Enhancement of aquaporin-3 by vasoactive intestinal polypeptide in a human colonic epithelial cell line.
BACKGROUND
Vasoactive intestinal polypeptide (VIP) plays an important role in water transport in the intestine. Several specialized channels termed aquaporins (AQP) facilitate water transport in the gastrointestinal tract. Aquaporin-3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms underlying the functions of AQP3 remain unclear. To characterize the regulation of AQP3 expression by VIP, we studied messenger (m)RNA expression, protein expression and DNA binding activity in a human colonic epithelial cell line, HT-29.


METHODS
Human colonic epithelial cells, HT-29, were incubated with VIP (10-12-10-7 M). The cells were treated with protein kinase-A (PK-A) inhibitors (H-89, H-9) or chloride channel-blockers (diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPD)). The expression of AQP3 mRNA and protein was determined by Northern blot and Western blot, respectively. The DNA-binding activities of cyclic adenosine monophosphate (cAMP) response elements/activating transcription factor (CRE/ATF)) in the nuclear extract were determined by electrophoretic mobility shift assay.


RESULTS
Aquaporin-3 mRNA was up-regulated at a concentration of 10-10 M VIP. The expression of AQP3 protein was enhanced at 3 h after addition of VIP. The PK-A inhibitors (H-89, H-9) inhibited the expression of AQP3 mRNA enhanced by VIP and cAMP. The gel shift assay of CRE/ATF in HT-29 cells revealed a single band.


CONCLUSIONS
These results indicate that VIP upregulated the expression of AQP3 mRNA and protein, and that a cAMP-dependent pathway mediated this effect in a human colonic epithelial cell line, HT-29.
Itoh
Akihiko
A
Division of Gastroenterology, Shiga University of Medical Science, Shiga, Japan.
Tsujikawa
Tomoyuki
T
Fujiyama
Yoshihide
Y
Bamba
Tadao
T
D051400:Aquaporin 3; D020346:Aquaporins / Q000235:genetics* / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D018118:Chloride Channels / Q000187:drug effects; D003106:Colon / Q000166:cytology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism; D015854:Up-Regulation; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
C496027:AQP3 protein, human; D020346:Aquaporins; D018118:Chloride Channels; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D018005:Receptors, Vasoactive Intestinal Peptide; D062367:ortho-Aminobenzoates; D051400:Aquaporin 3; D014660:Vasoactive Intestinal Peptide; C001209:fenamic acid; D017868:Cyclic AMP-Dependent Protein Kinases