Inhibition of p38 MAPK attenuates IL-1α induction of mRNAs encoding 11β-HSD1 and C/EBPβ and prevents IL-1α induced C/EBPβ phosphorylation.

<p>(<b>A</b>, <b>B</b>) qPCR measurement of levels of mRNA encoding 11β-HSD1 in A549 cells without inhibitor (-) or pre-incubated for 1h with inhibitors of p38 MAPK (p38, SB203580; 10µM), JNK II (SP600125; 20µM) or MEK I (PD0325901; 0.5µM) prior to 24h treatment with IL-1α (<b>A</b>) or TNFα (<b>B</b>) (black bars). White bars; no cytokine treatment. All data are expressed relative to levels in untreated cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n≥3. *, significantly different from IL-1α-stimulated control. *, p<0.05; **p<0.001. (<b>C</b>) qPCR measurements of levels of mRNA encoding C/EBPβ in cells treated for 24h with IL-1α with (p38 inhibitor), or without (-) prior addition of p38 MAPK inhibitor. Data are expressed relative to levels in untreated control cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n≥6. *, significantly different from IL-1α-stimulated control. *, p<0.05. #, significantly different from unstimulated cells. ##, p<0.001. (<b>D</b>) Western blot showing levels of phosphorylated C/EBPβ-LAP (P-C/EBPβ-LAP); upper image) in untreated A549 cells (-p38 MAPK, -IL-1α; white bar), or following 15min treatment with IL-1α alone (-p38 MAPK, +IL-1α; black bar) or with prior addition of p38 MAPK inhibitor for 1h (+p38 MAPK, +IL-1α; grey bar; SB203580, 50µM). The blot (20µg protein/lane) was stripped and reprobed with C/EBPβ antibody (bottom membrane) to detect total C/EBPβ-LAP. Protein bands were quantified using the Odyssey Infrared Imaging System and the ratio of P-C/EBPβ-LAP/total C/EBPβ-LAP calculated. Data are expressed relative to levels in untreated cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n=3. *, p<0.05; **, p<0.001. (<b>E</b>) Western blot showing increased levels of C/EBPβ-LAP and C/EBPβ-LIP isoforms after IL-1α treatment for up to 6h, compared to control untreated cells (C). (<b>F</b>) qPCR measurement of levels of mRNA encoding 11β-HSD1 in A549 cells incubated for 1h with CHX (+) or without CHX (-) followed by IL-1α/TNFα treatment for 4h or control (Con). Data are expressed relative to levels in untreated cells (no CHX, no cytokine), arbitrarily set to 1, and are mean ± SEM; n=3. *, p<0.05 significant effect of CHX. #, significantly different to untreated cells (no CHX, no cytokine). #, p<0.05; # #, p<0.001.