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

Brosig A, Kuhrt H, Wiedemann P, Kohen L, Bringmann A, Hollborn M - Mol. Vis. (2015)

Bottom Line: We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina.The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

Purpose: The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.

Methods: Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting.

Results: Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes.

Conclusions: The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

No MeSH data available.


Related in: MedlinePlus

Effects of viral RNA and viral/bacterial DNA on the gene expression of the transcription factor proteins HIF-1α, RELA (p65/NF-κB), STAT3, and NFAT5. A: Gene expression of transcription factor proteins in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B-D: The mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent (B, D) and confluent (C) cultures for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. The cells were stimulated with poly(I:C; 500 µg/ml; B, C) and CpG-ODN (500 nM; D), respectively. Each bar represents data obtained in 3 to 7 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate. Significant difference versus unstimulated controls: *p<0.05.
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f3: Effects of viral RNA and viral/bacterial DNA on the gene expression of the transcription factor proteins HIF-1α, RELA (p65/NF-κB), STAT3, and NFAT5. A: Gene expression of transcription factor proteins in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B-D: The mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent (B, D) and confluent (C) cultures for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. The cells were stimulated with poly(I:C; 500 µg/ml; B, C) and CpG-ODN (500 nM; D), respectively. Each bar represents data obtained in 3 to 7 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate. Significant difference versus unstimulated controls: *p<0.05.

Mentions: 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).


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

Brosig A, Kuhrt H, Wiedemann P, Kohen L, Bringmann A, Hollborn M - Mol. Vis. (2015)

Effects of viral RNA and viral/bacterial DNA on the gene expression of the transcription factor proteins HIF-1α, RELA (p65/NF-κB), STAT3, and NFAT5. A: Gene expression of transcription factor proteins in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B-D: The mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent (B, D) and confluent (C) cultures for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. The cells were stimulated with poly(I:C; 500 µg/ml; B, C) and CpG-ODN (500 nM; D), respectively. Each bar represents data obtained in 3 to 7 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate. Significant difference versus unstimulated controls: *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554413&req=5

f3: Effects of viral RNA and viral/bacterial DNA on the gene expression of the transcription factor proteins HIF-1α, RELA (p65/NF-κB), STAT3, and NFAT5. A: Gene expression of transcription factor proteins in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B-D: The mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent (B, D) and confluent (C) cultures for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. The cells were stimulated with poly(I:C; 500 µg/ml; B, C) and CpG-ODN (500 nM; D), respectively. Each bar represents data obtained in 3 to 7 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate. Significant difference versus unstimulated controls: *p<0.05.
Mentions: 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).

Bottom Line: We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina.The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

Purpose: The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.

Methods: Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting.

Results: Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes.

Conclusions: The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

No MeSH data available.


Related in: MedlinePlus