Figure 1.
Time course studies of p38 MAPK phosphorylation in NHEK treated with anisomycin (300 ng/ml) and IL-1β (10 ng/ml).
Western blotting of equivalent amounts of whole-cell protein extracts from NHEK treated with anisomycin or IL-1β for the indicated time periods. The proteins were separated by SDS-PAGE, blotted onto a nitrocellulose membrane and probed with an anti-phospho-p38 MAPK antibody. Equal loading was confirmed by incubating with anti-total-p38 MAPK. A representative gel of three different experiments is shown.
Figure 2.
Time course studies of IL-24 mRNA expression.
NHEK were incubated with (A) anisomycin (300 ng/ml) or (B) IL-1β (10 ng/ml) for the indicated time periods and IL-24 mRNA expression levels (relative to housekeeping gene 18s rRNA) were determined using quantitative RT-PCR. *p<0.05 compared with cells harvested before stimulation (0 hours) (A, n = 6 cultures, B, n = 5 cultures). Bars indicate mean ± SD.
Figure 3.
IL-24 protein expression in the supernatant of NHEK treated with medium control or anisomycin (300 ng/ml).
NHEK were cultured in the presence or absence of anisomycin for up to 24 hours. IL-24 protein levels in the supernatant were analyzed by ELISA. IL-24 protein concentrations were normalized to the calculated total protein concentration of the whole cell extracts.*p<0.05 compared with cells harvested before stimulation (0 hours) (n = 5 cultures). Points indicate mean ± SEM.
Figure 4.
Inhibition of p38 MAPK activation abrogates IL-24 expression.
(A) Whole cell extracts were prepared from NHEK incubated with or without (control) anisomycin (300 ng/ml) for 30 minutes in the absence or presence of the 38 MAPK inhibitor SB 202190 (10 µM). Proteins were separated by SDS-PAGE. After electroblotting, separated proteins were probed with anti-phospho-MK2 (recognizing the phosphorylated form of MK2 isoforms 1 and 2) and anti-total p38 MAPK. A gel representative of three different experiments is shown. NHEK were cultured with or without SB 202190 (10 µM) for 30 minutes before incubation with (B) anisomycin (300 ng/ml) or medium control for 4 hours or (C) IL-1β (10 ng/ml) or medium control for 2 hours. Total RNA was extracted and IL-24 mRNA (relative to housekeeping gene 18 s RNA) levels were determined. *p<0.05 compared with medium control (n = 5 cultures). Bars indicate mean ± SD. (D) NHEK were cultured in the presence or absence of SB 202190 (10 µM) with medium control or anisomycin (300 ng/ml) for 18 hours. Using ELISA IL-24 concentration in the supernatants was determined and subsequently normalized to the total protein concentration of whole cell extracts. *p<0.05 compared with medium control (n = 5 cultures). Bars indicate mean ± SD.
Figure 5.
p38 MAPK regulates IL-24 expression at a posttranscriptional level.
(A and B) NHEK were pre-stimulated with anisomycin (300 ng/ml) for 2 hours at which point medium (control), the transcription inhibitor actinomycin D, or SB 202190 were added to the cultures. Cells were then harvested at 30 minute intervals, total RNA extracted and (A) IL-24 and (B) TNFα-mRNA levels (relative to 18 s RNA) were determined. *p<0.05 compared with control (n = 5). Points indicate mean ± SD. (C) As control NHEK were pre-incubated with or without actinomycin D for one hour before treatment with medium or anisomycin (300 ng/ml) for another 2 hours. Total RNA was extracted IL-24 mRNA levels (relative to 18 s RNA) were determined. *p<0.05 compared with medium control (n = 4). Bars indicate mean ± SD.
Figure 6.
The 3′ UTR of IL-24 mRNA mediates rapid degradation and p38 MAPK-induced stabilization of a reporter mRNA.
(A) Scheme of IL-24 mRNA and insertion of its 3′ UTR into tet-off vector-expressed β-globin mRNA. The three AUUUA-motifs (indicated by arrows) are displayed. (B) HeLa cells were transfected with plasmids expressing the β-globin mRNA without insertion (β-G) or with the IL-24 3′ UTR inserted (β-G-IL24) and with empty vector or a plasmid expressing constitutively active MKK6 (MKK62E). Total RNA was isolated at the indicated times after stopping transcription with doxycycline (3 µg/ml). mRNA amounts were determined by qRT-PCR (means ± SD of triplicates) and expressed relative to the amount at the time of doxycycline addition ( = 100%). (C) mRNAs from a separate experiment carried out as described for (B) were analyzed by Northern blot with a β-globin antisense probe. Two species of the hybrid mRNA (β-G-IL24 a and b) were detected. Ethidium bromide staining of 28 S rRNA is shown as a loading control. Similar results were obtained in two separate experiments.