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Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5.

Hollborn M, Vogler S, Reichenbach A, Wiedemann P, Bringmann A, Kohen L - Mol. Vis. (2015)

Bottom Line: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration.The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells.

View Article: PubMed Central - PubMed

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

ABSTRACT

Purpose: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells.

Methods: Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA.

Results: Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.

Conclusions: Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

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Hyperosmolarity induces NFAT5 gene and protein expression, and the DNA binding of NFAT5, in RPE cells. The mRNA level (A, B) was determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The protein levels (C-E) were determined by western blot analysis of cytosolic (C) and nuclear extracts (D, E) of cells stimulated for 6 and 24 h, respectively. A. Effects of osmolarity changes, CoCl2 (150 µM; n=5), and cell culture in 1% O2 (n=4) on the level of NFAT5 mRNA. The hyperosmotic media were made up by adding 100 mM NaCl (n=5) and 100 mM sucrose (n=3), respectively. The hypoosmotic medium contained 60% of control osmolarity (n=5). Inset: Expression of β-actin and NFAT5 genes in RPE cells from different donors (1, 2) determined by RT–PCR. Negative controls (-) were done by adding double-distilled water instead of cDNA as a template. B. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. C. Effects of CoCl2 (150 µM), as well as of hyperosmotic (+ 100 mM NaCl) and hypoosmotic (Hypo) media, on the cellular level of the NFAT5 protein. Similar results were obtained in three independent experiments using cells from different donors. D. Hyperosmolarity (+ 50 and + 100 mM NaCl, respectively) increased dose-dependently the nuclear level of the NFAT5 protein. E. Hyperosmolarity (+ 100 mM NaCl) increased the nuclear levels of NFAT5 and p65/NF-κB proteins of RPE cells, while the nuclear level of STAT3 protein remained unchanged. As a control, the nuclear level of the histone H3 (HisH3) protein was determined. F. Hyperosmolarity (+ 100 mM NaCl) induces the DNA binding of NFAT5, as indicated by the appearance of complexes of NFAT5 protein and labeled oligonucleotides in EMSA that were not observed under isoosmotic conditions. An excess of unlabeled oligonucleotides (Competitor) abrogated the binding of NFAT5 protein to labeled oligonucleotides. Similar results were obtained in three independent experiments using cells from different donors. Bars represent means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
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f9: Hyperosmolarity induces NFAT5 gene and protein expression, and the DNA binding of NFAT5, in RPE cells. The mRNA level (A, B) was determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The protein levels (C-E) were determined by western blot analysis of cytosolic (C) and nuclear extracts (D, E) of cells stimulated for 6 and 24 h, respectively. A. Effects of osmolarity changes, CoCl2 (150 µM; n=5), and cell culture in 1% O2 (n=4) on the level of NFAT5 mRNA. The hyperosmotic media were made up by adding 100 mM NaCl (n=5) and 100 mM sucrose (n=3), respectively. The hypoosmotic medium contained 60% of control osmolarity (n=5). Inset: Expression of β-actin and NFAT5 genes in RPE cells from different donors (1, 2) determined by RT–PCR. Negative controls (-) were done by adding double-distilled water instead of cDNA as a template. B. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. C. Effects of CoCl2 (150 µM), as well as of hyperosmotic (+ 100 mM NaCl) and hypoosmotic (Hypo) media, on the cellular level of the NFAT5 protein. Similar results were obtained in three independent experiments using cells from different donors. D. Hyperosmolarity (+ 50 and + 100 mM NaCl, respectively) increased dose-dependently the nuclear level of the NFAT5 protein. E. Hyperosmolarity (+ 100 mM NaCl) increased the nuclear levels of NFAT5 and p65/NF-κB proteins of RPE cells, while the nuclear level of STAT3 protein remained unchanged. As a control, the nuclear level of the histone H3 (HisH3) protein was determined. F. Hyperosmolarity (+ 100 mM NaCl) induces the DNA binding of NFAT5, as indicated by the appearance of complexes of NFAT5 protein and labeled oligonucleotides in EMSA that were not observed under isoosmotic conditions. An excess of unlabeled oligonucleotides (Competitor) abrogated the binding of NFAT5 protein to labeled oligonucleotides. Similar results were obtained in three independent experiments using cells from different donors. Bars represent means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.

Mentions: In various cell systems, the transcriptional activity of NFAT5 is critical for cell survival under hyperosmotic conditions [37, 38]. We found that hyperosmotic media transiently increased the gene expression of NFAT5 in RPE cells while a hypoosmotic medium decreased the expression of NFAT5 (Figure 9A). The effect of high extracellular NaCl on the cellular NFAT5 mRNA level was dose-dependent (Figure 9B). Hyperosmolarity also increased the cellular (Figure 9C) and nuclear levels (Figure 9D, E) of the NFAT5 protein, while hypoosmolarity decreased the cellular level of NFAT5 protein (Figure 9C). In addition, hyperosmolarity increased the nuclear level of p65/NF-κB protein, while the nuclear level of STAT3 protein remained unchanged (Figure 9E). EMSA showed that hyperosmolarity induced the DNA binding of the NFAT5 protein (Figure 9F). Chemical hypoxia did not induce increased levels of NFAT5 mRNA (Figure 9A) and protein (Figure 9C). Similarly, hypoxic conditions induced by culturing the cells in 1% O2 did not alter the gene expression of NFAT5 (Figure 9A). In addition, oxidative stress induced by the addition of H2O2 (20 µM) did not increase the nuclear level of the NFAT5 protein (n=3; not shown). The data suggest that the expression of NFAT5 in RPE cells is regulated by osmotic changes, but not by hypoxia and oxidative stress.


Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5.

Hollborn M, Vogler S, Reichenbach A, Wiedemann P, Bringmann A, Kohen L - Mol. Vis. (2015)

Hyperosmolarity induces NFAT5 gene and protein expression, and the DNA binding of NFAT5, in RPE cells. The mRNA level (A, B) was determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The protein levels (C-E) were determined by western blot analysis of cytosolic (C) and nuclear extracts (D, E) of cells stimulated for 6 and 24 h, respectively. A. Effects of osmolarity changes, CoCl2 (150 µM; n=5), and cell culture in 1% O2 (n=4) on the level of NFAT5 mRNA. The hyperosmotic media were made up by adding 100 mM NaCl (n=5) and 100 mM sucrose (n=3), respectively. The hypoosmotic medium contained 60% of control osmolarity (n=5). Inset: Expression of β-actin and NFAT5 genes in RPE cells from different donors (1, 2) determined by RT–PCR. Negative controls (-) were done by adding double-distilled water instead of cDNA as a template. B. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. C. Effects of CoCl2 (150 µM), as well as of hyperosmotic (+ 100 mM NaCl) and hypoosmotic (Hypo) media, on the cellular level of the NFAT5 protein. Similar results were obtained in three independent experiments using cells from different donors. D. Hyperosmolarity (+ 50 and + 100 mM NaCl, respectively) increased dose-dependently the nuclear level of the NFAT5 protein. E. Hyperosmolarity (+ 100 mM NaCl) increased the nuclear levels of NFAT5 and p65/NF-κB proteins of RPE cells, while the nuclear level of STAT3 protein remained unchanged. As a control, the nuclear level of the histone H3 (HisH3) protein was determined. F. Hyperosmolarity (+ 100 mM NaCl) induces the DNA binding of NFAT5, as indicated by the appearance of complexes of NFAT5 protein and labeled oligonucleotides in EMSA that were not observed under isoosmotic conditions. An excess of unlabeled oligonucleotides (Competitor) abrogated the binding of NFAT5 protein to labeled oligonucleotides. Similar results were obtained in three independent experiments using cells from different donors. Bars represent means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
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Show All Figures
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f9: Hyperosmolarity induces NFAT5 gene and protein expression, and the DNA binding of NFAT5, in RPE cells. The mRNA level (A, B) was determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The protein levels (C-E) were determined by western blot analysis of cytosolic (C) and nuclear extracts (D, E) of cells stimulated for 6 and 24 h, respectively. A. Effects of osmolarity changes, CoCl2 (150 µM; n=5), and cell culture in 1% O2 (n=4) on the level of NFAT5 mRNA. The hyperosmotic media were made up by adding 100 mM NaCl (n=5) and 100 mM sucrose (n=3), respectively. The hypoosmotic medium contained 60% of control osmolarity (n=5). Inset: Expression of β-actin and NFAT5 genes in RPE cells from different donors (1, 2) determined by RT–PCR. Negative controls (-) were done by adding double-distilled water instead of cDNA as a template. B. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. C. Effects of CoCl2 (150 µM), as well as of hyperosmotic (+ 100 mM NaCl) and hypoosmotic (Hypo) media, on the cellular level of the NFAT5 protein. Similar results were obtained in three independent experiments using cells from different donors. D. Hyperosmolarity (+ 50 and + 100 mM NaCl, respectively) increased dose-dependently the nuclear level of the NFAT5 protein. E. Hyperosmolarity (+ 100 mM NaCl) increased the nuclear levels of NFAT5 and p65/NF-κB proteins of RPE cells, while the nuclear level of STAT3 protein remained unchanged. As a control, the nuclear level of the histone H3 (HisH3) protein was determined. F. Hyperosmolarity (+ 100 mM NaCl) induces the DNA binding of NFAT5, as indicated by the appearance of complexes of NFAT5 protein and labeled oligonucleotides in EMSA that were not observed under isoosmotic conditions. An excess of unlabeled oligonucleotides (Competitor) abrogated the binding of NFAT5 protein to labeled oligonucleotides. Similar results were obtained in three independent experiments using cells from different donors. Bars represent means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
Mentions: In various cell systems, the transcriptional activity of NFAT5 is critical for cell survival under hyperosmotic conditions [37, 38]. We found that hyperosmotic media transiently increased the gene expression of NFAT5 in RPE cells while a hypoosmotic medium decreased the expression of NFAT5 (Figure 9A). The effect of high extracellular NaCl on the cellular NFAT5 mRNA level was dose-dependent (Figure 9B). Hyperosmolarity also increased the cellular (Figure 9C) and nuclear levels (Figure 9D, E) of the NFAT5 protein, while hypoosmolarity decreased the cellular level of NFAT5 protein (Figure 9C). In addition, hyperosmolarity increased the nuclear level of p65/NF-κB protein, while the nuclear level of STAT3 protein remained unchanged (Figure 9E). EMSA showed that hyperosmolarity induced the DNA binding of the NFAT5 protein (Figure 9F). Chemical hypoxia did not induce increased levels of NFAT5 mRNA (Figure 9A) and protein (Figure 9C). Similarly, hypoxic conditions induced by culturing the cells in 1% O2 did not alter the gene expression of NFAT5 (Figure 9A). In addition, oxidative stress induced by the addition of H2O2 (20 µM) did not increase the nuclear level of the NFAT5 protein (n=3; not shown). The data suggest that the expression of NFAT5 in RPE cells is regulated by osmotic changes, but not by hypoxia and oxidative stress.

Bottom Line: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration.The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells.

View Article: PubMed Central - PubMed

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

ABSTRACT

Purpose: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells.

Methods: Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA.

Results: Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.

Conclusions: Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

Show MeSH
Related in: MedlinePlus