Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

Human material

The study followed the tenets of Declaration of Helsinki for the use of human subjects and the ARVO statement on human subjects. The use of human material was approved by the Ethics Committee of the University of Leipzig (approval #745, 07/25/2011). Eyes were obtained from post-mortem cornea donors without reported eye disease within 48 h of death. Written informed consent for the use of retinal cells in basic research was obtained from the relatives of all donors.

Materials

All cell culture materials were purchased from Gibco BRL (Paisley, UK). Recombinant human basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), hepatocyte growth factor (HGF), interleukin-1β (IL-1β), IL-1 receptor antagonist, platelet-derived growth factor-BB (PDGF), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNFα), and VEGF-A₁₆₅ were obtained from R&D Systems (Abingdon, UK). Recombinant human PlGF-2 was obtained from Reliatech (Braunschweig, Germany). Cyclosporin A, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), Gö6976, H-89, HIF inhibitor, LY294002, PD150606, PD98059, PP2, SP600125, SU1498, and U73122 were obtained from Calbiochem (Bad Soden, Germany). The following compounds were purchased from Tocris (Ellisville, MO): 666–15, A-438079, AR-C 118925XX, ARL-67156, caffeic acid phenethyl ester (CAPE), 8-(3-chlorostyryl) caffeine (CSC), GSK650394, MRS2179, SB203580, SR11302, the pannexin-blocking peptide ¹⁰panx, and the scrambled control peptide ¹⁰panxScr. Ac-YVAD-CMK, AG1478, and Stattic were from Enzo Life Science (Lausen, Switzerland). Dithiothreitol was obtained from Carl Roth (Karlsruhe, Germany), and PD173074 was kindly provided by Pfizer (Karlsruhe, Germany). Human-specific siRNA against NFAT5 and non-targeted control siRNA were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). AG1296, adenosine-5′-O-(α,β-methylene)-diphosphate (AOPCP), apyrase, indomethacin, N-acetyl-L-cysteine, N-nitrobenzylthioinosine (NBTI), 1,10-phenanthroline, ruthenium red, SB431542, and all other agents used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise.

The following antibodies were used: a rabbit anti-human β-actin (1:1,000; Cell Signaling, Frankfurt, Germany), a rabbit anti-c-Fos (1:1,000; Cell Signaling), a rabbit anti-c-Jun (1:1,000; Cell Signaling), a rabbit anti-phosphorylated c-Fos (1:1,000; ThermoFisher Scientific, Waltham, MA), a rabbit anti-phosphorylated c-Jun (1:1,000; Cell Signaling), a rabbit anti-cAMP response element-binding protein (CREB; 1:1,000; Cell Signaling), a rabbit anti-histone H3 (1:1,000; Cell Signaling), and anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:2,000; Cell Signaling).

Cell culture

The preparation and culture of RPE cells have been described previously [28]. After removing the vitreous and neural retina, RPE cells were mechanically harvested, separated by digestion with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA), and washed two times with phosphate-buffered saline (PBS). The cells were suspended in complete Ham F-10 medium containing 10% fetal bovine serum, glutamax II, and gentamycin, and were cultured in laminin-coated T-75 tissue culture flasks (Greiner, Nürtingen, Germany) in 95% air/5% CO₂ at 37 °C. Cell lines of passages 3–5 derived from a total of 34 Caucasian donors (mean ± standard deviation [SD] age, 76.4±11.3 years; 19 women, 15 men) were used; each line was used in three to ten different experiments. When a confluency of approximately 90% was achieved, the cells were cultured in serum-free medium for 16 h. Thereafter, test substances were added to the serum-free medium. Extracellular hyperosmolarity was induced with the addition of NaCl or sucrose to the culture medium. A decrease in the extracellular osmolarity to 60% of control was achieved with the addition of distilled water. Hypoxia was induced with the addition of the hypoxia mimetic CoCl₂ (150 µM) or with cell culture in a 0.2% O₂/5% CO₂ atmosphere. Pharmacological inhibitors were preincubated for 30 min.

RNA extraction and cDNA synthesis

Total RNA was extracted with the InviTrap Spin Universal RNA Mini Kit (Stratec Molecular, Berlin, Germany). The A₂₆₀/A₂₈₀ ratio of the optical density of the RNA samples (measured with NanoDrop 1000; peQLab, Erlangen, Germany) was between 1.95 and 2.05, indicating adequate RNA quality. The RNA samples were treated with DNase I, and cDNA was synthesized from 0.5 µg RNA with a reverse transcription kit (ThermoFisher Scientific).

Real-time RT–PCR analysis

Real-time reverse transcription (RT)–PCR was performed with the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Munich, Germany). The primer sequences are given in Table 1. The amplification reaction mixture (15 μl) consisted of 7.5 μl of 2×iQ Sybr Green Supermix (Bio-Rad), a specific primer set (0.2 µM each), and 1 μl (1.25 ng) cDNA. The following protocol was used: one cycle of denaturation at 95 °C for 3 min, 45 cycles denaturation at 95 °C for 30 s, annealing at 58 °C for 20 s, extension at 72 °C for 45 s, and melting curve at 55 °C with the temperature gradually increased for 0.5 °C up to 95 °C. To prove the correct lengths of the PCR products, the samples were analyzed with agarose gel electrophoresis. RT–PCR for β-actin mRNA was used as an internal control. The results were analyzed with the 2^-ΔΔCT method.

Western blot analysis

The cells were seeded at 5 × 10⁵ cells per well in 6-well plates and were cultured in fetal bovine serum (10%)-containing F-10 medium. When a confluency of 80–90% was achieved, the cells were growth arrested for 16 h in serum-free medium. Thereafter, NaCl (+ 100 mM) or CoCl₂ (150 µM) was added for a further 2, 4, or 6 h. After the medium was removed, the cells were washed twice with prechilled PBS (pH 7.4; Invitrogen, Paisley, UK, product number 20012-019) and scraped into 180 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail). The cell lysates were centrifuged at 20,124 ×g for 10 min. Equal amounts of cytosolic or nuclear protein (30 or 35 µg) were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Immunoreactive bands were probed with primary and secondary antibodies, and visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.

ELISA

Cells were seeded at 3 × 10³ cells per well in 12-well plates. After reaching a confluency of approximately 90%, the medium was changed to a serum-free medium for 16 h. NaCl (+ 100 mM) or CoCl₂ (150 µM) was added to the serum-free medium for 24 h. The levels of VEGF-A₁₆₅ and PlGF in the cultured media (100 µl) were quantified with enzyme-linked immunosorbent assay (ELISA; R&D Systems).

siRNA transfection

Cells were seeded at 7 × 10⁴ cells per well in 12-well plates. When a confluency of 60‒80% was achieved, NFAT5 siRNA or non-targeted siRNA (5 nM each) was transfected into the cells with HiPerFect reagent (Qiagen, Hilden, Germany) in serum (10%)-containing F-10 medium. After 48 h, the cells were cultured for 2 h in serum-free iso- or hyperosmotic medium (+ 100 mM NaCl). After RNA was extracted, the levels of the c-Fos and NFAT5 mRNAs were evaluated with real-time RT–PCR.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed with the DIG Gel Shift Kit 2nd Generation (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The following oligonucleotides that contained AP-1 binding sites (italics) were used: 5′-CGC TTG ATG ACT CAG CCG GAA-3′ and 5′-TTC CGG CTG AGT CAT CAA GCG-3′. The following oligonucleotides that contained CREB binding sites (italics) were used: 5′-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3′ and 5′-CTA GCT CTC TGA CGT CAG GCA ATC TCT-3′. The gel shift reaction was performed with 4 µg of nuclear extract in a total volume of 20 µl for 15 min at room temperature. To check the specificity of the DNA–protein interaction, unlabeled double-stranded oligonucleotides were used as the competitor in 200-fold excess. Electrophoresis was performed with a 6% non-denaturing PAGE; the separated oligonucleotide-protein complexes were transferred to a positively charged nylon membrane with electroblotting. DIG-labeled DNA fragments were detected with an anti-DIG antibody, and immunoreactive bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.

Statistical analysis

Each test involved at least three experiments using cell lines of different donors. Data are shown as means ± standard error of the mean (SEM). Statistical analysis was performed with Prism (GraphPad Software, San Diego, CA). Comparisons between groups were performed with one-way ANOVA followed by Bonferroni’s multiple comparison test and the Mann–Whitney U test. A p value of less than 0.05 was considered statistically significant.

Regulation of the expression of c-Fos and c-Jun genes

The transcription factor AP-1 is a heterodimer consisting of c-Fos and c-Jun [25]. Cultured human RPE cells expressed c-Fos and c-Jun genes (Figure 1A). To determine which pathogenic conditions induce expression of these genes, we stimulated RPE cells from different donors with the hypoxia mimetic CoCl₂ [29], high (25 mM) glucose, and hyper- and hypoosmotic media. The gene expression levels were determined with real-time RT–PCR analysis. As shown in Figure 1B, CoCl₂-induced chemical hypoxia induced statistically significant (p<0.05) increases in the expression of the c-Fos and c-Jun genes after 24 h of stimulation. Similar increases in the expression levels of both genes were found in cells that had been cultured in a low-O₂ atmosphere (Figure 1B). In the presence of high glucose, the levels of c-Fos and c-Jun mRNAs were decreased after 24 and 2 h, respectively (Figure 1B). Extracellular hyperosmolarity was induced with the addition of 100 mM NaCl or 200 mM sucrose (which caused equal increases in the extracellular osmolarity). The addition of 100 mM NaCl to the culture medium induced a strong increase in the expression of the c-Fos gene after 2 and 6 h of stimulation while the expression of the c-Jun gene remained unaltered (Figure 1B). The addition of 200 mM sucrose induced a moderate increase in the expression of the c-Fos gene after 2 h of stimulation and had no effect on the expression of the c-Jun gene (Figure 1C). Coadministration of NaCl and sucrose induced higher expression of the c-Fos gene than the addition of sucrose alone; there was a statistically significant (p<0.05) difference in the c-Fos mRNA level between cells cultured under both conditions after 6 h of stimulation (Figure 1C). The data indicate that the NaCl-induced expression of the c-Fos gene is mainly mediated by the alteration of the transmembrane NaCl gradient and less by the elevation of the extracellular osmolarity. The effect of high NaCl on the c-Fos mRNA level was dose-dependent; a statistically significant (p<0.05) increase was already found when 10 mM NaCl was added to the culture medium (Figure 1D). A hypoosmotic medium decreased the c-Fos and c-Jun mRNA levels in RPE cells (Figure 1B).

To determine whether the expression of the c-Fos gene in RPE cells is regulated by inflammatory and growth factors, we stimulated the cells with various cytokines and fetal calf serum, respectively. As shown in Figure 1E, exogenous bFGF and HB-EGF induced moderate increases in the c-Fos mRNA level after different time periods, PDGF and serum induced a biphasic regulation, and VEGF, TGF-β1, PlGF-2, IL-1β, and TNFα induced downregulation of the expression of the c-Fos gene after various time periods. The data indicate that the expression of the c-Fos gene in RPE cells is strongly induced by an elevation of the extracellular NaCl concentration while other factors, such as inflammatory and growth factors, induce moderate upregulation or downregulation of the expression of the c-Fos gene. In contrast, the expression of the c-Jun gene in RPE cells is not induced by high extracellular NaCl.

NaCl-induced expression of the genes of additional transcription factor proteins

AP-1 is a heterodimer composed of proteins belonging to various protein families. We investigated whether the gene expression of additional proteins belonging to the Jun, Fos, ATF, JDP, and CREB protein families (Figure 2A) is altered by high extracellular osmolarity induced with the addition of 100 mM NaCl to the medium. As shown in Figure 2B, the addition of high NaCl induced a strong increase in the expression of FOSB (Gene ID 2354; OMIM 164772) while the expression of the following genes remained unaltered or was moderately altered: JUND (Gene ID 3727; OMIM 165162), FOSL1 (Gene ID 8061; OMIM 136515), FOSL2 (Gene ID 2355; OMIM 601575), ATF2 (Gene ID 1386; OMIM 123811), ATF3 (Gene ID 467; OMIM 603148), JDP1 (Gene ID 55509; OMIM 612470), JDP2 (Gene ID 122953; OMIM 608657), and CREB1 (Gene ID 1385; OMIM 123810). The levels of the JUNB and BATF transcripts were below the detection threshold of the RT–PCR analysis.

Regulation of the expression of c-Fos and c-Jun proteins

To determine whether NaCl-induced extracellular hyperosmolarity and CoCl₂-induced chemical hypoxia induce alterations of the c-Fos and c-Jun protein levels in RPE cells, we determined the protein levels in cell lysates and nuclear extracts with western blot analysis. Under unstimulated control conditions, the cells contained a low level of c-Fos protein and a high level of c-Jun protein (Figure 3A), suggesting that c-Jun is constitutively produced by RPE cells. The addition of 100 mM NaCl to the culture medium induced a statistically significant (p<0.05) time-dependent increase in the cytosolic and nuclear levels of the c-Fos protein (Figure 3A,B,D). High NaCl had no effect on the cellular content of the c-Jun protein (Figure 3A,C). Chemical hypoxia did not alter the levels of the c-Fos and c-Jun proteins in RPE cells (Figure 3A–C).

Regulation of AP-1 activity

Phosphorylation of c-Jun by c-Jun NH₂-terminal kinases (JNKs) or extracellular signal-regulated kinases 1 and 2 (ERK1/2) stimulates the AP-1-mediated transcription of the target genes [30,31]. Therefore, the level of phosphorylated c-Jun protein reflects the level of AP-1 activity. We found that stimulation of RPE cells with high NaCl induced statistically significant (p<0.05) increases in the cytosolic and nuclear levels of phosphorylated c-Jun protein after 2 and 6 h of stimulation (Figure 3A,C,D). The level of the phosphorylated c-Jun protein was also increased within 2 h of stimulation with the hypoxia mimetic CoCl₂ (Figure 3C). The level of the phosphorylated c-Fos protein did not alter in response to high NaCl and CoCl₂ (Figure 3A,B). EMSA showed that hyperosmolarity induced increased DNA binding of the AP-1 protein but not increased DNA binding of CREB (Figure 4). The data suggest that NaCl-induced extracellular hyperosmolarity induces activation and DNA binding of AP-1 in RPE cells.

Intracellular signaling involved in NaCl-induced expression of the c-Fos gene

To investigate the intracellular signaling involved in mediating the NaCl-induced expression of the c-Fos gene in RPE cells, we tested pharmacological blockers of key intracellular signal transduction molecules in cell cultures that had been stimulated for 2 h with high (+ 100 mM) NaCl. As shown in Figure 5A, the expression of the c-Fos gene under control and high-NaCl conditions was statistically significantly (p<0.05) decreased by the JNK inhibitor SP600125. The expression of the c-Fos gene under high-NaCl conditions was statistically significantly (p<0.05) increased by the inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK), SB203580, and the inhibitor of phosphatidylinositol-3 kinase (PI3K)-related kinases, LY294002, while the inhibitor of ERK1/2, PD98059, had no effect (Figure 5A). SB203580 and PD98059 increased the expression of the c-Fos gene under control conditions (Figure 5A). The data suggest that the constitutive and NaCl-induced expression of the c-Fos gene in RPE cells is (at least in part) mediated by the JNK signal transduction pathway, while the p38 MAPK and PI3K signal transduction pathways inhibit the NaCl-induced expression of the c-Fos gene.

The NaCl-induced expression of the c-Fos gene in RPE cells was also decreased by inhibitors of phospholipase Cγ (PLCγ; U73122), calcium-binding proteins (ruthenium red), protein kinases C (PKC) α/β (Gö6976), protein kinase A (PKA; H-89), and Src tyrosine kinases (PP2), and nearly completely suppressed by the inhibitor of the serum and glucocorticoid-regulated kinase (SGK), GSK650394 (Figure 5B). The calpain inhibitor PD150606 had no effect (Figure 5B). In addition, the cyclooxygenase (COX) inhibitor indomethacin and the inhibitor of mitochondrial permeability transition, cyclosporin A, decreased the NaCl-induced expression of the c-Fos gene (Figure 5B). The cell-permeable reducing agent dithiothreitol and the reactive oxygen species inhibitor N-acetyl-L-cysteine had no effects (Figure 5B). The data suggest that the expression of the c-Fos gene under high-NaCl conditions is (in part) mediated by the activities of PLCγ, PKCα/β, PKA, Src tyrosine kinases, SGK, and COX, as well as by a loss of the mitochondrial integrity.

Extracellular signaling involved in NaCl-induced expression of the c-Fos gene

It has been shown that high extracellular NaCl induces the release of different growth factors, like VEGF, bFGF, and TGF-β1, from RPE cells [22,32]. Because various growth factors were found to induce expression of the c-Fos gene in RPE cells (Figure 1E), we determined whether autocrine/paracrine growth factor receptor signaling is required for the NaCl-induced expression of the c-Fos gene. We found that the NaCl-induced expression of the c-Fos gene in RPE cells was decreased by the inhibitor of TGF-β1 superfamily activin receptor-like kinase receptors, SB431542, and nearly completely suppressed by the inhibitor of the FGF receptor kinase, PD173074 (Figure 6A). Inhibition of the epidermal growth factor (EGF) receptor tyrosine kinase by AG1478 had no effect, while inhibition of the PDGF receptor tyrosine kinase by AG1296 and of the VEGF receptor-2 by SU1498 increased statistically significantly (p<0.05) the NaCl-induced expression of the c-Fos gene (Figure 6A). The broad-spectrum matrix metalloproteinase inhibitor 1,10-phenanthroline had no effect (Figure 6A), suggesting that shedding of growth factors from the extracellular matrix is not involved in mediating the NaCl-induced expression of the c-Fos gene. Furthermore, a caspase-1 inhibitor (Ac-YVAD-CMK) and a recombinant human IL-1 receptor antagonist had no effects (Figure 6A), suggesting that activation of the inflammasome [33] does not influence the NaCl-induced expression of the c-Fos gene. The data suggest that autocrine/paracrine activation of the FGF receptor kinase and (to a lesser extent) activation of TGF-β1 superfamily activin receptor-like kinase receptors are involved in mediating the NaCl-induced expression of the c-Fos gene in RPE cells. Autocrine/paracrine activation of the PDGF receptor tyrosine kinase and of the VEGF receptor-2 probably suppresses the NaCl-induced expression of the c-Fos gene.

It has been shown that high extracellular NaCl induces the release of adenosine 5′-triphosphate (ATP), a well-known cellular danger signal [34], from RPE cells [33]. To determine whether purinergic receptor signaling is involved in mediating the NaCl-induced expression of the c-Fos gene in RPE cells, we used pharmacological receptor antagonists. We found that the addition of the ATP-hydrolyzing enzyme apyrase to the culture medium decreased the NaCl-induced expression of the c-Fos gene in RPE cells (Figure 6B) to a similar extent as the inhibitor of PLCγ (U73122) and the blocker of PKCα/β (Gö6976), for example (Figure 5B). The NaCl-induced expression of the c-Fos gene was also statistically significantly (p<0.05) reduced by the adenosine A₁ receptor antagonist DPCPX, the antagonist of nucleoside transporters, NBTI, and the pannexin-blocking peptide ¹⁰panx while a scrambled control peptide had no effect (Figure 6B). Antagonists of P2Y₁ (MRS2179), P2Y₂ (AR-C 118925XX), P2X₇ (A-438079), and adenosine A_2A receptors (CSC), as well as the ecto-ATPase inhibitor ARL-67156, had no effects on the NaCl-induced expression of the c-Fos gene (Figure 6B). The data suggest that a pannexin-mediated release of ATP from RPE cells, extracellular degradation of ATP, a transporter-mediated release of adenosine, and autocrine/paracrine activation of adenosine A₁ receptors contribute to the full induction of the expression of the c-Fos gene under high-NaCl conditions.

Transcription factor activities involved in NaCl-induced expression of the c-Fos gene

It has been shown that hyperosmotic stress induces expression of various transcription factors in RPE cells, such as HIF-1α, NF-κB, and NFAT5 [22,24]. To investigate which transcription factors mediate the NaCl-induced expression of the c-Fos gene in RPE cells, we used pharmacological blockers. As shown in Figure 7A, the NaCl-induced expression of the c-Fos gene was not altered in the presence of the AP-1 inhibitor SR11302, the CREB inhibitor 666–15, and the signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic [35]. An HIF inhibitor [36] and the NF-κB inhibitor CAPE [37] increased statistically significantly (p<0.05) the expression of the c-Fos gene under the control and NaCl-stimulated conditions (Figure 7A), suggesting that HIF and NF-κB exert negative regulation of expression of the c-Fos gene in RPE cells. Negative regulation of the constitutive expression of the c-Fos gene exerted by various transcription factors, including AP-1, CREB, HIF, and NF-κB, is suggested by the statistically significant (p<0.05) increases in the c-Fos mRNA level under the unstimulated control conditions (Figure 7A).

In various cell systems, cellular survival in hyperosmotic stress depends on the transcriptional activity of NFAT5 [38]. It has been shown that high extracellular osmolarity increases the levels of the NFAT5 mRNA and protein, and induces DNA binding of the NFAT5 protein, in RPE cells [22]. To determine whether NFAT5 activity is involved in mediating the NaCl-induced expression of the c-Fos gene in RPE cells, we knocked down NFAT5 with transfection of the cells with NFAT5 siRNA. As negative control, non-targeted scrambled siRNA was used. As shown in Figure 7B, transfection with NFAT5 siRNA reduced the level of the NFAT5 transcripts by approximately 50% in the cells cultured under the control and high-NaCl conditions whereas transfection with non-targeted siRNA had no effect. Cells transfected with NFAT5 siRNA also displayed a statistically significant (p<0.05) lower level of c-Fos gene transcripts under high-NaCl conditions than cells transfected with non-targeted siRNA (Figure 7C). Knockdown of NFAT5 had no effect on the cellular level of the c-Fos gene transcripts under the control conditions (Figure 7C). The data suggest that the NaCl-induced expression of the c-Fos gene in RPE cells is (at least in part) mediated by the activity of NFAT5.

Role of AP-1 activity in NaCl-induced expression of genes

It has been shown that hyperosmotic stress induces expression of different genes in RPE cells, including genes that code for transcription factors, growth factors, NACHT, LRR and PYD domains-containing protein 3 (NLRP3), pro-IL-1β, aquaporin (AQP) water channels, COX, and aldose reductase [5,22,24,32,33,39]. To investigate which genes are transcriptionally activated by AP-1 activity in response to high extracellular NaCl, we stimulated RPE cells with high (+ 100 mM) NaCl in the absence and presence of the AP-1 inhibitor SR11302. As shown in Figure 8, the NaCl-induced expression of the the HIF-1α (Gene ID 3091; OMIM 603348) and NFAT5 (Gene ID 10725; OMIM 604708) genes was statistically significantly (p<0.05) reduced in the presence of SR11302. Inhibition of AP-1 activity also caused reductions in the NaCl-induced expression of the VEGF, PlGF, and TGF-β2 genes while the NaCl-induced expression of the bFGF (Gene ID 2247; OMIM 134920) and HB-EGF (Gene ID 1839; OMIM 126150) genes remained unaltered (Figure 8). Inhibition of AP-1 activity had no effects on the NaCl-induced expression of various other genes, including the NLRP3 (Gene ID 114548; OMIM 606416), pro-IL-1β (Gene ID 3553; OMIM 147720), AQP8 (Gene ID 343; OMIM 603750), AQP5 (Gene ID 362; OMIM 600442), COX-2 (Gene ID 5743; OMIM 600262), and aldose reductase genes (AKR1B1; Gene ID 231; OMIM 103880) genes (Figure 8). The data suggest that AP-1 activity is involved in mediating the NaCl-induced expression of various transcription factor (HIF-1α and NFAT5) and growth factor genes (VEGF, PlGF, and TGF-β2; Gene ID 7042; OMIM 190220).

Involvement of AP-1 activity in NaCl-induced secretion of PlGF

It has been shown that extracellular hyperosmolarity induces secretion of VEGF and PlGF from RPE cells [5,22]. To determine whether AP-1-mediated production of VEGF and PlGF contributes to the secretion of these factors, we inhibited AP-1 activity with SR11302. High extracellular NaCl induced the secretion of VEGF (Figure 9A) and PlGF (Figure 9B) from RPE cells, whereas chemical hypoxia induced the secretion of VEGF but not of PlGF (Figure 9A, B). SR11302 fully prevented the NaCl-induced secretion of PlGF (Figure 9B) and had no effects on the NaCl-induced and hypoxic secretion of VEGF (Figure 9A). However, the inhibitor of the FGF receptor kinase, PD173074, suppressed statistically significantly (p<0.05) the secretion of VEGF and PlGF under both conditions (Figure 9A, B). SR11302 increased the secretion of VEGF statistically significantly (p<0.05) under unstimulated control conditions (Figure 9A), suggesting that inhibition of the AP-1-mediated VEGF production may represent a stress condition for the cells.