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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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Involvement of signal transduction pathways in the hyperosmotic induction of VEGF in RPE cells. The mRNA level (A) was determined with real-time RT–PCR analysis in cells cultured 6 h under iso- (control) and hyperosmotic conditions (+ 100 mM NaCl), and is expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (B) was determined with ELISA in the media of cells cultured 24 h under iso- and hyperosmotic conditions, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 281.7±45.8 pg/ml VEGF). The cytosolic protein levels (C) were determined by western blot analysis. A. The hyperosmotic upregulation of VEGFA was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=6); the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=6); the JNK inhibitor SP600125 (10 µM; n=5); and the inhibitor of PI3K-related kinases, LY294002 (5 µM; n=6), respectively. The inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 µM; n=5), and the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM; n=5), did not inhibit the hyperosmotic upregulation of VEGFA. The vehicle control was made with DMSO (1%; n=3). B. The inhibitors of p38 MAPK (n=5), ERK1/2 (n=5), JNK (n=5), and PI3K (n=5) also decreased the secretion of VEGF induced by hyperosmotic stimulation. C. Stimulation of the cells for 20 min with CoCl2 (150 µM) or hyperosmotic medium (+ 100 mM NaCl) induced the phosphorylation of p38 and ERK1/2 proteins. PDGF (10 ng/ml) was used as a positive control. Amounts of total proteins are shown above; amounts of phosphorylated proteins are shown below. 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. Significant difference versus NaCl control: ●p<0.05.
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f3: Involvement of signal transduction pathways in the hyperosmotic induction of VEGF in RPE cells. The mRNA level (A) was determined with real-time RT–PCR analysis in cells cultured 6 h under iso- (control) and hyperosmotic conditions (+ 100 mM NaCl), and is expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (B) was determined with ELISA in the media of cells cultured 24 h under iso- and hyperosmotic conditions, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 281.7±45.8 pg/ml VEGF). The cytosolic protein levels (C) were determined by western blot analysis. A. The hyperosmotic upregulation of VEGFA was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=6); the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=6); the JNK inhibitor SP600125 (10 µM; n=5); and the inhibitor of PI3K-related kinases, LY294002 (5 µM; n=6), respectively. The inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 µM; n=5), and the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM; n=5), did not inhibit the hyperosmotic upregulation of VEGFA. The vehicle control was made with DMSO (1%; n=3). B. The inhibitors of p38 MAPK (n=5), ERK1/2 (n=5), JNK (n=5), and PI3K (n=5) also decreased the secretion of VEGF induced by hyperosmotic stimulation. C. Stimulation of the cells for 20 min with CoCl2 (150 µM) or hyperosmotic medium (+ 100 mM NaCl) induced the phosphorylation of p38 and ERK1/2 proteins. PDGF (10 ng/ml) was used as a positive control. Amounts of total proteins are shown above; amounts of phosphorylated proteins are shown below. 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. Significant difference versus NaCl control: ●p<0.05.

Mentions: The hyperosmotic increase in VEGF gene expression resulted from the stimulation of gene transcription (Figure 2A) and not from altered mRNA stability (Figure 2B). The hyperosmotic increase in VEGF gene transcription was nearly fully abrogated by inhibitors of p38 MAPK, ERK1/2, c-Jun NH2-terminal kinase (JNK), and phosphatidylinositol-3 kinase (PI3K)-Akt signal transduction pathways (Figure 3A). The inhibitors also decreased the hyperosmotic secretion of VEGF (Figure 3B). Selective antagonists of PDGF and epidermal growth factor (EGF) receptor tyrosine kinases, AG1296 and AG1478, did not inhibit the hyperosmotic expression of VEGF (Figure 3A), suggesting that the upregulation of VEGF was not mediated by autocrine/paracrine PDGF and EGF signaling. In agreement with a recent study [42], we found that hyperosmotic stimulation for 20 min induced increased phosphorylation of p38 MAPK protein (Figure 3C). In addition, hyperosmotic stimulation induced the phosphorylation of ERK1/2 proteins (Figure 3C). The phosphorylation of p38 and ERK1/2 was not stimulated by chemical hypoxia for 20 min (Figure 3C).


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)

Involvement of signal transduction pathways in the hyperosmotic induction of VEGF in RPE cells. The mRNA level (A) was determined with real-time RT–PCR analysis in cells cultured 6 h under iso- (control) and hyperosmotic conditions (+ 100 mM NaCl), and is expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (B) was determined with ELISA in the media of cells cultured 24 h under iso- and hyperosmotic conditions, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 281.7±45.8 pg/ml VEGF). The cytosolic protein levels (C) were determined by western blot analysis. A. The hyperosmotic upregulation of VEGFA was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=6); the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=6); the JNK inhibitor SP600125 (10 µM; n=5); and the inhibitor of PI3K-related kinases, LY294002 (5 µM; n=6), respectively. The inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 µM; n=5), and the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM; n=5), did not inhibit the hyperosmotic upregulation of VEGFA. The vehicle control was made with DMSO (1%; n=3). B. The inhibitors of p38 MAPK (n=5), ERK1/2 (n=5), JNK (n=5), and PI3K (n=5) also decreased the secretion of VEGF induced by hyperosmotic stimulation. C. Stimulation of the cells for 20 min with CoCl2 (150 µM) or hyperosmotic medium (+ 100 mM NaCl) induced the phosphorylation of p38 and ERK1/2 proteins. PDGF (10 ng/ml) was used as a positive control. Amounts of total proteins are shown above; amounts of phosphorylated proteins are shown below. 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. Significant difference versus NaCl control: ●p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Involvement of signal transduction pathways in the hyperosmotic induction of VEGF in RPE cells. The mRNA level (A) was determined with real-time RT–PCR analysis in cells cultured 6 h under iso- (control) and hyperosmotic conditions (+ 100 mM NaCl), and is expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (B) was determined with ELISA in the media of cells cultured 24 h under iso- and hyperosmotic conditions, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 281.7±45.8 pg/ml VEGF). The cytosolic protein levels (C) were determined by western blot analysis. A. The hyperosmotic upregulation of VEGFA was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=6); the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=6); the JNK inhibitor SP600125 (10 µM; n=5); and the inhibitor of PI3K-related kinases, LY294002 (5 µM; n=6), respectively. The inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 µM; n=5), and the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM; n=5), did not inhibit the hyperosmotic upregulation of VEGFA. The vehicle control was made with DMSO (1%; n=3). B. The inhibitors of p38 MAPK (n=5), ERK1/2 (n=5), JNK (n=5), and PI3K (n=5) also decreased the secretion of VEGF induced by hyperosmotic stimulation. C. Stimulation of the cells for 20 min with CoCl2 (150 µM) or hyperosmotic medium (+ 100 mM NaCl) induced the phosphorylation of p38 and ERK1/2 proteins. PDGF (10 ng/ml) was used as a positive control. Amounts of total proteins are shown above; amounts of phosphorylated proteins are shown below. 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. Significant difference versus NaCl control: ●p<0.05.
Mentions: The hyperosmotic increase in VEGF gene expression resulted from the stimulation of gene transcription (Figure 2A) and not from altered mRNA stability (Figure 2B). The hyperosmotic increase in VEGF gene transcription was nearly fully abrogated by inhibitors of p38 MAPK, ERK1/2, c-Jun NH2-terminal kinase (JNK), and phosphatidylinositol-3 kinase (PI3K)-Akt signal transduction pathways (Figure 3A). The inhibitors also decreased the hyperosmotic secretion of VEGF (Figure 3B). Selective antagonists of PDGF and epidermal growth factor (EGF) receptor tyrosine kinases, AG1296 and AG1478, did not inhibit the hyperosmotic expression of VEGF (Figure 3A), suggesting that the upregulation of VEGF was not mediated by autocrine/paracrine PDGF and EGF signaling. In agreement with a recent study [42], we found that hyperosmotic stimulation for 20 min induced increased phosphorylation of p38 MAPK protein (Figure 3C). In addition, hyperosmotic stimulation induced the phosphorylation of ERK1/2 proteins (Figure 3C). The phosphorylation of p38 and ERK1/2 was not stimulated by chemical hypoxia for 20 min (Figure 3C).

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