Osmotic and hypoxic induction of osteopontin in retinal pigment epithelial cells: Involvement of purinergic receptor signaling

Abstract

Purpose: Osteopontin (OPN) is a neuroprotective factor in the retina that improves photoreceptor survival. The aim of the present study was to investigate whether human RPE cells express and respond to OPN.

Methods: Hypoxia and chemical hypoxia were induced by cell culture in 0.25% O₂ and the addition of CoCl₂, respectively. Hyperosmolarity was produced by the addition of 100 mM NaCl or 200 mM sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA.

Results: The acutely isolated RPE cells and the cultured RPE cells expressed OPN. OPN gene expression was induced by hypoxia and hyperosmotic media, as well as by exogenous bFGF. High extracellular NaCl and hypoxia induced secretion of OPN. Hyperosmotic expression of the OPN gene was mediated by the p38 MAPK and ERK1/2 signal transduction pathways, and the transcriptional activities of CREB and NFAT5. The hypoxic expression of the OPN gene was mediated by the PI3K signal transduction pathway and caspase-mediated, necrosis-related pathways. Phospholipases A₂ were involved in mediating hyperosmotic and hypoxic OPN gene expression. Autocrine or paracrine P2Y₂ receptor signaling induced by extracellular ATP contributed to hyperosmotic expression of the OPN gene whereas activation of A₁ receptors by extracellularly formed adenosine contributed to thypoxic OPN gene expression. Autocrine or paracrine VEGF signaling exerted an inhibitory effect on expression of the OPN gene. Exogenous OPN induced expression and secretion of bFGF, but not of VEGF.

Conclusions: The data indicated that RPE cells produce and respond to OPN; OPN expression is, in part, induced by the cellular danger signal ATP. RPE-derived neuroprotective factors such as bFGF may contribute to the prosurvival effect of OPN on photoreceptor cells.

Figure 1

Regulation of OPN gene expression and OPN secretion in RPE cells. A: Presence of OPN gene transcripts in RPE cells. To confirm the correct lengths of the PCR products, agarose gel electrophoresis was performed using products obtained from acutely isolated RPE cells (a) and cultured RPE cell lines (c) derived from different post-mortem donors. Negative controls (0) were performed by adding double-distilled water instead of cDNA as the template. The β-actin (ACTB) mRNA level was used to normalize the OPN mRNA level in real-time reverse transcription (RT)–PCR. B: Expression level of the OPN gene in acutely isolated RPE cells and cultured RPE cell lines, as revealed with real-time RT–PCR. Each bar represents the cycle number necessary for the detection of the transcript. C‒F: The OPN mRNA level in cultured RPE cell lines was determined with real-time RT–PCR after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars), and is expressed as the fold of unstimulated control. C: Effects of chemical hypoxia and cell culture in a 0.25% O₂ atmosphere on OPN gene expression. Chemical hypoxia was induced with the addition of CoCl₂ (150 µM) to the culture medium. D: Effects of hyperglycemia induced by the addition of high (25 mM) glucose, extracellular hyperosmolarity induced by the addition of high (+ 100 mM) NaCl and sucrose (200 mM), respectively, and extracellular hypoosmolarity (60% osmolarity) on OPN gene expression. E: Dose-dependence of the effect of high extracellular NaCl on the OPN mRNA level. Ten millimoles to 100 mM NaCl were added to the culture medium, as indicated in the bars. Data were obtained in six independent experiments using cell lines from different donors. F: Effects of inflammatory and growth factors on the expression of the OPN gene. The following factors were tested: VEGF, bFGF, HB-EGF, PDGF-BB, TGF-β1, HGF, PlGF, IL-1β, and TNFα (each at 10 ng/ml). In addition, fetal calf serum (10%) was tested. G: Effects of purinergic receptor agonists on the expression of the OPN gene. The following agents were tested: ATP (50 µM), UTP (50 µM), the selective P2Y₁ agonist MRS2365 (200 nM), the selective P2Y₂ agonist MRS2768 (100 µM), adenosine (50 µM), and the selective A₁ agonist 2’-MeCCPA (100 nM). H: Presence of CD44 gene transcripts in RPE cells. Agarose gel electrophoresis was performed using products obtained from cultured RPE cell lines derived from different donors (1‒3). Negative controls (0) were performed by adding double-distilled water instead of cDNA as the template. I: Effects of a hyperosmotic medium (+ 100 mM NaCl) and chemical hypoxia induced with the addition of CoCl₂ (150 µM) on the expression of the CD44 gene. The numbers of independent experiments using cell lines from different donors are indicated in or above the bars. Statistically significant difference between acutely isolated and cultured cells: ^●p<0.05. Statistically significant difference versus unstimulated control: *p<0.05.

Figure 2

OPN protein in cultured RPE cells. A: Cell cultures were immunolabeled with an antibody against OPN (red). The cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). The cells were cultured for 24 h under unstimulated control conditions and in the presence of high (+ 100 mM) NaCl and the hypoxia mimetic CoCl₂ (150 µM), respectively. B: Double-immunolabeling of OPN (red) and cytokeratin (green). C: OPN secretion from RPE cells. The OPN protein level was determined with enzyme-linked immunosorbent assay (ELISA) in the media of cells cultured for 24 h in the presence of high (+ 100 mM) NaCl, sucrose (200 mM), and CoCl₂ (150 µM). The data are expressed as percent of unstimulated control (100%). The numbers of independent experiments using cell lines from different donors are indicated in the bars. Statistically significant difference versus unstimulated control: *p<0.05. Scale bars in A and B, 20 µm.

Figure 3

Intracellular signaling involved in osmotic and hypoxic expression of the OPN gene in RPE cells. The level of OPN mRNA was determined with real-time RT–PCR in cells cultured for 24 h in iso- (control; A) and hyperosmotic (+ 100 mM NaCl) media (B), and in the presence of CoCl₂ (150 µM; C), respectively. The following agents were tested: the inhibitor of p38 MAPK activation, SB203580 (10 µM), the inhibitor of ERK1/2 activation, PD98059 (20 µM), the JNK inhibitor SP600125 (10 µM), the inhibitor of PI3K-related kinases, LY294002 (5 µM), the JAK2 Inhibitor AG490 (10 µM), the inhibitor of protein kinase Cα/β, Gö6976 (1 µM), the inhibitor of store-operated calcium entry channels, inositol trisphosphate receptors, and transient receptor potential channels, 2-aminoethoxydiphenyl borate (2-APB; 100 μM), the protein kinase A inhibitor H-89 (1 µM), the inhibitor of Src tyrosine kinases, PP2 (100 nM), the reducing agent dithiothreitol (DTT; 3 mM), the PLA₂ inhibitor 4-bromophenacyl bromide (Bromo; 300 µM), the pan-caspase inhibitor z-VAD (30 µM), the caspase-1 inhibitor Ac-YVAD-CMK (YVAD; 500 nM), the inhibitor of programmed necrosis, necrostatin-1 (Nec-1; 30 µM), inactive necrostatin-1 (Nec-1i; 30 µM) which was tested as negative control, the caspase-3 inhibitor Ac-DEVD-CHO (DEVD; 100 µM), and the caspase-8 inhibitor Ac-IETD-CHO (IETD; 100 µM). Vehicle control was made with dimethyl sulfoxide (DMSO; 1:1000). The numbers of independent experiments using cell lines from different donors are indicated in the bars. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ^●p<0.05. Statistically significant difference versus CoCl₂ control: ^○p<0.05.

Figure 4

Receptor-mediated signaling involved in osmotic and hypoxic expression of the OPN gene in RPE cells. The level of OPN mRNA was determined with real-time reverse transcription (RT)–PCR in cells cultured for 24 h in iso- (control; A) and hyperosmotic (+ 100 mM NaCl) media (B), and in the presence of CoCl₂ (150 µM; C), respectively. The following agents were tested: the inhibitor of the VEGF receptor-2, SU1498 (10 µM), the inhibitor of the PDGF receptor tyrosine kinase, AG1296 (10 µM), the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM), the inhibitor of TGF-β1 superfamily activin receptor-like kinase receptors, SB431542 (10 µM), the FGF receptor kinase inhibitor, PD173074 (500 nM), the broad-spectrum metalloproteinase inhibitor 1,10-phenanthroline (1,10-Phen; 10 µM), and a human recombinant IL-1 receptor antagonist (IL-1RA; 1 µg/ml). The numbers of independent experiments using cell lines from different donors are indicated in or above the bars. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ^●p<0.05. Statistically significant difference versus CoCl₂ control: ^○p<0.05.

Figure 5

Purinergic receptor signaling involved in mediating osmotic and hypoxic expression of the OPN gene in RPE cells. The level of OPN mRNA was determined with real-time reverse transcription (RT)–PCR in cells cultured for 24 h in iso- (control; A) and hyperosmotic (+ 100 mM NaCl) media (B), and in the presence of CoCl₂ (150 µM; C), respectively. The following agents were tested: the ATP/ADP phosphohydrolase apyrase (10 U/ml), the P2X₇ receptor antagonist A-438079 (50 nM), the P2Y₁ receptor antagonist MRS2179 (30 µM), the P2Y₂ receptor antagonist AR-C 118925XX (AR-C; 10 µM), the ecto-ATPase inhibitor ARL-67156 (50 µM), the adenosine A₁ receptor antagonist DPCPX (50 nM), the adenosine A_2A receptor antagonist CSC (200 nM), and the antagonist of nucleoside transporters, NBTI (10 µM). The numbers of independent experiments using cell lines from different donors are indicated in or above the bars. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ^●p<0.05. Statistically significant difference versus CoCl₂ control: ^○p<0.05.

Figure 6

The transcriptional activities of CREB and NFAT5 contribute to induction of osmotic, but not hypoxic, expression of the OPN gene in RPE cells. The level of OPN mRNA was determined with real-time reverse transcription (RT)–PCR in cells cultured for 24 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and in the presence of CoCl₂ (150 µM), respectively. A: The following agents were tested: the STAT3 inhibitor Stattic (1 µM), the NF-κB inhibitor CAPE (5 µM), and a HIF-1 inhibitor (HIF Inh; 5 µM). B: Effects of the CREB inhibitor 666–15 (250 nM) and the AP-1 inhibitor SR11302 (5 µM) on the expression of the OPN gene. C, D: Effects of NFAT5 siRNA (siNFAT5; 5 nM) and nontargeted scrambled siRNA (siNon; 5 nM) on the levels of the NFAT5 mRNA (C) and OPN mRNA (D). The numbers of independent experiments using cell lines from different donors are indicated in the bars. Statistically significant difference versus unstimulated control: *p<0.05. Statistically significant difference versus NaCl control: ^●p<0.05. Statistically significant difference versus CoCl₂ control: ^○p<0.05. Statistically significant difference versus nontargeted siRNA: ^♦p<0.05.

Figure 7

Exogenous OPN stimulates expression and secretion of bFGF in RPE cells. A: Effects of exogenous OPN (10 ng/ml) on expression of the VEGF and bFGF genes. B: Effects of exogenous OPN (10 ng/ml) on the secretion of the VEGF-A₁₆₅ and bFGF proteins. The data were obtained in cells stimulated for 2, 6, and 24 h, as indicated in the panels of the bars. The mRNA levels were determined with real-time reverse transcription (RT)–PCR and are expressed as folds of unstimulated control. Protein levels in the cultured media were determined with enzyme-linked immunosorbent assay (ELISA) and are expressed as percent of unstimulated control (100%). The numbers of independent experiments using cell lines from different donors are indicated in the bars. Statistically significant difference versus unstimulated control: *p<0.05.

Figure 8

Schematic summary of signal transduction pathways that are implicated in regulation of expression of the OPN gene in cultured RPE cells under hyperosmotic and hypoxic conditions. A: High NaCl-induced extracellular hyperosmolarity induces release of VEGF, TGF-β1, FGF, IL-1β, and ATP from the cells. The first three factors inhibit, and IL-1β and ATP stimulate, OPN gene expression. The effect of ATP is mediated in part by autocrine or paracrine activation of the P2Y₂ receptors. Hyperosmotic OPN gene expression is mediated by the activities of p38 MAPK, ERK1/2, and PLA₂, and the transcriptional activities of CREB and NFAT5. Inhibitory effects on hyperosmotic expression of the OPN gene are mediated (in part) by JAK2 and AP-1. High NaCl, but not extracellular hyperosmolarity, also stimulates the secretion of the OPN protein from RPE cells. B: Hypoxia induces release of VEGF, IL-1β, and ATP from the cells. VEGF and IL-1β inhibit OPN gene expression. ATP is extracellularly degraded to adenosine which activates A₁ receptors resulting in stimulation of OPN gene expression. Hypoxic expression of the OPN gene is mediated (in part) by the activities of PI3K, PLA₂, and caspases. Inhibitory effects on hypoxic expression of the OPN gene are (in part) mediated by CREB and AP-1.