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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 ± 0.007versus 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. 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 ± 0.007versus 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. Hydration of the stratum corneum (SC), 1The abbreviations used for: SC, stratum corneum; PBS, phosphate-buffered saline; TEWL, transepidermal water loss; HPTLC, high-performance thin layer chromatography. 1The abbreviations used for: SC, stratum corneum; PBS, phosphate-buffered saline; TEWL, transepidermal water loss; HPTLC, high-performance thin layer chromatography. the non-viable outermost layer of skin, is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function (1Tagami H. Kobayashi H. Zhen X.S. Kikuchi K. J. Invest. Derm. Symp. Proc. 2001; 6: 87-94Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 2Blank I.H. J. Invest. Derm. 1965; 45: 249-256Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 3Scheuplein R.J. Blank I.H. Physiol. Rev. 1971; 51: 702-747Crossref PubMed Scopus (1181) Google Scholar). 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” (4Takenouchi M. Suzuki H. Tagami H. J. Invest. Derm. 1986; 87: 574-576Abstract Full Text PDF PubMed Scopus (84) Google Scholar). 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 (5Denda M. Hori J. Koyama J. Yoshida S. Nanba R. Takahashi M. Horii I. Yamamoto A. Arch. Derm. Res. 1992; 284: 363-367Crossref PubMed Scopus (63) Google Scholar, 6Jacobson T.M. Yuksel K.U. Geesin J.C. Gordon J.S. Lane A.T. Gracy R.W. J. Invest. Derm. 1990; 95: 96-300Crossref Scopus (59) Google Scholar). Decreased SC water content is found is a number of common skin diseases such as atopic dermatitis (7Watanabe M. Tagami H. Horii I. Takahashi M. Kligman A.M. Arch. Derm. 1991; 127: 1689-1692Crossref PubMed Scopus (181) Google Scholar), eczema (8Thune P. Acta Derm. Venereol. 1989; 144: 133-135Google Scholar), psoriasis (9Tagami H. Acta Derm. Venereol. 1994; 185: 29-33PubMed Google Scholar), senile xerosis (10Horii I. Nakayama Y. Obata M. Tagami H. Br. J. Derm. 1989; 121: 587-592Crossref PubMed Scopus (194) Google Scholar), and hereditary ichthyosis (11Hara M. Kato T. Tagami H. Acta Derm. Venereol. 1993; 73: 283-285PubMed Google Scholar). 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 (12Frigeri A. Gropper M.A. Umenishi F. Kawashima M. Brown D. Verkman A.S. J. Cell Sci. 1995; 108: 2993-3002Crossref PubMed Google Scholar) and then in human (13Sougrat R. Morand M. Gondran C. Barre P. Gobin R. Bonte F. Dumas M. Verbavatz J.M. J. Invest. Derm. 2002; 118: 678-685Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and mouse (14Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) 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 (14Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 new3H2O 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. AQP3 null mice, originally generated in a CD1 genetic background (15Ma T. Song Y. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4386-4391Crossref PubMed Scopus (337) Google Scholar), were back-crossed into the SKH1 hairless genetic background as described previously (14Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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. Skin samples were fixed in 2% glutaraldehyde and 0.2% ruthenium tetroxide (RuO4) in 0.1m 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). 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) (16Tagami H. Yoshikuni K. Arch. Derm. 1985; 121: 642-645Crossref PubMed Scopus (139) Google Scholar). 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 (17Agache P.G. Monneur C. Leveque J.L. De Rigal J. Arch. Derm. Res. 1980; 269: 221-232Crossref PubMed Scopus (503) Google Scholar, 18Fujimura T. Moriwaki S. Takema Y. Imokawa G. J. Derm. Sci. 2000; 24: 105-111Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Mice were injected3H2O 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. 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 (19Taljebini M. Warren R. Mao-Oiang M. Lane E. Elias P.M. Feingold K.R. Skin Pharmacol. 1996; 9: 111-119Crossref PubMed Scopus (32) Google Scholar). TEWL was measured at 2, 6, and 24 h after tape stripping. 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 (= πr2) and expressed as the percentage of initial wound area. 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 (20Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-919Crossref PubMed Scopus (42807) Google Scholar) and subjected to high-performance thin layer chromatography (HPTLC) as described (21Holleran W.M. Uchida Y. Halkier-Sorensen L. Haratake A. Hara M. Epstein J.H. Elias P.M. Photoderm. Photoimmunol. Photomed. 1997; 13: 117-128Crossref PubMed Scopus (113) Google Scholar). 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 were using in with the After removal of the SC by tape were incubated in 10 mm for amino measurement or in water for content acids were using a as described previously S. S. Y. K. Tagami H. S. Skin Res. 2000; 6: PubMed Scopus Google Scholar). content was using chromatography Japan) and Japan) using mm phosphate at 1 Mice were body 1 mCi/ml, Skin was removed at 1 and epidermal were from dermis by in 10 mm at °C for 60 min and in 10 of of was and the was with The was extracted at °C for min in A. Uchida Y. K. Elias P.M. W.M. J. Invest. Derm. 1997; 108: Full Text PDF PubMed Scopus Google Scholar). radioactivity in was measured by liquid scintillation was using and as described Uchida Y. Brown Elias P.M. W.M. J. Invest. Derm. Full Text Full Text PDF PubMed Scopus Google Scholar). was expressed as radioactivity total The SC was collected by nine tape and the were in PBS to glycerol distribution the SC, from first to to to were were from the dermis by in 10 mm at °C for 60 min and in 10 of PBS using a were at for 10 min at 4 °C to and the mice, was from the and serum was used for glycerol content in SC, epidermis, and serum was using a on analysis SC collected by tape stripping were for lactic acid, and using acid, in was measured using a Bio-Rad DC protein assay kit. SC hydration was in the hairless mice used for and biochemical studies. that high-frequency skin surface conductance was in AQP3 null mice wild-type mice in with (14Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). However, without evidence that high-frequency surface conductance is to the water content of the superficial stratum (16Tagami H. Yoshikuni K. Arch. Derm. 1985; 121: 642-645Crossref PubMed Scopus (139) Google Scholar, K. Takahashi K. Tagami H. Acta Derm. Venereol. 1993; 73: Google Scholar). is to be skin SC or content or SC SC water content 3H2O was into mice, to and in SC after removal by tape stripping. 3H2O in SC over min 1 by a that is to be The amount of SC radioactivity at 60 min a of SC water By serum versus SC radioactivity at 60 min the SC of wild-type mice water by 1 ∼50% reduced 3H2O content in SC of AQP3 null mice water by providing evidence for reduced SC hydration. Skin elasticity was measured by in displacement of the skin surface was measured in response to of a for 2 s followed by of the suction. 2 the of parameters and displacement for wild-type and AQP3 null mice The parameters and Uf, Ue, and skin elasticity, and skin or H. Skin Res. 2000; 6: PubMed Scopus Google Scholar). 2 Uf, Ue, and for a of wild-type and AQP3 null mice. The elasticity parameters Ue, and were significantly in AQP3 null mice, the was not differences in SC mechanical properties could account for the elasticity properties, were done after SC removal by tape stripping. 2 Uf, Ue, and in wild-type and AQP3 null mice after tape that AQP3 deletion the mechanical properties of the SC and not of the underlying epidermis and dermis. were also done to exposure to external humidity for 24 h could the elasticity defect in AQP3 null mice. 2 that the reduced Uf, Ue, and in AQP3 null mice after exposure to a humidified atmosphere, with results that reduced superficial skin conductance could not be corrected by a humidified (14Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). to a reduced Uf, Ue, and in wild-type mice to that in AQP3 null mice. these results that reduced SC water content in AQP3 null mice results in reduced skin function, as by TEWL, was in intact skin of wild-type and AQP3 null mice ± and ± mice the kinetics of recovery in TEWL after removal of of the SC by tape stripping. standard was used in repeated tape stripping was done to TEWL 10-fold over that measured under basal conditions (19Taljebini M. Warren R. Mao-Oiang M. Lane E. Elias P.M. Feingold K.R. Skin Pharmacol. 1996; 9: 111-119Crossref PubMed Scopus (32) Google Scholar). The in the percentage barrier recovery in TEWL is to tape stripping and after tape stripping. The number of tape strippings was not wild-type and AQP3 null mice ± and ± recovery after tape stripping was significantly reduced at 2 and h in AQP3 null mice ± 4 and ± at Another of skin biosynthetic function is wound healing. standard wound was used in the in wound area was measured daily after of a wound by punch through the epidermis and dermis E. E. A. J.M. J. J.L. M.A. J. 2001; PubMed Scopus Google Scholar). a of the wound after and at and wound on to a epidermis, but significantly delayed wound in AQP3 null mice on 2 through ± versus ± at SC morphology and composition were to investigate structural or biochemical differences account for the functional in skin of AQP3 null mice. differences in lipid or SC structure as examined by electron microscopy with 4 a number of SC and SC in wild-type and AQP3 null mice. study the in epidermis, was using 4 of epidermal in wild-type and AQP3 null mice. The lipid composition of the SC was by the principal SC lipids and in barrier function and free acids. results from a of mice are in B. The lipid composition and the amount of lipids were not significantly by AQP3 deletion. the content of thought to be in SC hydration free amino lactic acid, and ions and significant differences were the profile of free amino acids in SC was and significant differences were of SC composition ± mice free amino acids ± ± of total free amino ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± not significant wild-type versus AQP3 in a not significant wild-type versus AQP3 AQP3 is a transporter of water and the glycerol content of SC, epidermis, and serum were significantly reduced glycerol content in SC and epidermis in AQP3 null mice 5.5 ± 0.4 versus 2.3 ± 0.7 nmol/μg ± versus 0.022 ± 0.005 nmol/μg as measured by a assay with a using glycerol with SC reduced glycerol content in SC and epidermis, was significant in the glycerol content of dermis and serum of wild-type versus AQP3 null mice. that glycerol was significantly reduced the of the SC in AQP3 null mice. AQP3 is a water/glycerol transporter expressed in the basal layer of keratinocytes in mammalian We that SC water and glycerol content are significantly reduced in AQP3 null mice, as for the in skin mechanical and biochemical functions. differences were found in wild-type versus AQP3 null mice in SC lipid composition, amino composition, and the content of ions and small osmolytes for glycerol was not affected by AQP3 nor was the glycerol content in dermis. The for the reduced glycerol content in epidermis and SC of AQP3 null mice is glycerol transport across the basal layer of epidermis in response to a glycerol glycerol is a or reduced SC glycerol content may be the principal cause of reduced SC water content in AQP3 null mice. high-frequency skin conductance is the used to study SC hydration (16Tagami H. Yoshikuni K. Arch. Derm. 1985; 121: 642-645Crossref PubMed Scopus (139) Google Scholar). However, the SC in the conductance is and and high-frequency surface conductance is in to SC structure and composition. Although skin conductance may be for in and analysis of comparative of SC water content may not be in normal versus skin or in wild-type versus mice. that used to SC moisture include total A. M. F. PubMed Scopus Google Scholar, Arch. Derm. Res. 1985; 277: PubMed Scopus Google Scholar), J. Invest. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), S. 1996; PubMed Scopus Google Scholar), and G. H. M. J. Invest. Derm. 1991; Full Text PDF PubMed Scopus Google Scholar). We used a method, on 3H2O to water content the SC. The on 3H2O with in the SC was measured from the radioactivity of We found ∼2-fold radioactivity in the SC of AQP3 null mice, reduced SC hydration. results the first measurement of the percentage of water in the SC, is that of by in human SC, the analysis the of and of water (4Takenouchi M. Suzuki H. Tagami H. J. Invest. Derm. 1986; 87: 574-576Abstract Full Text PDF PubMed Scopus (84) Google Scholar, G. H. M. J. Invest. Derm. 1991; Full Text PDF PubMed Scopus Google Scholar). of functional skin were in AQP3 null mice that were to be to SC water glycerol The mechanical properties of skin and skin hydration are H. Skin Res. 2000; 6: PubMed Scopus Google Scholar). mechanical properties such as elasticity in human skin Y. Y. M. Imokawa G. Br. J. Derm. 1994; PubMed Scopus Google Scholar), in epidermis of mice T. Moriwaki S. Takema Y. Imokawa G. J. Derm. Sci. 2000; 24: 105-111Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), in rat and mouse skin K. Takema Y. Moriwaki S. T. S. Imokawa G. Br. J. Derm. 2001; 144: PubMed Scopus Google Scholar, Y. C. A. K. M. R. J. Invest. Derm. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and after of in human skin H. Skin Res. 2000; 6: PubMed Scopus Google Scholar). We used an cutometry to skin mechanical properties in the kinetics of skin displacement was measured in response to and of a The principal finding was reduced elasticity parameters Ue, in AQP3 null mice, after removal of the SC by tape stripping or by the SC for 24 h by exposure to a atmosphere. in elasticity parameters after exposure to a for 24 These that reduced SC water content is responsible for reduced elasticity in AQP3 null mice. after barrier used as a of the biosynthetic function of the epidermis P.M. Feingold K.R. Skin Pharmacol. Skin Physiol. 2001; PubMed Scopus Google Scholar). water loss a of SC barrier Although basal barrier function (in unperturbed was not by AQP3 the recovery of barrier function, the of SC was delayed in AQP3 null mice. on studies of skin E. W.M. Elias P.M. Feingold K.R. Br. J. Derm. 1993; PubMed Scopus Google Scholar), reduced lipid from reduced epidermal glycerol content is the cause of delayed barrier recovery after tape stripping. lipid may also be responsible for the wound healing in AQP3 null mice, as assessed by was not AQP3 deletion in mice in reduced SC water content, skin elasticity, delayed barrier recovery after SC and delayed wound healing. analysis of SC morphology and composition selective reduction in SC and epidermal glycerol content in AQP3 null mice, appear to account for of the functional abnormalities. results functional evidence for an important of glycerol transport through an We Tagami for in analysis of electron and for and for mouse and
Hara et al. (Fri,) studied this question.