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J. Biol. Chem., Vol. 277, Issue 48, 46616-46621, November 29, 2002

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Selectively Reduced Glycerol in Skin of Aquaporin-3-deficient Mice May Account for Impaired Skin Hydration, Elasticity, and Barrier Recovery^*

Mariko Hara^§, Tonghui Ma, and A. S. Verkman^¶

From the  Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521 and ^§ Basic Research Laboratory, Kanebo Ltd., Odawara, Japan

Received for publication, September 4, 2002, and in revised form, September 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by ~2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel ³H₂O distribution method, SC water content was reduced by ~50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with ~50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an ~2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also ~2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na⁺, K⁺, Ca²⁺, Mg²⁺) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/µg protein: 5.5 ± 0.4 versus 2.3 ± 0.7 in SC; 0.037 ± 0.007 versus 0.022 ± 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hydration of the stratum corneum (SC),¹ the non-viable outermost layer of skin, is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function (1-3). Water is continuously exchanged among the SC, the underlying viable epidermis, and the external atmosphere. SC water content depends on external humidity, the capacity of the epidermis to replace evaporative water losses, and the intrinsic SC "water holding capacity" (4). The determinants of SC water holding capacity are thought to include SC structure and composition, particularly the content of small molecule osmolytes or "humectants" such as free amino acids (5, 6). Decreased SC water content is found is a number of common skin diseases such as atopic dermatitis (7), eczema (8), psoriasis (9), senile xerosis (10), and hereditary ichthyosis (11).

The water/glycerol transporting protein aquaporin-3 (AQP3) is expressed in the basal (innermost) layer of keratinocytes in mammalian epidermis as originally shown in rat skin (12) and then in human (13) and mouse (14) skin. AQP3 facilitates the transmembrane transport of water in response to osmotic gradients and the transport of glycerol in response to glycerol gradients. We recently tested the hypothesis that AQP3-facilitated water transport is important in SC water content by comparative phenotype studies in wild-type and AQP3 null mice (14). SC water content, as assessed indirectly by high-frequency superficial skin conductance, was reduced by ~2-fold in the AQP3 null mice. However, contrary to expectations, the reduction in skin conductance in AQP3 null mice was not corrected by occlusion or exposure to a humidified atmosphere, suggesting an intrinsic defect in SC water holding capacity. Thus, the water transporting function of AQP3 did not appear to be responsible for the reduced superficial skin conductance in AQP3 null mice. We proposed, by exclusion, that the glycerol-transporting function of AQP3 may be important in SC hydration.

The purpose of this study was to investigate the etiology of reduced superficial skin conductance in AQP3 null mice, as well as the possibility that other functional properties of AQP3-deficient skin are abnormal. We found significant reduction in SC water content in AQP3-deficient mice, as measured using a new ³H₂O distribution method, as well as impairment in skin elasticity, barrier recovery, and wound healing. The morphology and composition of the SC of wild-type versus AQP3 null mice were systematically examined to identify structural or biochemical differences that could account for the functional abnormalities. The principal finding was selectively reduced glycerol content in SC and epidermis of AQP3 null mice without reduced serum glycerol concentration. We propose that reduced glycerol transport across the AQP3-deficient epidermis lowers SC glycerol content and is responsible for the impairment in SC hydration, skin mechanical properties, and biosynthetic functions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mice-- AQP3 null mice, originally generated in a CD1 genetic background (15), were back-crossed into the SKH1 hairless genetic background as described previously (14). Six- to 10-week-old mice were used for functional and biochemical studies. The mice were maintained in air-filtered cages and fed normal mouse chow in the U.C.S.F. Animal Care facility. All procedures were approved by the U.C.S.F. Committee on Animal Research.

Electron Microscopy-- Skin samples were fixed in 2% glutaraldehyde and 0.2% ruthenium tetroxide (RuO₄) in 0.1 M sodium cacodylate buffer. After fixation, samples were dehydrated in graded ethanol solutions and embedded in an Epon-epoxy mixture. Ultra-thin sections were examined in an electron microscope (JEOL JEM 100S).

Skin Conductance and Elasticity Measurements-- Stratum corneum hydration (water content) was measured under standardized condition (external temperature 22 ± 2 °C, humidity 40 ± 3%) by high-frequency surface electrical conductance using a Skicon-200 (IBS Co., Hamamatsu, Japan) (16). Mechanical properties were measured using a Cutometer (model SEM474, Courage and Khazaka, Koln, Germany) under standardized conditions after exposure to low (10%) and high (90%) external humidity for 24 h and after removal of SC by tape stripping. The kinetics of skin displacement (2-mm diameter probe) were measured over 2 s in response to a 50 mbar suction, followed by a 2-s relaxation period after terminating the suction. The key parameters of skin elasticity, immediate distention (Ue), final distention (Uf), immediate retraction (Ur), and delayed distention (Uv), were calculated from the distension kinetics as described (17, 18).

SC Water Content-- Mice were injected ³H₂O water intraperitoneally (10 µl/g body weight, 1 mCi/ml, PerkinElmer Life Sciences). The SC was collected by nine tape strippings with D-squame (CuDerm, TX) at 5, 30, 60, 120, 150, or 240 min after injection. Tapes were incubated in PBS, radioactivity was measured by liquid scintillation spectrometry, and total protein was measured using a Bio-Rad DC protein assay kit.

Barrier Recovery after Tape Stripping-- Cutaneous barrier function was evaluated by measurement of transepidermal water loss (TEWL) using a Meeco moisture analyzer (Meeco Inc., PA). The SC barrier was disrupted by repeated tape stripping with cellophane tape until the TEWL increased 10-fold above baseline as described previously (19). TEWL was measured at 2, 6, and 24 h after tape stripping.

Wound Healing-- Two full-thickness punch biopsies extending through the epidermis and dermis (5 mm in diameter) were done on the back of wild-type and AQP3 null mice. Wound repair was monitored daily by measurement of wound area (= r²) and expressed as the percentage of initial wound area.

Lipid Analysis-- Full-thickness skin was heat-split at 60 °C for 10 s to remove intact epidermis from dermis. Nucleated epidermal cells were removed from the SC by floating full-thickness skin, basal side downward, in 0.5% trypsin in PBS overnight at 4 °C, followed by vortexing. Lipids were extracted using standard procedures (20) and subjected to high-performance thin layer chromatography (HPTLC) as described (21). After solvent fractionation, dried plates were sprayed with charring solution (10% cupric sulfate in 10% phosphate buffer) and then heated to 160 °C for 20 min. Plates were scanned using NIH image software, and lipid fractions were quantified using standards run in parallel with the experimental samples.

Amino Acid and Ionic Content-- After removal of the SC by tape stripping, tapes were incubated in 10 mM HCl for amino acid measurement or in distilled water for ionic content determination. Amino acids were analyzed using a Hitachi Amino Acid Analyzer L-8800 as described previously (22). Ionic content was analyzed using ion chromatography IC-8010 (TOSOH, Tokyo, Japan) and Shodex packed column HPLC YK-421 (Showa Denko, Tokyo, Japan) using 3 mM phosphate buffer at 1 ml/min flow.

DNA Synthesis-- Mice were injected [³H]thymidine (1 µCi/g body weight intraperitoneal, 1 mCi/ml, ICN, CA). Skin was removed at 1 h, and epidermal sheets were separated from dermis by incubation in 10 mM EDTA/PBS at 37 °C for 60 min and homogenized in 10 volume of PBS. An equal volume of cold trichloroacetic acid (20 wt %) was added, and the pellet was rinsed twice with 5% trichloroacetic acid. The DNA-containing fraction was extracted at 90 °C for 15 min in 1 N NaOH (23). ³H radioactivity in aliquots was measured by liquid scintillation spectrometry. Total cellular DNA was assayed using Hoechst buffer and bisbenzimidazole as described (24). DNA synthesis was expressed as [³H]thymidine radioactivity per total cellular DNA.

Glycerol Assay-- The SC was collected by nine tape strippings, and the tapes were soaked in PBS to solubilize polar molecules including glycerol. To determine glycerol distribution within the SC, soluble fractions from 3 tapes (upper layer, first to third tapes; middle layer, fourth to sixth; lower layer, seventh to ninth) were pooled. Epidermal sheets were separated from the dermis by incubation in 10 mM EDTA/PBS at 37 °C for 60 min and homogenized in 10 volume of PBS using a Polytron homogenizer. Homogenates were centrifuged at 3500 g for 10 min at 4 °C to give epidermal- and dermal-soluble fraction. In the same mice, blood was sampled from the heart and serum was used for glycerol assay. Glycerol content in SC, epidermis, dermis, and serum was determined using a Glycerol Assay kit based on enzymatic analysis (Roche Molecular Biochemicals).

Lactic Acid, Urea, Glucose, and Triglycerol Assays-- SC water-soluble fractions collected by tape stripping were assayed for lactic acid, urea, and glucose using commercial kits (lactic acid, Roche Molecular Biochemicals; urea, Sigma; glucose, Molecular Probes). Protein concentration in SC-soluble fractions was measured using a Bio-Rad DC protein assay kit.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reduced Water Content in SC of AQP3 Null Mice-- SC hydration was analyzed in the hairless mice used for functional, morphological, and biochemical studies. Fig. 1A shows that high-frequency skin surface conductance was remarkably lower in AQP3 null mice than matched wild-type mice in agreement with our previous data (14). However, it has been assumed without direct evidence that high-frequency surface conductance is proportional to the water content of the very superficial stratum coreum (16, 25). This assumption is unlikely to be valid when comparing skin having different SC ionic or dipolar solute content or different SC ultrastructure. To measure SC water content directly, ³H₂O was injected into mice, allowed to equilibrate throughout all tissues, and assayed in SC after removal by tape stripping. ³H₂O equilibrated in SC over ~60 min (Fig. 1B) by a diffusional exchange mechanism that is expected to be unstirred-layer limited. The amount of SC radioactivity at 60 min thus provides a direct measure of SC water content. By comparing serum versus SC radioactivity at 60 min (expressed per tissue volume), the SC of wild-type mice contains ~18% water by volume. Fig. 1C shows ~50% reduced ³H₂O content in SC of AQP3 null mice (~9% water by volume), providing direct evidence for reduced SC hydration.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Reduced stratum corneum water content in AQP3 null mice. A, high-frequency superficial skin surface conductance in dorsal skin of hairless wild-type and AQP3 null mice (mean ± S.E., 20 mice per group). *, p < 0.001. B, time course of 3H2O content in SC of wild-type mice at indicated times after intraperitoneal 3H2O injection (mean ± S.E., 2-5 mice per group). C, 3H2O content in SC at 60 min after intraperitoneal injection of 3H2O (mean ± S.E., 5 mice per group). *, p < 0.001

Impaired Skin Elasticity, Barrier Recovery, and Wound Healing in AQP3 Null Mice-- Skin elasticity was measured by cutometry, in which displacement of the skin surface was measured in response to application of a mild suction force for 2 s followed by release of the suction. Fig. 2A shows the definition of parameters (top) and representative displacement curves for wild-type and AQP3 null mice (bottom). The deduced parameters include: Uf (final distension), Ue (immediate distension), Uv (delayed distention), Ur (immediate retraction), and Ur/Uf. Uf, Ue, and Ur provide information about skin elasticity, and Uv about skin viscosity or viscoelasticity (26). Fig. 2B summarizes averaged Uf, Ue, Uv, Ur, and Ur/Uf for a series of wild-type and AQP3 null mice. The elasticity parameters (Uf, Ue, Ur, and Ur/Uf) were decreased significantly in AQP3 null mice, although the viscosity parameter Uv was not changed.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Reduced skin elasticity in AQP3 null mice. A, elasticity measured in dorsal skin using a 2-mm diameter suction probe and 50 mbar pressure transient. Skin elasticity parameters shown (top): immediate distention (Ue), final distention (Uf), immediate retraction (Ur), and delayed distention (Uv). Bottom, representative displacement curves for wild-type (black curve) and AQP3 null (gray curve) mice. B, elasticity parameters (Uf, Ue, Uv, Ur, and Ur/Uf) measured in wild-type and AQP3 null mice (mean ± S.E., seven mice per group). **, p < 0.01. *, p < 0.05. C, Elasticity parameters (Uf, Ue, Ur) measured after removal of SC by tape stripping (mean ± S.E., four mice per group). D, Uf, Ue, and Ur measured after 24 h exposure to dry (10% relative humidity) or moist (90% relative humidity) atmosphere (mean ± S.E., four mice per group). *, p < 0.01.

To determine whether differences in SC mechanical properties could account for the altered elasticity properties, measurements were done after SC removal by tape stripping. Fig. 2C shows similar Uf, Ue, and Ur in wild-type and AQP3 null mice after tape stripping, indicating that AQP3 deletion affects the mechanical properties of the SC and not of the underlying epidermis and dermis. Experiments were also done to determine whether exposure to altered external humidity for 24 h could correct the elasticity defect in AQP3 null mice. Fig. 2D shows that the reduced Uf, Ue, and Ur in AQP3 null mice persisted after exposure to a humidified atmosphere, consistent with previous results showing that reduced superficial skin conductance could not be corrected by a humidified atmosphere (14). Exposure to a dry atmosphere reduced Uf, Ue, and Ur, in wild-type mice to that in AQP3 null mice. Together these results suggest that reduced SC water content in AQP3 null mice results in reduced skin elasticity.

Barrier function, as deduced by TEWL, was similar in intact (unperturbed) skin of wild-type and AQP3 null mice (11.1 ± 0.8 and 9.7 ± 1.0 µg H₂O/min/cm², respectively, S.E., n = 5 mice per group). Fig. 3A shows the kinetics of recovery (reduction) in TEWL after removal of most of the SC by tape stripping. A standard protocol was used in which repeated tape stripping was done to increase TEWL 10-fold over that measured under basal conditions (19). The ordinate in Fig. 3A shows the percentage barrier recovery in which TEWL is normalized to 100% before tape stripping and 0% just after tape stripping. The number of tape strippings was not different between wild-type and AQP3 null mice (9.1 ± 0.3 and 8.7 ± 0.2, respectively). Barrier recovery after tape stripping was significantly reduced at 2 and 6 h in AQP3 null mice (75 ± 4 and 54 ± 3% at 6 h, respectively).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Delayed recovery of barrier function after barrier disruption and wound healing in AQP3 null mice. A, barrier recovery after disruption. The permeability barrier was disrupted by tape stripping to remove most of the SC. TEWL was measured as an index of barrier function at 0, 2, 6, and 24 h after tape stripping. Data normalized to prestripping TEWL (100%) and TEWL just after striping (0% recovery) (mean ± S.E., six mice per group). **, p < 0.01; *, p < 0.05. B, wound healing. Two full-thickness punch biopsies (5-mm diameter) were performed on the back (left photo), and the area was measured. Representative photographs of wound closure at 1, 3, and 5 days in wild-type and AQP3 null mice (left panels). Scale bar, 5 mm. Right, data as percentage of initial wound area (mean ± S.E., five mice per group). *, p < 0.01.

Another index of skin biosynthetic function is wound healing. A standard wound closure model was used in which the decrease in wound area was measured daily after creation of a circular wound by punch biopsy through the epidermis and dermis (27). Fig. 3B (left) shows a photograph of the wound just after creation and at 1, 3, and 5 days. Fig. 3B (right) shows similar wound closure on day 1, prior to generating a new epidermis, but significantly delayed wound closure in AQP3 null mice on days 2 through 5 (58 ± 9 versus 28 ± 4% at day 5).

Reduced SC Glycerol Content in AQP3 Null Mice-- SC morphology and composition were analyzed to investigate whether structural or biochemical differences might account for the functional abnormalities in skin of AQP3 null mice. Fig. 4A shows no differences in lipid lamellar (a, b) or SC (c, d) structure as examined by electron microscopy with RuO₄ postfixation. Fig. 4B shows a similar averaged number of SC layers and SC thickness in wild-type and AQP3 null mice. To study the cell proliferation rate in epidermis, DNA synthesis was determined using [³H]thymidine incorporation. Fig. 4C shows similar rates of epidermal cell proliferation in wild-type and AQP3 null mice.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   SC morphology and epidermal DNA synthesis. A, lipid lamellar structure (a, b) and stratum corneum layer (c, d) examined by electron microscopy with RuO4 postfixation. Wild-type mice, a and c; AQP3 null mice, b and d. Scale bars: 0.1 µm (a, b) and 1 µm (c, d). B, number of SC layers and SC thickness (mean ± S.E.) measured in multiple electron micrographs (four mice per group). C, [3H]thymidine incorporation into epidermis (mean ± S.E., five mice per group).

The lipid composition of the SC was determined by HPTLC. Fig. 5A shows representative chromatograms, which resolve the principal SC lipids comprising lamellar membranes and involved in barrier function including ceramides, cholesterol, and free fatty acids. Averaged results from a series of mice are summarized in Fig. 5B. The lipid composition and the amount of different lipids were not significantly altered by AQP3 deletion.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   SC lipid analysis. A, representative HPTLC (left) and lipid content analysis (ceramides, cholesterol, and free fatty acids, right). B, averaged SC content of ceramides, cholesterol, and free fatty acids (mean ± S.E., five mice per group).

Table I summarizes the averaged content of "natural moisturizing factors" thought to be involved in SC hydration including free amino acids, lactic acid, sugar (glucose), urea, and ions (sodium, potassium, magnesium, and calcium). No significant differences were found. In addition, the profile of free amino acids in SC was fully analyzed and no significant differences were found.

Because AQP3 is a transporter of both water and glycerol, the glycerol content of SC, epidermis, dermis, and serum were determined. Fig. 6A shows significantly reduced glycerol content in SC and epidermis in AQP3 null mice (SC: 5.5 ± 0.4 versus 2.3 ± 0.7 nmol/µg protein; epidermis: 0.035 ± 0.007 versus 0.022 ± 0.005 nmol/µg protein) as measured by a glycerol-specific enzymatic assay with colorimetric detection. Fig. 6A (right) shows a calibration curve using glycerol standards along with SC data. Despite remarkably reduced glycerol content in SC and epidermis, there was no significant difference in the glycerol content of dermis and serum of wild-type versus AQP3 null mice. Fig. 6B shows that glycerol was significantly reduced throughout the thickness of the SC in AQP3 null mice.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Reduced glycerol content in SC and epidermis of AQP3 null mice. A, glycerol content in SC, epidermis, dermis, and serum (mean ± S.E., five to six mice per group). *, p < 0.01. Right, representative glycerol enzymatic assay calibration (read-out optical density at 340 nm) showing glycerol standards (filled squares) and SC measurements (circles). B, glycerol distribution in stratum corneum. Stratum corneum layers were collected by tape stripping (nine times). Three tapes were pooled for indicated layers (upper layer: first to third tapes; middle layer: fourth to sixth tapes; lower layer: seventh to ninth tapes) (mean ± S.E., four mice per group). C, schematic of skin showing SC, epidermal, and dermal layers with AQP3 in basal epidermal cells. Right, hypothesis showing reduced epidermal and SC glycerol as a result of reduced glycerol transport through basal epidermal cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

AQP3 is a water/glycerol transporter expressed in the basal layer of keratinocytes in mammalian epidermis. We find here that SC water and glycerol content are significantly reduced in AQP3 null mice, which as explained below probably accounts for the abnormalities in skin mechanical and biochemical functions. No differences were found in wild-type versus AQP3 null mice in SC morphology, lipid composition, amino acid composition, and the content of ions and small non-polar osmolytes (except for glycerol). Serum glycerol concentration was not affected by AQP3 deletion, nor was the glycerol content in dermis. The simplest explanation for the reduced glycerol content in epidermis and SC of AQP3 null mice is slowed glycerol transport across the relatively glycerol-impermeable basal layer of epidermis in response to a steady-state dermal-to-epidermal glycerol gradient (Fig. 6C). Since glycerol is a water-retaining osmolyte or humectant, reduced SC glycerol content may be the principal cause of reduced SC water content in AQP3 null mice.

Superficial high-frequency skin conductance is the most commonly used method to study SC hydration (16). However, the SC depth probed in the conductance method is uncertain and probably very small, and high-frequency surface conductance is in principle sensitive to SC structure and ion/small dipolar solute composition. Although skin conductance may be useful for serial measurements in humans and analysis of cosmetic effects, comparative measurements of SC water content may not be valid in normal versus disease skin or in wild-type versus genetically modified mice. Other indirect biophysical methods that have been used to assess SC moisture include infrared attenuated total reflection spectroscopy (28, 29), confocal Raman microspectroscopy (30), magnetic resonance imaging (31), and differential scanning calorimetry (32). We used here a simple method, based on steady-state ³H₂O distribution, to measure water content throughout the SC. The method relied on relatively rapid ³H₂O equilibration following intraperitoneal injection (compared with renal clearance). ³H₂O in the SC was measured from the radioactivity of tape-stripped samples. We found ~2-fold reduced ³H₂O radioactivity in the SC of AQP3 null mice, indicating reduced SC hydration. Also, our results provide the first direct measurement of the percentage of water in the SC, ~18 volume %. This value is lower than that of ~30 weight % estimated by differential scanning calorimetry in human SC, where the analysis assumed the presence of "primary," "secondary" (bound), and "free" types of water (4, 32).

A series of functional skin measurements were carried out in wild-type versus AQP3 null mice that were predicted to possibly be sensitive to altered SC water and/or glycerol content. The mechanical properties of skin and skin hydration are related (26). Altered mechanical properties such as elasticity have been reported in aged human facial skin (33), in thick epidermis of mice (18), in UV-B irradiated rat and mouse skin (34, 35), and after application of moisturizers in human skin (26). We used an established cutometry method to assess skin mechanical properties in which the kinetics of skin displacement was measured in response to application and release of a brief suction force. The principal finding was reduced elasticity parameters (Uf, Ue, Ur) in AQP3 null mice, which became similar after removal of the SC by tape stripping or by dehydrating the SC for 24 h by exposure to a dry atmosphere. Reduction in elasticity parameters persisted after exposure to a moist atmosphere for 24 h. These findings indicate that reduced SC water content is responsible for reduced elasticity in AQP3 null mice.

Recovery after barrier disruption has been used extensively as a measure of the biosynthetic function of the epidermis (36). Transepidermal water loss provides a quantitative measure of SC barrier function. Although basal barrier function (in unperturbed skin) was not altered by AQP3 deletion, the recovery of barrier function, representing largely the biosynthesis of SC lipids, was remarkably delayed in AQP3 null mice. Based on studies of skin biosynthesis (37), reduced lipid synthesis resulting from reduced epidermal glycerol content is likely the cause of delayed barrier recovery after tape stripping. Reduced lipid biosynthesis may also be responsible for the slowed wound healing in AQP3 null mice, since cell proliferation as assessed by [³H]thymidine incorporation was not impaired.

In summary, AQP3 deletion in mice resulted in reduced SC water content, impaired skin elasticity, delayed barrier recovery after SC removal, and delayed wound healing. Systematic analysis of SC morphology and composition revealed selective reduction in SC and epidermal glycerol content in AQP3 null mice, which appear to account for each of the functional abnormalities. Our results provide functional evidence for an important physiological role of glycerol transport through an aquaglyceroporin.

	ACKNOWLEDGEMENTS

We thank Prof. Hachiro Tagami for help in analysis of electron micrographs, Shintaro Inoue, Shingo Sakai, and Noriaki Nakagawa for technical support, and Liman Qain for mouse breeding and genotype analysis.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK35124, EB00415, EY13574, HL60288, and HL59198, a grant from the Cystic Fibrosis Foundation, and a gift from Kanebo, Ltd. of Japan.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 Inst., 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.

Published, JBC Papers in Press, September 21, 2002, DOI 10.1074/jbc.M209003200

	ABBREVIATIONS

The abbreviations used are: SC, stratum corneum; PBS, phosphate-buffered saline; TEWL, transepidermal water loss; HPTLC, high-performance thin layer chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				T. Hibuse, N. Maeda, T. Funahashi, K. Yamamoto, A. Nagasawa, W. Mizunoya, K. Kishida, K. Inoue, H. Kuriyama, T. Nakamura, et al. From The Cover: Aquaporin 7 deficiency is associated with development of obesity through activation of adipose glycerol kinase PNAS, August 2, 2005; 102(31): 10993 - 10998. [Abstract] [Full Text] [PDF]

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