CCN1, a Pro-Inflammatory Factor, Aggravates Psoriasis Skin Lesions by Promoting Keratinocyte Activation  Yue Sun, Jie Zhang, Zhou Zhou, Pinru Wu, Rongfen Huo, Beiqing Wang, Zhengyu Shen, Huidan Li, Tianhang Zhai, Baihua Shen, Xiangdong Chen, Ningli Li  Journal of Investigative Dermatology  Volume 135, Issue 11, Pages 2666-2675 (November 2015) DOI: 10.1038/jid.2015.231 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CCN1 expression is increased in the skin samples from psoriasis patients. (a) CCN1 expression was evaluated with real-time PCR (left panel) and western blotting analysis (right panel) of the skin biopsies isolated from psoriasis patients and healthy donors. Each point represents a skin sample obtained from a healthy donor (○), non-lesional skin (●), or corresponding lesional skin of one psoriasis patient (▲). (b) CCN1 expression in separated epidermis and dermis, respectively, detected in non-lesional and lesional skin samples from psoriasis patients treated with dispase (50 U ml-1) at 4 °C overnight. (c) Representative immunohistochemical staining of CCN1 expression in the normal skin, non-lesional skin, and lesional psoriatic plaque skin. Bar=100 μm. Values represent the means±SD.*P<0.05, **P<0.01. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CCN1 expression knocked down by lentiviral vector ameliorates psoriasis symptoms in imiquimod (IMQ)-induced psoriasis-like mice. (a) CCN1 expression in the back skins of control and IMQ-treated mice detected by real-time PCR and western blotting. Each point represents a skin sample obtained from control (○) or IMQ-treated (mice. (b) Representative immunofluorescent staining of CCN1 in the back skins of control and IMQ-treated mice. Bar=20 μm. (c) Representative histological sections of the skins infected with control viruses and CCN1-targeting viruses examined by hematoxylin and eosin staining. Bar=100 μm. (d) Epidermal thickness and (e) infiltrated cells in the dermis were measured from the back skins of mice. (f) Expression of tumor necrosis factor (TNF)-α and IL-17 in the back skins examined by real-time PCR. For d–f, each point represents a skin sample obtained from pLV-iCon virus-treated (○) or pLV-iCCN1 virus-treated ( mice. *P<0.05, **P<0.01, ***P<0.001. The data represent one of three independent experiments. DAPI, 4,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CCN1 expression knocked down by lentiviral vector relieves psoriasis symptoms in IL-23-treated mice. (a) CCN1 expression in ear skins of control (○) and IL-23-challenged (mice detected by real-time PCR. (b) Representative immunohistochemical staining of CCN1 expression in the ear skins of control and IL-23-challenged mice. Bar=50 μm. (c) CCN1 expression in virus-treated ear skins detected by real-time PCR. (d) Ear thickness of virus-treated mice. (e) Representative histological sections of ear skins examined by hematoxylin and eosin staining. Bar=100 μm. (f) Epidermal thickness and (g) infiltrated cells in the dermis were measured in the ear skins of virus-treated mice. (h) Expression of tumor necrosis factor (TNF)-α and IL-17 in ear skins examined by real-time PCR. For c, d, f–h, each point represents a skin sample obtained from pLV-iCon virus-treated (○) or pLV-iCCN1 virus-treated (mice. *P<0.05, **P<0.01. The data represent one of three independent experiments. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Blocking CCN1 alleviates epidermal hyperplasia in imiquimod (IMQ)-induced mice. IMQ-challenged mice were treated with anti-CCN1 antibody (093G9) or control IgG. (a) CCN1 expression in the back skins of mice treated with 093G9 or IgG evaluated by real-time PCR and western blotting analysis. (b) Histological sections of mice treated with 093G9 or IgG. Bar=100 μm. (c) Epidermal thickness and (d) infiltrated cells in the dermis were measured from the back skin of 093G9- or IgG-treated mice. (e) Tumor necrosis factor (TNF)-α and IL-17 expression was detected by real-time PCR. For a, c–e, each point represents a skin sample obtained from IgG- (○) or 093G9-treated ( mice, respectively. **P<0.01, ***P<0.001. The data represent one of three independent experiments. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 CCN1 provokes keratinocyte activation. To identify the activation of keratinocytes promoted by CCN1, keratinocyte activation–associated hallmarks were assessed in primary cultured keratinocytes. (a) The proliferation of keratinocytes stimulated with different doses of CCN1 (0, 1.25, 2.5, 5, 10 μg ml-1) was evaluated with a CCK8 (Cell Counting Kit 8) assay. (b) Keratinocyte proliferation was assessed for Ki67 staining using flow cytometric analysis. (c) The division of carboxyfluorescein succinimidyl ester (CFSE)-labeled keratinocytes determined by flow cytometry. (d) The frequency and mean fluorescence intensity (MFI) of ICAM+, HLA-ABC+, and HLA-DR+ keratinocytes estimated with flow cytometry. (e) The expression of ICAM, HLA-ABC, and HLA-DR in keratinocytes detected by real-time PCR. For b–e, 5 μg ml-1 of CCN1 (black bar) or BSA (open bar) was used to stimulate keratinocytes. The values are presented as the means±SD. *P<0.05, **P<0.01. The data represent one of three independent experiments. CCL20, C-C chemokine ligand 20. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The α6β1/PI3K/Akt/NF-κB pathway is activated by CCN1 in keratinocytes. (a) The expression of integrin α and β genes in primary cultured keratinocytes stimulated with CCN1 (black bar) or BSA (open bar) was detected by real-time PCR. (b) The expression of integrin α and β genes in skin samples derived from healthy donors (open bar) and psoriasis patients (black bar) was detected by real-time PCR. (c) ICAM mRNA in CCN1-stimulated HaCaT cells treated with specific α6β1 small interfering RNA (siRNA). (d) Phosphorylation of PI3K (phosphoinositide-3 kinase), Akt, and NF-κB p65 in CCN1-stimulated HaCaT cells detected by western blotting analysis. (e) Nuclear translocation of NF-κB in CCN1-stimulated HaCaT cells monitored by immunofluorescence. NF-κB was detected by FITC-anti-p65 (green). Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole; blue). This merged picture shows NF-κB translocation into the nucleus. Bar=20 μm. CCN1 of 5 μg ml-1 was used to stimulate keratinocytes. *P<0.05. The data represent one of three independent experiments. Journal of Investigative Dermatology 2015 135, 2666-2675DOI: (10.1038/jid.2015.231) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions