Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.
OBJECTIVE
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.
Shankardas
Jwalitha
J
Department of Biomedical Sciences, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Patil
Rajkumar V
RV
Vishwanatha
Jamboor K
JK
D051398:Aquaporin 1 / Q000235:genetics* / Q000378:metabolism; D051402:Aquaporin 5 / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D003315:Cornea / Q000166:cytology*; D015536:Down-Regulation / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
C496020:AQP1 protein, human; C496036:AQP5 protein, human; D051402:Aquaporin 5; D034741:RNA, Small Interfering; D051398:Aquaporin 1; D048049:Extracellular Signal-Regulated MAP Kinases
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