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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 on the phosphorylation levels of ERK1/2, p38 MAPK, Akt, and STAT3 proteins in RPE cells. Near-confluent cultures were stimulated for 20 min and 1 h, respectively, with 100 and 500 µg/ml of poly(I:C). PDGF (10 ng/ml) was used as a positive control, and β-actin as a control for equal protein loading. Amounts of total proteins are shown above, while amounts of phosphorylated proteins are shown below. Similar results were obtained in three independent experiments using RPE cell lines from different human eye donors.
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f7: Effects of viral RNA on the phosphorylation levels of ERK1/2, p38 MAPK, Akt, and STAT3 proteins in RPE cells. Near-confluent cultures were stimulated for 20 min and 1 h, respectively, with 100 and 500 µg/ml of poly(I:C). PDGF (10 ng/ml) was used as a positive control, and β-actin as a control for equal protein loading. Amounts of total proteins are shown above, while amounts of phosphorylated proteins are shown below. Similar results were obtained in three independent experiments using RPE cell lines from different human eye donors.

Mentions: 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.


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 on the phosphorylation levels of ERK1/2, p38 MAPK, Akt, and STAT3 proteins in RPE cells. Near-confluent cultures were stimulated for 20 min and 1 h, respectively, with 100 and 500 µg/ml of poly(I:C). PDGF (10 ng/ml) was used as a positive control, and β-actin as a control for equal protein loading. Amounts of total proteins are shown above, while amounts of phosphorylated proteins are shown below. Similar results were obtained in three independent experiments using RPE cell lines from different human eye donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Effects of viral RNA on the phosphorylation levels of ERK1/2, p38 MAPK, Akt, and STAT3 proteins in RPE cells. Near-confluent cultures were stimulated for 20 min and 1 h, respectively, with 100 and 500 µg/ml of poly(I:C). PDGF (10 ng/ml) was used as a positive control, and β-actin as a control for equal protein loading. Amounts of total proteins are shown above, while amounts of phosphorylated proteins are shown below. Similar results were obtained in three independent experiments using RPE cell lines from different human eye donors.
Mentions: 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.

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