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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

Effect of siRNA mediated AQP5 down-regulation (AQP5DR) on cell proliferation in CEPI17 cells. A: SRB assay shows that AQP5DR increased cell proliferation in CEPI17 cells (significant at 3 and 5 days p<0.05, 95% CI as determined by the paired T-Test, [n=3]). There was no significant difference in phosphorylated ERK 96 h post transfection in CEPI17 cells when compared to the controls as shown by western blot analysis (B).
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f6: Effect of siRNA mediated AQP5 down-regulation (AQP5DR) on cell proliferation in CEPI17 cells. A: SRB assay shows that AQP5DR increased cell proliferation in CEPI17 cells (significant at 3 and 5 days p<0.05, 95% CI as determined by the paired T-Test, [n=3]). There was no significant difference in phosphorylated ERK 96 h post transfection in CEPI17 cells when compared to the controls as shown by western blot analysis (B).

Mentions: AQP5 siRNA was used to study the effect of down regulation of AQP5 on migration of CEPI17 cells. CEPI17 cells were transfected with AQP5 siRNA and wound-healing assay was performed to study the effect of AQP5 siRNA on the migration of CEPI17 cells as described earlier. As seen in Figure 5A, AQP5 down regulation resulted in significant increase in migration of CEPI17 cells as determined by the in vitro wound-healing assay. However, when control siRNA was used to transfect the CEPI17 cells it had no effect on cell migration (Figure 5A). The area of the wound over time was measured using image J software and statistical analysis was performed. There was significant increase in cell migration 18 h after the scratch was created (Figure 5A,B). The scratch assay was also performed in the presence of 0.04% Mitomycin C, an inhibitor of cell proliferation, and similar results were obtained (data not shown). To study the effect of AQP5 down regulation on cell proliferation, SRB assay was performed as described earlier. Significant increase in cell proliferation was also observed on AQP5 down-regulation as determined by SRB proliferation assay (Figure 6A). This was significant after 3 and 5 days following transfection with AQP5 siRNA. ERK phosphorylation was monitored over time as a marker for cell proliferation. There was, however, no significant change in levels of phosphorylated ERK protein in AQP5 down-regulated cells when compared to the controls at different time points (96 h time point shown in Figure 6B, repeated three times). This was determined by western blot analysis using phosphorylated ERK antibody. Expression levels of total ERK was used as a loading control.


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

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

Effect of siRNA mediated AQP5 down-regulation (AQP5DR) on cell proliferation in CEPI17 cells. A: SRB assay shows that AQP5DR increased cell proliferation in CEPI17 cells (significant at 3 and 5 days p<0.05, 95% CI as determined by the paired T-Test, [n=3]). There was no significant difference in phosphorylated ERK 96 h post transfection in CEPI17 cells when compared to the controls as shown by western blot analysis (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Effect of siRNA mediated AQP5 down-regulation (AQP5DR) on cell proliferation in CEPI17 cells. A: SRB assay shows that AQP5DR increased cell proliferation in CEPI17 cells (significant at 3 and 5 days p<0.05, 95% CI as determined by the paired T-Test, [n=3]). There was no significant difference in phosphorylated ERK 96 h post transfection in CEPI17 cells when compared to the controls as shown by western blot analysis (B).
Mentions: AQP5 siRNA was used to study the effect of down regulation of AQP5 on migration of CEPI17 cells. CEPI17 cells were transfected with AQP5 siRNA and wound-healing assay was performed to study the effect of AQP5 siRNA on the migration of CEPI17 cells as described earlier. As seen in Figure 5A, AQP5 down regulation resulted in significant increase in migration of CEPI17 cells as determined by the in vitro wound-healing assay. However, when control siRNA was used to transfect the CEPI17 cells it had no effect on cell migration (Figure 5A). The area of the wound over time was measured using image J software and statistical analysis was performed. There was significant increase in cell migration 18 h after the scratch was created (Figure 5A,B). The scratch assay was also performed in the presence of 0.04% Mitomycin C, an inhibitor of cell proliferation, and similar results were obtained (data not shown). To study the effect of AQP5 down regulation on cell proliferation, SRB assay was performed as described earlier. Significant increase in cell proliferation was also observed on AQP5 down-regulation as determined by SRB proliferation assay (Figure 6A). This was significant after 3 and 5 days following transfection with AQP5 siRNA. ERK phosphorylation was monitored over time as a marker for cell proliferation. There was, however, no significant change in levels of phosphorylated ERK protein in AQP5 down-regulated cells when compared to the controls at different time points (96 h time point shown in Figure 6B, repeated three times). This was determined by western blot analysis using phosphorylated ERK antibody. Expression levels of total ERK was used as a loading control.

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