The aquaporins (AQPs) are small membrane proteins forming water channels and transporters for smal solutes like glycerol. The AQPs play important roles in transmembrane or transcellular transports in various cells, like kidney cells, but also in skin epidermis keratinocytes. The skin epidermis is a pluristratified epithelium, undergoing continuous renewal. Extracellular calcium plays an important role in the differentiation of keratinocytes.In this work, we demonstrate that during calcium-induced differentiation of human keratinocytes, aquaporin-3 (AQP3) is translocated from the endoplasmic reticulum to plasma membranes. In order to study the kinetics and the molecular bases of this regulation, our goal was to produce stable clones of a human keratinocyte cell line (HaCat) expressing a fluorescent AQP3. Despite several trials, i was not able to obtain such clones. Thus i pursued with another epithelial cell line: MDCK cells. We have produced two lines of MDCK cells stably expressing fluorescent AQPs: AQP3-GFP and AQP2mCherry. Interestingly in MDCK cells, AQP3-GFP reproduced the regulated intracellular trafficking observed in human keratinocytes; in MDCK cells grown in a medium containing 0.15 mM Ca2+,, AQP3-GFP was localized in the endoplasmic reticulum. After extracellular Ca2+ was raised to 1.5 mM, AQP3-GFP was seen in plasma membranes. In the same conditions, AQP2-mCherry remained intracellular throughtout the experiment. With calcium-switch experiments, when have then studied the kinetics of AQP3 trafficking. We have shown that targeting of AQP3 to plasma membranes is a slow process (at least 6h) and seems dependent not only of cell differentiation, but also on the establishment of cell polarity. Using inhibitors of PLC and PKC, we have shown the implication of this signalling pathway, which is dependent on calcium, in AQP3 trafficking. In addition we found that plasma membrane expression of AQP3 is dependent on actin cytoskeleton.In conclusion, we show for the first time a regulation of intracelluar trafficking of an aquaporin in calcium-induced differentiation and after establishment of epithelial cell polarity. This regulation likely allows human skin epidermis hydration whithout compromising the permeability barrier of skin.
