Host Defense (Antimicrobial) Peptide, Human β-Defensin-3, Improves the Function of the Epithelial Tight-Junction Barrier in Human Keratinocytes  Chanisa Kiatsurayanon, François Niyonsaba, Rithee Smithrithee, Toshihiro Akiyama, Hiroko Ushio, Mutsuko Hara, Ko Okumura, Shigaku Ikeda, Hideoki Ogawa  Journal of Investigative Dermatology  Volume 134, Issue 8, Pages 2163-2173 (August 2014) DOI: 10.1038/jid.2014.143 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effects of human β-defensins (hBDs) on the expression of tight-junction (TJ) mRNAs and proteins in keratinocytes. (a) Keratinocytes were incubated with 5–20 μg ml-1 of hBDs or medium alone for 24–48 hours. Following the incubation, total RNA was extracted and reverse-transcribed into complementary DNA, and quantitative real-time reverse-transcriptase–PCR was performed. The values represent fold-increases in gene expression relative to those of controls stimulated with medium alone (Med). *P<0.05, **P<0.01, ***P<0.001, n=5. (b) Keratinocytes were stimulated with 2.5–20 μg ml-1 of hBD-3 or the diluent (Med) for 48 hours. The levels of claudin-1, claudin-3, claudin-4, claudin-14, and claudin-23 in cell lysates were determined by western blot. The results of one representative experiment of three separate experiments yielding similar results are shown. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Human β-defensin-3 (hBD-3) enhances immunolocalization of certain claudins. Keratinocytes grown to confluence on collagen I-coated chamber slides were stimulated with 20 μg ml-1 hBD-3 for 72 hours. The cells were then fixed in methanol, processed for immunofluorescence with antibodies against claudins overnight, and then visualized using confocal laser scanning microscopy. The results of one representative experiment of three separate experiments yielding similar results are shown. Scale bar=10 μm. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Human β-defensin-3 (hBD-3) enhances the tight-junction (TJ) barrier function. (a, b) Keratinocyte layers were stimulated with 2.5–20 μg ml-1 hBD-3, and the transepithelial electrical resistance (TER) and paracellular flux were determined as described in Materials and Methods. The values obtained using stimulated and nonstimulated cells were compared (Med). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=5. (c) Keratinocyte layers were pre-treated with 0.5 and 1 nM ochratoxin A (OTA) or 0.1% DMSO for 24 hours, stimulated with 20 μg ml-1 hBD-3 or diluent for 72 hours, and then the TER and the paracellular flux were measured. The values obtained using stimulated and nonstimulated cells (Med, OTA) or with the presence and absence of inhibitor were compared. **P<0.01, ***P<0.001, n=5. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Human β-defensin-3 (hBD-3) enhances the tight-junction (TJ) barrier function through CCR6. (a) Keratinocyte layers were pre-treated with 50 μg ml-1 anti-CCR6 antibody for 24 hours, stimulated with 20 μg ml-1 hBD-3 for 72 hours, and then the transepithelial electrical resistance (TER) and the paracellular flux were measured. *P<0.05, **P<0.01, n=5, compared with the nonstimulated cells, and in the presence and absence of CCR6 antibody. (b) Keratinocyte layers were transfected with 400 nM CCR6 small interfering RNA (siRNA) or control siRNA for 48 hours, stimulated with 20 μg ml-1 hBD-3 for 72 hours, and the TER and the paracellular flux were measured. *P<0.05, **P<0.01, n=5, compared with the nonstimulated cells, and between CCR6 siRNA-transfected cells and control siRNA-transfected cells. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Human β-defensin-3 (hBD-3) enhances the tight-junction (TJ) function through activation of atypical protein kinase C (aPKC)ζ/λ and guanosine triphosphate (GTP)-Rac1. Keratinocytes were incubated with 20 μg ml-1 hBD-3, and lysates were separated by SDS-PAGE using antibodies against phosphorylated aPKCζ/λ and aPKC (a), or quantified for GTP-Rac1 using a Rac1 activation assay kit (c). One representative experiment of three separate experiments yielding similar results is shown. Keratinocyte layers were pre-treated with GF 109203X (b), NSC23766 (d), or 0.1% DMSO for 48 hours and stimulated with 20 μg ml-1 hBD-3 for 72 hours, and the transepithelial electrical resistance (TER) and paracellular flux were determined. The values obtained using stimulated and nonstimulated cells or with the presence and absence of inhibitor were compared. *P<0.05, **P<0.01, ***P<0.001, n=4. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Human β-defensin-3 (hBD-3) enhances the tight-junction (TJ) function through glycogen synthase kinase-3 (GSK-3)α/β and phosphatidylinositol 3 kinase (PI3K) activation. Keratinocytes were incubated with 20 μg ml-1 hBD-3, and lysates were separated by SDS-PAGE using antibodies against phosphorylated GSK-3α/β (Y279/Y216) and GSK-3α/β (a), or phosphorylated PI3K (p85/p55) and PI3K (c). The results of one representative experiment of the three separate experiments yielding similar results are shown. Keratinocyte layers were pre-treated with SB 415286 (SB) (b), wortmannin (Wort) (d), or 0.1% DMSO for 48 hours and stimulated with 20 μg ml-1 hBD-3 for 72 hours, and the transepithelial electrical resistance (TER) and paracellular flux were determined. The values obtained using stimulated and nonstimulated cells or with the presence and absence of inhibitor were compared. *P<0.05, **P<0.01, ***P<0.001, n=5. Journal of Investigative Dermatology 2014 134, 2163-2173DOI: (10.1038/jid.2014.143) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions