2-(-2-Benzofuranyl)-2-imidazoline reciprocally regulates Th17/Treg balance induced by ischemic stroke in rats.

Our group previously showed that 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) is a potent neuroprotective agent in the treatment of ischemic stroke in rats. As its mode of action was not well defined, we determined if its therapeutic effect includes altering an immune response to experimental ischemic stroke in rats. In the current study, 2-BFI significantly reduced stroke-induced brain infarct volume and it also decreased neurological deficits. Its anti-immune effects were determined based on flow cytometry measurements of both the 2-BFI-induced changes in the Th17/ Treg cell balance ratio and ELISA measurements of proinflammatory IL-17A and anti-inflammatory IL-10 cytokine expression levels in the brain and peripheral blood following ischemic strokes. 2-BFI blunted the stroke-induced increases in this ratio, which resulted from suppression of the rises in the Th17 cell number whereas the proportion of Treg cells increased. Stroke also induced increases in IL-17A expression levels whereas the IL-10 expression levels declined. 2-BFI treatment inhibited the rises in IL-17A expression levels whereas the corresponding declines in IL-10 were suppressed by this agent. Therefore, one of the neuroprotective effects of 2-BFI in the treatment of cerebral strokes stems from its suppression of rises in the Th17/Treg balance along with corresponding changes in related cytokines modulating development of this condition.

Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury.

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

NF-κB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells.

Corneal epithelial injury induces release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) agonists. We determined the functional contributions by CB1 and TRPV1 activation to eliciting responses underlying wound healing in human corneal epithelial cells (HCEC). Both the selective CB1 and TRPV1 agonists (i.e., WIN55,212-2 [WIN] and capsaicin [CAP], respectively) induced EGFR phosphorylation whereas either inhibition of its tyrosine kinase activity with AG1478 or functional blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF (HB-EGF). CB1-induced Ca(2+) transients were reduced during exposure to either the CB1 antagonist, AM251 or AG1478. Both CAP and WIN induced transient increases in Erk1/2, p38, JNK1/2 MAPK and Akt/PI-3K phosphorylation status resulting in cell proliferation and migration increases which mirrored those elicited by EGF. Neither EGF nor WIN induced any increases in IL-6 and IL-8 release. On the other hand, CAP-induced 3- and 6-fold increases, which were fully attenuated during exposure to CPZ, but AG1478 only suppressed them by 21%. The mixed CB1 and TRPV1 antagonist, AM251, enhanced the CAP-induced rise in IL-8 release to a higher level than that elicited by CAP alone. In conclusion, CB1 and TRPV1 activation induces increases in HCEC proliferation and migration through EGFR transactivation leading to global MAPK and Akt/PI-3K pathway stimulation. On the other hand, the TRPV1-mediated increases in IL-6 and IL-8 release are elicited through both EGFR dependent and EGFR-independent signaling pathways.

The corneal epithelium: clinical relevance of cytokine-mediated responses to maintenance of corneal health: [review] / O epitélio da córnea: relevância clínica das respostas mediadas por citocinas para manter a saúde da córnea: [revisão]

We review the growth factor receptor-mediated cell signaling events that induce the responses required for the maintenance of corneal epithelial health. Our focus is to show how such responses contribute to sustaining corneal transparency and deturgescence, so basic to the pathogenesis of corneal diseases. Furthermore, we point out how alterations of receptor-mediated control of these responses account for losses in corneal transparency. In particular, the roles of growth factors in the mediation of normal corneal function, including epithelial cell proliferation, prevention of compromise of the barrier function of the cornea, and maintenance of normal renewal processes are discussed in relation to clinical entities involving the cornea.

Revimos os eventos de sinalização celular mediados por receptores de fatores de crescimento, usados para manter a saúde do epitélio da córnea. O objetivo é mostrar como essas respostas contribuem para manter a transparência e a deturgescência da córnea, críticos na patogênese das doenças da córnea. Mais ainda, enfatizamos como alterações no controle mediado por receptor dessas respostas contribuem na transparência da córnea. Especificamente, o papel dos fatores de crescimento na mediação do controle funcional normal da córnea, incluindo proliferação epitelial, prevenção da quebra da função de barreira, manutenção do processo de renovação são discutidos em relação às entidades clínicas envolvidas na córnea.

The corneal epithelium: clinical relevance of cytokine-mediated responses to maintenance of corneal health.

We review the growth factor receptor-mediated cell signaling events that induce the responses required for the maintenance of corneal epithelial health. Our focus is to show how such responses contribute to sustaining corneal transparency and deturgescence, so basic to the pathogenesis of corneal diseases. Furthermore, we point out how alterations of receptor-mediated control of these responses account for losses in corneal transparency. In particular, the roles of growth factors in the mediation of normal corneal function, including epithelial cell proliferation, prevention of compromise of the barrier function of the cornea, and maintenance of normal renewal processes are discussed in relation to clinical entities involving the cornea.

Characterization of regulatory volume behavior by fluorescence quenching in human corneal epithelial cells.

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), tau(c) = 19 min. In contrast, a hypotonic challenge induced a rapid (tau(c) = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24 degrees C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 microM), ouabain (1 mM), DIDS (1 mM), EIPA (100 microM), or Na(+)-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K+, Cl- channel and K(+)-Cl(-) cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 microM), high K+ (20 mM) or Cl(-)-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K+ and Cl- channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na(+)-K(+)-2Cl(-) cotransporter (Na(+)-K(+)-2Cl(-)) and the Na(+)-K(+) pump. Inhibition of K+ and Cl- channels and KCC but not Na(+)-K(+)-2Cl(-) affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.

Functional and molecular characterization of a volume-activated chloride channel in rabbit corneal epithelial cells.

We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light-scattering measurements evaluated temporal cell-volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 microM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 microM), whereas inhibition by both NPPB (100 microM) and niflumic acid (500 microM) was voltage-independent. VACC was insensitive to glibenclamide (250 microM), verapamil (500 microM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.

EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca2+]i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca2+]i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 microM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca2+ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn2+ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 microM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 microM UO126 (MEK-1/2 inhibitor) or 10 microM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca2+]i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP3R isoforms 1-3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [3H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erkl/2 activity, whereas PKA stimulation suppresses these effects of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

Hypertonicity-induced p38MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity.

In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90- and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30-40% of their basal values. SB203580, a specific p38 inhibitor, dose dependently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery.

Modulation of rabbit corneal epithelial cell proliferation by growth factor-regulated K(+) channel activity.

We characterized the dependence of the mitogenic response by rabbit corneal epithelial (RCE) cells to serum containing growth factors on K(+) channel activation. Using both cell-attached and nystatin-perforated patch-clamp configurations, a K(+) channel was identified whose current-voltage relationship is linear with a single-channel conductance of 31 pS. Its activity was barely detectable following 24 h serum starvation. Exposure of starved cells to either 10% FBS, 5 ng/ml epidermal growth factor (EGF) or 2 n M endothelin-1 (ET-1) continuously increased its activity within 30 min by 40%, 54% and 29%, respectively. EGF and ET-1 in combination had additive effects on such activity. Application of 100 micro M 4-aminopyridine (4-AP), a K(+) channel blocker, inhibited serum-stimulated K(+) channel activity by 85%. DNA synthesis was markedly stimulated by serum, whereas incubation with either 4-AP (200 micro M) or Ba(2+) (1 m M) suppressed this increase by 51% and 23%, respectively, whereas 5 m M tetra ethyl ammonium (TEA) had no effect. Taken together, growth factor-induced increases in proliferation are dependent on K(+) channel stimulation. As the increases in K(+) channel activity induced by ET-1 and EGF were additive, these mitogens may stimulate K(+) channel activity through different signaling pathways linked to their cognate receptors.

Characterization of human taurine transporter expressed in insect cells using a recombinant baculovirus.

A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases.

Differential expression of Na:K:2Cl cotransporter, glucose transporter 1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues.

Little is known about whether culturing corneal limiting layers causes changes in the expression of their membrane transporter proteins from those present in fresh tissues. Accordingly, we compared mRNA abundance of three well-described types of transporters: water channel aquaporin 1 (AQP1), glucose transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC protein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, and NKCC was quantified by a lysate nuclease protection assay. NKCC transcription was further characterized by Northern blotting. All data were normalized to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Expression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. After the third passage, the endothelial cell mRNA abundance in each case decreased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there was a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA probe hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins with apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, membrane transporter function in culture may not be always indicative of their role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithelial and endothelial cells, there is expression of two different proteins and mRNAs that possibly encode for secretory (NKCC1) and absorptive (NKCC2) isoforms.

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells.

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 microm RG-13022) and cPLA2 activity (10 microm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 microg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 microm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 microm H-89); 2) Erk1/2 (15 microm PD98059) or 3) p38 (15 microm SB203580) activity. Stimulation protein kinase C activity (0.1 microm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 microm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.

Activation of a CFTR-mediated chloride current in a rabbit corneal epithelial cell line.

PURPOSE: To determine whether there is gene expression and functional activity of cystic fibrosis transmembrane conductance regulator protein (CFTR) in an SV40-immortalized rabbit corneal epithelial cell line, tRCE. METHODS: Both whole-cell and cell-attached patch-clamp techniques were used to examine the biophysical characteristics of the cAMP-dependent chloride current. The molecular identity of this conductance was evaluated using RT-PCR analysis. RESULTS: In whole-cell patch-clamp studies, a cAMP-dependent chloride conductance was further facilitated by the known CFTR activator genistein (20 microM). Kinetic analysis of cell-attached patches containing few channels ascertained that genistein increased the chloride channel activity by increasing channel open probability (via an increased channel open time and a decreased channel closed time). In addition, in the presence of a reduced forskolin concentration (i.e., 100 nM), the chloride conductance generated could be augmented by the nonspecific phosphodiesterase enzyme inhibitor, IBMX (100 microM), implicating the importance of intracellular cAMP in the regulation of this conductance. Furthermore, this conductance exhibited voltage-dependent inhibition in the presence of the CFTR chloride channel blocker glibenclamide (250 microM), but was DIDS insensitive (500 microM). Consistent with the presence of a CFTR-mediated chloride conductance, the expression of CFTR-mRNA was detected using RT-PCR. Sequence analysis of the product revealed 99.4% homology to that described for rabbit CFTR. CONCLUSIONS: In tRCE cells, there is gene expression and functional CFTR activity. Its presence may have important therapeutic implications in corneal epithelial diseases resulting from declines in transepithelial secretory and fluid transport activity.

Corneal epithelial wound healing.

One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. Furthermore, the smooth optical properties of the corneal epithelial surface are sustained through this renewal process. The rate of renewal is dependent on a highly integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. One experimental approach to characterize these three aspects of the renewal process has been to study the kinetics and dynamics of corneal re-epithelialization in a wound-healing model. This effort has employed in vivo and in vitro studies. From such studies it is evident that the appropriate integration and coordination of corneal epithelial proliferation, adhesion, migration, and cell demise is dependent on the actions of a myriad of cytokines. Our goal here is to provide an overview into how these mediators and environmental factors elicit control of cellular proliferation, adhesion, migration, and apoptosis. To this end we review the pertinent literature dealing with the receptor and the cell signaling events that are responsible for mediating cytokine control of corneal epithelial renewal. It is our hope that a better appreciation can be obtained about the complexity of the control processes that are responsible for assuring continuous corneal epithelial renewal in health and disease.

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport.

Although Na(+)-K(+)-2Cl(-) cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [(3)H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s(-1). Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na(+)-K(+)-2Cl(-) cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.

Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression.

PURPOSE: To determine in SV40-immortalized rabbit corneal epithelial cells (RCE), whether there is conservation of parent tissue serum growth-factor-stimulated cytokine receptor activation and downstream intracellular signaling events mediating control of cell cycle progression and differentiation. METHODS: Immunostaining and Western blot analysis were used to measure cytokeratin K3 and K12 expression with AE5 and AK12 antibodies. Karyotype analysis was performed based on comparison of the RCE chromosomal complement with its parent tissue. EGF receptor activation was evaluated based on immunochemistry and Western blot analyses of EGF receptor dimerization and phosphorylation. Functional status of EGF receptor was determined through measurements of EGF-induced stimulation of ERK-2 activity, which is a component of the mitogen-activated protein kinase cascade (MAPK). This was done by immunocomplex and kinase assay using anti-ERK antibodies and a specific substrate. EGF-induced increases in proliferation and cell cycle progression were determined based on measurements of [(3)H]-thymidine incorporation, G(2)-specific cyclin B1 expression and cell cycle mapping. RESULTS: From days 7 to 14, K12 expression increased based on marked rises in the levels of a 55 kD band. At day 14, a 64 kD band also appeared indicative of K3 expression. Karyotype analysis showed that there were no chromosomal losses due to SV-40 transformation. Upon exposure to EGF (5 ng/ml) for 1 min, EGF receptors were activated and formed clusters indicating that autophosphorylation and multimerization of the EGF receptor were occurred. In the presence of serum growth factors or EGF, ERK-2 kinase activity was markedly increased with a bell-shaped time-dependent activation pattern. Cell cycle progression was analyzed in G(1)/S boundary synchronized RCE cells. After releasing the cells into modified Supplemented Hormonal Epithelium Medium containing 10% serum and DMEM/F-12 medium, 80% of the cells had entered the S phase within 2 h. In addition, time dependent changes in [(3)H]-hymidine incorporation over 8 h confirmed RCE passage through the G(1)/S checkpoint. There were more RCE cells entered the G(2)/M phase of cell cycle in the 6-8 h interval after their release. Another indication of cell cycle progression into the G(2)/M phase was that at 8-10 h cyclin B(1) expression reached its maximal level. CONCLUSIONS: RCE in passage number 12-20 are a physiologically relevant model for studies on growth factor receptor mediated control of cell cycle progression and differentiation in its parent tissue as each of these phenomena were conserved: 1) EGF-induced EGF receptor activation; 2) EGF-activated ERK signaling; 3) expression of cornea-specific differentiation markers; 4) karyotype profile; and 5) cell cycle control and progression.

Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells.

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1-100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 microM) decreased cell proliferation by 65% and the distribution of cells in the G1 phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.

Adaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity.

We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 microm bumetanide, proliferation measured as (3)H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive (86)Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC.