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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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The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels (A, B, D-G) were 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 (A, B, D) and NaCl control (E-G), respectively. The level of VEGF-A165 protein (C, H) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [C] and 324.2±35.1 pg/ml VEGF [H], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 (A; n=7) and VEGF (B; n=3), as well as the hyperosmotic secretion of VEGF (C; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic (D; n=5) and hyperosmotic (+ 100 mM NaCl) media (E; n=3). Data shown in (D) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively (E). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 (F; n=6) and VEGF mRNAs (G; n=6) in cells cultured for 24 (F) and 6 h (G), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (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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f10: The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels (A, B, D-G) were 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 (A, B, D) and NaCl control (E-G), respectively. The level of VEGF-A165 protein (C, H) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [C] and 324.2±35.1 pg/ml VEGF [H], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 (A; n=7) and VEGF (B; n=3), as well as the hyperosmotic secretion of VEGF (C; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic (D; n=5) and hyperosmotic (+ 100 mM NaCl) media (E; n=3). Data shown in (D) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively (E). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 (F; n=6) and VEGF mRNAs (G; n=6) in cells cultured for 24 (F) and 6 h (G), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (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: To determine whether the hyperosmotic expression of AQP5 and VEGF depends on the transcriptional activity of NFAT5, we tested the NFAT5 inhibitor rottlerin [51]. Rottlerin inhibited the hyperosmotic gene expression of AQP5 (Figure 10A) and VEGF (Figure 10B), as well as the hyperosmotic secretion of VEGF (Figure 10C). To confirm the involvement of NFAT5 activity in the hyperosmotic induction of AQP5 and VEGF with another method, we used siRNA to knock down NFAT5. The inhibitory activity of the siRNA was confirmed in cells cultured under isoosmotic (Figure 10D) and hyperosmotic conditions (Figure 10E). RPE cells transfected with NFAT5 siRNA displayed a significantly (p<0.05) reduced upregulation of AQP5 (Figure 10F) and VEGF expression (Figure 10G), and a significantly (p<0.05) reduced secretion of VEGF (Figure 10H) under hyperosmotic conditions compared to non-transfected cells. Transfection of the cells with nontargeted negative control siRNA had no significant effects on the hyperosmotic induction of AQP5 and VEGF gene expression (Figure 10F,G), or on the hyperosmotic secretion of VEGF from RPE cells (Figure 10H).


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)

The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels (A, B, D-G) were 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 (A, B, D) and NaCl control (E-G), respectively. The level of VEGF-A165 protein (C, H) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [C] and 324.2±35.1 pg/ml VEGF [H], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 (A; n=7) and VEGF (B; n=3), as well as the hyperosmotic secretion of VEGF (C; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic (D; n=5) and hyperosmotic (+ 100 mM NaCl) media (E; n=3). Data shown in (D) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively (E). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 (F; n=6) and VEGF mRNAs (G; n=6) in cells cultured for 24 (F) and 6 h (G), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (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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f10: The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels (A, B, D-G) were 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 (A, B, D) and NaCl control (E-G), respectively. The level of VEGF-A165 protein (C, H) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [C] and 324.2±35.1 pg/ml VEGF [H], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 (A; n=7) and VEGF (B; n=3), as well as the hyperosmotic secretion of VEGF (C; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic (D; n=5) and hyperosmotic (+ 100 mM NaCl) media (E; n=3). Data shown in (D) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively (E). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 (F; n=6) and VEGF mRNAs (G; n=6) in cells cultured for 24 (F) and 6 h (G), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (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: To determine whether the hyperosmotic expression of AQP5 and VEGF depends on the transcriptional activity of NFAT5, we tested the NFAT5 inhibitor rottlerin [51]. Rottlerin inhibited the hyperosmotic gene expression of AQP5 (Figure 10A) and VEGF (Figure 10B), as well as the hyperosmotic secretion of VEGF (Figure 10C). To confirm the involvement of NFAT5 activity in the hyperosmotic induction of AQP5 and VEGF with another method, we used siRNA to knock down NFAT5. The inhibitory activity of the siRNA was confirmed in cells cultured under isoosmotic (Figure 10D) and hyperosmotic conditions (Figure 10E). RPE cells transfected with NFAT5 siRNA displayed a significantly (p<0.05) reduced upregulation of AQP5 (Figure 10F) and VEGF expression (Figure 10G), and a significantly (p<0.05) reduced secretion of VEGF (Figure 10H) under hyperosmotic conditions compared to non-transfected cells. Transfection of the cells with nontargeted negative control siRNA had no significant effects on the hyperosmotic induction of AQP5 and VEGF gene expression (Figure 10F,G), or on the hyperosmotic secretion of VEGF from RPE cells (Figure 10H).

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