oxozeaenol (5z-OX). In the JNK1 subline, CAP failed to increase IL-6/8 release, but still stimulated NF-κB by 50%. In the NF-κB1 subline, these IL-6/8 responses were absent, JNK1 activation was attenuated and there was a concomitant increase in DUSP1 expression compared to the control. In the DUSP1 subline, JNK1 phosphorylation was enhanced and prolonged and accompanied by larger increases in IL-6/8 release.TRPV1 induced increases in IL-6/IL-8 release occur through TAK1 activation of JNK1-dependent and JNK1-independent signaling pathways. Their joint activation is required for NF-κB to elicit sufficient positive feedback control of JNK1/2 phosphorylation to elicit increases in IL-6/8 release. Such regulation depends on NF-κB modulation of DUSP1 expression levels and associated changes in PKCδ protein levels.
An analysis is presented of how the permeability coefficient/octanol:water partition coefficient ratio for 33 different chemical substances crossing egg lecithin bilayers depends on the molecular volume of the substances. From this analysis we conclude that bilayers made from egg lecithin behave as soft polymers in their discrimination between permeants of different sizes and shapes.
