Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fiber formation without inducing epithelial-to-mesenchymal transition

Abstract

Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.

Aquaporins differentially regulate cell‐cell adhesion in MDCK cells

“…Epithelial cells undergoing EMT gain motility and invasiveness, which are characteristic features of metastatic cells (57), and AQP5 knockdown in various cancer cells indicated that AQP5 overexpression promotes EMT (9,58). However, our data indicate that AQP5 regulation of adhesion proteins in MDCK cells, a noncancerous cell line, is independent of EMT (10). Although the role of AQPs in cancer cell motility is well established, the full extent of the cellular changes triggered by AQP overexpression as well as the mechanisms inducing cancer cell motility is still elusive.…”

“…It has been reported that overexpression of some AQPs in cancer cells, especially AQP5, aggravated epithelial-mesenchymal transition (EMT) and increased cancer cell migration (7)(8)(9). However, we recently showed that overexpression of AQP5 in Madin-Darby canine kidney (MDCK) cells, a noncancerous epithelial cell line, did not increase levels of the mesenchymal marker vimentin, even though it facilitated a decrease of the epithelial marker E-cadherin (10). Thus, the AQP5-mediated decrease in E-cadherin levels in MDCK cells was not associated with EMT, highlighting discrepancies in AQP5associated phenotypes between normal epithelial cells and ABBREVIATIONS: 3T3, 3-d transfer, inoculum 3 3 10 5 ; AQP, aquaporin; AQP3 LD5 , AQP3 with an AQP5 loop D substitution; AQP3 Tail5 , AQP3 with an AQP5 tail substitution; BSA, bovine serum albumin; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; EMT, epithelial-mesenchymal transition; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; HEK, human embryonic kidney; IRES, internal ribosome entry site; MDCK, Madin-Darby canine kidney; PFA, paraformaldehyde; PIV, particle image velocimetry; SSC, saline sodium citrate; ZO1, zonula occludens-1 different cancer cells.…”

“…Human embryonic kidney (HEK) 293T cells were grown at 5% CO 2 at 37°C in DMEM high glucose (4.5 g/L D-glucose; Thermo Fisher Scientific), 10% FBS (Thermo Fisher Scientific), and antibiotics. Both cell lines were allowed to grow to a maximum of 80% confluency and were passaged every 3 d. EGFP-MDCK-, AQP5-EGFP-MDCK-, and AQP5 S156A -EGFP-stable (MDCK cell stably expressing AQP5 S156A -EGFP point mutant) cell lines were published in a previous study (10). Cell lines stably expressing AQP1, -P3, and -P4 were generated as previously described (10,19).…”

“…Both cell lines were allowed to grow to a maximum of 80% confluency and were passaged every 3 d. EGFP-MDCK-, AQP5-EGFP-MDCK-, and AQP5 S156A -EGFP-stable (MDCK cell stably expressing AQP5 S156A -EGFP point mutant) cell lines were published in a previous study (10). Cell lines stably expressing AQP1, -P3, and -P4 were generated as previously described (10,19). For experiments involving transient transfections, 1 mg of plasmid was transfected into cells with Lipofectamine 2000 (Thermo Fisher Scientific) upon seeding on collagen-coated glass coverslips, and cells were subsequently grown for 48 h as previously described.…”

“…Cell-cell junctions are often affected during carcinogenesis, whereby adhesion proteins are down-regulated, facilitating metastasis (35)(36)(37). We recently showed that AQP5 expression in MDCK cells promoted lower levels of Ecadherin (10). To determine whether AQP5 affects other cell-cell adhesion-associated proteins than Ecadherin, we analyzed the levels of several junctional proteins by immunofluorescence microscopy.…”