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Periostin Controls Keratinocyte Proliferation and Differentiation by Interacting with the Paracrine IL-1α/IL-6 Loop  Kazuto Taniguchi, Kazuhiko Arima,

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Presentation on theme: "Periostin Controls Keratinocyte Proliferation and Differentiation by Interacting with the Paracrine IL-1α/IL-6 Loop  Kazuto Taniguchi, Kazuhiko Arima,"— Presentation transcript:

1 Periostin Controls Keratinocyte Proliferation and Differentiation by Interacting with the Paracrine IL-1α/IL-6 Loop  Kazuto Taniguchi, Kazuhiko Arima, Miho Masuoka, Shoichiro Ohta, Hiroshi Shiraishi, Kanako Ontsuka, Shoichi Suzuki, Masako Inamitsu, Ken-ichi Yamamoto, Olga Simmons, Shuji Toda, Simon J. Conway, Yuhei Hamasaki, Kenji Izuhara  Journal of Investigative Dermatology  Volume 134, Issue 5, Pages (May 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Identification of soluble factors in air–liquid interface (ALI) cocultures of keratinocytes with wild-type (WT) or Postn−/− fibroblasts. (a) A scheme of the three-dimensional (3D) organotypic ALI coculture system. (b) Histology (hematoxylin and eosin (H&E)) and immunohistochemistry (proliferating cell nuclear antigen (PCNA), cytokeratin-14 (CK14), and cytokeratin-10 (CK10)) of the ALI cocultures. (c) Numbers of PCNA-positive or -negative cells in basal areas in (b). Data are shown as mean±SD in five views of 200μm length. (d) Histology (H&E) of the ALI cocultures with indicated fibroblasts. POSTN was retrovirally transduced. (b, d) Bars=20μm. (e) Results of antibody array analyses. AU, arbitrary units. (f) ELISA analyses of the indicated cytokines in the supernatants of the ALI cocultures with WT and Postn−/− fibroblasts. KGF, keratinocyte growth factor; Postn, periostin. *P<0.05; **P<0.01; and NS, not significant. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Functional validation of IL-6 as a major keratinocyte proliferation and differentiation factor. (a, b) Histology (hematoxylin and eosin (H&E)) and immunohistochemistry with anti-CK10 and anti-CK14 antibodies of the air–liquid interface (ALI) cocultures. CK, cytokeratin; Postn, periostin. (a) ALI cocultures of keratinocytes and wild-type (WT) fibroblasts in the presence of neutralizing antibodies against either IL-6, GM-CSF, or G-CSF, or control IgG. (b) ALI cocultures of keratinocytes with Postn−/− fibroblasts in the presence of recombinant IL-6 (rIL-6) at the indicated concentration. (a, b) Bars=20μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Determining the cellular source of IL-6 in the air–liquid interface (ALI) cocultures. (a) A schematic diagram of the experimental system. Conditioned medium (CM) was collected from the ALI coculture of keratinocytes and wild-type (WT) fibroblasts at days 3, 5, and 7 (D3, D5, and D7). Keratinocytes or fibroblasts were cultured in a 1:2 mixture of CM and fresh medium (Med) for 24hours and subjected to quantitative PCR analysis. As control, fresh medium or 10ngml−1 tumor necrosis factor (TNF) was also used to culture the cells. (b) Relative mRNA abundance of IL-6, as well as other keratinocyte growth–related factors, determined by quantitative PCR. AU, arbitrary units; 3D, three dimensional; MIP-2, macrophage inflammatory protein-2. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Identifying major IL-6-inducing factors in the air–liquid interface (ALI) coculture system. (a, b) Production of (a) IL-6 and (b) IL-1α in the presence of indicated neutralizing antibodies in the ALI coculture of keratinocytes with either wild-type (WT) or Postn−/− fibroblasts at day 5 determined by ELISA. *P<0.05 versus control IgG–treated coculture with WT fibroblasts. Postn, periostin; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin. (c) Histology (hematoxylin and eosin (H&E)) of the keratinocytes cocultured either with WT fibroblasts in the absence (phosphate-buffered saline (PBS)) or presence of anti-IL-1α antibodies or control IgG1 or with Postn−/− fibroblasts in the presence of control IgG1. Bars=20μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Cooperation of periostin (Postn) and IL-1α on IL-6 production in fibroblasts. (a) IL-6 secretion and relative mRNA abundance of indicated (b) cytokines/chemokines in wild-type (WT) and Postn−/− fibroblasts. (c, d) IL-6 production in periostin-overexpressed NIH3T3 cells. (c) Periostin expression (left, ELISA and right, quantitative PCR) and (d) IL-6 production. (e) Effect of fibroblast-derived periostin on IL-6 production. Newly prepared WT (above) or Postn−/− (below) fibroblasts were cultured in a 1:1 mixture of fresh medium and conditioned medium (CM) from either WT or Postn−/− fibroblast culture. The cells were stimulated with IL-1α for 24hours (a, d, e) or 20hours (b). (a–e) *P<0.05; **P<0.01; and ***P< (f) Western blotting in IL-1α-stimulated WT and Postn−/− fibroblasts. ERKI/2, extracellular signal–regulated kinase-1/2; KGF, keratinocyte growth factor; MCP-1, monocyte chemoattractant protein-1; MIP-2, macrophage inflammatory protein-2; RANTES, regulated on activation, normal T cell expressed and secreted; STAT3, signal transducer and activator of transcription 3. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 The autocrine loop of periostin (Postn) controls the paracrine loop composed of IL-1α and IL-6 in the epithelial–mesenchymal interaction. A schematic model of the epithelial–mesenchymal interaction shown in this study. The paracrine loop composed of IL-1α and IL-6 derived from keratinocytes and fibroblasts, respectively, in the epithelial–mesenchymal interactions regulates proliferation and differentiation of keratinocytes that crucially depend on an autocrine loop of periostin within fibroblasts. Periostin cooperates with IL-1α on induction of robust IL-6 production in fibroblasts by synergistically activating the NF-κB pathway. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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