Osmotic induction of cyclooxygenase-2 in RPE cells: Stimulation of inflammasome activation

Abstract

Purpose: Systemic hypertension is a risk factor of age-related macular degeneration, a disease associated with chronic retinal inflammation. The main cause of acute hypertension in the elderly is consumption of dietary salt (NaCl) resulting in increased extracellular osmolarity. The aim of the present study was to determine whether extracellular osmolarity regulates the expression of cyclooxygenase (COX) genes in cultured human retinal pigment epithelial (RPE) cells, and whether COX activity is involved in mediating the osmotic expression of key inflammatory (NLRP3 and IL1B) and angiogenic factor (VEGFA) genes.

Methods: Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Gene expression was determined with real-time reverse transcription (RT)-PCR. Cytosolic interleukin-1β (IL-1β) and extracellular vascular endothelial growth factor (VEGF) levels were evaluated with enzyme-linked immunosorbent assay (ELISA).

Results: Extracellular hyperosmolarity induced a dose-dependent increase in COX2 gene expression when >10 mM NaCl was added to the culture medium, while COX1 gene expression was increased at higher doses (>50 mM of added NaCl). Extracellular hypo-osmolarity decreased COX2 gene expression. High extracellular osmolarity also induced increases in the COX2 protein level. NaCl-induced expression of COX2 was mediated by various intracellular signal transduction molecules (p38 mitogen-activated protein kinase [p38 MAPK], extracellular signal-regulated kinases 1 and 2 [ERK1/2], and phosphatidylinositol-3 kinase [PI3K]), intracellular calcium signaling involving activation of phospholipase Cγ (PLCγ) and protein kinase Cα/β (PKCα/β), and the activity of nuclear factor of activated T cell 5 (NFAT5). Inhibition of fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and interleukin-1 (IL-1) receptor activities decreased NaCl-induced COX2 gene expression. Selective inhibition of COX2 activity decreased osmotic expression of the VEGFA, IL1B, and NLRP3 genes, and blocked the NaCl-induced increase in the cytosolic IL-1β level.

Conclusions: The expression of COX2 in RPE cells is osmoresponsive, and depends on NFAT5. COX2 activity stimulates hyperosmotic expression of angiogenic (VEGFA) and inflammatory factor (IL1B and NLRP3) genes, and activation of the NLRP3 inflammasome in RPE cells.

Figure 1

Regulation of cyclooxygenases -1 and -2 (COX1 and COX2) gene expression in retinal pigment epithelial (RPE) cells. A: Presence of COX1 and COX2 transcripts in RPE cells. To confirm the correct lengths of the PCR products, agarose gel electrophoresis was performed using products obtained from two RPE cell lines (1, 2) derived from different post-mortem donors. Negative controls (0) were done by adding double-distilled water instead of cDNA as the template. The β-actin (ACTB) mRNA level was used to normalize the COX1 and COX2 mRNA levels. B–H: COX1 and COX2 mRNA levels, as determined with real-time reverse transcription (RT)–PCR after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars). The mRNA levels are expressed as folds of unstimulated control. B: Effects of extracellular hyperosmolarity induced by addition of high (+ 100 mM) NaCl on the COX1 and COX2 gene expression levels. C, D: Dose-dependencies of the effects of high extracellular NaCl on the COX1 and COX2 mRNA levels. Ten to 100 mM NaCl were added to the culture medium, as indicated in the bars. The data were obtained after stimulation of the cells for 6 h (C) and 24 h (D). E: Effect of extracellular hypo-osmolarity (Hypo; 60% osmolarity) on the COX2 gene expression level. F: Effect of the addition of 200 mM sucrose on the COX2 gene expression level. G: Effects of hypoxia induced by cell culture in 1% O₂, and by addition of 150 µM CoCl₂, on the COX2 gene expression level. H: Effects of inflammatory and growth factors on the expression of COX2. The following factors were tested: endothelial growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), placental growth factor-2 (PlGF-2), pigment epithelium-derived factor (PEDF), interleukin-1β (IL-1β), and matrix metalloproteinase-2 (MMP-2). Each factor was applied at 10 ng/ml. Each bar represents data obtained in three to ten independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to six donors; in total, cell lines from 21 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05.

Figure 2

Regulation of cyclooxygenase-2 (COX2) protein expression in retinal pigment epithelial (RPE) cells. The cells were stimulated for 6 and 24 h with high (+ 100 mM) NaCl, 200 mM sucrose, and the hypoxia mimetic CoCl₂ (150 µM), respectively. Protein levels were determined with western blotting of cell lysates. A: Examples of western blotting of two cell lines (above and below). The levels of the β-actin and COX2 proteins are shown. B: Mean ± standard error of the mean (SEM) cytosolic level of the COX2 protein. The data are normalized to the level of β-actin protein, and are expressed as a percent of the unstimulated control (100%). Each bar represents data obtained in four to five independent experiments with cell lines from different donors. Each experiment was performed with cell lines from four to five donors. Statistically significant difference versus unstimulated control: *p<0.05.

Figure 3

Intracellular signaling involved in mediating NaCl-induced expression of cyclooxygenase-2 (COX2) in retinal pigment epithelial (RPE) cells. The mRNA level was determined with real-time reverse transcription (RT)–PCR in cells cultured 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and is expressed as folds of the unstimulated control. The following compounds were tested: the inhibitor of p38 mitogen-activated protein kinase (p38 MAPK) activation, SB203580 (10 µM), the inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, PD98059 (20 µM), the inhibitor of phosphatidylinositol-3 kinase (PI3K)-related kinases, LY294002 (5 µM), the c-Jun NH₂-terminal kinase (JNK) inhibitor SP600125 (10 µM), the cell-permeable calcium chelator BAPTA/AM (5 µM), the phospholipase Cγ (PLCγ) inhibitor U73122 (4 µM), the phospholipase D1 (PLD1) inhibitor VU0359595 (150 nM), the phospholipase D2 (PLD2) inhibitor VU0285655–1 (500 nM), the inhibitor of protein kinase Cα/β (PKCα/β), Gö6976 (1 µM), the serum and glucocorticoid-regulated kinase (SGK) inhibitor GSK650394 (GSK; 1 µM), the protein kinase A inhibitor H-89 (1 µM), the inhibitor of Src tyrosine kinases, PP2 (100 nM), the caspase-1 inhibitor Ac-YVAD-CMK (500 nM), the reducing agent dithiothreitol (DTT; 3 mM), the inhibitor of mitochondrial permeability transition, cyclosporin A (CsA; 1 µM), the anti-inflammatory glucocorticoid triamcinolone acetonide (Triam; 50 µM), and the COX2 antagonist NS-398 (50 µM). Each bar represents data obtained in three to 20 independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to seven donors; in total, cell lines from 26 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05.

Figure 4

Receptor-mediated signaling involved in mediating NaCl-induced expression of cyclooxygenase-2 (COX2) in retinal pigment epithelial (RPE) cells. The mRNA level was determined with real-time reverse transcription (RT)–PCR in cells cultured 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and is expressed as folds of the unstimulated control. The following agents were tested: the inhibitor of the fibroblast growth factor (FGF) receptor kinase, PD173074 (500 nM), the blocker of vascular endothelial growth factor (VEGF) receptor-2, SU1498 (10 µM), the blocker of the endothelial growth factor (EGF) receptor tyrosine kinase, AG1478 (600 nM), the inhibitor of the platelet-derived growth factor (PDGF) receptor tyrosine kinase, AG1296 (10 µM), the inhibitor of transforming growth factor-β1 (TGF-β1) superfamily activin receptor-like kinase receptors, SB431542 (10 µM), the broad-spectrum matrix metalloproteinase inhibitor 1,10-phenanthroline (1,10-Phen; 10 µM), and a recombinant human interleukin-1 receptor antagonist (IL-1RA; 1 µg/ml). Vehicle control was made with dimethyl sulfoxide (DMSO; 1:1000). Each bar represents data obtained in three to 24 independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to seven donors; in total, cell lines from 23 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05.

Figure 5

Transcription factor activity involved in mediating NaCl-induced expression of cyclooxygenase-2 (COX2) in retinal pigment epithelial (RPE) cells. The mRNA level was determined with real-time reverse transcription (RT)–PCR in cells cultured 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and is expressed as folds of the unstimulated control. A: The following compounds were tested: a hypoxia-inducible transcription factor (HIF)-1 inhibitor (HIF-Inh; 5 µM), the signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic (1 µM), the nuclear factor (NF)-κB inhibitor CAPE (5 µM), the activator protein-1 (AP-1) inhibitor SR11302 (5 µM), and the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM). B: Knocking down nuclear factor of activated T cell 5 (NFAT5) with small interfering RNA (siRNA) reduced the level of COX2 mRNA under hyperosmotic conditions. Nontargeted siRNA (siNon) had no effects. Each bar represents data obtained in three to nine independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to six donors; in total, cell lines from 21 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05. Statistically significant difference versus nontargeted siRNA: ○p<0.05.

Figure 6

Effects of cyclooxygenase-2 (COX2) inhibition by NS-398 (50 μM) and the anti-inflammatory glucocorticoid triamcinolone acetonide (Triam; 50 μM) on NaCl-induced expression of the vascular endothelial growth factor A (VEGFA), nucleotide-binding oligomerization domain receptors-like receptor protein 3 (NLRP3), interleukin-1ß (IL1B), and transforming growth factor-ß2 (TGFB2) genes in retinal pigment epithelial (RPE) cells. The mRNA levels were determined with real-time reverse transcription (RT)–PCR in cells cultured 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and are expressed as folds of the unstimulated control. Vehicle control was made with dimethyl sulfoxide (DMSO; 1:1,000). Each bar represents data obtained in four to ten independent experiments with cell lines from different donors. Each experiment was performed with cell lines from four to six donors; in total, cell lines from nine donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05.

Figure 7

Effects of cyclooxygenase-2 (COX2) inhibition on NaCl-induced vascular endothelial growth factor A₁₆₅ (VEGF-A₁₆₅) and mature interleukin-1ß (IL-1ß) protein levels. A: Effects of COX2 inhibition on NaCl-induced increases of the extracellular level of VEGF-A₁₆₅ protein and (B) the cytosolic IL-1ß protein level. The cells were stimulated for 24 (A) and 6 h (B) with high (+ 100 mM) NaCl in the absence and presence of the COX2 antagonist NS-398 (50 µM) and the anti-inflammatory glucocorticoid triamcinolone acetonide (Triam; 50 µM), respectively. Vehicle control was made with dimethyl sulfoxide (DMSO; 1:1,000). The protein levels in the cultured media (A) and in the cell lysates (B) were determined with enzyme-linked immunosorbent assay (ELISA). Each bar represents data obtained in three to five independent experiments with cell lines from different donors. Each experiment was performed with cell lines from four to five donors. Statistically significant difference versus unstimulated control (100%): *p<0.05. Statistically significant difference versus NaCl control: ●p<0.05.

Figure 8

Schematic summary of the intracellular signal transduction pathways implicated in mediating NaCl-induced cyclooxygenase-2 (COX2) gene expression and the effects of COX2 activity in retinal pigment epithelial (RPE) cells. High extracellular NaCl induces release of inflammatory and growth factors from RPE cells; autocrine or paracrine activation of fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and interleukin-1 (IL-1) receptors increases NaCl-induced expression of the COX2 gene. NaCl-induced expression of the COX2 gene is dependent on intracellular calcium signaling mediated by phospholipase Cγ (PLCγ) and IP₃-induced release of calcium from the endoplasmic reticulum (ER), as well as phosphopolipase A₂ (PLA₂) activity. These events are likely implicated in activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and phosphatidylinositol-3 kinase (PI3K) signal transduction pathways that contribute to the expression of the osmosensitive transcription factor nuclear factor of activated T cell 5 (NFAT5). NFAT5 activity is involved in mediating NaCl-induced COX2 gene expression. COX2 activity stimulates the hyperosmotic expression of angiogenic (VEGFA) and inflammatory factor (NLRP3 and IL1B) genes, and IL-1β production by the activated NLRP3 inflammasome.