Integrin-β4–TNS4–Focal Adhesion Kinase Signaling Mediates Keratinocyte Proliferation in Human Skin  Eun Young Seo, Seon-Pil Jin, Yeon Kyung Kim, Hanon Lee, Sangbum Han, Dong Hun Lee, Jin Ho Chung  Journal of Investigative Dermatology  Volume 137, Issue 3, Pages 763-766 (March 2017) DOI: 10.1016/j.jid.2016.10.039 Copyright © 2016 The Authors Terms and Conditions

Figure 1 TNS4 promotes keratinocyte proliferation via FAK and ERK. (a) TNS4 (green) and K14 (red) immunofluorescence staining of normal human skin specimens. Scale bar = 50 μm. The leftmost image is the negative control with normal IgG as a primary antibody. (b) Keratinocytes (70% confluency) were treated with 1.2 mmol/L CaCl2 for the indicated times to induce differentiation and harvested for immunoblotting. (c) Efficacy of TNS4 siRNA. Keratinocytes were treated with TNS4 siRNA or control scrambled siRNA for 48 hours; the relative cell numbers were determined. (d) After TNS4 siRNA transfection, proliferative cells were detected by BrdU immunostaining. (e) Immunoblot analysis using the indicated antibodies after TNS4 siRNA transfection. (f) After infection with adenoviruses expressing GFP-TNS4 or GFP control for 24 hours, cells were treated with FAK-I14 (FAK inhibitor) or PD98059 (ERK inhibitor) for 24 hours, and relative cell numbers were assessed using a hemocytometer. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ctrl, control; FAK, focal adhesion kinase; FAK-I14, FAK inhibitor 14; GFP, green fluorescent protein; IB, immunoblotting; INV, involucrin; IP, immunoprecipitation; K, keratin-14; ns, nonsignificant; p-ERK, phosphorylated ERK; p-FAK, phosphorylated FAK; siCtrl, control scrambled siRNA; siRNA, small interfering RNA; siTNS4, TNS4 siRNA; t-ERK, total ERK; t-FAK, total FAK. Journal of Investigative Dermatology 2017 137, 763-766DOI: (10.1016/j.jid.2016.10.039) Copyright © 2016 The Authors Terms and Conditions

Figure 2 TNS4 interacts with integrin β4 (ITGB4) and FAK upon integrin activation in vitro and promotes epidermal proliferation in vivo. (a) Expression of ITGB4, TNS4, FAK, and ERK signaling proteins. Keratinocytes were treated with TNS4 siRNA or scrambled siRNA and placed on gelatin- or laminin-332–coated dishes with EGF. (b) Co-immunoprecipitation of ITGB4, FAK, and TNS4 from keratinocytes. (c) Eight-week-old C57BL/6 mice injected intradermally 3 times a week with 10 μl of GFP-TNS4 (n = 5)- or GFP control (n = 5)-expressing adenovirus (109 particles) were killed after 7 days, and skin samples were stained with hematoxylin and eosin, anti-GFP, anti-CD45 (pan-leukocyte marker), or anti-keratin 6 (marker for inflamed and hyperproliferative epidermis). Scale bars = 100 μm. Epidermal thickness was measured by using ImageJ software (National Institutes of Health, Bethesda, MD). Error bars denote standard error of the mean. (d) Immunohistochemical staining with anti-Ki67. Quantitative analysis of Ki-67 staining from four independent experiments. The number of Ki-67–positive cells was calculated by counting positive cells per 50 μm length of basement membrane. (e) Role of the ITGB4-TNS4-FAK pathway in keratinocyte proliferation. ∗P < 0.05. Ctrl, control; EGF, epidermal growth factor; ERK, extracellular-regulated kinase; FAK, focal adhesion kinase; G, gelatin; GFP, green fluorescent protein; H&E, hematoxylin and eosin; IB, immunoblotting; IP, immunoprecipitation; ITGB4, integrin β4; K6, keratin 6; Ki67, keratinocyte 67; Ln, laminin-332; p-ERK, phosphorylated ERK; p-FAK, phosphorylated FAK; siCtrl, control scrambled siRNA; siRNA, small interfering RNA; siTNS4, TNS4 siRNA; t-ERK, total ERK; t-FAK, total FAK. Journal of Investigative Dermatology 2017 137, 763-766DOI: (10.1016/j.jid.2016.10.039) Copyright © 2016 The Authors Terms and Conditions