Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress.

PURPOSE: Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation. METHODS: ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites. RESULTS: In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38α and p38ß) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation. CONCLUSIONS: Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.

Modification of aquaporin expression in response to fenretinide-induced transdifferentiation of ARPE-19 cells into neuronal-like cells.

PURPOSE: The goal of this study was to investigate the modifications of aquaporin (AQP) expression in ARPE-19 cells in response to fenretinide-induced transdifferentiation into neuronal-like cells METHODS: ARPE-19 cells were treated daily for 7 days with 3 µm fenretinide or dimethyl sulphoxide as control. mRNA and protein expression were evaluated by real-time quantitative PCR, Western blot analysis and immunofluorescence. RESULTS: Control ARPE-19 cells expressed AQP1, AQP4, AQP6 and AQP11 at the mRNA level, but only AQP4, AQP6 and AQP11 at the protein level. Fenretinide induced the transdifferentiation of ARPE-19 cells into neuronal-like cells. Indeed, fenretinide induced morphological changes similar to neurons characterized by elongated cell body and the formation of neurite branching. Moreover, ARPE-19 cells transdifferentiated to neuron-like cells were characterized by significant decrease in retinal pigmented epithelium markers, for example cytokeratin 8 and cellular retinaldehyde-binding protein, as well as an increase in neuronal markers such as synaptophysin and calretinin. AQP4 expression, at both mRNA and protein levels, and AQP6 expression, only at protein level, were significantly decreased in ARPE-19 cells transdifferentiated into neuronal-like cells. CONCLUSIONS: The expression of AQP4 and AQP6 is downregulated during fenretinide-induced transdifferentiation.

Origins and consequences of hyperosmolar stress in retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is composed of retinal pigmented epithelial cells joined by tight junctions and represents the outer blood-retinal barrier (BRB). The inner BRB is made of endothelial cells joined by tight junctions and glial extensions surrounding all the retinal blood vessels. One of the functions of the RPE is to maintain an osmotic transepithelial gradient created by ionic pumps and channels, avoiding paracellular flux. Under such physiological conditions, transcellular water movement follows the osmotic gradient and flows normally from the retina to the choroid through the RPE. Several diseases, such as diabetic retinopathy, are characterized by the BRB breakdown leading to leakage of solutes, proteins, and fluid from the retina and the choroid. The prevailing hypothesis explaining macular edema formation during diabetic retinopathy incriminates the inner BRB breakdown resulting in increased osmotic pressure leading in turn to massive water accumulation that can affect vision. Under these conditions, it has been hypothesized that RPE is likely to be exposed to hyperosmolar stress at its apical side. This review summarizes the origins and consequences of osmotic stress in the RPE. Ongoing and further research advances will clarify the mechanisms, at the molecular level, involved in the response of the RPE to osmotic stress and delineate potential novel therapeutic targets and tools.

Aquaporin-1 expression in proliferative vitreoretinopathy and in epiretinal membranes.

PURPOSE: Aquaporin-1 (AQP1) is involved in cell migration and proliferation; therefore, the purpose of the study was to investigate its expression in proliferative vitreoretinopathy (PVR) and epiretinal membranes (ERM). METHODS: 19 membranes from PVR and ERM were collected following eye surgery. AQP1 mRNA and protein expressions were determined by RT-qPCR and immunofluorescence in the membranes from PVR and ERM. RESULTS: AQP1 mRNA and protein were expressed in both PVR and ERM as shown by RT-qPCR and immunofluorescence. AQP1 protein expression was heterogeneous among and between PVR and ERM and colocalized with alpha-smooth muscle actin ( α SMA) and with glial fibrillary acidic protein (GFAP). There were a higher percentage of cells coexpressing AQP1 and α SMA than AQP1 and GFAP. GFAP and α SMA did not colocalize. CONCLUSION: Our data show for the first time AQP1 expression in both PVR and ERM. AQP1 is expressed mostly by the α SMA-positive cells, presumably myofibroblasts, but also by GFAP-positive cells, assumed to be glial cells. These original findings warrant further functional investigations aiming at studying the potential role of AQP1 in cell migration and proliferation occurring during the development of PVR and ERM.