J' R' Kaczvinsky, Ph'D', K' M ' Lammers, M'S', S' Xie, Ph'D', A' B' Newland, B'S', C' C' Bascom, Ph'

1
P419
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
METHODS

• In vivo Expression of AQP 3
• 20 Caucasian female subjects, 18-55
• years old
• 10 with clinically dry leg skin
• (grades gt 2.0 on 4 pt scale)
• 10 with non-dry leg skin (grades lt 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 GeneChip assay

• 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)

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 1990s (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.

• ELISA for In Vitro AQP Expression
• Cultured human neonatal keratinocytes were
  cultured were seeded in 96-well plates and
  incubated at 37C 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.

• References
• Preston G. M., Carroll T.P., Guggino W. P. and
  Agre P., Science 256, 385-387 (1992).
• Sougrat R., Morand M., Gondran C., Barre P.,
  Gobin R., Bonte F., Dumas M., and Verbavatz J.,
  J. Invest. Derm., 118(4), 678-685 (2002).
• Sugiyama Y., Ota Y., Hara M., and Inoue S.,
  Biochim. Biophys. Acta 1522, 82-88 (2001).
• Hara-Chikuma M. and Verkman A. S., Biol Cell,
  97(7), 479-486 (2005).
• Zheng X. and Bollinger Bollag W., J. Invest.
  Derm., 121(6), 1487-1495 (2003).

This work was supported in its entirety by The
Procter Gamble Company