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Hyun Jeong Park, Hee Jung Kim, Jun Young Lee, Baik Kee Cho, Richard L

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Presentation on theme: "Hyun Jeong Park, Hee Jung Kim, Jun Young Lee, Baik Kee Cho, Richard L"— Presentation transcript:

1 Adrenocorticotropin Hormone Stimulates Interleukin-18 Expression in Human HaCaT Keratinocytes 
Hyun Jeong Park, Hee Jung Kim, Jun Young Lee, Baik Kee Cho, Richard L. Gallo, Dae Ho Cho  Journal of Investigative Dermatology  Volume 127, Issue 5, Pages (May 2007) DOI: /sj.jid Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Effects of ACTH on the mRNA expression and production of IL-18 in HaCaT keratinocytes. (a) HaCaT cells were harvested after ACTH treatment for 6hours. Total RNA was extracted, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to β-actin by densitometry. (b) Cells were treated with indicated concentration of ACTH for 24hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean±SD. *P<0.05 versus control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Caspase-1 is essential for the ACTH-induced IL-18 production in HaCaT keratinocytes. (a) HaCaT cells were pretreated with 40μM of the caspase-1 inhibitor Ac-YVAD-CHO for 1hour, and then challenged with 1nM ACTH. The IL-18 concentration in the culture supernatants was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1nM ACTH for 6hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer's instruction. The data normalized to the negative control are shown as fold increases in caspase activity. (c) Effects of Z-FA-fmk on ATCH-induced IL-18 production. HaCaT cells were pretreated with 20μM of Z-FA-FMK for 2hours, and then stimulated with 1nM ACTH. The IL-18 concentration in the supernatant was measured by ELISA, each performed in triplicate. Data are mean±SD. *P<0.05 versus control, §P<0.05 versus ACTH treated group. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Effects of the p38 MAPK inhibitor SB203580, or the ERK inhibitor PD98059 on ACTH-induced IL-18 expression in HaCaT cells. HaCaT cells were pretreated with SB (10μM) or PD98059 (10μM) for 2hours before addition of 1nM of ACTH. (a) Total RNA was extracted at 6hours following ACTH treatment, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to β-actin by densitometry. (b) After cells were harvested for 24hours, IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean±SD. *P<0.05 versus control, §P<0.05 versus ACTH treated group. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effects of ACTH on activation of MAPKs in HaCaT cells. HaCaT cells were treated with 1nM of ACTH for indicated time periods. Phosphorylation of (a) p38 kinase and (b) ERK were analyzed by immunoprecipitation and Western blot as described in Materials and methods. After cells were harvested for 5minutes, phosphorylation of p38 kinase and ERK were analyzed by immunoprecipitation and Western blot. The band intensities were quantitated. Data are mean±SD. *P<0.05 versus control, §P<0.05 versus ACTH treated group. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Involvement of MC1R and MC2R in ACTH-induced IL-18 production. (a) HaCaT cells were treated with 1nM of ACTH. After that, cells were harvested for 24hours. The expression level of MC1R and MC2R were analyzed by Western blot. (b) Cells were pretreated with anti-MC1R (2μg/mL) or anti-MC2R antibody (2μg/mL) for 1hour and then incubated with 1nM of ACTH for 24hours. The supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean±SD. *P<0.05 versus control, §P<0.05 versus ACTH treated group. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Effects of ACTH on the production of IL-18 in primary human epidermal keratinocytes. (a) Primary human epidermal keratinocytes were pretreated with or without Ac-YVAD-CHO (40μM) for 2hours before addition of 1nM of ACTH for 24hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1nM ACTH for 6hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer's instruction. The data, normalized to the negative control, are shown as fold increases in caspase activity. (c) Cells were treated with or without SB (10μM) or PD98059 (10μM) for 2hours before addition of 1nM of ACTH for 24hours. Supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean±SD. *P<0.05 versus control, §P<0.05 versus ACTH treated group. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

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