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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

Intracellular signaling involved in poly(I:C; 500 µg/ml)-induced gene expression in RPE cells. The cellular levels of the following gene transcripts were determined: TLR3 (A), bFGF (B), TNFα (C), IL-1β (D), and CFB (E) mRNAs. mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent cultures for 2 h (TNFα and IL-1β mRNAs) and 6 h (TLR3, bFGF, and CFB mRNAs), respectively, and are expressed as folds of unstimulated controls. The following blocking agents were tested: the inhibitor of ERK1/2 activation, PD98059 (20 µM); the inhibitor of p38 MAPK activation, SB203580 (10 µM); the JNK inhibitor SP600125 (10 µM); and the inhibitor of PI3K-related kinases, LY294002 (5 µM). Vehicle control was made with DMSO (DMSO; 1:1000). Each bar represents data obtained in 3 to 9 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. Significant difference versus poly(I:C) controls: ●p<0.05.
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f8: Intracellular signaling involved in poly(I:C; 500 µg/ml)-induced gene expression in RPE cells. The cellular levels of the following gene transcripts were determined: TLR3 (A), bFGF (B), TNFα (C), IL-1β (D), and CFB (E) mRNAs. mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent cultures for 2 h (TNFα and IL-1β mRNAs) and 6 h (TLR3, bFGF, and CFB mRNAs), respectively, and are expressed as folds of unstimulated controls. The following blocking agents were tested: the inhibitor of ERK1/2 activation, PD98059 (20 µM); the inhibitor of p38 MAPK activation, SB203580 (10 µM); the JNK inhibitor SP600125 (10 µM); and the inhibitor of PI3K-related kinases, LY294002 (5 µM). Vehicle control was made with DMSO (DMSO; 1:1000). Each bar represents data obtained in 3 to 9 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. Significant difference versus poly(I:C) controls: ●p<0.05.

Mentions: 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.


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)

Intracellular signaling involved in poly(I:C; 500 µg/ml)-induced gene expression in RPE cells. The cellular levels of the following gene transcripts were determined: TLR3 (A), bFGF (B), TNFα (C), IL-1β (D), and CFB (E) mRNAs. mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent cultures for 2 h (TNFα and IL-1β mRNAs) and 6 h (TLR3, bFGF, and CFB mRNAs), respectively, and are expressed as folds of unstimulated controls. The following blocking agents were tested: the inhibitor of ERK1/2 activation, PD98059 (20 µM); the inhibitor of p38 MAPK activation, SB203580 (10 µM); the JNK inhibitor SP600125 (10 µM); and the inhibitor of PI3K-related kinases, LY294002 (5 µM). Vehicle control was made with DMSO (DMSO; 1:1000). Each bar represents data obtained in 3 to 9 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. Significant difference versus poly(I:C) controls: ●p<0.05.
© Copyright Policy - open-access
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f8: Intracellular signaling involved in poly(I:C; 500 µg/ml)-induced gene expression in RPE cells. The cellular levels of the following gene transcripts were determined: TLR3 (A), bFGF (B), TNFα (C), IL-1β (D), and CFB (E) mRNAs. mRNA levels were determined with real-time RT–PCR analysis after stimulation of near-confluent cultures for 2 h (TNFα and IL-1β mRNAs) and 6 h (TLR3, bFGF, and CFB mRNAs), respectively, and are expressed as folds of unstimulated controls. The following blocking agents were tested: the inhibitor of ERK1/2 activation, PD98059 (20 µM); the inhibitor of p38 MAPK activation, SB203580 (10 µM); the JNK inhibitor SP600125 (10 µM); and the inhibitor of PI3K-related kinases, LY294002 (5 µM). Vehicle control was made with DMSO (DMSO; 1:1000). Each bar represents data obtained in 3 to 9 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. Significant difference versus poly(I:C) controls: ●p<0.05.
Mentions: 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.

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