FIGURES AND TABLES
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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 and Dae Ho Cho
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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 6 hours. Total RNA was extracted, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to 
-actin by densitometry. (b) Cells were treated with indicated concentration of ACTH for 24 hours. 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.
Figure 2.
Caspase-1 is essential for the ACTH-induced IL-18 production in HaCaT keratinocytes. (a) HaCaT cells were pretreated with 40 
M of the caspase-1 inhibitor Ac-YVAD-CHO for 1 hour, and then challenged with 1 nM ACTH. The IL-18 concentration in the culture supernatants was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. 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 20
M of Z-FA-FMK for 2 hours, and then stimulated with 1 nM ACTH. The IL-18 concentration in the supernatant was measured by ELISA, each performed in triplicate. Data are meanSD. *
P<0.05 versus control, §
P<0.05 versus ACTH treated group.
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 SB203580 (10
M) or PD98059 (10
M) for 2 hours before addition of 1 nM of ACTH. (a) Total RNA was extracted at 6 hours following ACTH treatment, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to -actin by densitometry. (b) After cells were harvested for 24 hours, IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. *
P<0.05 versus control, §
P<0.05 versus ACTH treated group.
Figure 4.
Effects of ACTH on activation of MAPKs in HaCaT cells. HaCaT cells were treated with 1 nM 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 5 minutes, phosphorylation of p38 kinase and ERK were analyzed by immunoprecipitation and Western blot. The band intensities were quantitated. Data are meanSD. *
P<0.05 versus control, §
P<0.05 versus ACTH treated group.
Figure 5.
Involvement of MC1R and MC2R in ACTH-induced IL-18 production. (a) HaCaT cells were treated with 1 nM of ACTH. After that, cells were harvested for 24 hours. The expression level of MC1R and MC2R were analyzed by Western blot. (b) Cells were pretreated with anti-MC1R (2 g/mL) or anti-MC2R antibody (2 g/mL) for 1 hour and then incubated with 1 nM of ACTH for 24 hours. The supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. *
P<0.05 versus control, §
P<0.05 versus ACTH treated group.
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 (40
M) for 2 hours before addition of 1 nM of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. 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 SB203580 (10
M) or PD98059 (10
M) for 2 hours before addition of 1 nM of ACTH for 24 hours. Supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are meanSD. *
P<0.05 versus control, §
P<0.05 versus ACTH treated group.
