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Reduced Expression of Epidermal Growth Factor Receptor, E-Cadherin, and Occludin in the Skin of Flaky Tail Mice Is Due to Filaggrin and Loricrin Deficiencies

2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2012.06.005

1525-2191

Kozo Nakai, Kozo Yoneda, Yoichiro Hosokawa, Tetsuya Moriue, Richard B. Presland, Padraic G. Fallon, Kenji Kabashima, Hiroaki Kosaka, Yasuo Kubota,

Advancements in Transdermal Drug Delivery

Disruption of skin barrier function leads to increases in the percutaneous transfer of allergens and the incidence of atopic dermatitis. Flaky tail (Flgft) mice have been used as a model of atopic dermatitis with skin barrier dysfunction. Although Flgft mice are known to have filaggrin mutation, the mechanism responsible for the skin barrier dysfunction that they display needs to be determined, especially for the roles of epidermal adhesion and junction proteins. Herein, we report the decreased expression of epidermal growth factor receptor (EGFR), E-cadherin, occludin, and SIRT1 in the skin of Flgft mice, compared with those in C57BL/6J mice. Administration of N-acetyl-L-cysteine, an antioxidant, in the drinking water improved these protein expressions in the skin of Flgft mice. Notably, we discovered that loricrin expression was suppressed in Flgft mice. In vitro experiments showed that filaggrin small interfering RNA, loricrin small interfering RNA, or SIRT1 inhibitor sirtinol suppressed the expression levels of EGFR, E-cadherin, and occludin in a human immortalized keratinocyte cell line (HaCaT cells). Our findings suggest that the observed reductions in EGFR, E-cadherin, and occludin expression were due to filaggrin deficiency accompanied with subsequent loricrin deficiency and disruption of the SIRT1 pathway in the skin of Flgft mice. Disruption of skin barrier function leads to increases in the percutaneous transfer of allergens and the incidence of atopic dermatitis. Flaky tail (Flgft) mice have been used as a model of atopic dermatitis with skin barrier dysfunction. Although Flgft mice are known to have filaggrin mutation, the mechanism responsible for the skin barrier dysfunction that they display needs to be determined, especially for the roles of epidermal adhesion and junction proteins. Herein, we report the decreased expression of epidermal growth factor receptor (EGFR), E-cadherin, occludin, and SIRT1 in the skin of Flgft mice, compared with those in C57BL/6J mice. Administration of N-acetyl-L-cysteine, an antioxidant, in the drinking water improved these protein expressions in the skin of Flgft mice. Notably, we discovered that loricrin expression was suppressed in Flgft mice. In vitro experiments showed that filaggrin small interfering RNA, loricrin small interfering RNA, or SIRT1 inhibitor sirtinol suppressed the expression levels of EGFR, E-cadherin, and occludin in a human immortalized keratinocyte cell line (HaCaT cells). Our findings suggest that the observed reductions in EGFR, E-cadherin, and occludin expression were due to filaggrin deficiency accompanied with subsequent loricrin deficiency and disruption of the SIRT1 pathway in the skin of Flgft mice. Recent reports have suggested that filaggrin is essential for skin barrier function which prevents the body from the entry of foreign substances that would otherwise trigger aberrant immune responses. Decreased filaggrin expression partially accounts for the impaired epidermal barrier observed in ichthyosis vulgaris and atopic dermatitis.1Hudson T.J. Skin barrier function and allergic risk.Nat Genet. 2006; 38: 399-400Crossref PubMed Scopus (184) Google Scholar The disruption of skin barrier function due to filaggrin deficiency has been reported in studies that used flaky tail (Flgft) mice.2Fallon P.G. Sasaki T. Sandilands A. Campbell L.E. Saunders S.P. Mangan N.E. Callanan J.J. Kawasaki H. Shiohama A. Kubo A. Sundberg J.P. Presland R.B. Fleckman P. Shimizu N. Kudoh J. Irvine A.D. Amagai M. McLean W.H. A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming.Nat Genet. 2009; 41: 602-608Crossref PubMed Scopus (380) Google Scholar, 3Scharschmidt T.C. Man M.Q. Hatano Y. Crumrine D. Gunathilake R. Sundberg J.P. Silva K.A. Mauro T.M. Hupe M. Cho S. Wu Y. Celli A. Schmuth M. Feingold K.R. Elias P.M. Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.J Allergy Clin Immunol. 2009; 124 (506 e491-496): 496-506Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 4Moniaga C.S. Egawa G. Kawasaki H. Hara-Chikuma M. Honda T. Tanizaki H. Nakajima S. Otsuka A. Matsuoka H. Kubo A. Sakabe J. Tokura Y. Miyachi Y. Amagai M. Kabashima K. 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 (98) Google Scholar However, cellular pathogenesis of the skin barrier defects caused by the decreased filaggrin expression remains unclear. Cell–cell interactions might have fundamental effects on the barrier function of the epidermis. The interactions are primarily mediated by four types of plasma membrane structure: gap junctions, tight junctions, desmosomes, and adherens junctions. Tight junctions, in which occludin5Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions.J Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar and claudins6Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. Claudin-1 and −2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.J Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1730) Google Scholar are integral membrane proteins, act as important apical barriers that regulate paracellular permeability and separate the apical and basolateral membrane regions, thereby inducing polarity.7Anderson J.M. Van Itallie C.M. Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol. 1995; 269: G467-G475PubMed Google Scholar, 8Tsukita S. Furuse M. Itoh M. Molecular dissection of tight junctions.Cell Struct Funct. 1996; 21: 381-385Crossref PubMed Scopus (54) Google Scholar, 9Citi S. The molecular organization of tight junctions.J Cell Biol. 1993; 121: 485-489Crossref PubMed Scopus (170) Google Scholar Adherens junctions and desmosomes mediate cell–cell adhesion via members of the cadherin family such as E-cadherin, which controls adherens junctions in the epidermis.10Young P. Boussadia O. Halfter H. Grose R. Berger P. Leone D.P. Robenek H. Charnay P. Kemler R. Suter U. E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles.EMBO J. 2003; 22: 5723-5733Crossref PubMed Scopus (114) Google Scholar A recent study found defects in tight junction claudin-1 and claudin-23 expression in patients with atopic dermatitis.11De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. Berger A.E. Zhang K. Vidyasagar S. Yoshida T. Boguniewicz M. Hata T. Schneider L.C. Hanifin J.M. Gallo R.L. Novak N. Weidinger S. Beaty T.H. Leung D.Y. Barnes K.C. Beck L.A. Tight junction defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (773-786e771-777)PubMed Google Scholar The dissolution of tight junctions may be followed by the down-regulation of E-cadherin expression and the dissolution of adherens junctions,12Thiery J.P. Sleeman J.P. Complex networks orchestrate epithelial-mesenchymal transitions.Nat Rev Mol Cell Biol. 2006; 7: 131-142Crossref PubMed Scopus (3248) Google Scholar which are concomitantly regulated with epidermal growth factor receptor (EGFR).13Muller E.J. Williamson L. Kolly C. Suter M.M. Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?.J Invest Dermatol. 2008; 128: 501-516Crossref PubMed Scopus (133) Google Scholar We hypothesized that these cell–cell interactions and EGFR expression could be disturbed by filaggrin deficiency. Silent mating type information regulation 2 homolog 1 (SIRT1) is a redox-sensitive nicotinamide adenine dinucleotide-dependent deacetylase that is modified by oxidants and carbonyl stress, and its deacetylase levels are decreased by aging and in chronic inflammatory conditions such as chronic obstructive pulmonary disease in which excess reactive oxygen species formation occurs.14Caito S. Rajendrasozhan S. Cook S. Chung S. Yao H. Friedman A.E. Brookes P.S. Rahman I. SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress.FASEB J. 2010; 24: 3145-3159Crossref PubMed Scopus (241) Google Scholar Because SIRT1 is known to inhibit proliferation and to promote differentiation in in vitro skin keratinocytes,15Blander G. Bhimavarapu A. Mammone T. Maes D. Elliston K. Reich C. Matsui M.S. Guarente L. Loureiro J.J. SIRT1 promotes differentiation of normal human keratinocytes.J Invest Dermatol. 2009; 129: 41-49Crossref PubMed Scopus (101) Google Scholar SIRT1 can be concomitantly involved in the regulation of adhesion molecules and EGFR in skin keratinocytes. Because atopic dermatitis is a chronic inflammatory skin condition, it is also possible that SIRT1 expression is modified in the filaggrin-deficient skin of Flgft mice, which denote human atopic dermatitis. In the present study, we examined whether the expression levels of EGFR, E-cadherin, occludin, and SIRT1 are reduced in Flgft mice, compared with expression levels in C57BL/6J (B6) mice. We next used an antioxidant, N-acetyl-L-cysteine (NAC), for the purpose of recovering these protein expressions by ameliorating inflammatory/oxidative conditions in the skin of Flgft mice. Because loricrin is a major protein of the epidermal cornified cell envelope, which is an essential structure for skin barrier function, and it has been reported that loricrin expression is down-regulated in patients with atopic dermatitis,16Kim B.E. Leung D.Y. Boguniewicz M. Howell M.D. Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.Clin Immunol. 2008; 126: 332-337Crossref PubMed Scopus (385) Google Scholar we examined whether the expression levels of loricrin are reduced or not in Flgft mice. Because in vivo skin is heterogeneous, and Flgft mouse is a mixed strain of filaggrin mutation and matted mutation, we examined the effects of filaggrin or loricrin knockdown with small interfering RNA (siRNA) on the protein expression levels of EGFR, E-cadherin, occludin, and SIRT1 in a human epidermal keratinocyte cell line (HaCaT cells). The effects of the SIRT1 inhibitor sirtinol were also examined. NAC, Dulbecco's modified Eagle's medium, PBS, and Nω-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma-Aldrich (St. Louis, MO). AG1478 was purchased from Calbiochem-Novabiochem (San Diego, CA). Sirtinol was purchased from Enzo Life Sciences (Plymouth Meeting, PA). The antibodies against EGFR, E-cadherin, SIRT1, occludin, and β-actin were purchased from Cell Signaling Technology (Danvers, MA). The antibody against filaggrin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), loricrin was obtained from Covance (Emeryville, CA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from EnoGene Biotech (New York, NY). All animal experiments were conducted in accordance with the institutional guidelines for the care and use of laboratory animals. The Flgft mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and the C57BL/6J (B6) mice were obtained from CLEA Japan (Megro, Tokyo, Japan). The mice were housed in a room at the Health Science Center of Kagawa University under controlled temperature (23°C) and humidity (55%) and specific pathogen free conditions. The mice had free access to water and chow (Oriental Yeast Co., Itabashi, Tokyo, Japan). We administered NAC dissolved in drinking water at a concentration of 7 mg/mL (neutralized to pH 7.4 with NaOH) to some Flgft mice from birth.17Cotter M.A. Thomas J. Cassidy P. Robinette K. Jenkins N. Florell S.R. Leachman S. Samlowski W.E. Grossman D. N-acetylcysteine protects melanocytes against oxidative stress/damage and delays onset of ultraviolet-induced melanoma in mice.Clin Cancer Res. 2007; 13: 5952-5958Crossref PubMed Scopus (73) Google Scholar The rest of the Flgft mice and B6 mice served as the untreated group. At 8 weeks of age, the mice were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg) injection, and full-thickness sections of the dorsal skin were dissected. We obtained the skin of the neonatal and 8-week-old Flgft mice that were backcrossed five generations onto a B6 strain background from Kyoto University, Kyoto, Japan. We also obtained 8- to10-week-old Flgft mice that were backcrossed 10 generations onto a B6 strain background from Trinity College Dublin, Dublin, Ireland. Skin samples (1 to 2 cm2) were removed from the mid-dorsum of the sacrificed mice and fixed directly in 10% buffered formalin. The tissues were dehydrated and embedded in paraffin, and then 5-μm sections were cut, deparaffinized in xylene, rehydrated, and immunostained with primary antibodies with the use of the Histofine simple stain reagent (Nichirei, Tokyo, Japan), according to the manufacturer's protocol. Briefly, endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 5 minutes. After being washed with PBS, the sections were incubated with the primary antibodies at room temperature for 1 hour, washed in PBS, and then incubated with peroxidase-conjugated secondary antibodies. After being washed, the sections were incubated with 3-3′-diaminobenzidine tetrahydrochloride solution and counterstained with Mayer's hematoxylin. For the histologic portion of the study, skin sections were visualized with routine H&E staining. Tumor necrosis factor (TNF)α/IL-6 levels in the mouse serum and skin tissue were determined by enzyme-linked immunosorbent assay (ELISA) kit of Gen-Probe Incorporated (San Diego, CA). IL-1β/IL-17 levels in the mouse serum and skin tissue were determined by ELISA Kit of RayBiotech Inc. (Norcross, GA). The vascular endothelial growth factor (VEGF) levels in the mouse serum and skin tissue were determined by Quantikine Mouse VEGF Immunoassay (R&D Systems, Minneapolis, MN). In accordance with the manufacturer's protocol, the avidin–horseradish peroxidase color reaction was measured by using a microplate reader. The values of skin tissue TNFα/IL-6/IL-1β/IL-17/VEGF concentration were normalized to the protein concentration of the skin tissue. HaCaT cells (a generous gift from Professor N.E. Fusenig, German Cancer Research Center, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 and 95% air.18Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar For the raft culture studies, LabCyte EPI-MODEL (J-TEC, Gamagori, Aichi, Japan) was used with modification. Briefly, HaCaT cells were grown on cell culture inserts (0.4-μm pore diameter; BD, Franklin Lakes, NJ) in LabCyte EPI-MODEL assay medium. The cell culture inserts were placed in a 24-well plate that had been filled with the assay medium. The medium in the cell culture inserts was aspirated 24 hours later, and HaCaT cells were grown at the air–liquid interface. The medium in the 24-well plates was changed twice a week. Cells were cultured for 7 days. To knockdown filaggrin and loricrin in HaCaT cells, we used stealth siRNA (Invitrogen, Carlsbad, CA). Target sequences of the gene-specific stealth siRNA sequences were as indicated for filaggrin (NM-001016, nt1941, 5′-GAGGUGGUCUGGGUCUGCUUCCAGA-3′) and loricrin (NM-000427, nt31, 5′-GGCUCUCCUUCCUUCUCAGACAAGA-3′). The stealth siRNA transfection was performed according to the manufacturer's protocol. HaCaT cells were plated in 6-well plates. OPTI-MEM I Reduced Serum Medium (500 μL) was mixed with 5 μL of Lipofectamine 2000 and 5 μL of a 20-μmol/L stealth siRNA solution or the control RNA solution. After incubation at room temperature for 30 minutes, the solution was added to 1.5 mL of OPTI-MEM I Reduced Serum Medium and transfected to the HaCaT cells. HaCaT cells were then incubated for 24 hours before seeding onto cell culture inserts. The cell proliferation assay was conducted with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), in which WST-8 (2-(2-methoxy-4-nitrophenyl)−3-(4-nitrophenyl)- 5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) is converted to formazan by cellular dehydrogenase. The absorbance of formazan, which is proportional to the cell number, was determined at 450 nm. Skin tissue specimens were homogenized with a Polytron homogenizer in RIPA buffer containing 1% v/v NP40, 20 mmol/L Tris (pH 7.7), 150 mmol/L NaCl, 1 mmol/L EDTA, and a mixture of protease inhibitors (Calbiochem, San Diego, CA). The cells were washed with PBS and then treated with RIPA buffer. After incubation at 4°C for 20 minutes, the samples were sonicated on ice and centrifuged at 14,000 rpm for 10 minutes. The protein concentrations of the supernatant fluids were analyzed with the bicinchoninic acid protein assay (Pierce, Rockford, IL). An equal amount of protein was separated on reducing NuPAGE 4% to 12% Bis-Tris or 7% Tris-acetate gel (Invitrogen) and transferred to a nitrocellulose membrane. Alternatively, the protein was separated on SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was then probed with antibodies against various primary antibodies. The membrane-bound primary antibodies were visualized with appropriate secondary antibodies conjugated to horseradish peroxidase and a chemiluminescent substrate (Pierce). The immunoreactive signals from the chemiluminescent substrate were visualized by exposing them to standard x-ray films. The images were subjected to densitometric analysis with the use of Image J software verison 1.38 (NIH, Bethesda, MD). Total RNA was extracted from skin tissue or cultured HaCaT cells with the use of TRIzol reagent (Invitrogen). Reverse transcription was performed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative RT-PCR (qRT-PCR) was performed with the Taqman gene expression assay system (Applied Biosystems). The probes used were Mm00433023_m1 (mouse EGFR), Mm01247357_m1 (mouse E-cadherin), Mm00500912_m1 (mouse occludin), Mm00490758_m1 (mouse SIRT1), Mm01219285_m1 (mouse loricrin), and Mm999999_g1 (mouse GAPDH). Probes for GAPDH were used as endogenous control. qRT-PCR was performed with an ABI 7500 Real-Time PCR System. Gene expression values were calculated on the basis of the comparative threshold cycle method, normalized to the expression values of GAPDH, and displayed as fold induction relative to control. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance, followed by the Student's t-test, Welch's t-test, or Mann–Whitney's U-test. P values of <0.05 were considered to denote statistical significance. Apparent abnormal histologic changes were not observed in the dorsal skin of 8-week-old Flgft mice (Figure 1A, upper panels). However, the formation of N-terminal peptide (32 kDa) and filaggrin monomer (28 kDa) was barely detectable by Western blot analysis in the dorsal skin of Flgft mice compared with that in the B6 mice (Figure 1B) Immunohistologic analysis found the relative absence of filaggrin in the epidermis of Flgft mice compared with that of the B6 mice (Figure 1A, middle panels). Unexpectedly, loricrin protein expression was barely detectable by Western blot analysis in the dorsal skin of Flgft mice compared with that of B6 mice (Figure 1, C and D). Immunohistologic analysis found the complete absence of loricrin from the epidermis of Flgft mice compared with that of B6 mice (Figure 1A, bottom panels). We have recently reported that the expression of a gain-of-function mutant loricrin caused activation of EGFR in a cellular model of loricrin keratoderma.19Yoneda K. Demitsu T. Nakai K. Moriue T. Ogawa W. Igarashi J. Kosaka H. Kubota Y. Activation of vascular endothelial growth factor receptor 2 in a cellular model of loricrin keratoderma.J Biol Chem. 2010; 285: 16184-16194Crossref PubMed Scopus (15) Google Scholar Conversely, we hypothesized that a decrease in the expression of loricrin protein would affect the expression of EGFR. As shown in Figure 2B, we found that the expression of EGFR protein was suppressed in the dorsal skin of Flgft mice compared with that of age-matched B6 mice. Immunohistologic analysis found the relative absence of EGFR from the epidermis of Flgft mice compared with that of age-matched B6 mice (Figure 2A, upper panels). E-cadherin, a cell adhesion molecule, has been reported to associate/cooperate with EGFR to modulate the proliferation and differentiation of keratinocytes.13Muller E.J. Williamson L. Kolly C. Suter M.M. Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?.J Invest Dermatol. 2008; 128: 501-516Crossref PubMed Scopus (133) Google Scholar Western blot analysis found that the expression of E-cadherin protein was suppressed in the dorsal skin of Flgft mice compared with that of age-matched B6 mice (Figure 2B). Immunohistologic analysis found the relative absence of E-cadherin from the epidermis of Flgft mice compared with that of age-matched B6 mice (Figure 2A). As for the expression of the tight junction molecule occludin, its protein level was suppressed in the dorsal skin of Flgft mice compared with the age-matched B6 mice, as shown in Figure 2B. SIRT1 protein expression was also suppressed in the skin of Flgft mice (Figure 2B). As shown in Figure 2C, mRNA expression of EGFR, E-cadherin, occludin, SIRT1, and loricrin was suppressed in the skin of Flgft mice. Reduced SIRT1 expression indicated deranged inflammation and/or oxidative stress.14Caito S. Rajendrasozhan S. Cook S. Chung S. Yao H. Friedman A.E. Brookes P.S. Rahman I. SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress.FASEB J. 2010; 24: 3145-3159Crossref PubMed Scopus (241) Google Scholar To ameliorate the inflammatory/oxidative condition in the skin, we administrated NAC water, an antioxidant solution, to Flgft mice. Notably, NAC supplementation for 8 weeks partially restored EGFR, E-cadherin, occludin, and SIRT1 protein expression in Flgft mice (Figure 2B). However, NAC did not restore loricrin protein expression (Figure 2B) and occludin, SIRT1, and loricrin mRNA expression (Figure 2C). To exclude the issue of variable genetic background of Flgft mice, we examined the skin of Flgft mice that were backcrossed to B6 mice. The formation of N-terminal peptide (32 kDa) was reduced, and the formation of filaggrin monomer (28 kDa) was barely detectable in the dorsal skin of 8-week-old backcrossed Flgft mice compared with that in the B6 mice (Figure 3A). The high expression levels of loricrin were detected in the skin of neonatal backcrossed Flgft mice, but the expression levels were markedly reduced in 8-week-old backcrossed Flgft mice compared with B6 mice (Figure 3B). As shown in Figure 3C, protein expression of EGFR, E-cadherin, occludin, SIRT1, and loricrin was suppressed in the skin of 8-week-old backcrossed Flgft mice compared with that in the B6 mice. In addition, mRNA expression of EGFR, E-cadherin, occludin, SIRT1, and loricrin was suppressed in the skin of 8-week-old backcrossed Flgft mice compared with that in the B6 mice (Figure 3D). These results are consistent with those of non-backcrossed Flgft mice. To examine the effects of NAC on immune status, we measured TNFα, IL-6, IL-1β, and IL-17 protein levels in the serum and the skin of Flgft mice. The serum protein levels of TNFα, IL-6, and IL-1β were below the detection limits of ELISA in both Flgft and B6 mice. The serum IL-17 protein levels and the skin tissue TNFα/IL-1β protein levels were significantly increased in Flgft mice compared with B6 mice. However, NAC did not alter these proinflammatory cytokine levels in the serum and the skin of the Flgft mice (Figure 4, A and B). VEGF interacts with EGFR and other related molecules in human epidermal keratinocytes.19Yoneda K. Demitsu T. Nakai K. Moriue T. Ogawa W. Igarashi J. Kosaka H. Kubota Y. Activation of vascular endothelial growth factor receptor 2 in a cellular model of loricrin keratoderma.J Biol Chem. 2010; 285: 16184-16194Crossref PubMed Scopus (15) Google Scholar NAC supplementation significantly increased the VEGF concentration in the skin of Flgft mice (Figure 4C). Generally, in vivo skin is heterogeneous, and Flgft mouse is a mixed strain of filaggrin mutation plus matted mutation. We therefore performed in vitro experiments by using a human epidermal keratinocyte cell line (HaCaT cells). We cultured HaCaT cells at the air–liquid interface, where HaCaT cells can differentiate and develop a multilayered epithelium. We knocked down filaggrin or loricrin expression in HaCaT cells with siRNA. As shown in Figure 4, A and B, filaggrin siRNA inhibited the expression of loricrin as well as filaggrin in HaCaT cells grown at the air–liquid interface. However, loricrin siRNA blocked loricrin protein expression without affecting filaggrin protein expression in HaCaT cells grown at the air–liquid interface (Figure 5, A and B). Both filaggrin siRNA and loricrin siRNA reduced the protein expression levels of EGFR, E-cadherin, occludin, and SIRT1 in HaCaT cells grown at the air–liquid interface (Figure 5, C and D). These results indicate that the reduced protein expression of EGFR, E-cadherin, SIRT1, and occludin was due to loricrin deficiency, which was inducible by filaggrin deficiency. Because SIRT1 is known to inhibit proliferation and to promote differentiation,15Blander G. Bhimavarapu A. Mammone T. Maes D. Elliston K. Reich C. Matsui M.S. Guarente L. Loureiro J.J. SIRT1 promotes differentiation of normal human keratinocytes.J Invest Dermatol. 2009; 129: 41-49Crossref PubMed Scopus (101) Google Scholar we hypothesized that SIRT1 is involved in the adherens and EGFR expression of epidermal keratinocytes. To verify our hypothesis, we investigated whether sirtinol, a SIRT1 inhibitor, altered the expression of EGFR and E-cadherin in HaCaT cells. The EGFR inhibitor AG1478 (10 μmol/L) suppressed the protein expression levels of EGFR and E-cadherin in monolayer cultured HaCaT cells (Figure 6, A and B), suggesting that AG1478 suppressed E-cadherin expression by inhibiting EGFR activity and expression. Similarly, sirtinol (100 μmol/L) suppressed the protein expression levels of EGFR and E-cadherin in monolayer cultured HaCaT cells. We further examined the effects of sirtinol on the cellular proliferation of monolayer cultured HaCaT cells. As shown in Figure 6C, HaCaT cells that were treated with AG1478 (10 μmol/L) for 48 hours displayed reduced proliferation. Similar effects were observed in the HaCaT cells treated with sirtinol (100 μmol/L), suggesting that SIRT1 is involved in the proliferation of HaCaT cells. Although nitric oxide might interfere with EGFR or E-cadherin expression, L-NAME (1 mmol/L), a nitric oxide synthase inhibitor, did not affect the expression levels of these proteins (Figure 6, A and B) or proliferation (Figure 6C) in HaCaT cells. We next investigated the effects of sirtinol on differentiating HaCaT cells by culturing the cells at the air–liquid interface. Sirtinol (100 μmol/L) suppressed occludin expression in HaCaT cells grown at the air–liquid interface (Figure 7, A and B).Sirtinol (100 μmol/L) also suppressed EGFR and E-cadherin expression in HaCaT cells grown at the air–liquid interface (Figure 7, A and B). These results suggest that SIRT1 affects occludin expression in differentiating epidermal keratinocytes. It is generally accepted that atopic dermatitis is caused partially by the induction of skin barrier disruption due to decreased filaggrin expression. To examine the mechanism by which filaggrin deficiency compromises skin barrier, we investigated the expression of cellular junction-related proteins in Flgft mice, a model of atopic dermatitis with skin barrier dysfunction. As a result, we observed decreased EGFR, E-cadherin, occludin, and SIRT1 expression in Flgft mice. NAC supplementation partially restored the expression of these proteins in Flgft mice. To the best of our knowledge, we found for the first time that loricrin expression was suppressed in Flgft mice. In vitro experiments found that filaggrin siRNA, loricrin siRNA, and the SIRT1 inhibitor sirtinol suppressed the expression of EGFR, E-cadherin, and occludin in HaCaT cells. Filaggrin siRNA inhibited the expression of loricrin as well as filaggrin in HaCaT cells grown at the air–liquid interface, although loricrin siRNA did not block filaggrin protein expression. Flgft mice arose spontaneously in 1958 at The Jackson Laboratory and were first reported in 1972.20Lane P.W. Two new mutations in linkage group XVI of the house mouse Flaky tail and varitint-waddler-J.J Hered. 1972; 63: 135-140Crossref PubMed Scopus (50) Google Scholar A filaggrin deficiency in their epidermis was found in 2000.21Presland R.B. Boggess D. Lewis S.P. Hull C. Fleckman P. Sundberg J.P. Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.J Invest Dermatol. 2000; 115: 1072-1081Crossref PubMed Scopus (154) Google Scholar Recent reports have suggested that Flgft mice display different phenotypes. Some Flgft mice show spontaneous dermatitis in the absence of treatments such as allergen administration,4Moniaga C.S. Egawa G. Kawasaki H. Hara-Chikuma M. Honda T. Tanizaki H. Nakajima S. Otsuka A. Matsuoka H. Kubo A. Sakabe J. Tokura Y. Miyachi Y. Amagai M. Kabashima K. 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 (98) Google Scholar whereas others have no dermatitis.2Fallon P.G. Sasaki T. Sandilands A. Campbell L.E. Saunders S.P. Mangan N.E. Callanan J.J. Kawasaki H. Shiohama A. Kubo A. Sundberg J.P. Presland R.B. Fleckman P. Shimizu N. Kudoh J. Irvine A.D. Amagai M. McLean W.H. A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming.Nat Genet. 2009; 41: 602-608Crossref PubMed Scopus (380) Google Scholar, 3Scharschmidt T.C. Man M.Q. Hatano Y. Crumrine D. Gunathilake R. Sundberg J.P. Silva K.A. Mauro T.M. Hupe M. Cho S. Wu Y. Celli A. Schmuth M. Feingold K.R. Elias P.M. Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.J Allergy Clin Immunol. 2009; 124 (506 e491-496): 496-506Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar We did not find any abnormal histologic changes in the dorsal skin of 8-week-old Flgft mice. Moniaga et al4Moniaga C.S. Egawa G. Kawasaki H. Hara-Chikuma M. Honda T. Tanizaki H. Nakajima S. Otsuka A. Matsuoka H. Kubo A. Sakabe J. Tokura Y. Miyachi Y. Amagai M. Kabashima K. 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 (98) Google Scholar suggested that these discrepancies depend on the presence of the matted mutation or variations in the genetic backgrounds of the different strains used as well as on environmental factors. Previously, Presland et al21Presland R.B. Boggess D. Lewis S.P. Hull C. Fleckman P. Sundberg J.P. Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.J Invest Dermatol. 2000; 115: 1072-1081Crossref PubMed Scopus (154) Google Scholar reported that loricrin expression was slightly increased in the skin of newborn Flgft mice compared with that of B6 mice. In the present study, we found that the expression levels of loricrin were decreased in the dorsal skin of 8-week-old Flgft mice. Although no reports have suggested that loricrin expression is decreased in patients with ichthyosis vulgaris, it has been reported that loricrin expression was decreased in both the lesional and nonlesional skin of patients with atopic dermatitis.16Kim B.E. Leung D.Y. Boguniewicz M. Howell M.D. Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.Clin Immunol. 2008; 126: 332-337Crossref PubMed Scopus (385) Google Scholar Therefore, the down-regulation of loricrin expression could be a key step in atopic dermatitis deterioration due to filaggrin deficiency. Because previous reports have found that TNFα or type 2 helper T-cell cytokines suppressed loricrin mRNA expression,16Kim B.E. Leung D.Y. Boguniewicz M. Howell M.D. Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.Clin Immunol. 2008; 126: 332-337Crossref PubMed Scopus (385) Google Scholar, 22Kim B.E. Howell M.D. Guttman E. Gilleaudeau P.M. Cardinale I.R. Boguniewicz M. Krueger J.G. Leung D.Y. TNF-alpha downregulates filaggrin and loricrin through c-Jun N-terminal kinase: role for TNF-alpha antagonists to improve skin barrier.J Invest Dermatol. 2011; 131: 1272-1279Crossref PubMed Scopus (152) Google Scholar loricrin might be reduced by these factors in Flgft mice. Tight junctions exist just below the stratum corneum and regulate the paracellular permeability of the epidermis. De Benedetto et al11De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. Berger A.E. Zhang K. Vidyasagar S. Yoshida T. Boguniewicz M. Hata T. Schneider L.C. Hanifin J.M. Gallo R.L. Novak N. Weidinger S. Beaty T.H. Leung D.Y. Barnes K.C. Beck L.A. Tight junction defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (773-786e771-777)PubMed Google Scholar reported that the expression of the tight junction proteins claudin-1 and claudin-23 is reduced in patients with atopic dermatitis. Gruber et al23Gruber R. Elias P.M. Crumrine D. Lin T.K. Brandner J.M. Hachem J.P. Presland R.B. Fleckman P. Janecke A.R. Sandilands A. McLean W.H. Fritsch P.O. Mildner M. Tschachler E. Schmuth M. Filaggrin genotype in ichthyosis vulgaris predicts abnormalities in epidermal structure and function.Am J Pathol. 2011; 178: 2252-2263Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar reported the reduced expression of the tight junction proteins occludin and ZO-1 in filaggrin-deficient human skin. We have found here that the expression levels of the tight junction protein occludin and the adherens junction protein E-cadherin were suppressed in the skin of Flgft mice. Because molecular cross talk between E-cadherin and EGFR has been reported in human NCI-H292 airway epithelial cells24Kim S. Schein A.J. Nadel J.A. E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1049-L1060Crossref PubMed Scopus (41) Google Scholar and HaCaT cells,25Pece S. Gutkind J.S. Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation.J Biol Chem. 2000; 275: 41227-41233Crossref PubMed Scopus (280) Google Scholar we examined EGFR expression and found that it was decreased in the skin of Flgft mice. We further found that the SIRT1 expression was decreased in the skin of Flgft mice. NAC partially restored EGFR, E-cadherin, occludin, and SIRT1 protein expression in the skin of Flgft mice. NAC also restored EGFR and E-cadherin mRNA expression in the skin of Flgft mice. The biological effects of NAC on the skin might include antioxidant and anti-inflammatory effects as well as the modulation of redox signaling pathways.26Parasassi T. Brunelli R. Costa G. De Spirito M. Krasnowska E. Lundeberg T. Pittaluga E. Ursini F. Thiol redox transitions in cell signaling: a lesson from N-acetylcysteine.ScientificWorldJournal. 2010; 10: 1192-1202Crossref PubMed Scopus (73) Google Scholar Although NAC has been reported to modify immunoreactions,27Bengtsson A. Lundberg M. Avila-Carino J. Jacobsson G. Holmgren A. Scheynius A. Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells.Clin Exp Immunol. 2001; 123: 350-360Crossref PubMed Scopus (36) Google Scholar in our study, TNFα, IL-6, IL-1β, and IL-17 concentrations were not altered by NAC in the skin and the serum of Flgft mice. Considering that NAC restored SIRT1 expression in the skin of Flgft mice, NAC might increase VEGF, EGFR, E-cadherin, occludin, and SIRT1 by reducing the levels of oxidative stress in the skin of Flgft mice. Other possible effects of NAC have been suggested in clinical reports. The favorable therapeutic effects of topical NAC against lamellar ichthyosis suggest that NAC directly improves filaggrin-related skin barrier dysfunction28Redondo P. Bauza A. Topical N-acetylcysteine for lamellar ichthyosis.Lancet. 1999; 354: 1880Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar because patients with lamellar ichthyosis display abnormal keratinization due to mutations in their transglutaminase 1 gene and a differential pattern of filaggrin expression. We could not conclude that the reduced protein expressions observed in Flgft mice were completely due to filaggrin deficiency, because Flgft mice are a mixed strain of filaggrin mutation plus matted mutation. We therefore performed in vitro siRNA experiments to gain insights into the molecular mechanisms responsible for the suppressed expression of EGFR, E-cadherin, SIRT1, and occludin in epidermis. Monolayer cultured HaCaT cells represent subbasal spinous keratinocytes, but they can develop a multilayered epithelium such as subcorneal keratinocytes when cultured at the air–liquid interface. Our results suggest that loricrin, which needs filaggrin for its expression, is required for the EGFR, SIRT1, E-cadherin, and occludin expressions in human skin epidermis. Our results also suggest that SIRT1 is required for the expressions of EGFR, E-cadherin, and occludin in human skin epidermis. In summary, we have shown that loricrin, EGFR, E-cadherin, occludin, and SIRT1 expression were down-regulated in Flgft mice. We also performed in vitro experiments, which provided a mechanism for the skin barrier dysfunction observed in atopic dermatitis. NAC supplementation reversed the observed reductions in the expression levels of the above-mentioned proteins in Flgft mice, indicating that NAC could be considered as a therapeutic agent to restore the impaired barrier function of filaggrin-deficient skin. We thank Fumiko Nishiyama and Miho Kato for their technical help.