Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

Materials

Tissue culture components and solutions were purchased from Gibco BRL (Paisley, UK). Platelet-derived growth factor (PDGF)-BB was purchased from R&D Systems (Abingdon, UK). The inhibitor of hypoxia-inducible transcription factor 1 (HIF-1) 3-[2-(4-adamantan-1-yl-phenoxy)-acetylamino]-4-hydroxybenzoic acid methyl ester, LY294002, PD98059, and SP600125 were obtained from Calbiochem (Bad Soden, Germany). Stattic was from Enzo Life Science (Lörrach, Germany). Caffeic acid phenethyl ester and SB203580 were from Tocris (Ellisville, MO). ODN 2006 (ODN 7909) was obtained from InvivoGen (San Diego, CA). Poly(I:C) and all other agents used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise.

The following antibodies were used: a rabbit anti-human zonula occludens (ZO)-1 (1:50; Zymed Laboratory, San Francisco, CA); a rabbit anti-human occludin (1:50; Zymed Laboratory); a monoclonal anti-cytokeratin pan-FITC antibody that recognizes cytokeratins 4, 5, 6, 8, 10, 13, and 18 (1:50, Sigma-Aldrich); a Cy3-conjugated goat anti-rabbit IgG (1:200; Jackson Immuno Research, Suffolk, UK); a rabbit anti-human β-actin (1:2000, Cell Signaling, Frankfurt/M., Germany); rabbit anti-human extracellular signal-regulated kinases 1 and 2 (ERK1/2, p44/p42; 1:1000; Cell Signaling, Frankfurt/M., Germany); a rabbit anti-phosphorylated ERK1/2 (1:1000; Cell Signaling); a rabbit anti-human p38 mitogen-activated protein kinase (p38 MAPK; 1:1000; Cell Signaling); a rabbit anti-human phosphorylated p38 MAPK (1:750; Cell Signaling); a rabbit anti-human protein kinase B (Akt; Cell Signaling; 1:1000); a rabbit anti-human phosphorylated Akt (Cell Signaling; 1:1000); a rabbit anti-human signal transducer and activator of transcription 3 (STAT3; Cell Signaling; 1:1000); a rabbit anti-human phosphorylated STAT3 (Cell Signaling; 1:1000); and anti-rabbit IgG conjugated with alkaline phosphatase (1:2000; Chemicon, Hofheim, Germany).

Cell culture

The study followed the tenets of the Declaration of Helsinki for the use of human subjects. The use of human material was approved by the Ethics Committee of the University of Leipzig (approval #745, 07/25/2011). Human donor eyes were obtained from 12 females and 29 males within 48 h of death with written informed consent from relatives of the donors. The ages of the donors varied between 19 and 85 years (means ± SD, 62.7±19.7 years for females, and 59.3±19.0 years for males). There were no significant differences between data obtained in cells from younger and aged donor eyes, and in cells from both sexes (not shown).

RPE cells were prepared and cultured as following. After removing the vitreous and the retina, RPE cells were mechanically harvested, separated by digestion with 0.05% trypsin and 0.02% EDTA, and washed two times with phosphate-buffered saline. Cells were suspended in complete Ham F-10 medium containing 10% fetal bovine serum, glutamax II, and penicillin/streptomycin, and were cultured in tissue culture flasks (Greiner, Nürtingen, Germany) in 95% air/5% CO₂ at 37 °C. Cells of passages 3 to 5 were used. Near-confluent cultures (confluency 80%–90%, reached after 4 days of cultivation) or confluent cultures (5 days of cultivation) were growth-arrested in medium without serum for 16 h, and subsequently, serum-free media with and without test substances were added. The cells were preincubated with the pharmacological inhibitors for 30 min. The confluency of the cultures was evaluated microscopically.

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 by agarose gel electrophoresis. The A₂₆₀/A₂₈₀ ratio of the optical density was measured using the NanoDrop 1000 device (Peqlab, Erlangen, Germany), and was between 1.8 and 2.1 for all RNA samples, indicating sufficient quality. After treatment with DNase I (Roche, Mannheim, Germany), cDNA was synthesized from 1 µg of total RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Roth, Germany).

RT–PCR

PCR was performed using the Taq PCR Master Mix kit (Qiagen), and the primer pairs are described in Table 1. One µl of the first-strand mixture and 0.25 µM of each gene-specific sense and anti-sense primer 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 (BioRad, Munich, Germany) using the primer pairs described in Table 1. The PCR solution contained 1 µl of cDNA, a specific primer set (0.2 µM each), and 7.5 µl of a 2×mastermix (iQ SYBR Green Supermix, BioRad) 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 for 45 cycles at 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 45 s; and a melting curve, 55 °C with the temperature gradually increased (0.5 °C) up to 95 °C. The amplified samples were analyzed by standard agarose gel electrophoresis. The mRNA expression was normalized to the level of β-actin mRNA. The changes in mRNA expression were calculated according to the 2^-ΔΔCT method (CT, cycle threshold), with ΔCT=CT_{target gene} - CT_actb and ΔΔCT=ΔCT_treatment - ΔCT_control.

Western blot analysis

Cells were seeded at 5×10⁵ cells per well in six-well plates in 1.5 ml of complete medium, and were allowed to growth up to a confluency of ~80%. After growth arrest for 16 h, the cells were treated with test substances for 20 min and 1 h, respectively. Then, the medium was removed, the cells were washed twice with prechilled phosphate-buffered saline (pH 7.4; Invitrogen, Paisley, UK), and the monolayer was scraped into 150 µl of lysis buffer (Mammalian Cell Lysis-1 Kit; Sigma). Total cell lysates were centrifuged at 10,000 × g for 10 min, and the supernatants were analyzed by immunoblots. Equal amounts of protein (30 µg) were separated by 10% SDS–PAGE. Immunoblots were probed with primary and secondary antibodies, and immunoreactive bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.

ELISA

Cells were stimulated with poly(I:C; 500 µg/ml) in a serum-free medium. The supernatants were collected after 6 and 24 h, and the levels of VEGF-A₁₆₅, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and tumor necrosis factor α (TNFα), respectively, in the cultured media (200 µl) were determined with ELISA (R&D Systems).

Immunocytochemistry

For immunolabeling, the cultures were fixed in 1% paraformaldehyde for 20 min on ice. After several washing steps in buffered saline, the cultures were incubated for 15 min in buffered saline containing 1% DMSO and 0.3% Triton X-100. Blocking was performed in buffered saline containing 5% normal goat serum, 1% DMSO, and 0.3% Triton X-100 for 2 h at room temperature, and subsequently, in the primary antibody overnight at 4 °C. After washing in saline plus 1% DMSO and 0.3% Triton X-100, the secondary antibody and Hoechst 33258 were applied for 1 h at room temperature. Images were taken with a confocal laser scanning microscope (LSM 510 Meta; Zeiss, Oberkochen, Germany).

Statistics

For each test, at least three independent experiments using cells from different donors were performed. Data are expressed as means ± SEM. Statistical analysis was made using Prism (Graphpad Software, San Diego, CA). Significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test and the Mann–Whitney U test, respectively, and was accepted at p<0.05.

Cultures of human RPE cells

The experiments were performed with near-confluent and confluent cultures of human RPE cells. The epithelial nature of the cells was identified by immunolabeling for cytokeratins (Figure 1). In different near-confluent and confluent cultures, 70%–85% of all cells investigated displayed immunoreactivity for cytokeratins. The expression of the tight junction proteins ZO-1 and occludin (Figure 1) suggests that the cells form tight junctions. The tight junction proteins were distributed in a clustered fashion in the plasma membranes and the nuclei of the cells (Figure 1).

Expression of TLR genes

To determine whether viral RNA and viral/bacterial DNA induce alterations in the gene expression patterns of RPE cells, we stimulated the cells with the synthetic viral dsRNA analog poly(I:C) and the viral/bacterial DNA analog CpG-ODN. To examine the effects on the gene expression of pattern recognition receptors, we determined the cellular levels of TLR2, TLR3, and TLR9 mRNAs (Figure 2A). As shown in Figure 2B,C, poly(I:C) induced a significant (p<0.05) time-dependent increase in the expression of the TLR3 gene. In near-confluent cultures, the levels of TLR2 and TLR9 mRNAs were moderately altered in the presence of poly(I:C). In confluent cultures, poly(I:C) induced a significant (p<0.05) increase in the expression of the TLR2 gene and no alteration in the expression of the TLR9 gene (Figure 2C). On the other hand, CpG-ODN did not significantly (p>0.05) alter the cellular levels of TLR2, TLR3, and TLR9 mRNAs (Figure 2D). The data suggest that viral RNA induces upregulation of TLR3 and, to a lower degree, TLR2 in RPE cells.

Expression of transcription factor genes

To determine whether viral RNA and viral/bacterial DNA induce activation of RPE cells, we examined the cellular levels of various transcription factor genes (Figure 3A). As shown in Figure 3B,C, stimulation of the cells with the synthetic viral RNA analog poly(I:C) induced time-dependent increases in the gene expression levels of HIF-1α, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA, the p65 subunit of nuclear factor [NF]-κB), and the nuclear factor of activated T cell 5 (NFAT5). Stimulation with poly(I:C) had no significant (p>0.05) effect on the expression of the STAT3 gene (Figure 3B). Synthetic viral/bacterial DNA (CpG-ODN) induced moderate alterations in the expression of RELA and NFAT5 genes, with up- and downregulation after 6 and 24 h of stimulation, respectively (Figure 3D). The data suggest that both viral RNA and viral/bacterial DNA induce alterations in the transcriptional activation of various transcription factor genes in RPE cells, including HIF-1α (viral RNA), p65/NF-κB (RNA and DNA), and NFAT5 (RNA and DNA).

Expression of angiogenic growth factor genes

Progression of AMD to the neovascular stage is driven by angiogenic growth factors such as VEGF, bFGF, and HB-EGF, which are produced, for example, by RPE cells [29-31]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of these factors in RPE cells (Figure 4A). As shown in Figure 4B,C, the synthetic analog of viral RNA poly(I:C) induced a significant (p<0.05) increase in the expression of the bFGF gene in RPE cells and had no effect on the expression of the VEGF gene. Poly(I:C) also induced a moderate increase in the expression of the HB-EGF gene in non-confluent cultures (Figure 4B). The administration of poly(I:C) also induced a significant (p<0.05) increase in the secretion of the bFGF protein from RPE cells, while it had no effect on the secretion of VEGF protein (Figure 4D). We did not find detectable amounts of soluble HB-EGF protein with ELISA in the media of cells cultured for 6 or 24 h in the absence and presence of poly(I:C; 500 µM; not shown). Stimulation of RPE cells with the synthetic analog of viral/bacterial DNA, CpG-ODN, did not alter the cellular levels of VEGF, bFGF, and HB-EGF mRNAs (Figure 4E). The data suggest that viral RNA induces expression and secretion of bFGF.

Expression of proinflammatory cytokine genes

AMD is associated with systemic and local inflammation [1]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of proinflammatory cytokines in RPE cells (Figure 5A). As shown in Figure 5B,C, stimulation of the cells with the synthetic analog of viral RNA poly(I:C) induced significant (p<0.05) increases in the gene expression of interleukin (IL)-1β, IL-6, TNFα, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2. The time-dependency of the poly(I:C)-induced gene expression differed among various inflammatory cytokines. While the increases in the IL-1β, TNFα, and MIP-2 mRNA levels were largest after 2 h of stimulation with poly(I:C), the increases in the expression of IL-6 and MCP-1 genes were more pronounced after 6 h of stimulation (Figure 5B). Poly(I:C) also induced a strong increase in the secretion of TNFα protein from RPE cells (Figure 5D). The analog of viral/bacterial DNA, CpG-ODN did not induce significant (p>0.05) alterations in the gene expression of inflammatory cytokines (Figure 5E). The data suggest that viral RNA induces gene expression of various inflammatory cytokines in RPE cells, and the secretion of TNFα.

Expression of complement factor genes

Age-related retinal inflammation is associated with local activation of the alternative complement cascade [2,3] and subretinal deposition of activated complement proteins such as C3a and C5a [32]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of various complement proteins in RPE cells (Figure 6A). As shown in Figure 6B, the synthetic viral RNA analog poly(I:C) induced significant (p<0.05) time-dependent increases in the expression of C5, C9, CFB, and CFH genes, and had no effect on the expression of the C3 gene. The effect of poly(I:C) was more pronounced in respect to the CFB gene than to the other complement factor genes investigated (Figure 6B,C). Stimulation of RPE cells with the viral/bacterial DNA analog CpG-ODN induced increased expression of C5 and C9 genes, while it had no effect on the expression of C3, CFB, and CFH genes (Figure 6D). The data suggest that both viral RNA and viral/bacterial DNA induce the expression of distinct complement factor genes in RPE cells.

Activation of intracellular signal transduction molecules

We found that the synthetic analog of viral RNA, poly(I:C), induces the expression of various different genes in RPE cells. To determine whether poly(I:C) also induces activation of intracellular signal transduction pathways, we examined with western blot analysis the phosphorylation levels of ERK1/2, p38 MAPK, and Akt proteins. As shown in Figure 7, poly(I:C) induced dose-dependent increases in the phosphorylation levels of ERK1/2 and p38 MAPK proteins. The effects of poly(I:C) were more pronounced after 1 h than after 20 min of stimulation (Figure 7). Poly(I:C) also induced a moderate increase in the phosphorylation level of the Akt protein, which was apparent after 20 min, but not after 1 h of stimulation (Figure 7). In contrast, poly(I:C) did not induce an alteration in the phosphorylation level of STAT3 protein (Figure 7). In agreement with previous studies [33,34], the positive control, PDGF, induced alterations in the phosphorylation level of all proteins investigated (Figure 7). The data suggest that viral RNA induces activation of various signal transduction pathways in RPE cells.

Intracellular signaling involved in poly(I:C)-induced gene expression

We found that the synthetic analog of viral RNA, poly(I:C), induced activation of different signal transduction molecules in RPE cells (Figure 7). To determine the intracellular signaling that regulates the poly(I:C)-induced gene expression, we tested pharmacological blockers of key intracellular signal transduction molecules. Inhibition of ERK1/2 activation by PD98059 significantly (p<0.05) reduced the poly(I:C)-induced expression of TLR3 (Figure 8A), TNFα (Figure 8C), IL-1β (Figure 8D), and CFB (Figure 8E) genes, while it had no effect on the poly(I:C)-induced expression of the bFGF gene (Figure 8B). The inhibition of p38 MAPK activity by SB203580 resulted in a significant (p<0.05) decrease of the poly(I:C)-induced expression of the following genes: TLR3 (Figure 8A), bFGF (Figure 8B), TNFα (Figure 8C), and CFB (Figure 8E). The inhibition of p38 MAPK activity had no effect on the poly(I:C)-induced expression of the IL-1β gene (Figure 8D). Inhibition of the c-Jun NH₂-terminal kinase (JNK) activity by SP600125 resulted in significant (p<0.05) reduction of the poly(I:C)-induced expression of TLR3 (Figure 8A), TNFα (Figure 8C), and CFB (Figure 8E) genes, while it had no effect on the poly(I:C)-induced expression of bFGF (Figure 8B) and IL-1β (Figure 8D) genes. The inhibitor of the phosphatidylinositol-3 kinase (PI3K)-Akt pathway, LY294002, reduced significantly (p<0.05) the poly(I:C)-induced expression of TLR3 (Figure 8A), TNFα (Figure 8C), and IL-1β (Figure 8D) genes, and had no effect on the poly(I:C)-induced expression of bFGF (Figure 8B) and CFB (Figure 8E) genes. The data suggest that the poly(I:C)-induced expression of various genes in RPE cells is differentially regulated by distinct signal transduction pathways.

Transcription factor activities involved in poly(I:C)-induced gene expression

Because poly(I:C) also induced the expression of various transcription factor genes (Figure 3B), we examined the involvement of transcription factor activities in the poly(I:C)-induced gene expression. To determine the role of HIF-1 activity, we tested an HIF inhibitor [35]. The HIF inhibitor decreased significantly (p<0.05) the poly(I:C)-induced transcriptional activation of the TLR3 gene (Figure 9A), and had no effect on the poly(I:C)-induced expression of TNFα (Figure 9C), IL-1β (Figure 9D), MCP-1 (Figure 9E), and CFB (Figure 9F) genes. The HIF inhibitor increased significantly (p<0.05) the expression of the bFGF gene under control and poly(I:C)-stimulated conditions (Figure 9B). The NF-κB inhibitor, caffeic acid phenethyl ester [36], decreased significantly (p<0.05) the poly(I:C)-induced expression of TLR3 (Figure 9A), bFGF (Figure 9B), and IL-1β (Figure 9D) genes, and had no effect on the poly(I:C)-induced expression of TNFα (Figure 9C), MCP-1 (Figure 9E), and CFB (Figure 9F) genes. The STAT3 inhibitor Stattic [37] reduced significantly (p<0.05) the poly(I:C)-induced expression of TLR3 (Figure 9A), TNFα (Figure 9C), IL-1β (Figure 9D), MCP-1 (Figure 9E), and CFB (Figure 9F) genes. Stattic had no effect on the poly(I:C)-induced expression of the bFGF gene (Figure 9B). The data suggest that the poly(I:C)-induced gene expression in RPE cells is differentially regulated by distinct transcription factor activities.