For panel C, curcumin (Cur; 50?M) or apigenin (Api; 40?M) were added to the cells 1?h before the addition of IL-1 and DMSO was used as a vehicle control. role for NF-B in the IL-1-induced expression of C/EBP, and thereby downstream APP genes regulated by this transcription factor. test with responses (Hiron et al., 1992; Ramji et al., 1993a; Foka et al., 2009; Coulouarn et al., 2004, 2005 and recommendations therein). For example, the hepatocyte origin and pro-inflammatory cytokine responsiveness of mRNAs that were found to be upregulated in acute systemic inflammation, as judged by complete coverage of the human liver transcriptome, has been confirmed in these cells (Coulouarn et al., 2004). In addition, Rabbit Polyclonal to ALK genome-wide studies in these cells in relation to the actions of pro-inflammatory cytokines have shown proper kinetics of changes in mRNA abundances for cytokines and their receptors, transcription factors and APPs, with the overall percentage of regulated mRNAs (7%) being similar to the number of liver mRNAs regulated during the APR in mouse or humans (Coulouarn et al., 2005). We have previously analysed the effect of IL-1 on C/EBP expression in SKA-31 J774.2 macrophages and glomerular mesangial cells (Tengku-Muhammad et al., 2000; Granger et al., 2000) but not in Hep3B cells. This was therefore investigated by time course RT-PCR and western blot analysis. For RT-PCR, sequence analysis confirmed the specific amplification of C/EBP. Fig. 1A shows that IL-1 induces C/EBP mRNA expression that peaks at 3?h and remains at comparable or slightly reduced levels throughout the 24?h incubation period. Such an SKA-31 induction was due to IL-1 and not because of a nonspecific effect of harvesting the cells at the various time-points. The expression of the C/EBP protein was also induced by IL-1 at 3? h post-treatment and was then expressed at reduced levels over the rest of the 24?h period (Fig. 1B). Statistical analysis of the data from three impartial experiments showed that this IL-1-induced expression of the C/EBP protein (normalized to -actin) was significant (kinase assay kit in which the ability of immunoprecipitated JNKs to phosphorylate c-Jun, its key downstream target, is determined. This assay was therefore employed to determine the action of IL-1 on JNK activity in the absence or the presence of pharmacological inhibitors. Consistent with the pattern of phospho-JNK levels (Fig. 3A), IL-1 induced the activity of the enzyme (Fig. 3B) ( em P /em ? ?0.05 at 15?min and 30?min). In addition, curcumin and SP600125, but not apigenin, attenuated the IL-1-induced JNK activity (Fig. 3C) ( em P /em ? ?0.001 for both curcumin and SP600125). 3.4. siRNA-mediated knockdown of NF-B attenuates the IL-1-induced expression of C/EBP siRNA-mediated knockdown of JNK-1 SKA-31 and -2, the two prominent isoforms expressed in hepatocytes, or its downstream target c-Jun did not attenuate the IL-1-induced expression of the C/EBP protein (Figs. IIA-IIB in supplementary data). Similarly, siRNA-mediated knockdown of CK2- and -, two of the three catalytic subunits of this enzyme (Singh and Ramji, 2008) had no effect (Fig. IIC in supplementary data). For JNK and CK2, the findings were confirmed at the level of C/EBP mRNA expression (data not shown). Although the precise reason(s) for these results are currently unclear, it is possible SKA-31 that this could be because of functional redundancy between the different SKA-31 pathways and/or sufficient amount of residual activity being present following siRNA-mediated knockdown. Because it was not possible to simultaneously knockdown all the JNK and CK2 isoforms due to potential toxicity effects from the use of large quantities of siRNA that would be required, the role of the downstream transcription factor NF-B was investigated. The expression of both the p50 and p65 NF-B subunits, the two most abundant isoforms that play a key role in IL-1 signalling (Pereira and Oakley, 2008), was knocked down by transfection of Hep3B cells with siRNA against these two subunits but not GAPDH (Fig. 4A and B). The IL-1-induced expression of the C/EBP protein was attenuated by knockdown of p50 and.
