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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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Dependence of the hyperosmotic induction of AQP5 in RPE cells on the activation of signal transduction pathways. The mRNA level was measured with real-time RT–PCR analysis. The cells were cultured for 24 h in isoosmotic (control) and hyperosmotic (+ 100 mM NaCl) media. The hyperosmotic elevation of AQP5 expression was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=8), and the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=10), respectively. Inhibitors of JNK activation (SP600125; 10 µM; n=10), of the catalytic subunits of PI3K-related kinases (LY294002; 5 µM; n=8), of the PDGF receptor tyrosine kinase (AG1296; 10 µM; n=8), of the EGF receptor tyrosine kinase (AG1478; 600 nM; n=8), and of the VEGF receptor-2 (SU1498; 10 µM; n=8) did not decrease the hyperosmotic induction of AQP5. The vehicle control was made with DMSO (1%; n=6). Data are 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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f7: Dependence of the hyperosmotic induction of AQP5 in RPE cells on the activation of signal transduction pathways. The mRNA level was measured with real-time RT–PCR analysis. The cells were cultured for 24 h in isoosmotic (control) and hyperosmotic (+ 100 mM NaCl) media. The hyperosmotic elevation of AQP5 expression was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=8), and the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=10), respectively. Inhibitors of JNK activation (SP600125; 10 µM; n=10), of the catalytic subunits of PI3K-related kinases (LY294002; 5 µM; n=8), of the PDGF receptor tyrosine kinase (AG1296; 10 µM; n=8), of the EGF receptor tyrosine kinase (AG1478; 600 nM; n=8), and of the VEGF receptor-2 (SU1498; 10 µM; n=8) did not decrease the hyperosmotic induction of AQP5. The vehicle control was made with DMSO (1%; n=6). Data are 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: Hyperosmotic AQP5 expression was induced by the stimulation of gene transcription (Figure 6A) and not by altered mRNA stability (Figure 6B). The hyperosmotic expression of AQP5 was significantly (p<0.05) decreased by the inhibitors of p38 MAPK and ERK1/2 signal transduction pathways (Figure 7). Inhibitors of the PI3K signal transduction pathway and JNK activation had no effects (Figure 7). Furthermore, antagonists of PDGF and EGF receptor tyrosine kinases, and of the VEGF receptor-2, did not inhibit the hyperosmotic upregulation of AQP5 (Figure 7). The data suggest that hyperosmotic AQP5 gene expression is in part dependent on the activation of p38 MAPK and ERK1/2 signal transduction pathways.


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)

Dependence of the hyperosmotic induction of AQP5 in RPE cells on the activation of signal transduction pathways. The mRNA level was measured with real-time RT–PCR analysis. The cells were cultured for 24 h in isoosmotic (control) and hyperosmotic (+ 100 mM NaCl) media. The hyperosmotic elevation of AQP5 expression was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=8), and the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=10), respectively. Inhibitors of JNK activation (SP600125; 10 µM; n=10), of the catalytic subunits of PI3K-related kinases (LY294002; 5 µM; n=8), of the PDGF receptor tyrosine kinase (AG1296; 10 µM; n=8), of the EGF receptor tyrosine kinase (AG1478; 600 nM; n=8), and of the VEGF receptor-2 (SU1498; 10 µM; n=8) did not decrease the hyperosmotic induction of AQP5. The vehicle control was made with DMSO (1%; n=6). Data are 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

License
Show All Figures
getmorefigures.php?uid=PMC4390809&req=5

f7: Dependence of the hyperosmotic induction of AQP5 in RPE cells on the activation of signal transduction pathways. The mRNA level was measured with real-time RT–PCR analysis. The cells were cultured for 24 h in isoosmotic (control) and hyperosmotic (+ 100 mM NaCl) media. The hyperosmotic elevation of AQP5 expression was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=8), and the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=10), respectively. Inhibitors of JNK activation (SP600125; 10 µM; n=10), of the catalytic subunits of PI3K-related kinases (LY294002; 5 µM; n=8), of the PDGF receptor tyrosine kinase (AG1296; 10 µM; n=8), of the EGF receptor tyrosine kinase (AG1478; 600 nM; n=8), and of the VEGF receptor-2 (SU1498; 10 µM; n=8) did not decrease the hyperosmotic induction of AQP5. The vehicle control was made with DMSO (1%; n=6). Data are 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: Hyperosmotic AQP5 expression was induced by the stimulation of gene transcription (Figure 6A) and not by altered mRNA stability (Figure 6B). The hyperosmotic expression of AQP5 was significantly (p<0.05) decreased by the inhibitors of p38 MAPK and ERK1/2 signal transduction pathways (Figure 7). Inhibitors of the PI3K signal transduction pathway and JNK activation had no effects (Figure 7). Furthermore, antagonists of PDGF and EGF receptor tyrosine kinases, and of the VEGF receptor-2, did not inhibit the hyperosmotic upregulation of AQP5 (Figure 7). The data suggest that hyperosmotic AQP5 gene expression is in part dependent on the activation of p38 MAPK and ERK1/2 signal transduction pathways.

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