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J. Biol. Chem., Vol. 277, Issue 19, 17147-17153, May 10, 2002
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From the
Received for publication, January 29, 2002, and in revised form, February 27, 2002
The water and solute transporting
properties of the epidermis have been proposed to be important
determinants of skin moisture content and barrier properties. The
water/small solute-transporting protein aquaporin-3 (AQP3) was found by
immunofluorescence and immunogold electron microscopy to be expressed
at the plasma membrane of epidermal keratinocytes in mouse skin. We
studied the role of AQP3 in stratum corneum (SC) hydration by
comparative measurements in wild-type and AQP3 null mice generated in a
hairless SKH1 genetic background. The hairless AQP3 null mice had
normal perinatal survival, growth, and serum chemistries but were
polyuric because of defective urinary concentrating ability. AQP3
deletion resulted in a >4-fold reduced osmotic water permeability and
>2-fold reduced glycerol permeability in epidermis. Epidermal, dermal,
and SC thickness and morphology were not grossly affected by AQP3
deletion. Surface conductance measurements showed remarkably reduced SC
water content in AQP3 null mice in the hairless genetic background
(165 ± 10 versus 269 ± 12 microsiemens
(µS), p < 0.001), as well as in a CD1 genetic
background (209 ± 21 versus 469 ± 11 µS).
Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels
(90-100 µS) after a 24-h exposure to a dry atmosphere, but the
difference was increased when surface evaporation was prevented by
occlusion or exposure to a humidified atmosphere (179 ± 13 versus 441 ± 34 µS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC
in AQP3 null mice. Water sorption-desorption experiments indicated
reduced water holding capacity in the SC of AQP3 null mice. The
impaired skin hydration in AQP3 null mice provides the first functional
evidence for the involvement of AQP3 in skin physiology. Modulation of
AQP3 expression or function may thus alter epidermal moisture content
and water loss in skin diseases.
The water content of the stratum corneum is an important
determinant of the appearance, physical properties, and barrier
function of the skin (1-3). The stratum corneum, the most superficial layer of skin, consists of layers of flattened corneocytes (dead epidermal cells) embedded in a lipid-rich matrix containing specialized proteins and lipids (4). Abnormalities of stratum corneum hydration are
seen in a variety of hereditary and acquired skin diseases such as
atopic dermatitis (5), eczema (6), psoriasis (7), senile xerosis (8),
and hereditary ichthyosis (9). Hydration of the stratum corneum could
in principle be determined by a number of factors including the
concentration of water-retaining osmolytes, the water and solute
transporting properties of the underlying layers of viable epidermal
keratinocytes, and the barrier properties of the stratum corneum. There
is evidence for a high concentration of solutes (Na+,
K+, and Cl The integral membrane protein AQP3 has been proposed to be a
potentially important transporter of water and solutes across epidermal
keratinocytes (14-16). AQP3 was initially cloned from rat kidney (17,
18) and is a member of a family of homologous aquaporin water channels
expressed widely in mammalian epithelia and endothelia that facilitate
fluid transport. Phenotype studies in aquaporin knockout mice have
implicated the involvement of aquaporins in the urinary concentrating
mechanism in kidney (19), water movement in lung (20, 21), cerebral
water balance (22), exocrine gland secretion (23, 24), and
mechano-electric signal transduction (25). AQP3 is a member of a
subclass of aquaporins, called aquaglyceroporins, which transport not
only water but also glycerol and possibly other small solutes.
AQP3-mediated water permeability was reported to be
pH-dependent, decreasing at low pH (26). The physiological
role of AQP3-mediated glycerol transport has been the subject of
considerable speculation but remains unknown. We first reported by
immunocytochemistry the expression of AQP3 protein in epidermal
keratinocytes in rat skin (15), and subsequently AQP3 was localized in
human keratinocytes (14), and in moist noncornified barriers such as
the mucous membranes in the mouth (16). It was reported recently that
water permeability of human epidermal keratinocytes was inhibited by
mercurials and low pH, consistent with the involvement of AQP3 (14).
Additional indirect evidence supporting a role for AQP3 in skin
physiology includes the regulation of epidermal cell AQP3
expression by extracellular osmolality, barrier perturbation, and
exposure to a dry atmosphere (27, 28).
The purpose of this study was to investigate whether AQP3 is involved
in stratum corneum hydration. Because humans with AQP3 deficiency have
not been identified and nontoxic inhibitors of AQP3 are not yet
available, we assessed skin phenotype in transgenic mice lacking AQP3.
For studies in skin, the AQP3 null genotype, originally created in a
CD1 genetic background (29), was transferred to the hairless SKH1
background. We found remarkably reduced stratum corneum water content
in the skin of AQP3 null mice and investigated possible mechanisms for
this defect. An interesting and unexpected finding was that AQP3
deletion remarkably impaired the ability of stratum corneum to become
hydrated even when water loss from the skin surface was prevented by
occlusion or exposure to a humidified atmosphere.
Mice--
The AQP3 null genotype originally generated in CD1
genetic background (29) was transferred to a hairless background by
back-cross breeding of heterozygous AQP3 CD1 mice with SKH1 hairless
mice. Hairless AQP3 heterozygous founder mice were bred to generate wild-type, heterozygous, and AQP3 null hairless mice. The mice were
maintained in air-filtered cages and fed normal mouse chow in the
University of California, San Francisco, Animal Care Facility. All
procedures were approved by the UCSF Committee on Animal Research.
Urine Output, Osmolarity, and Serum Chemistries--
24-h urine
output was measured using metabolic cages (Harvard Apparatus). Urine
osmolality was measured by freezing-point osmometry (Precision Systems
Inc.). Serum chemistries were measured by the UCSF Clinical Laboratory.
Immunofluorescence, Light Microscopy, and
RT-PCR--
Immunofluorescence was done on frozen sections of mouse
skin fixed in PBS containing 2% paraformaldehyde using a 1:200
dilution of affinity-purified anti-AQP3 rabbit polyclonal antibody.
Slides were washed three times in PBS and incubated for 40 min at room temperature in PBS/bovine serum albumin containing fluorescein isothiocyanate-coupled anti-rabbit antibody (10 µg/ml). Micrographs were obtained with a cooled CCD camera in an Olympus fluorescence microscope. For morphological examination by light microscopy, skin was
fixed in PBS containing 4% paraformaldehyde, and 2 µm plastic
sections were stained with toluidine blue. For RT-PCR analysis, total
RNA was isolated from the epidermis of wild-type and AQP3 null mice.
RT-PCR was performed with sequence-specific sense and antisense
oligonucleotide primers for mouse aquaporins 1-9 as described
previously (23).
Electron Microscopy--
Skin samples were fixed in 2%
glutaraldehyde and embedded in Epon. 70-nm thick sections were cut on a
Reichert-E ultramicrotome and collected on Formvar-coated electron
microscopy grids. Sections were stained in 5% uranyl acetate
for 3 min and then in lead citrate for 1 min, dried, and observed by
electron microscopy (Philips CM 400). For immunogold labeling, skin
samples were post-fixed in 2% paraformaldehyde + 0.1% glutaraldehyde
and embedded in Unicryl. 70-nm thick sections were preincubated in 20 mM Tris buffer (pH 7.4) containing 0.1% bovine serum
albumin, 0.1% fish gelatin, and 0.05% Tween 20 (TBuffer) followed by
a 1-h incubation in affinity-purified anti-AQP3 antibody (1:50
dilution) in TBuffer. Grids were incubated in a 1:25 dilution of 10-nm
gold-coupled anti-rabbit antibody (Amersham Biosciences) for 1 h,
washed six times, stained in 5% uranyl acetate for 3 min and then in
lead citrate for 1 min, dried, and observed by electron microscopy.
Water and Glycerol Permeability Measurements--
For
measurement of water permeability, epidermal sheets were freshly
isolated from 6-8-week-old wild-type and AQP3 null mice by digesting
full thickness skin fragments in 50 units/ml dispase solution (BD
Biosciences) at room temperature for 1 h. After washing in cold
PBS, the epidermis was peeled off the dermis. Small fragments of the
epidermal sheets (~12-mm diameter) were immobilized (surface facing
downward) on 18-mm-diameter round coverglasses using superglue. The
coverglass was mounted in a perfusion chamber (exchange time < 0.2 s) for measurement of osmotic water permeability by spatial filtering microscopy as described previously for studies in cultured cell layers and bladder sheets (30) and in kidney tubules (29). The
time course of transmitted light intensity was measured in response to
changing between perfusate osmolalities of 300 (PBS) and 600 mosM (PBS containing 300 mM sucrose).
For measurement of glycerol permeability, viable keratinocytes were
isolated from epidermal sheets (prepared as above) by digestion in
0.5% trypsin solution for 10 min at room temperature with gentle
shaking. After neutralizing with fetal bovine serum (10% final
concentration), the cell suspension was centrifuged three times for 10 min at 800 × g. More than 95% of the cells were
viable as judged by trypan blue exclusion. The keratinocyte cell
suspension (~106 cells/ml) was incubated for specified
times with PBS containing tracer quantities of
[3H]glycerol (Amersham Biosciences) at room temperature.
After separating on glass fiber filters and washing three times
with ice-cold PBS in a suction filtration apparatus, cells were
disrupted with 1 M NaOH. Cell-associated radioactivity was
determined by scintillation counting. Protein concentration was
measured using a Bio-Rad DC protein assay kit.
Skin Conductance Measurements--
Stratum corneum hydration was
determined by high frequency electrical conductance using a Skicon-200
skin surface hygrometer (IBS). Three independent measurements
from the same area of skin were averaged for each value. Measurements
were performed on mice under normal conditions (external temperature
22 ± 2 °C, humidity 40 ± 3%) or on mice treated with
various maneuvers including exposure to low (10%) and high (90%)
external humidity, tape stripping, and topical occlusion. Water
sorption-desorption studies were done as described (7, 31), where
conductance was measured before and at 30-s intervals after pipetting
20 µl of distilled water onto the skin and blotting after 10 s.
In some experiments, conductance measurements were conducted on shaved
skin of wild-type and AQP3 null mice in the (hairy) CD1 genetic background.
Analysis of Stratum Corneum Protein Content--
Stratum corneum
layers were stripped and collected using Scotch cellophane tape. The
stripped stratum corneum layers on the tape were dissolved in 1 M NaOH for 1 h. After neutralization with 1 M HCl, total protein concentration was measured using a Bio-Rad DC protein assay kit (Richmond, CA).
To study skin phenotype, the AQP3 null mutation generated in CD1
mice was transferred to a SKH1 hairless genetic background by
back-cross breeding. The AQP3 null hairless mice generated by breeding
of heterozygous founder mice had normal perinatal survival and growth
compared with wild-type littermates. However, the daily urine output of
the AQP3 null mice was ~10-fold greater than in wild-type mice (Fig.
1, A, top, and
B). Urine osmolality in the AQP3 null hairless mice was very
low (132 ± 22 versus 1974 ± 123 mosM in wild-type mice) when given free access to
water (Fig., 1A, bottom). After a 24-h water
deprivation, urine osmolality increased submaximally (481 ± 11 versus 3792 ± 204 mosM in wild-type mice)
(Fig. 1A, bottom), indicating a urinary
concentrating defect as found for AQP3 null mice in a CD1 genetic
background (29). Analysis of serum chemistries showed no differences in
electrolyte concentrations, creatinine, and liver function tests,
except for mild elevation in blood urea nitrogen (45 ± 4 versus 29 ± 3 mg/dl) and decreased triglyceride
concentration (110 ± 12 versus 166 ± 16 mg/dl).
Immunofluorescence showed strong AQP3 protein expression in wild-type
mice in a membrane pattern in the keratinocyte layer just below the
stratum corneum (Fig. 2A).
Immunogold electron microscopy indicated plasma membrane localization
of AQP3 protein in epidermal keratinocytes (Fig. 2B).
Antibody staining was absent in AQP3 null mice. RT-PCR analysis of
mRNA isolated from epidermis using primers specific for aquaporins
0-9 revealed only AQP3 transcript in wild-type mice and no aquaporins
in AQP3 null mice (Fig. 2C).
Impaired Stratum Corneum Hydration in Mice Lacking Epidermal
Water Channel Aquaporin-3*
§,¶,
,, and
Departments of Medicine and Physiology,
Cardiovascular Research Institute, University of California, San
Francisco, California 94143-0521, ¶ Basic Research Laboratory,
Kanebo Ltd., Odawara 250-0002, Japan, and Service de
Biologie Cellulaire, CEA, F-91191 Saclay, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and a low concentration of water
(13-35%, Ref. 10) in the superficial stratum corneum, producing in
the steady-state gradients of both solutes and water from the skin
surface to the viable epidermal keratinocytes (11-13). Although
transepithelial fluid transporting properties have been studied
extensively in various mammalian epithelia, the molecular mechanisms of
fluid transport across epidermal keratinocyte layers remain poorly
understood, as is the relationship between keratinocyte fluid transport
and stratum corneum hydration. It has been proposed that aquaporin-3 (AQP3)1 might facilitate
transepidermal water permeability to protect the stratum corneum
against desiccation by evaporative water loss from the skin surface
and/or to dissipate water gradients in the epidermal keratinocyte cell
layer (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (46K):
[in a new window]
Fig. 1.
Urinary concentrating defect in hairless AQP3
knockout mice. A, 24-h urine output (top) in
wild-type (+/+) and AQP3 null ( 
/) mice. Urine osmolality before and
after a 36-h water deprivation (means ± S.E., n = 6). B, photograph showing 24-h urine collection in
hairless SKH1 (left) and CD1 (right) wild-type
and AQP3 null mice.
View larger version (61K):
[in a new window]
Fig. 2.
AQP3 localization and morphology of mouse
skin. A, immunofluorescence showing strong AQP3
staining in epidermal cells of hairless wild-type mice. E,
epidermis; D, dermis; sc, stratum corneum.
B, immunogold labeling of AQP3 protein in plasma membranes
of epidermal cells in wild-type hairless mice (bottom).
Arrowheads, gold particles; d, desmosomes. No
labeling is seen in the epidermis of AQP3 null mice (top).
C, RT-PCR analysis of aquaporin expression in epidermis
isolated from wild-type and AQP3 null mice. Transcripts for portions of
the coding sequences of the indicated mouse aquaporins were
PCR-amplified using specific primers. Rows labeled "+/+" and
" /" correspond to amplifications done using epidermal cDNA
as template, and the row labeled "control " corresponds
to amplifications done using a mixture of cDNAs from brain, lung,
liver, and kidney, which contain all mouse aquaporins. D,
toluidine blue-stained thin plastic sections from two wild-type and two
AQP3 null hairless mice. Scale bar, 20 µm. E,
thickness of indicated skin layers (means ± S.E.) measured in
plastic sections from n = 4 mice. Differences in
thickness of the stratum corneum, epidermis, and dermis not
significant.
The general structure of the epidermis was studied by light and electron microscopy. Light microscopy of plastic-embedded sections of epidermis stained with toluidine blue revealed similar thickness and structure of the stratum corneum and epidermal cell layers (Fig. 2D). Thickness measurements on a series of sections showed no significant differences in the stratum corneum, epidermal, or dermal layers (Fig. 2E). The thickness of the innermost fat layer was reduced in the AQP3 null mice (p < 0.01), which may be related to the relative serum hypotriglyceridemia.
Transmission electron microscopy showed no apparent differences in the
structures of the stratum corneum and keratinocytes in wild-type (Fig.
3, A and C) and
AQP3 null (Fig. 3, B and D) mice. At the apex of
the epidermis (Fig. 3, A and B), the stratum corneum contained five layers in both wild-type and AQP3 null mice,
with each layer of a similar thickness. Keratohyalin granules seen in
the superficial epidermal cells were of similar size in wild-type and
AQP3 null mice. As expected, keratinocytes at the base of the epidermis
(Fig. 3, C and D) were taller but showed no
differences between genotypes. Despite the possible consequences of
AQP3 deficiency on water movement, intercellular spaces were not
dilated in AQP3 null mice from the base to the surface of the
epidermis.
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Osmotic water and glycerol permeability were measured to investigate
the functionality of plasma membrane AQP3 in epidermal keratinocytes.
Osmotic water permeability was measured by spatial filtering light
microscopy using fragments of epidermis (isolated by dispase
digestion), which were mounted in a perfusion chamber with epidermal
keratinocytes facing upward. Fig.
4A (left) shows the
reversible time course of keratinocyte layer shrinking and swelling in
response to the changing of perfusate osmolality between 300 and 600 mosM. Fitted reciprocal exponential time constants (proportional to water permeability; Fig. 4A,
right) were significantly reduced by ~4-fold in epidermis
of AQP3 null mice. Because of the possible unstirred layer effects in
the multilayered epidermis, which limits observed water permeability,
the 4-fold difference formally represents a lower limit to the
difference in epidermal water permeability in wild-type
versus AQP3 null mice.
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Glycerol permeability was measured from the uptake of radiolabeled [3H]glycerol in suspensions of freshly isolated keratinocytes. Measurements in keratinocytes from wild-type mice indicated approximately linear [3H]glycerol uptake for 10 min (not shown). Fig. 4B shows that the average [3H]glycerol uptake at 90 s (after subtraction of bound [3H]glycerol uptake by 0 time measurement) was significantly reduced in keratinocytes from AQP3 null mice. The ~2-fold reduction in glycerol permeability represents a lower limit to the difference in epidermal glycerol permeability in wild-type versus AQP3 null mice because of possible effects of the protease treatment (needed to release keratinocytes) on AQP3 function. These results establish the functionality of AQP3 as a water/glycerol transporter in mouse epidermis.
Stratum corneum water content was measured using a well established
surface conductance method (32). Surface electrical conductance is
approximately linearly related to percentage water content of the outer
stratum corneum (33). Fig. 5A
shows remarkably reduced skin conductance in different areas of the
skin in adult hairless AQP3 null mice measured under standard
atmospheric conditions (22 °C, 40% relative humidity). The greatest
differences was found in the upper abdominal skin, with ~50% reduced
conductance in the AQP3 null mice. To confirm that these observations
are not unique to the hairless genetic background, skin conductance was measured at 1 day after shaving the mid-back skin of CD1 mice. Although
skin conductance was greater in the CD1 than the hairless background,
there remained a large effect of AQP3 deletion (209 ± 21 versus 469 ± 11 µS in wild-type mice).
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Skin conductance was measured in mice of different ages to determine when the defect in stratum corneum hydration is first manifest. As summarized in Fig. 5B, skin conductance was low and similar at 1 and 2 days after birth but was significantly lower by day 3 in AQP3 null mice compared with wild-type mice. Skin conductance increased substantially during the first month in wild-type mice, to a much greater extent than in AQP3 null mice.
To test the hypothesis that decreased stratum corneum hydration in AQP3 null mice is due to impaired replacement of surface evaporative losses by water transport across epidermal keratinocytes, conductance measurements were performed after subjecting mice to different external humidity conditions and after skin surface occlusion. Exposure to 10% humidity increases evaporative water loss, whereas exposure to 90% humidity or surface occlusion prevents evaporative water loss. The prediction is that the defective stratum corneum hydration in AQP3 null mice would be corrected by preventing evaporation but exaggerated by exposure to a 10% humidity atmosphere. Fig. 5C summarizes conductance measurements. Exposure to 90% humidity or occlusion for 24 h resulted in increased stratum corneum hydration in wild-type mice, but contrary to expectations, the impaired hydration in AQP3 null mice was not corrected. Also, contrary to expectations, skin conductance of the wild-type and AQP3 null mice became similar after exposure to 10% humidity. Fig. 5D shows the full time courses of skin conductance after changing external humidity. In both cases the new steady-state conductance was achieved in 2-4 h but with a small undershoot for exposure to 10% humidity.
To investigate whether the impaired hydration occurs in deeper layers
of the stratum corneum of AQP3 null mice, conductance measurements were
made after layers of the stratum corneum were removed progressively by
cellophane tape (tape stripping). Fig. 6A shows that the accumulated
total protein was approximately linear with the number of tape
strippings and that the amount of protein removed was similar in
wild-type and AQP3 null mice. Fig. 6B shows the correlation
between skin conductance and total accumulated protein for a series of
wild-type and AQP3 null mice subjected to serial tape stripping.
Hydration increased progressively as deeper layers of the stratum
corneum were exposed by tape stripping, which supports the notion of a
water gradient from deep to superficial stratum corneum (12, 34). Skin
conductance was lower in AQP3 null mice, but the steepness of the water
gradient was increased. These results are consistent with the notion
that a water gradient is established from the well hydrated epidermal
keratinocytes through the relatively water-poor and watertight
stratum corneum.
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The results shown in Figs. 5 and 6 provide evidence against the
hypothesis that impaired hydration in the stratum corneum of AQP3 null
mice results from decreased epidermal water permeability. We tested
whether the stratum corneum of AQP3 null mice has an intrinsic defect
in its ability to be hydrated and in its "water holding capacity,"
as found in diseases associated with dry skin. An established
sorption-desorption test was used in which skin conductance is measured
before and at different times after a 10-s exposure of the skin to
distilled water (32). Fig. 6C shows that skin conductance
increased immediately after water exposure and then recovered over a
few min. The initial increase in skin conductance, which was
significantly greater in wild-type mice, has been taken as a measure of
the ability of the stratum corneum to become hydrated. More
importantly, the recovery after hydration has been taken as a measure
of the water holding capacity of the stratum corneum. The area under
the recovery curve (after subtraction of prehydration conductance) has
been used as a single parameter describing water holding capacity (10).
The recovery area parameter was remarkably reduced in AQP3 null mice
(8.2 ± 1.7 × 104 versus 4.5 ± 1.9 × 104 µS·s, p < 0.005) (see
"Discussion").
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The goal of this study was to determine whether AQP3 plays a role in skin physiology. As discussed in the Introduction, previous findings of AQP3 expression and regulation in epidermal keratinocytes provided indirect evidence for a role of AQP3 in skin function. To facilitate the study of stratum corneum hydration, the AQP3 null genotype was transferred to a SKH1 hairless background. The hairless AQP3 null mice manifested a urinary concentrating defect with polyuria and reduced urinary osmolality, as found previously for AQP3 null mice in the CD1 background (29). AQP3 null CD1 mice develop progressive renal failure with dilatation of the kidneys and urinary bladder by 8 weeks of life (35) but have unimpaired function of the airways (36), another major site of AQP3 expression. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries, except for a mild elevation in blood urea nitrogen and a decrease in serum triglyceride concentration. We do not believe that these mild alterations in blood chemistry cause the abnormalities in stratum corneum hydration in AQP3 null mice. The decreased stratum corneum hydration was found in AQP3 null mice at 4 weeks of age before changes in blood urea nitrogen were detected, and AQP1 null mice have an even greater degree of serum hypotriglyceridemia (37) but do not manifest altered stratum corneum hydration.
Adequate stratum corneum hydration is important in maintaining skin plasticity and barrier integrity. The stratum corneum gains water from underlying viable layers of epidermis and dermis to maintain its proper hydration status in the relatively dry external environment. There are several possible mechanisms by which AQP3 deletion might affect stratum corneum hydration. Reduced epidermal water permeability might impair water transport into the stratum corneum, which would lead to decreased stratum corneum water content when surface evaporation occurs but not when the skin surface is occluded or exposed to a humidified environment. Reduced permeability of the epidermal cell layer to glycerol or other small solutes might produce alternations in the composition and/or structure of the stratum corneum that alter its ability to hold water even when surface evaporation is blocked. High aquaporin-dependent water permeability in the epidermal cell layer might prevent water gradients within the viable keratinocytes, preserving their normal biosynthetic functions. However, it seems unlikely that water gradients are present in the layer of viable keratinocytes because water permeability of the stratum corneum is orders of magnitude lower than in the keratinocyte layer (38).
Transport measurements showed significantly reduced water and glycerol permeability in AQP3 null mice. These results are consistent with localization of AQP3 in the plasma membrane of keratinocytes, as shown by immunofluorescence and immunogold electron microscopy, and with the absence of the compensatory expression of another aquaporin in epidermis of AQP3 null mice, as shown by RT-PCR. The finding of functional expression of AQP3 in mouse epidermis agrees with the conclusion of a recent study showing that apparent water permeability in human epidermis was inhibited by mercurials and low pH (14). Utilizing a spatial filtering approach to measure osmotic water permeability in freshly isolated, intact epidermal sheets, we found that AQP3 deletion produced an ~4-fold reduction in water permeability. This approach preserves the normal epidermal anatomy, and unlike keratinocyte isolation procedures, the isolation of epidermal sheets does not require digestion with proteases. In other studies we have found up to 10-fold reductions in water permeability after aquaporin deletion, for example in erythrocytes (35), the proximal tubule (19), and the thin descending limb of Henle (39) of AQP1 null mice and in the lungs of AQP1 and AQP5 null mice (20, 21). The relatively small reduction in epidermal water permeability in AQP3 null mice may be related to the relatively weak intrinsic water transporting activity of AQP3 compared with other aquaporins (40). In addition, unstirred layer effects in the multilayered epidermal sheet would decrease apparent osmotic water permeability and blunt the effects of AQP3 deletion. Nevertheless, the results shown here prove that AQP3 is functional as a plasma membrane water/glycerol transporter in the epidermis, but they provide only a lower limit to the contribution of AQP3 to total water permeability.
Conductance measurements showed remarkably reduced stratum corneum water content in AQP3 null mice in most areas of the skin, despite grossly normal morphology by light and electron microscopy. Reduced stratum corneum hydration was also seen in AQP3 null mice in the CD1 genetic background. The defect in stratum corneum hydration was seen as early as 3 days after birth in the hairless AQP3 null mice, and from tape stripping experiments it appeared to involve the full stratum corneum thickness.
Exposure of the skin to moist and dry humidity conditions produced fairly prompt changes in stratum corneum hydration as a new steady state was reached for surface evaporative water loss versus water movement into the stratum corneum through the epidermal cell layer. The surprising observation was that stratum corneum hydration in AQP3 null mice increased little in a humidified environment and with surface occlusion, despite a marked increase in hydration in the wild-type mice. In a dry environment, stratum corneum hydration decreased to comparable levels in wild-type and AQP3 null mice. These results provide evidence against the hypothesis that a major function of epidermal AQP3 is to facilitate water movement into the stratum corneum. Instead, the AQP3 null mice appear to manifest an intrinsic defect in the stratum corneum that limits its ability to accumulate water. Water sorption-desorption studies supported the conclusion that the stratum corneum of AQP3 null mice has an intrinsic defect in its ability to absorb and hold water.
The determination of the molecular mechanism by which AQP3 deletion impairs hydration and water holding in the stratum corneum will require analysis of the peptide, lipid, and carbohydrate composition of the stratum corneum and epidermis, as well as high resolution structural studies. In addition, the identification of specific proteins in the stratum corneum and keratinocytes that are up-regulated in AQP3 deficiency may be informative, especially if they are involved in the biosynthetic pathways for generation of water-retaining osmolytes or specialized barrier lipids. Although direct evidence is not yet available, we suspect that defective glycerol/small solute transport in the epidermis of AQP3 null mice is the basis of their abnormal hydration. Studies of non-skin phenotypes in aquaporin-deficient mice support the conclusion that epidermal cell water permeability is probably not a major determinant of stratum corneum hydration. Water movement across the stratum corneum is very slow compared with other tissues, such as the proximal tubule and secretory epithelia, where aquaporins are important. Also, water movement into and through the stratum corneum is likely to be limited by unstirred layers rather than the intrinsic water permeability of the epidermal cell layer. Further studies are needed to determine whether water and/or glycerol transport by AQP3 is essential for normal hydration of the stratum corneum.
In summary, the impaired stratum corneum hydration in AQP3 null mice
provides the first functional evidence for the involvement of AQP3 in
skin physiology. Although the exact mechanism for impaired stratum
corneum water holding remains to be established, we propose that
pharmacological modulation of AQP3 expression or function may alter
epidermal moisture content and water loss. Controlled modulation of
skin moisture content and barrier function may thus be useful in the
treatment of skin disorders associated with abnormally wet, dry, or
permeable skin.
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ACKNOWLEDGEMENTS |
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We thank Martin Behne, Peter Elias, and Shintaro Inoue for helpful advice and discussions and Liman Qain for mouse breeding and genotype analysis.
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FOOTNOTES |
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* This work was supported by Grants DK35124, DK43840, EB00415, EY13574, HL60288, and HL59198 from the National Institutes of Health, a grant from the Cystic Fibrosis Foundation, a gift from Kanebo, Ltd. of Japan, and Contract ERBFMRXCT97-0128.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: tonghui@itsa.ucsf.edu.
Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M200925200
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ABBREVIATIONS |
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The abbreviations used are: AQP3, aquaporin-3; RT-PCR, reverse transcription PCR; PBS, phosphate-buffered saline; µS, microsiemens.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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