Annika Sanfridson | Search PubMed

PubMed2006

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation.

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Inflammatory pathway analysis using a high content screening platform.

High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response.

Anthracenes↗