Determination of Aquaporin Expression in Keratinocytes and Human Tissue
J. R. Kaczvinsky, Ph.D., K. M . Lammers, M.S., S. Xie, Ph.D., A. B. Newland, B.S., C. C. Bascom, Ph.D., J. P . Tiesman, Ph.D., T. D. Reichling, M.S., G. J. Hurley, M. S., K. D. Juhlin, Ph.D., R. M. Osborne, Ph.D., L. A. Mullins, B.S. The Procter and Gamble Company, Cincinnati, Ohio USA
Introduction
Aquaporins are a class of membrane proteins that regulate the transport of water and other small solutes across plasma membranes. First reported in the early 1990's (1), their discoverer was recently awarded a Nobel Prize for this work. To date, 13 different aquaporins have been identified in a variety of mammalian tissues involved in water distribution. Two, AQP-3 and AQP-9, are expressed in human skin cells (2,3). AQP-3 in particular has been implicated as playing a key role in the transport and distribution of epidermal water and glycerol (3,4), and the regulation of keratinocyte differentiation (5), critical processes for maintaining skin hydration, barrier function and overall skin health.
Objective
Determine AQP expression levels in dry and non-dry skin and develop in vitro methods to identify compounds capable of stimulating AQP-3 expression in human skin cells.
Methods
In vivo Expression of AQP 3 •20 Caucasian female subjects, 18-55 years old •10 with clinically dry leg skin (grades > 2.0 on 4 pt scale) •10 with non-dry leg skin (grades < 1.0) •One week washout with mild soap •Two 4-mm punch biopsies from each subject taken from upper calf (near knee) •One 4-mm punch biopsy taken from front of hip (non-exposed/non-dry control site) •RNA isolated from hip biopsies & 1 leg biopsy •AQP RNA expression quantitated via GeneChipTM assay
ELISA for In Vitro AQP Expression •Cultured human neonatal keratinocytes were cultured were seeded in 96-well plates and incubated at 37°C for 48-96 hrs with culture medium containing test compound •Fixed, dried cells were incubated with anti-AQP3 antibody, then a peroxidase coupled secondary antibody. •AQP3 protein content was quantitated at 450 nm after treatment with Horseradish Peroxidase Chromogen TMB and acidic stop. •AQP3 protein in lysed human Epiderm culture tissue and leg biopsy tissues were similarly analyzed by ELISA.
Results
AQP3 Expression In Vivo •Mean visual dryness grades: Dry - legs 2.8, hips 0.7 Non-dry - legs 0.7, hips 0.3 •Hip samples were adequate control sites •Dry skin control (hip) samples had lower AQP3 RNA levels than non-dry controls •Non-dry leg samples had significantly lower AQP3 expression than hip samples •Dry leg biopsies had higher relative AQP3 RNA expression than would be predicted from hip comparisons •More AQP3 protein also found in the legs of dry skin subjects •Age could not be excluded as a cofactor in AQP3 expression (Mean ages: Dry - 51.1 yrs, Non-dry - 38.9 yrs) In Vitro Expression of AQP3 •Both niacinamide and caffeine stimulated AQP3 expression by cultured keratinocytes •Enhanced expression was dose-dependent •Caffeine also stimulated AQP3 expression in stratified keratinocytes from an Epiderm model system (p=0.108)
Conclusion
•AQP3 expression was quantitated in skin and varied with body site, skin condition and/or age •Not visibly dry control samples from dry skin subjects showed lower AQP3 expression than in non-dry subjects (genetic factor?) •In non-dry subjects, AQP-3 varied by site; expression was significantly lower in legs than hips •AQP3 RNA and protein may be over expressed in dry legs to compensate for the effect of environmental insult or due to protein functionality issues •Larger-base studies are needed to confirm these findings •. •AQP3 expression by epidermal keratinocytes can be stimulated by niacinamide and caffeine
