Cornulin Is Induced in Psoriasis Lesions and Promotes Keratinocyte Proliferation via Phosphoinositide 3-Kinase/Akt Pathways  Changji Li, Lei Xiao, Jinjing Jia, Fan Li, Xin Wang, Qiqi Duan, Huiling Jing, Peiwen Yang, Caifeng Chen, Qiong Wang, Jiankang Liu, Yongping Shao, Nanping Wang, Yan Zheng  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 71-80 (January 2019) DOI: 10.1016/j.jid.2018.06.184 Copyright © 2018 The Authors Terms and Conditions

Figure 1 Upregulation of CRNN protein expression in patients with psoriasis and in IMQ-induced psoriasis-like skin inflammation in mice. Formalin-fixed paraffin-embedded tissue sections from psoriasis patients (Psoriasis) and healthy individuals (Normal) were examined by histopathologic assay. (a) Representative IHC staining for CRNN in serial sections of psoriatic lesional skin or healthy controls. Scale bar = 100 μm. (b) The statistical analysis of IHC staining for CRNN in psoriatic lesional skin (72 patients) or healthy controls (57 subjects). (c) Western blot analysis of CRNN protein level in psoriatic lesional skin or normal controls. ∗∗P < 0.01 versus Normal, which indicates a statistically significant difference. (d) Mice were treated with IMQ or vehicle for 7 days. Representative images of mice were shown. For each group, n = 10. (e) Formalin-fixed biopsies were paraffin-embedded and stained for CRNN in vehicle mice and lesional skin from mice was treated with IMQ. Scale bar = 100 μm. (f) CRNN mRNA levels in the epithelium of IMQ or vehicle-treated mice were assessed by qRT-PCR (∗∗P < 0.01 vs. vehicle, n = 6). (g) CRNN protein expression levels and quantification of CRNN levels in the epithelium of IMQ or vehicle-treated mice were assessed by Western blot (∗∗P < 0.01 vs. vehicle, n = 6). IHC, immunohistochemical; IMQ, imiquimod; qRT-PCR, real-time quantitative PCR. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Upregulation of CRNN protein expression in cell models of psoriasis. (a) HEKa cells were seeded in six-cell plates and stimulated with 0, 2.5, and 5 ng/ml M5 for 24 hours before measuring CRNN mRNA level by qRT-PCR (n = 6). (b) HEKa cells were seeded in six-cell plates and stimulated with M5 (2.5 ng/ml) for different time points (0, 3, 6, 12, 24, and 72 hours) before measuring CRNN mRNA level by qRT-PCR (n = 3). (c) HaCaT cells were seeded in six-cell plates and stimulated with 0, 2.5, and 5 ng/ml M5 for 24 hours before measuring CRNN mRNA level by qRT-PCR (n = 6). (d) HaCaT cells were seeded in six-cell plates and stimulated with M5 (2.5 ng/ml) for different time points (0, 3, 6, 12, 24, and 72 hours) before measuring CRNN mRNA level by qRT-PCR (n = 3). (e) HEKa cells were seeded in six-cell plates and stimulated with 0, 2.5, and 5 ng/ml M5 for 48 hours before measuring CRNN protein level by Western blot (n = 3). (f) HEKa cells were seeded in six-cell plates and stimulated with M5 (2.5 ng/ml) for different time points (0, 3, 6, 12, 24, and 72 hours) before measuring CRNN protein level and quantification of CRNN level by Western blot (n = 3). (g) HaCaT cells were seeded in six-cell plates and stimulated with 0, 2.5, and 5 ng/ml M5 for 48 hours before measuring CRNN protein level and quantification of CRNN level by Western blot (n = 3). (h) HaCaT cells were seeded in six-cell plates and stimulated with M5 (2.5 ng/ml) for different time points (0, 3, 6, 12, 24, and 72 hours) before measuring CRNN protein level and quantification of CRNN level by Western blot (n = 3). ∗P < 0.05, ∗∗P < 0.01 vs. Control, which indicates a statistically significant difference. M5, combination of IL-17A, IL-22, IL-1α, oncostatin M, and TNF-α; qRT-PCR, real-time quantitative PCR. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions

Figure 3 CRNN positively regulated keratinocyte proliferation. (a) HEKa (left) and HaCaT (right) cells were seeded in 96-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 24, 48, and 72 hours before measuring keratinocyte proliferation by MTT assay (n = 3). (b) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 48 hours before measuring cell cycle profiles by flow cytometry (n = 3). (c) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 48 hours before measuring CRNN and cyclin D1 protein levels by Western blot (n = 3). (d) HEKa (left) and HaCaT (right) cells were seeded in 96-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours before measuring keratinocyte proliferation by MTT assay (n = 3). HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours before measuring cell cycle profiles by flow cytometry (e) or CRNN and cyclin D1 protein levels by Western blot (f) (n = 3). ∗P < 0.05, ∗∗P < 0.01 versus Control group (VEC or Ctrl siRNA), which indicates a statistically significant difference. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NS, not significant; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions

Figure 3 CRNN positively regulated keratinocyte proliferation. (a) HEKa (left) and HaCaT (right) cells were seeded in 96-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 24, 48, and 72 hours before measuring keratinocyte proliferation by MTT assay (n = 3). (b) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 48 hours before measuring cell cycle profiles by flow cytometry (n = 3). (c) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 48 hours before measuring CRNN and cyclin D1 protein levels by Western blot (n = 3). (d) HEKa (left) and HaCaT (right) cells were seeded in 96-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours before measuring keratinocyte proliferation by MTT assay (n = 3). HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours before measuring cell cycle profiles by flow cytometry (e) or CRNN and cyclin D1 protein levels by Western blot (f) (n = 3). ∗P < 0.05, ∗∗P < 0.01 versus Control group (VEC or Ctrl siRNA), which indicates a statistically significant difference. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NS, not significant; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions

Figure 4 CRNN activated Akt and ERK in keratinocytes. (a) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours. The protein levels of Akt, phospho-Akt, ERK1/2, phospho-ERK1/2, and CRNN levels were analyzed by Western blot analysis (n = 3). (b) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN (#1 and #2) or Control (Ctrl) siRNA for 48 hours before measuring Akt, phospho-Akt, ERK1/2, phospho-ERK1/2, and CRNN protein levels by Western blot (n = 3). ∗P < 0.05, ∗∗P < 0.01 versus Control group (VEC or Ctrl siRNA), which indicates a statistically significant difference. ERK, extracellular signal–regulated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NS, not significant; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Inhibition of Akt, but not ERK, abrogated the effects of M5 on keratinocyte proliferation. (a) HEKa (left) and HaCaT (right) cells were seeded in 96-cell plates and pretreated with MK2206 for 6 hours, and then stimulated with M5 (2.5 ng/ml) for 24, 48, and 72 hours before measuring cells proliferation by MTT assay (n = 5). (b) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and pretreated with MK2206 for 6 hours, and then stimulated with M5 (2.5 ng/ml) for 48 hours before measuring the protein levels of cyclin D1, Akt, and phospho-Akt by Western blot analysis (n = 3). (c) HEKa (left) and HaCaT (right) cells were seeded in six-cell plates and transfected with CRNN overexpressing lentivirus (CRNN) or Control (VEC) for 48 hours before exposure to LY29402 (100 μM). Protein levels of cyclin D1, Akt, and phospho-Akt levels were measured by Western blot (n = 3). ∗P < 0.05, ∗∗P < 0.01 versus Control group (DMSO or VEC), which indicates a statistically significant difference. ERK, extracellular signal–regulated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NS, no significant. Journal of Investigative Dermatology 2019 139, 71-80DOI: (10.1016/j.jid.2018.06.184) Copyright © 2018 The Authors Terms and Conditions