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• W2020777300 abstract "Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed in keratinocytes of the epidermis. We previously showed that AQP3-mediated transport of water and glycerol is involved in keratinocyte migration and proliferation, respectively. However, the involvement of AQP3 in epidermal hyperplasia in skin diseases, such as atopic dermatitis (AD), is unknown. In this study, we found significantly increased AQP3 transcript and protein expression in the epidermis of human AD lesions. The upregulation of AQP3 expression in human keratinocytes by transfection with human AQP3 DNA plasmid was associated with increased cellular glycerol and ATP, as well as increased cell proliferation. Among several cytokines and chemokines produced in the skin, CCL17, which is highly expressed in AD, was found to be a strong inducer of AQP3 expression and enhanced keratinocyte proliferation. In mouse AD models, AQP3 was strongly overexpressed in the epidermis in wild-type mice. Epidermal hyperplasia was reduced in AQP3-deficient mice, with a decreased number of proliferating keratinocytes. These results suggest the involvement of AQP3 in epidermal hyperplasia by a mechanism involving upregulated AQP3 expression and consequent enhancement of keratinocyte proliferation. Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed in keratinocytes of the epidermis. We previously showed that AQP3-mediated transport of water and glycerol is involved in keratinocyte migration and proliferation, respectively. However, the involvement of AQP3 in epidermal hyperplasia in skin diseases, such as atopic dermatitis (AD), is unknown. In this study, we found significantly increased AQP3 transcript and protein expression in the epidermis of human AD lesions. The upregulation of AQP3 expression in human keratinocytes by transfection with human AQP3 DNA plasmid was associated with increased cellular glycerol and ATP, as well as increased cell proliferation. Among several cytokines and chemokines produced in the skin, CCL17, which is highly expressed in AD, was found to be a strong inducer of AQP3 expression and enhanced keratinocyte proliferation. In mouse AD models, AQP3 was strongly overexpressed in the epidermis in wild-type mice. Epidermal hyperplasia was reduced in AQP3-deficient mice, with a decreased number of proliferating keratinocytes. These results suggest the involvement of AQP3 in epidermal hyperplasia by a mechanism involving upregulated AQP3 expression and consequent enhancement of keratinocyte proliferation. atopic dermatitis aquaporin normal human keratinocyte ovalbumin oxazolone proliferating cell nuclear antigen thymus and activation-regulated chemokine transepidermal water loss tumor necrosis factor-α wild type The aquaporins (AQPs, named AQP0–12) are a family of transmembrane channels that transport water, and in some cases small solutes such as glycerol (Carbrey and Agre, 2009Carbrey J.M. Agre P. Discovery of the aquaporins and development of the field.Handb Exp Pharmacol. 2009; 190: 3-28Crossref PubMed Scopus (161) Google Scholar; Verkman, 2009Verkman A.S. Aquaporins: translating bench research to human disease.J Exp Biol. 2009; 212: 1707-1715Crossref PubMed Scopus (113) Google Scholar). AQP3 is one such water/glycerol-transporting protein, which is expressed in keratinocytes of the epidermis (Ma et al., 2002Ma T. Hara M. Sougrat R. et al.Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3.J Biol Chem. 2002; 277: 17147-17153Crossref PubMed Scopus (193) Google Scholar). Our previous studies using AQP3 knockout mice and human keratinocytes showed that AQP3-mediated water and glycerol transport is involved in keratinocyte migration and proliferation, respectively, which were implicated to have important roles in cutaneous wound healing and tumorigenesis (Hara-Chikuma and Verkman, 2008aHara-Chikuma M. Verkman A.S. Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing.J Mol Med. 2008; 86: 221-231Crossref PubMed Scopus (194) Google Scholar, Hara-Chikuma and Verkman, 2008bHara-Chikuma M. Verkman A.S. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.Mol Cell Biol. 2008; 28: 326-332Crossref PubMed Scopus (174) Google Scholar). We have also shown that AQP3 deficiency has little effect on differentiation markers in human keratinocytes, suggesting that AQP3 is not involved in keratinocyte differentiation (Hara-Chikuma et al., 2009Hara-Chikuma M. Takahashi K. Chikuma S. et al.The expression of differentiation markers in aquaporin-3 deficient epidermis.Arch Dermatol Res. 2009; 301: 245-252Crossref PubMed Scopus (31) Google Scholar). Previous conflicting studies had suggested that AQP3 is involved in early differentiation, but not in proliferation, of keratinocytes (Zheng and Bollag, 2003Zheng X. Bollag W.B. Aquaporin 3 colocates with phospholipase d2 in caveolin-rich membrane microdomains and is downregulated upon keratinocyte differentiation.J Invest Dermatol. 2003; 121: 1487-1495Crossref PubMed Scopus (74) Google Scholar; Bollag et al., 2007Bollag W.B. Xie D. Zheng X. et al.A potential role for the phospholipase D2-aquaporin-3 signaling module in early keratinocyte differentiation: production of a phosphatidylglycerol signaling lipid.J Invest Dermatol. 2007; 127: 2823-2831Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The development and maintenance of the mature epidermis rely on balanced keratinocyte proliferation and terminal differentiation. Hyperproliferation and disturbed differentiation are associated with certain pathological conditions, such as atopic dermatitis (AD), ichthyosis, and psoriasis (Fuchs and Raghavan, 2002Fuchs E. Raghavan S. Getting under the skin of epidermal morphogenesis.Nat Rev Genet. 2002; 3: 199-209Crossref PubMed Scopus (541) Google Scholar; Jensen et al., 2004Jensen J.M. Fölster-Holst R. Baranowsky A. et al.Impaired sphingomyelinase activity and epidermal differentiation in atopic dermatitis.J Invest Dermatol. 2004; 122: 1423-1431Crossref PubMed Scopus (244) Google Scholar). With regard to keratinocyte proliferation, multiple studies have shown that several growth factors and cytokines, including tumor necrosis factor-α (TNF-α), IFN-γ, IL-1, and GM-CSF, are involved in the growth of keratinocyte in skin disorders, such as epidermal barrier disruption and wound healing (Wood et al., 1992Wood L.C. Jackson S.M. Elias P.M. et al.Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.J Clin Invest. 1992; 90: 482-487Crossref PubMed Scopus (387) Google Scholar; Segre, 2006Segre J.A. Epidermal barrier formation and recovery in skin disorders.J Clin Invest. 2006; 116: 1150-1158Crossref PubMed Scopus (310) Google Scholar; Barrientos et al., 2008Barrientos S. Stojadinovic O. Golinko M.S. et al.Growth factors and cytokines in wound healing.Wound Repair Regen. 2008; 16: 585-601Crossref PubMed Scopus (2008) Google Scholar). Recent studies have revealed that both IL-21 and IL-23 mediate keratinocyte proliferation and epidermal hyperplasia, which was found to be implicated in the pathogenesis of psoriasis (Chan et al., 2006Chan J.R. Blumenschein W. Murphy E. et al.IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.J Exp Med. 2006; 203: 2577-2587Crossref PubMed Scopus (514) Google Scholar; Caruso et al., 2009Caruso R. Botti E. Sarra M. et al.Involvement of interleukin-21 in the epidermal hyperplasia of psoriasis.Nat Med. 2009; 15: 1013-1015Crossref PubMed Scopus (156) Google Scholar). The aim of this study was to investigate the hypothesis that AQP3 upregulation is involved in keratinocyte proliferation and epidermal hyperplasia in skin disorders. Motivated by the observation that AQP3 expression was increased in the AD skin (Olsson et al., 2006Olsson M. Broberg A. Jernås M. et al.Increased expression of aquaporin 3 in atopic eczema.Allergy. 2006; 61: 1132-1137Crossref PubMed Scopus (91) Google Scholar), we used human keratinocytes and murine AD models in AQP3-null mice. We found that upregulation of AQP3 enhanced proliferation of human keratinocytes, which was involved in epidermal hyperplasia during AD development. Our data suggest that AQP3 inhibition by topical agents may be beneficial for the treatment of epidermal hyperplasia in AD. A previous report showed increased AQP3 transcript expression in the whole skin affected by atopic eczema (Olsson et al., 2006Olsson M. Broberg A. Jernås M. et al.Increased expression of aquaporin 3 in atopic eczema.Allergy. 2006; 61: 1132-1137Crossref PubMed Scopus (91) Google Scholar). To verify AQP3 protein expression in the epidermis of AD lesions, we performed immunostaining with anti-AQP3 antibody. Figure 1a shows remarkably greater AQP3 protein expression on the plasma membrane of keratinocytes in AD lesions than in the healthy epidermis. AQP3 was broadly distributed throughout the AD lesions. To quantify AQP3 transcript expression, we isolated the epidermis from the AD skin (lesion and non-lesion) and assayed AQP3 mRNA by quantitative reverse transcription-PCR. Figure 1b shows approximately four-fold increased AQP3 transcript expression in the epidermis of AD lesions versus controls. We determined whether increased AQP3 expression could enhance keratinocyte proliferation. Normal human keratinocytes (NHKs) were transfected with either empty vector or plasmid expressing human AQP3. Figure 2a shows that transfection of AQP3 plasmid produced at least 3.2-fold increase in AQP3 mRNA. Immunoblot analysis showed an approximately six-fold increase in AQP3 protein expression, with the expected molecular size of 28kDa (Figure 2a, right). Keratins 5 and 14, markers of basal proliferating cells, were significantly increased in AQP3-overexpressing NHKs compared with empty vector-transfected cells (Figure 2b). We found no significant differences in keratins 1 and 10, markers of early differentiation. Measurement of cell growth using the modified MTT assay showed that AQP3 upregulation enhanced cell growth (Figure 2c). We have previously proposed that AQP3-facilitated glycerol transport is an important determinant of keratinocyte proliferation and cellular ATP generation (Hara-Chikuma and Verkman, 2008aHara-Chikuma M. Verkman A.S. Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing.J Mol Med. 2008; 86: 221-231Crossref PubMed Scopus (194) Google Scholar, Hara-Chikuma and Verkman, 2008bHara-Chikuma M. Verkman A.S. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.Mol Cell Biol. 2008; 28: 326-332Crossref PubMed Scopus (174) Google Scholar). Levels of cellular glycerol and ATP were increased in NHKs with upregulated AQP3 expression (Figure 2d and e). These findings show that increased AQP3 expression enhances keratinocyte proliferation. It has been reported that various Th1 and Th2 cytokines/chemokines are altered in the epidermis in AD, which are proposed to be involved in epidermal hyperplasia and AD pathogenesis (Novak et al., 2003Novak N. Bieber T. Leung D.Y. Immune mechanisms leading to atopic dermatitis.J Allergy Clin Immunol. 2003; 112: S128-S139Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). We asked whether cytokines/chemokines could affect AQP3 expression in keratinocytes during the development of AD. Human keratinocytes (HaCaT) were used in this study to examine the effect of cytokines/chemokines on AQP3 expression, because the expressions of cytokine/chemokine receptors are more stable in HaCaT than in NHKs. Cells were incubated with cytokines/chemokines for 2 days, and AQP3 expression was quantified by immunoblotting. We found that TARC (thymus and activation-regulated chemokine)/CCL17, a Th2 chemotactic chemokine, increased AQP3 expression (Figure 3a). TNF-α significantly decreased AQP3 expression, which is consistent with previous data in a human squamous cell carcinoma cell line (DJM-1) (Horie et al., 2009Horie I. Maeda M. Yokoyama S. et al.Tumor necrosis factor-alpha decreases aquaporin-3 expression in DJM-1 keratinocytes.Biochem Biophys Res Commun. 2009; 387: 564-568Crossref PubMed Scopus (31) Google Scholar). Figure 3b shows that CCL17 increased AQP3 expression in a dose-dependent manner. CCL17 is produced by dendritic cells, T cells, and keratinocytes (Reiss et al., 2001Reiss Y. Proudfoot A.E. Power C.A. et al.CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.J Exp Med. 2001; 194: 1541-1547Crossref PubMed Scopus (434) Google Scholar). It has been reported that stimulation with IFN-γ and TNF-α synergistically induced CCL17 production in HaCaT cells (Vestergaard et al., 2000Vestergaard C. Bang K. Gesser B. et al.A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin.J Invest Dermatol. 2000; 115: 640-646Crossref PubMed Scopus (277) Google Scholar). To elucidate the mechanism of CCL17-facilitated AQP3 upregulation, HaCaT keratinocytes were incubated with TNF-α and INF-γ, and assayed for CCL17 and AQP3 mRNA expression. Figure 3c shows that addition of TNF-α/INF-γ increased intrinsic CCL17 in keratinocytes approximately nine-fold, without effect on AQP3 expression. In contrast, incubation with CCL17 increased AQP3 expression, whereas intrinsic CCL17 expression was not altered. These results suggest that exogenous CCL17 affects AQP3 expression. We next studied whether exogenous CCL17 could affect cell signaling, resulting in increased AQP3 expression. Cells were treated with various cell signaling inhibitors following incubation with CCL17 for 1 day, after which AQP3 mRNA expression was assessed. Figure 3d shows that mitogen-activated protein kinase and protein kinase C inhibitors (U0126 and R03-2432) suppressed CCL17-facilitated AQP3 upregulation, suggesting the involvement of CCL17-dependent mitogen-activated protein kinase and/or protein kinase C cell signaling in increased AQP3 expression. We determined the effect of CCL17 on keratinocyte proliferation. HaCaT keratinocytes were starved for 1 day, and treated with CCL17 for 6hours. AQP3 mRNA expression was increased 2.6-fold in CCL17-treated cells (Figure 4a, left). CCL17 also significantly increased the expressions of proliferation markers, keratins 5 and 14, but not those of differentiation markers, keratins 1 and 10 (Figure 4a, right). To examine the effect of CCL17 on cell growth, cells were treated with CCL17 for 2 days in the starved medium, after which cell proliferation was induced by replacing the medium with 0.1 or 2% fetal bovine serum. As shown in Figure 4b, cell proliferation, assessed by BrdU incorporation, was significantly increased in CCL17-treated cells as compared with control cells. Finally, cell growth was assayed in controls and AQP3 knockdown keratinocytes to determine the involvement of AQP3 in CCL17-induced cell proliferation. Transfection of small-interfering RNA-AQP3 into HaCaT cells consistently reduced AQP3 mRNA expression by ∼90% (9.1±1.4% of controls). Figure 4c shows reduced CCL17-induced cell proliferation in AQP3 knockdown keratinocytes. Taken together, these findings suggest that exogenous CCL17 increases AQP3 expression and enhances keratinocyte proliferation. To investigate the requirement of AQP3 for the development of AD, we applied an established murine model of AD in wild-type (WT) and AQP3-null mice. Dermatitis was induced by repeated epicutaneous application of ovalbumin (OVA) in a patch to tape-stripped skin, as described previously (Spergel et al., 1998Spergel J.M. Mizoguchi E. Brewer J.P. et al.Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (499) Google Scholar). Hematoxylin and eosin staining showed that the OVA-treated epidermis in WT mice was thicker than that in AQP3-null mice (Figure 5a, left), with the representative data summarized in Figure 5a (right). Immunostaining showed strong expression of the AQP3 protein on the plasma membranes in the OVA-applied epidermis of WT mice (Figure 5b). Immunoblot analysis confirmed that repeated OVA sensitization significantly increased AQP3 expression (Figure 5c), supporting the utility of the OVA-AD model in investigating the role of AQP3 in AD pathogenesis. To quantify keratinocyte proliferation, immunostaining with anti-proliferating cell nuclear antigen (PCNA) was performed (Supplementary Figure S1 online). Figure 5d shows that OVA sensitization resulted in an ∼10-fold increase in PCNA-positive cells in the WT epidermis, while the number of PCNA-positive cells was much lower in the AQP3-null than in the WT-OVA-treated epidermis. To determine epidermal permeability, transepidermal water loss (TEWL) was measured on the OVA- and saline-treated skin. After five treatments with OVA, TEWL was significantly elevated in the WT-OVA treated skin compared with the control- and saline-treated skin (Figure 5e). Download .pdf (6.72 MB) Help with pdf files Supplementary Information Total IgE and OVA-specific IgE were significantly higher in OVA sensitization than saline application in both WT and AQP3-null mice, indicating that repeated OVA application induced comparable allergic sensitization in WT and AQP3-null mice (Supplementary Figure S2 online). These findings suggest that AQP3 deficiency suppresses OVA-induced keratinocyte hyperproliferation, which may be responsible for epidermal hyperplasia and barrier disruption during AD development. To confirm the involvement of AQP3 expression in epidermal hyperplasia and barrier disruption during AD development, we investigated a different, hapten-induced mouse model of AD (Man et al., 2008Man M.Q. Hatano Y. Lee S.H. et al.Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.J Invest Dermatol. 2008; 128: 79-86Crossref PubMed Scopus (188) Google Scholar). WT and AQP3-null mice were challenged 10 times with an application of oxazolone (Ox) after 1 week of sensitization. Figure 6a shows that Ox-treated WT mice developed mild erythema and a rough-textured skin surface, whereas there were only minor changes in AQP3-null mice, suggesting that WT mice are more susceptible to atopic disorders than AQP3-null mice. TEWL values were much greater in Ox-applied WT than in the AQP3-null skin, indicating that AQP3 deficiency prevented the barrier disruption induced by Ox applications (Figure 6b). Hematoxylin and eosin staining showed that AQP3 deficiency sustained hapten-induced epidermal hyperplasia compared with the WT epidermis (Figure 6c). Immunostaining with AQP3 showed strong expression in the thickened epidermis of Ox-treated WT mice (Figure 6d). Immunoblotting verified an ∼10-fold increase in AQP3 expression in the WT AD epidermis (Figure 6e). CCL17 was comparably elevated in Ox-applied WT and the AQP3-null epidermis as assessed by ELISA assay (Figure 6f). Figure 6g showed that total IgE was significantly elevated in both WT and AQP3-null mice, indicating that allergic sensitization occurred in both WT and AQP3-null mice. These data from the Ox model are in agreement with those from the OVA-AD model: AQP3 expression is required for epidermal hyperplasia, which might contribute to barrier disruption during AD development. We found that AQP3 upregulation enhanced keratinocyte proliferation, which may be responsible for epidermal hyperplasia found in a number of skin disorders. We previously suggested the involvement of AQP3 in keratinocyte proliferation, in view of the observation that AQP3 deficiency impaired keratinocyte proliferation and reduced cellular glycerol and ATP content (Hara-Chikuma and Verkman, 2008cHara-Chikuma M. Verkman A.S. Roles of aquaporin-3 in the epidermis.J Invest Dermatol. 2008; 128: 2145-2151Crossref PubMed Scopus (137) Google Scholar). We proposed that AQP3-mediated glycerol transport is an important determinant of keratinocyte proliferation, in which glycerol works as a key regulator of cellular ATP energy. In this study, we showed that increased AQP3 expression by plasmid-DNA transfection or the AQP3 inducer CCL17 enhanced keratinocyte proliferation with increased proliferation markers, and increased cellular glycerol and ATP content. Although further studies are required to elucidate the exact mechanisms by which AQP3 expression increases cell proliferation, AQP3 upregulation might be one of the determinants of keratinocyte hyperproliferation in several skin diseases, such as AD. In this study, we showed in two different murine models that AQP3-null mice exhibit defective epidermal hyperplasia with suppressed barrier disruption during AD development. Repeated application of OVA or Ox induced AD-like skin lesions with irregularly acanthotic epidermis, high TEWL, and increased AQP3 expression in WT mice. These data provide evidence for the involvement of AQP3 in excessive keratinocyte proliferation and disturbed barrier function during the development of AD. Enhanced keratinocyte proliferation might induce disturbed differentiation and barrier function, as there is no sufficient time for normal differentiation or development of a functional epidermal barrier during accelerated cell renewal. AD is a common chronic inflammatory skin disease, which is classified into extrinsic and intrinsic types according to the presence or absence of sensitization toward environmental allergens (Tokura, 2010Tokura Y. Extrinsic and intrinsic types of atopic dermatitis.J Dermatol Sci. 2010; 58: 1-7Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The extrinsic and allergic AD lesions display impaired epidermal barrier function by inherited and acquired factors, which not only enhances allergen sensitization but also leads to systemic allergic responses (Spergel et al., 1998Spergel J.M. Mizoguchi E. Brewer J.P. et al.Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (499) Google Scholar; Novak et al., 2003Novak N. Bieber T. Leung D.Y. Immune mechanisms leading to atopic dermatitis.J Allergy Clin Immunol. 2003; 112: S128-S139Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar; Elias and Steinhoff, 2008Elias P.M. Steinhoff M. “Outside-toinside” (and now back to “outside”) pathogenic mechanisms in atopic dermatitis.J Invest Dermatol. 2008; 128: 1067-1070Crossref PubMed Scopus (214) Google Scholar; O'Regan et al., 2008O'Regan G.M. Sandilands A. McLean W.H. et al.Filaggrin in atopic dermatitis.J Allergy Clin Immunol. 2008; 122: 689-693Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Recent human genetic studies have shown that loss-of-function mutation in filaggrin was associated with impaired skin barrier function in AD patients (Palmer et al., 2006Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2088) Google Scholar; Morar et al., 2007Morar N. Cookson W.O. Harper J.I. et al.Filaggrin mutations in children with severe atopic dermatitis.J Invest Dermatol. 2007; 127: 1667-1672Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Coincidentally, the flaky-tail mouse, which exhibited low filaggrin gene expression, showed barrier abnormality with epidermal hyperplasia like a severe AD (Scharschmidt et al., 2009Scharschmidt T.C. Man M.Q. Hatano Y. et al.Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.J Allergy Clin Immunol. 2009; 124: 496-506Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar; Moniaga et al., 2010Moniaga C.S. Egawa G. Kawasaki H. et al.Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.Am J Pathol. 2010; 176: 2385-2393Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although further studies are necessary, it is expected that AQP3 expression might be increased in the other murine AD model, including filaggrin deficiency. The application of the AQP3 inhibitor will provide a, to our knowledge, previously unreported therapeutic strategy for controlling unwanted increased keratinocyte proliferation in the AD epidermis. The acute-phase AD skin displayed an allergen-derived Th2 cell-dominant infiltrate of T lymphocytes and increased Th2-type cytokine secretion, which induced an elevation in serum IgE and inflammation. In the chronic phase, AD lesions displayed infiltration with both Th1 and Th2 cells. The screening assay was performed based on the hypothesis that some Th1 and/or Th2 cell-derived chemokines/cytokines might increase AQP3 expression, which enhances keratinocyte proliferation during AD development. We found that the CCL17 Th2 chemotactic chemokine increased AQP3 expression in keratinocytes through mitogen-activated protein kinase and/or protein kinase C cell signaling. Indeed, we verified that CCL17 activated extracellular signal-regulated kinase of mitogen-activated protein kinase (not shown). It has been reported that CCL17 is produced by dendritic cells, T cells, and keratinocytes, and induces Th2-type T-cell migration (Reiss et al., 2001Reiss Y. Proudfoot A.E. Power C.A. et al.CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.J Exp Med. 2001; 194: 1541-1547Crossref PubMed Scopus (434) Google Scholar). CCL17 was found to be increased to a greater extent in the AD serum and epidermis than in healthy controls (Kakinuma et al., 2001Kakinuma T. Nakamura K. Wakugawa M. et al.Thymus and activation-regulated chemokine in atopic dermatitis: serum thymus and activation-regulated chemokine level is closely related with disease activity.J Allergy Clin Immunol. 2001; 107: 535-541Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar; Saeki and Tamaki, 2006Saeki H. Tamaki K. Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases.J Dermatol Sci. 2006; 43: 75-84Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Consistent with previous observations, we found significant elevation of CCL17 in both the WT and the AQP3-null AD epidermis as assessed by ELISA assay. Our findings implicate the involvement of CCL17 in the increased AQP3 expression in the AD epidermis, although further studies are required to establish the mechanisms of AQP3 upregulation by CCL17. CCL17-induced AQP3 upregulation might accelerate keratinocyte proliferation during AD development. In conclusion, our data provide several lines of evidence that AQP3 is involved in keratinocyte proliferation and epidermal hyperplasia. We propose that upregulated AQP3 expression enhances keratinocyte proliferation, which is involved in hyperplasia and barrier disruption in AD. Our findings suggest that AQP3 suppression by topical drugs may be useful for treatment of skin diseases associated with excessive epidermal proliferation. A total of 7 patients with AD (lesion, n=7; non-lesion, n=4) and 6 healthy non-AD volunteers were enrolled in this study. AD was diagnosed according to the consensus criteria as described previously (Williams et al., 1994Williams H.C. Burney P.G. Hay R.J. et al.The U.K. Working Party's diagnostic criteria for atopic dermatitis. I. Derivation of a minimum set of discriminators for atopic dermatitis.Br J Dermatol. 1994; 131: 383-396Crossref PubMed Scopus (767) Google Scholar). Informed consent was obtained from all subjects involved in this study. The study was approved by the Ethics Committee of the Kyoto University and was conducted according to the Declaration of Helsinki Principles. None of the patients had received local or systemic treatment with glucocorticoids or immunosuppressants within 1 week before the study. Skin biopsies were analyzed with real-time PCR and immunohistochemistry. For real-time PCR, the skin was first separated into the epidermis and the dermis by incubation in 0.25% trypsin-EDTA (Invitrogen, Carlsbad, CA) at 37°C for 1hour, and total RNA was extracted from the epidermis as described below. The AQP3-null mice (hairless genetic background) were generated by targeted gene disruption as described previously (Ma et al., 2002Ma T. Hara M. Sougrat R. et al.Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3.J Biol Chem. 2002; 277: 17147-17153Crossref PubMed Scopus (193) Google Scholar). All animal experiments were approved by the Committee on Animal Research of the Kyoto University. Mice aged 6–8 weeks were treated with OVA (Sigma-Aldrich, St Louis, MO) or Ox (Sigma-Aldrich), as described previously (Spergel et al., 1998Spergel J.M. Mizoguchi E. Brewer J.P. et al.Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (499) Google Scholar; Man et al., 2008Man M.Q. Hatano Y. Lee S.H. et al.Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.J Invest Dermatol. 2008; 128: 79-86Crossref PubMed Scopus (188) Google Scholar). In brief, for the OVA model, the dorsal skin was tape stripped six times, and OVA (100μg in 100μl saline) or saline alone (100μl) was placed on a round patch (16mm in diameter) (Torii Pharmaceutical, Tokyo, Japan), which was secured to the skin with an elastic tape (Alcare, Tokyo, Japan). Each mouse was treated with five 4-day periods of epicutaneous application of OVA or saline under occlusion at 3-day intervals. For the Ox model, each mouse was sensitized by one topical treatment on the dorsal skin with 60μl of 2.5% Ox (in ethanol). One week later, the mouse was treated topically with 120μl of 0.1% Ox on the dorsal area once every other day for an additional 3 weeks. TEWL was measured with a Tewameter Vapo Scan (Asahi Biomed, Tokyo, Japan). CCL17 level was assayed in the epidermal homogenate by ELISA (R&D Systems, Minneapolis, MN). Normal human epidermal keratinocytes (Kurabo, Osaka, Japan) were grown in Humedia-KG2 medium (Kurabo). HaCaT cells (a kind gift of Dr Fusenig, German Cancer Research Center, Heidelberg, Germany) were cultured in low-glucose DMEM (Invitrogen) with 10% fetal bovine serum (Funakoshi, Tokyo, Japan). After the cells grew to 80–90% confluence, they were treated with 10ngml-1 IFN-γ (R&D Systems), 10ngml-1 TNF-α (Miltenyi Biotec, Bergisch Gladbach, Germany), 1ngml-1 transforming growth factor-β1 (PeproTeck, Rocky Hill, NJ), 50ngml-1 IL-4 (PeproTeck), 50ngml-1 IL-13 (PeproTeck), 20ngml-1 CCL27 (PeproTeck), and 20–200ngml-1 CCL17 (Miltenyi Biotec), respectively. For treatment with cell signaling inhibitors, cells were incubated for 1hour with 10μM U73122 (Cayman Chemical, Ann Arbor, MI), 50μM LY294002 (Jena Bioscience, Jena, Germany), 10μM U0126 (Cell Signaling Technology, Danvers, MA), or 10μM R03-2432 (Enzo Life Sciences, Plymouth Meeting, PA), followed by treatment with CCL17 (20ngml-1). Experiments were performed 6–48hours after incubation for quantitative reverse transcription-PCR, immunoblot analysis, and cell growth assay. The constructs yielding human AQP3 (NM_004925) and control vector pCMV6-XL4 were obtained from Origene TrueClone (Rockville, MD). NHKs were transfected with purified plasmid DNA using Lipofectamine 2000 (5–25ng per 8 × 103 cells; Invitrogen). HaCaT cells were transfected with AQP3 small-interfering RNA or non-targeting small-interfering RNAs (Dharmacon, Lafayette, CO) at 40–50% confluence using Lipofectamine 2000. Cell proliferation was analyzed using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan) or the BrdU Cell Proliferation Assay kit (Calbiochem, San Diego, CA). Cell homogenates (3,500g, 10minutes, 4°C) were assayed for glycerol and ATP using commercial kits (glycerol, Sigma-Aldrich; ATP, Roche, Basel, Switzerland). Paraffin-embedded sections were stained with hematoxylin and eosin or immunostained with anti-AQP3 (Millipore, Billerica, MA) or anti-PCNA (Dako, Glostrup, Denmark) with biotinylated IgG and horseradish peroxidase-conjugated ABC reagent (Vector Laboratories, Burlingame, CA). Epidermal thickness and PCNA-positive cells per 100μm were measured at three locations per mouse. The epidermis of each mouse was separated from the dermis by incubation in phosphate-buffered saline solution at 60°C for 20seconds. The epidermis and cultured HaCaT cells were lysed with extraction buffer containing 250mM sucrose, 1mM EDTA, and 1% protein inhibitor cocktail (Sigma-Aldrich). For immunoblot analysis, polyclonal AQP3 antibody (Millipore) and horseradish peroxidase-conjugated secondary anti-rabbit IgG antibody (Cell Signaling Technology) were used for detection by ECL (GE Healthcare, Piscataway, NJ). Total RNA was isolated using RNeasy kits and digested with DNase I (Qiagen, Hilden, Germany). The cDNA was reverse transcribed from total RNA samples using the Prime Script RT reagent kit (Takara Bio, Otsu, Japan). Quantitative reverse transcription-PCR was performed using SYBR Green I (Takara Bio) and primers listed in Supplementary Table S1 online using the Light Cycler real-time PCR apparatus (Roche). Statistical analysis was performed using the two-tailed Student's t-test or analysis of variance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and NIH grant DK35124 to ASV. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid" @default.
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• W2020777300 title "Upregulation of Aquaporin-3 Is Involved in Keratinocyte Proliferation and Epidermal Hyperplasia" @default.
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