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Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.

Shankardas J, Patil RV, Vishwanatha JK - Mol. Vis. (2010)

Bottom Line: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK).In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane.AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Purpose: The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively.

Methods: AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay.

Results: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.

Conclusions: AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.

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Related in: MedlinePlus

Effective silencing of AQP1 and AQP5 using siRNA. Down-regulation of AQP1 expression in HCEC cells using siRNA as determined by (A) RT–PCR and (B) western blot analysis. Down-regulation of AQP5 expression in CEPI17 cells using siRNA as determined by (C) RT–PCR, (D) western blot analysis. Panel E shows down-regulation of AQP5 (green) in CEPI17 cells, 96 h post trasnfection with AQP5 siRNA. AQP5 down regulation was determined by Immunocytochemical analysis (DAPI was used to stain the nucleus).
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f2: Effective silencing of AQP1 and AQP5 using siRNA. Down-regulation of AQP1 expression in HCEC cells using siRNA as determined by (A) RT–PCR and (B) western blot analysis. Down-regulation of AQP5 expression in CEPI17 cells using siRNA as determined by (C) RT–PCR, (D) western blot analysis. Panel E shows down-regulation of AQP5 (green) in CEPI17 cells, 96 h post trasnfection with AQP5 siRNA. AQP5 down regulation was determined by Immunocytochemical analysis (DAPI was used to stain the nucleus).

Mentions: HCEC cells were transfected with AQP1 siRNA and scrambled control siRNA using lipofectamine. Significant down-regulation (98%) in the AQP1 mRNA level was observed in 48 h (lane 3, Figure 2A) following transfection with AQP1 siRNA as determined by RT–PCR analysis. The mRNA levels, however, increased at the 96 h time point and hence all the downstream experiments were conducted 72 to 96 h after transfection. Significant down-regulation (97%) in AQP1 protein synthesis was also observed 72 h (lane 5, Figure 2B) after transfection with AQP1 siRNA. This down-regulation was determined by western blot analysis using AQP1 antibody and the experiments were repeated three times (n=3).


Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.

Shankardas J, Patil RV, Vishwanatha JK - Mol. Vis. (2010)

Effective silencing of AQP1 and AQP5 using siRNA. Down-regulation of AQP1 expression in HCEC cells using siRNA as determined by (A) RT–PCR and (B) western blot analysis. Down-regulation of AQP5 expression in CEPI17 cells using siRNA as determined by (C) RT–PCR, (D) western blot analysis. Panel E shows down-regulation of AQP5 (green) in CEPI17 cells, 96 h post trasnfection with AQP5 siRNA. AQP5 down regulation was determined by Immunocytochemical analysis (DAPI was used to stain the nucleus).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2925904&req=5

f2: Effective silencing of AQP1 and AQP5 using siRNA. Down-regulation of AQP1 expression in HCEC cells using siRNA as determined by (A) RT–PCR and (B) western blot analysis. Down-regulation of AQP5 expression in CEPI17 cells using siRNA as determined by (C) RT–PCR, (D) western blot analysis. Panel E shows down-regulation of AQP5 (green) in CEPI17 cells, 96 h post trasnfection with AQP5 siRNA. AQP5 down regulation was determined by Immunocytochemical analysis (DAPI was used to stain the nucleus).
Mentions: HCEC cells were transfected with AQP1 siRNA and scrambled control siRNA using lipofectamine. Significant down-regulation (98%) in the AQP1 mRNA level was observed in 48 h (lane 3, Figure 2A) following transfection with AQP1 siRNA as determined by RT–PCR analysis. The mRNA levels, however, increased at the 96 h time point and hence all the downstream experiments were conducted 72 to 96 h after transfection. Significant down-regulation (97%) in AQP1 protein synthesis was also observed 72 h (lane 5, Figure 2B) after transfection with AQP1 siRNA. This down-regulation was determined by western blot analysis using AQP1 antibody and the experiments were repeated three times (n=3).

Bottom Line: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK).In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane.AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Purpose: The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively.

Methods: AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay.

Results: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.

Conclusions: AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.

Show MeSH
Related in: MedlinePlus