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

Materials

Cell culture components and solutions were purchased from Gibco BRL (Paisley, UK). Recombinant human VEGF-A₁₆₅, bFGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), hepatocyte growth factor (HGF), IL-1 receptor antagonist, OPN, platelet-derived growth factor (PDGF)-BB, transforming growth factor β1 (TGF-β1), and tumor necrosis factor-α (TNFα) were purchased from R&D Systems (Abingdon, UK). Recombinant human interleukin-1β (IL-1β) and placental growth factor (PlGF)-2 were from Reliatech (Braunschweig, Germany). 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), Gö6976, H-89, the inhibitor of hypoxia-inducible transcription factor-1 (HIF-1), LY294002, necrostatin-1, inactive necrostatin-1, PD98059, PP2, SP600125, and SU1498 were obtained from Calbiochem (Bad Soden, Germany). Ac-DEVD-CHO, Ac-IETD-CHO, Ac-YVAD-CMK, AG1478, Stattic, and z-VAD were from Enzo Life Science (Lausen, Switzerland). 666–15, A-438079, adenosine, adenosine 5′-triphosphate (ATP), AG490, AR-C 118925XX, ARL-67156, caffeic acid phenethyl ester (CAPE), 8-(3-chlorostyryl) caffeine (CSC), 2-chloro-N-cyclopentyl-2’-methyladenosine (2'-MeCCPA), MRS2179, MRS2365, MRS2768, SB203580, and SR11302 were from Tocris (Ellisville, MO). Uridine 5′-triphosphate (UTP) and dithiothreitol were from Carl Roth (Karlsruhe, Germany), and PD173074 was kindly provided by Pfizer (Karlsruhe, Germany). Human-specific small interfering RNA (siRNA) against nuclear factor of activated T cell 5 (NFAT5) and nontargeted scrambled siRNA were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). 4’,6-Diamidino-2-phenylindole (DAPI) was from Invitrogen (Paisley, UK). AG1296, 2-aminoethoxydiphenyl borate, apyrase, 4-bromophenacyl bromide, N-nitrobenzylthioinosine (NBTI), 1,10-phenanthroline, SB431542, and all other agents used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. The following antibodies were used for immunocytochemistry: rabbit anti-human OPN (1:100; HPA027541; Sigma-Aldrich), fluorescein isothiocyanate-coupled monoclonal anti-pan cytokeratin (1:50; Sigma-Aldrich), and Alexa Fluor 568-conjugated goat anti-rabbit (1:400; A11036; Invitrogen).

Cell culture

The study followed the tenets of the Declaration of Helsinki for research involving human subjects and the ARVO statement for the use of human subjects. The use of human material was approved by the Ethics Committee of the University of Leipzig (#745, 07/25/2011). Post-mortem eyes from human cornea donors without reported eye disease were obtained within 48 h of death with written informed consent from the donors’ relatives for the use of retinal tissue in basic science. The RPE cells were prepared and cultured as described [47]. Cell lines derived from different donors were used in passages 3 to 5. Near-confluent cultures were growth arrested in medium without serum for 16 h, and subsequently, serum-free media with and without test substances were added. Hyperosmotic media were made up by addition of NaCl (100 mM) or sucrose (200 mM). The hypoosmotic medium (60% osmolarity) was made up by adding distilled water. The cells were preincubated with pharmacological inhibitors 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 quality of the RNA was analyzed with agarose gel electrophoresis. The A₂₆₀/A₂₈₀ ratio of the optical density was measured using the NanoDrop 1000 device (peQLab, Erlangen, Germany), and was between 1.95 and 2.03 for all RNA samples, indicating sufficient quality. After treatment with DNase I (Roche, Mannheim, Germany), cDNA was synthesized from 1 µg total RNA using the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas, St. Leon-Roth, Germany).

RT–PCR

Reverse transcription (RT)–PCR was performed with the Taq PCR Master Mix kit (Qiagen, Hilden, Germany) and the primer pairs described in Table 1. One microliter of the first-strand mixture and 0.25 µM of each gene-specific sense and anti-sense primers were used for amplification in a final volume of 20 µl. Amplification was performed for 40 cycles with the PTC-200 Thermal Cycler (MJ Research, Watertown, MA). Each cycle consisted of 30 s at 94 °C, 60 s at 58 °C, and 1 min at 72 °C.

Real-time RT–PCR

Real-time RT–PCR was performed with the Single-Color Real-Time PCR Detection System (Bio-Rad, Munich, Germany) using the primer pairs described in Table 1. The PCR solution contained 1 µl cDNA, a specific primer set (0.2 µM each), and 7.5 µl of a 2X mastermix (iQ SYBR Green Supermix; Bio-Rad) in a final volume of 15 µl. The following conditions were used: initial denaturation and enzyme activation (one cycle at 95 °C for 3 min); denaturation, amplification, and quantification, 45 cycles at 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 45 s; melting curve, 55 °C with the temperature gradually (0.5 °C) increased up to 95 °C. The amplified samples were analyzed with standard agarose gel electrophoresis. mRNA expression was normalized to the level of β-actin (ACTB, Gene ID: 60; OMIM: 102630) mRNA. The changes in mRNA expression were calculated according to the 2^-ΔΔCT method [48].

ELISA

The cells were stimulated with iso- and hyperosmotic media (+ 100 mM NaCl, 200 mM sucrose), with CoCl₂ (150 µM)-containing, serum-free medium, or with OPN (10 ng/ml) in serum-free medium. The supernatants were collected after 6 and 24 h, and the levels of OPN, VEGF-A₁₆₅, and bFGF in the cultured media (100 µl) were determined with enzyme-linked immunosorbent assay (ELISA; R&D Systems).

siRNA transfection

Cells were seeded at 7 × 10⁴ cells per well in 12-well culture plates and were allowed to grow to a confluency of 60–80%. Thereafter, the cells were transfected with NFAT5 siRNA (5 nM) and nontargeted siRNA (5 nM), respectively, using HiPerfect reagent (Qiagen) in F-10 medium containing 10% fetal bovine serum according to the manufacturer’s instructions. After 48 h, the medium was removed, and fresh medium without serum was added for 5 h. Thereafter, serum-free medium containing high (+ 100 mM) NaCl and CoCl₂ (150 µM), respectively, was added for 24 h.

Immunocytochemistry

The cultures were fixed with 4% paraformaldehyde for 15 min on ice. After several washing steps in prechilled PBS (1X; 155 mM NaCl, 1.54 mM KH₂PO₄, 2.71 mM Na₂HPO₄-7H₂O pH 7.2; Invitrogen), the cultures were incubated in PBS containing 0.3% Triton X-100 for 15 min at room temperature (RT). Blocking of nonspecific antibody binding was performed with PBS containing 10% normal goat serum and 0.3% Triton X-100 for 2 h at RT. Primary antibodies diluted in blocking solution were incubated at 4 °C overnight. After several washing steps in PBS plus 0.3% Triton X-100, the secondary antibody was applied for 1 h at RT. After additional washing steps, DAPI (1:10,000) was added for 15 min at RT. The coverslips were mounted with Fluorescence Mounting Medium (DakoCytomation, Glostrup, Denmark). Images were taken with an epifluorescence microscope (Olympus BX40, Olympus, Essex, UK), a charge-coupled device (CCD) camera (Olympus XM10), and cellSens software (Olympus).

Statistical analysis

For each test, at least three independent experiments using cells from different donors were performed. Data are expressed as means ± standard error of the mean (SEM). Statistical analysis was performed with Prism (GraphPad Software, San Diego, CA). Statistical significance was determined with one-way analysis of variance (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.

OPN gene expression in RPE cells

RT–PCR was performed to compare the levels of OPN (Gene ID: 6696; OMIM: 166490) mRNA in RPE cells that had been acutely isolated from the eyes of post-mortem donors without reported eye disease and in cultured human RPE cell lines derived from different donors. As shown in Figure 1A, the acutely isolated cells and the cultured cells contained OPN gene transcripts. The level of OPN mRNA was strongly reduced in the cultured cells compared to the acutely isolated cells, as indicated by the statistically significantly (p<0.05) increased cycle number necessary for the detection of the transcript (Figure 1B).

Regulation of OPN gene expression

To investigate which pathogenic conditions induce the expression of the OPN gene in RPE cells, we stimulated cultured cell lines from different donors with the hypoxia mimetic CoCl₂ [49], high (25 mM) glucose, and hyper- and hypoosmotic media, and performed real-time RT–PCR. As shown in Figure 1C, CoCl₂-induced chemical hypoxia induced a statistically significant (p<0.05) increase in OPN gene expression after 24 h of stimulation. Cell culture in a 0.25% O₂ atmosphere induced biphasic regulation of OPN gene expression with down- and upregulation after 2 and 6‒24 h of stimulation, respectively (Figure 1C). Treatment of the cells with CoCl₂ caused a higher increase in the OPN mRNA level than cell culture in 0.25% O₂ (Figure 1C). The data suggest that CoCl₂-induced expression of the OPN gene is mediated by several mechanisms that may include hypoxia, alteration of the metal ion homeostasis, and oxidative stress. Expression of the OPN gene remained unaltered in the presence of high glucose (Figure 1D). Extracellular hyperosmolarity was induced with the addition of 100 mM NaCl or 200 mM sucrose (which caused equal increases in the osmolarity). As shown in Figure 1D, hyperosmotic media induced statistically significant (p<0.05) increases in OPN gene expression after 24 h of stimulation. The amplitudes and time-dependencies of the effects of high NaCl and sucrose were similar (Figure 1D). This result suggests that the NaCl-induced expression of the OPN gene was caused by the elevation of the extracellular osmolarity, but not by the alterations of the transmembrane sodium and chloride gradients. The effect of high NaCl on the OPN mRNA level was dose-dependent; statistically significant (p<0.05) increases were found when more than 30 mM NaCl was added to the culture medium (Figure 1E). The hypoosmotic medium did not alter the cellular level of OPN mRNA (Figure 1D). The data suggest that the OPN gene in RPE cells is transcriptionally activated by hypoxia and extracellular hyperosmolarity.

To investigate whether the expression of the OPN gene in RPE cells is regulated by inflammatory and growth factors, we stimulated the cells with various cytokines. As shown in Figure 1F, exogenous VEGF, HB-EGF, PDGF, HGF, and PlGF did not alter the level of OPN transcripts in RPE cells. bFGF induced biphasic regulation of the expression of the OPN gene, with down- and upregulation after 2 and 24 h of stimulation, respectively (Figure 1F). The cellular level of the OPN gene transcripts was time-dependently decreased during stimulation with TGF-β1, IL-1β, TNFα, and fetal calf serum (Figure 1F).

Extracellular ATP is an endogenous danger signal which is released in large quantities by stressed cells [50]. In many cell systems, such as retinal glial cells [50], autocrine or paracrine purinergic signaling is mediated by osmo- or mechanosensitive release of ATP, and subsequent activation of purinergic receptors is involved in the early cellular response to osmotic stress. Extracellular ATP acts at the purinergic metabotropic (P2Y) and ionotropic (P2X) receptors. RPE cells are known to express multiple purinergic receptor subtypes, including P2Y, P2X, and adenosine receptors [51,52]. The expression level of the OPN gene in the RPE cells was increased by exogenous ATP, but not UTP (Figure 1G). The selective P2Y₁ receptor agonist MRS2365 also induced an increase in the OPN mRNA level in RPE cells while the selective P2Y₂ receptor agonist MRS2768 induced a transient increase (Figure 1G). In addition, exogenous adenosine and the selective A₁ receptor agonist 2’-MeCCPA stimulated the expression of the OPN gene in RPE cells (Figure 1G).

OPN is a ligand of CD44 receptor variants [26,27]. RPE cells contained CD44 (Gene ID: 960; OMIM: 107269) gene transcripts (Figure 1H). The expression level of the CD44 gene was unaltered under hyperosmotic conditions and upregulated after 24 h of stimulation with the hypoxia mimetic CoCl₂ (Figure 1I).

Regulation of OPN secretion

Cultured RPE cells were labeled with an antibody against OPN (Figure 2A). The punctate OPN immunoreactivities were arranged in lines along the borders of the cell somata and processes (Figure 2A). The distribution of the OPN immunoreactivity was not altered in response to high extracellular NaCl and the hypoxia mimetic CoCl₂ compared to control (Figure 2A). There was no colocalization of OPN and cytokeratin immunoreactivities (Figure 2B), suggesting that OPN was mainly localized near the plasma membrane.

High extracellular NaCl, but not sucrose-induced extracellular hyperosmolarity, stimulated the secretion of OPN from RPE cells (Figure 2C). The data suggest that alterations of the transmembrane sodium and chloride gradients, but not the increase in extracellular osmolarity, induce the secretion of OPN. Chemical hypoxia caused a moderate increase in the OPN protein level in the cultured media (Figure 2C).

Intracellular signaling involved in osmotic and hypoxic expression of the OPN gene

Extracellular hyperosmolarity and hypoxia induced delayed upregulation of OPN gene expression in RPE cells (Figure 1C,D). To investigate which signal transduction pathways are involved in mediating OPN gene expression, we tested different inhibitory agents in cells that were stimulated for 24 h with high (+ 100 mM) NaCl and hypoxia mimetic CoCl₂, respectively. The agents tested did not alter statistically significantly (p>0.05) OPN gene expression under unstimulated control conditions (Figure 3A). As shown in Figure 3B, hyperosmotic OPN gene expression was statistically significantly (p<0.05) decreased by the inhibitor of p38 mitogen-activated protein kinase (p38 MAPK) activation, SB203580, the inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, PD98059, and the inhibitor of phospholipases A₂ (PLA₂), 4-bromophenacyl bromide. Many other inhibitors of various signaling molecules, including inhibitors of phosphatidylinositol-3 kinase (PI3K)-related kinases (LY294002) and c-Jun NH₂-terminal kinase (JNK; SP600125), did not alter hyperoosmotic expression of the OPN gene (Figure 3B).

Hypoxic activation of the OPN gene was statistically significantly (p<0.05) decreased by the inhibitor of PI3K-related kinases, LY294002, and the inhibitor of PLA₂, 4-bromophenacyl bromide (Figure 3C). The inhibitor of Janus kinase 2 (JAK2), AG490, induced a statistically significant (p<0.05) increase in OPN gene expression under hypoxic conditions (Figure 3C). We also found that hypoxic expression of the OPN gene was statistically significantly (p<0.05) suppressed by a broad-spectrum caspase inhibitor, z-VAD, and an inhibitor of programmed necrosis, necrostatin-1 [53] (Figure 3C). Although the caspase-3-specific inhibitor Ac-DEVD-CMK increased the hypoxic activation of the OPN gene, caspase-1- and caspase-8-specific inhibitors, and the inactive derivative of necrostatin-1, had no effects (Figure 3C). The data indicate that hyperosmotic and hypoxic expression of the OPN gene in RPE cells is mediated by activation of different signal transduction pathways. Hyperosmotic expression of the OPN gene is mediated by activation of p38 MAPK and ERK1/2 signal transduction pathways, but hypoxic expression of the OPN gene is mediated by the PI3K signal transduction pathway and caspase-mediated, necrosis-related pathways. Activation of PLA₂ is involved in mediating OPN gene expression under both conditions.

Extracellular signaling involved in osmotic and hypoxic expression of the OPN gene

It was shown that extracellular hyperosmolarity induces release of various growth factors, such as VEGF, bFGF, and TGF-β1 from RPE cells [54,55]. To investigate whether autocrine or paracrine growth factor signaling is required for hyperosmotic and hypoxic expression of the OPN gene, we tested inhibitors of the VEGF receptor-2 (SU1498), the PDGF receptor tyrosine kinase (AG1296), the epidermal growth factor (EGF) receptor tyrosine kinase (AG1478), of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), and the FGF receptor kinase (PD173074), as well as an IL-1 receptor antagonist. As shown in Figure 4A, inhibitors of VEGF receptor-2 (SU1498) and TGF-β1 superfamily activin receptor-like kinase receptors (SB431542) induced statistically significant (p<0.05) increases in OPN gene expression under unstimulated control conditions. The data suggest that constitutive secretion of VEGF and TGF-β1 inhibits OPN gene expression in RPE cells. Hyperosmotic expression of the OPN gene was statistically significantly (p<0.05) decreased by the IL-1 receptor antagonist, and increased in the presence of inhibitors of VEGF receptor-2, TGF-β1 superfamily activin receptor-like kinase receptors, and the FGF receptor kinase (Figure 4B). Inhibitors of PDGF and EGF receptor tyrosine kinases (AG1296, AG1478) had no effects on hyperosmotic expression of the OPN gene (Figure 4B). In addition, the broad-spectrum metalloproteinase inhibitor 1,10-phenanthroline had no effect, suggesting that metalloproteinase-mediated release of growth factors from the extracellular matrix plays no role in hyperosmotic expression of the OPN gene. Under hypoxic conditions, the antagonist of VEGF receptor-2, SU1498, and the IL-1 receptor antagonist induced strong increases in OPN gene expression while the other inhibitory agents tested had no effects (Figure 4C). The data suggest that autocrine or paracrine VEGF signaling exerts an inhibitory effect on OPN gene expression under control, hyperosmotic, and hypoxic conditions. Autocrine or paracrine TGF-β1 signaling inhibits expression of the OPN gene under control and hyperosmotic conditions, while autocrine or paracrine IL-1 signaling stimulates hyperosmotic expression and inhibits hypoxic expression of the OPN gene.

We found that extracellular ATP and adenosine stimulated OPN gene expression in RPE cells under control conditions (Figure 1G). To investigate whether purinergic receptor signaling is involved in mediating hyperosmotic and hypoxic expression of the OPN gene, we tested pharmacological receptor antagonists. The agents tested did not alter OPN gene expression under unstimulated control conditions (Figure 5A). We found that hyperosmotic and hypoxic expression of the OPN gene was statistically significantly (p<0.05) decreased in the presence of the ATP/ADP phosphohydrolase apyrase (Figure 5B,C). Because apyrase removes extracellular ATP and ADP by enzymatic hydrolyzation, the data suggest that the release of endogenous ATP is required for the full induction of OPN gene expression in RPE cells. Hyperosmotic expression of the OPN gene was also decreased by the selective inhibitor of P2Y₂ receptors, AR-C 118925XX (Figure 5B). The following inhibitory agents had no effects on hyperosmotic OPN gene expression: the P2X₇ receptor antagonist A-438079, the P2Y₁ receptor antagonist MRS2179, the ecto-ATPase inhibitor ARL-67156, the adenosine A₁ receptor antagonist DPCPX, the adenosine A_2A receptor antagonist CSC, and the antagonist of nucleoside transporters, NBTI (Figure 5B). The data suggest that extracellular ATP and autocrine or paracrine P2Y₂ receptor signaling is required for the full induction of OPN gene expression under hyperosmotic conditions.

Hypoxic expression of the OPN gene was statistically significantly (p<0.05) decreased by ATP/ADP phosphohydrolase apyrase, the ecto-ATPase inhibitor ARL-67156 (which blocks the conversion of ATP to ADP/AMP), and the antagonist of adenosine A₁ receptors, DPCPX (Figure 5C). All other inhibitory agents tested had no effects. The data suggest that the release of endogenous ATP, extracellular degradation of ATP, and autocrine or paracrine activation of A₁ receptors by adenosine are involved in mediating the hypoxic expression of the OPN gene in RPE cells.

Transcription factor activities involved in hyperosmotic expression of the OPN gene

Hyperosmotic stress was shown to induce expression and activation of various transcription factors in RPE cells, including HIF-1α, nuclear factor-κB (NF-κB), and NFAT5 [54]. To investigate which transcription factors mediate hyperosmotic and hypoxic expression of the OPN gene, we tested inhibitory agents in cells stimulated with high NaCl or the hypoxia mimetic CoCl₂. As shown in Figure 6A, hyperosmotic and hypoxic expression of the OPN gene remained unaltered in the presence of the inhibitor of signal transducer and activator of transcription 3 (STAT3), Stattic [56], the NF-κB inhibitor CAPE [57], and a HIF-1 inhibitor [58]. Hyperosmotic expression of the OPN gene was statistically significantly (p<0.05) decreased by the CREB inhibitor 666–15, and increased by the AP-1 inhibitor SR11302 (Figure 6B). Both inhibitors increased statistically significantly (p<0.05) hypoxic expression of the OPN gene (Figure 6B). The data suggest that hyperosmotic expression of the OPN gene in RPE cells is (in part) mediated by the transcriptional activity of CREB while the transcriptional activity of AP-1 inhibits hyperosmotic OPN gene expression. Hypoxic expression of the OPN gene is inhibited by the transcriptional activities of CREB and AP-1. Stattic, 666–15, SR11302 induced statistically significant (p<0.05) increases in OPN gene expression under unstimulated control conditions (Figure 6A,B), suggesting that the transcriptional activities of STAT3, AP-1, and CREB inhibit constitutive expression of the OPN gene in RPE cells.

To investigate whether the activity of the transcription factor NFAT5, which is critically involved in cellular adaptation to hyperosmotic stress in various cell systems [59], contributes to expression of the OPN gene, we knocked down NFAT5 with siRNA. Transfection with NFAT5 siRNA reduced the level of NFAT5 (Gene ID10725; OMIM 604708) gene transcripts by 50‒60% in cells cultured in control and hyperosmotic media, and in the presence of CoCl₂, respectively (Figure 6C). We found no differences in the level of OPN gene transcripts between cells transfected with NFAT5 siRNA and nontargeted scrambled siRNA cultured under unstimulated control and hypoxic conditions (Figure 6D). Under hyperosmotic conditions, cells transfected with NFAT5 siRNA displayed a statistically significantly (p<0.05) lower level of OPN mRNA compared to cells transfected with nontargeted siRNA (Figure 6D). The data suggest that hyperosmotic, but not the hypoxic, expression of the OPN gene is mediated by the transcriptional activities of CREB and NFAT5.

OPN stimulates the expression and secretion of bFGF

To investigate whether OPN influences the production of VEGF and bFGF in RPE cells, we stimulated cultured cells with exogenous OPN. As shown in Figure 7A, administration of OPN increased statistically significantly (p<0.05) expression of the bFGF (Gene ID: 2247; OMIM: 134920) gene after 2 h of stimulation and had no effect on expression of the VEGF (Gene ID: 7422; OMIM: 192240) gene. Exogenous OPN also stimulated statistically significantly (p<0.05) secretion of the bFGF protein from RPE cells after 6 h of stimulation and had no effect on secretion of VEGF (Figure 7B). The data indicate that exogenous OPN stimulates production and secretion of bFGF, but not VEGF, in RPE cells.