Yuangang Liu, James P. Lagowski, Shangpu Gao, James H

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Regulation of the Psoriatic Chemokine CCL20 by E3 Ligases Trim32 and Piasy in Keratinocytes Yuangang Liu, James P. Lagowski, Shangpu Gao, James H. Raymond, Clifton R. White, Molly F. Kulesz-Martin Journal of Investigative Dermatology Volume 130, Issue 5, Pages 1384-1390 (May 2010) DOI: 10.1038/jid.2009.416 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Elevated Trim32 expression in human psoriasis. (a) Immunohistochemical analysis of Trim32 expression in psoriasis specimens. Trim32 was visualized in paraffin sections using 0.2μgml−1 chicken anti-Trim32 antibody and stained by the ABC technique. Trim32 level was elevated in the epidermis of psoriatic lesions relative to that in uninvolved skin ( × 200 magnification). (b) Indirect immunofluorescence analysis of Trim32 expression in psoriasis specimens. Trim32 was visualized with chicken anti-Trim32 antibody followed by Texas red secondary antibody ( × 600 magnification). The nucleus was stained with Hoechst 33342. The micrographs are representative of 20 Trim32-positive specimens out of 33 psoriatic skin specimens. Bar=50μm. Journal of Investigative Dermatology 2010 130, 1384-1390DOI: (10.1038/jid.2009.416) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Regulation of CCL20 expression in keratinocytes by Trim32 and Piasy. (a) CCL20 mRNA expression in the mouse keratinocyte line 291 infected with adenovirus expressing GFP, Trim32, or Piasy. (b) Analysis of CCL20 mRNA in primary mouse keratinocytes isolated from Trim32-null mice (Trim32−/-) and wild-type littermates (Wt). CCL20 mRNA was normalized to GAPDH. The data are representative of three independent experiments for Figure 1a and two independent experiments for Figure 1b (**P< 0.01; ***P< 0.001). Journal of Investigative Dermatology 2010 130, 1384-1390DOI: (10.1038/jid.2009.416) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Trim32 and CCL20 expression in human psoriasis. Paraffin-embedded psoriatic lesional tissues from 33 patients were sectioned serially and stained with chicken anti-Trim32 antibody and goat anti-CCL20 antibody ( × 100 magnification). The results are summarized in the table. Correlation of Trim32 overexpression with CCL20 expression was statistically significant (*two-tailed Fisher's exact test, P-value=0.00015). Bar=50μm. Journal of Investigative Dermatology 2010 130, 1384-1390DOI: (10.1038/jid.2009.416) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Regulation of Th17 induced CCL20 by Trim32 and Piasy. (a) CCL20 mRNA was measured by quantitative real-time PCR in mouse 291 keratinocytes treated with the indicated cytokines and LPS. (b) CCL20 mRNA was measured by quantitative real-time PCR in mouse 291 keratinocytes infected overnight with adenovirus expressing GFP, Trim32, or Piasy, then treated with indicated cytokines for 4hours. CCL20 mRNA was normalized to GAPDH. (c) CCL20 protein was measured by ELISA in mouse 291 keratinocytes infected overnight with adenovirus expressing GFP, Trim32, or Piasy, then treated with indicated cytokines for 6hours. (d) The human keratinocyte line HEKnV was infected with adenovirus expressing GFP, Trim32, or Piasy, and then treated with the indicated cytokines for 6hours. CCL20 protein was measured from the culture supernatant by ELISA. Data are representative of three independent experiments. Statistical analysis was conducted by one-way analysis of variance followed by Bonferroni post-test (**P< 0.01; ***P< 0.001). Journal of Investigative Dermatology 2010 130, 1384-1390DOI: (10.1038/jid.2009.416) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Piasy regulates CCL20 through p65/RelA subunit of NF-κB. (a) CCL20 mRNA was measured in 291 keratinocytes transfected with plasmids expressing GFP, p65, Trim32+p65, or Piasy+p65. Data are representative of two independent experiments. Statistical analysis of Trim32 and Piasy effect was conducted relative to p65 alone control (*P<0.05). (b) Piasy interacts with endogenous p65 in keratinocytes. Flag-tagged Piasy and Flag-tagged Erk (negative control) were transfected into 291 keratinocytes. Bound p65 was immunoprecipitated with an anti-Flag antibody and visualized by immunoblotting with an anti-p65 antibody. Journal of Investigative Dermatology 2010 130, 1384-1390DOI: (10.1038/jid.2009.416) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions