Osmotic regulation of NFAT5 expression in RPE cells: The involvement of purinergic receptor signaling

Purpose: Systemic hypertension is a risk factor for age-related neovascular retinal diseases. The major condition that induces hypertension is the intake of dietary salt (NaCl) resulting in increased extracellular osmolarity. High extracellular NaCl was has been shown to induce angiogenic factor production in RPE cells, in part via the transcriptional activity of nuclear factor of activated T cell 5 (NFAT5). Here, we determined the signaling pathways that mediate the osmotic expression of the NFAT5 gene in RPE cells. Methods: Cultured human RPE cells were stimulated with high (+100 mM) NaCl. Alterations in gene and protein expression were determined with real-time reverse transcriptase (RT)-PCR and western blot analysis, respectively. Results: NaCl-induced NFAT5 gene expression was fully inhibited by calcium chelation and blockers of inositol triphosphate (IP3) receptors and phospholipases C and A(2). Blockers of phospholipases C and A(2) also prevented the NaCl-induced increase of the cellular NFAT5 protein level. Inhibitors of multiple intracellular signaling transduction pathways and kinases, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), phosphatidylinositol-3 kinase (PI3K), protein kinases A and C, Src tyrosine kinases, and calpains, as well as cyclooxygenase inhibitors, decreased the NaCl-induced expression of the NFAT5 gene. In addition, autocrine purinergic signaling mediated by a release of ATP and a nucleoside transporter-mediated release of adenosine, activation of P2X(7), P2Y(1), P2Y(2), and adenosine A(1) receptors, but not adenosine A(2A) receptors, is required for the full expression of the NFAT5 gene under hyperosmotic conditions. NaCl-induced NFAT5 gene expression is in part dependent on the activity of nuclear factor kappa B (NF-kappa B). The NaCl-induced expression of NFAT5 protein was prevented by inhibitors of phospholipases C and A(2) and an inhibitor of NF-kappa B, but it was not prevented by a P2Y(1) inhibitor. Conclusions: The data suggest that in addition to calcium signaling and activation of inflammatory enzymes, autocrine/paracrine purinergic signaling contributes to the stimulatory effect of hyperosmotic stress on the expression of the NFAT5 gene in RPE cells. It is suggested that high intake of dietary salt induces RPE cell responses, which may contribute to age-related retinal diseases.