Molecular and genomic investigations of the role of MAP3K8 in IL-1β-induced response in respiratory epithelial cells

Mitogen Activated Protein 3 Kinase 8 (MAP3K8) is a member of the serine/threonine protein kinase family and has been demonstrated to be involved in the mitogen activated protein kinase (MAPK) pathway. The latter pathway plays an important role in many aspects of the immune mediated inflammatory response. Inhibition of MAP3K8 in primary human cell types decreases the production of TNF-α and other pro-inflammatory mediators during inflammatory events. Pharmacologic inhibition of MAP3K8 has been shown to be an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases. Inhibition of MAP3K8 may therefore potentially be a new therapeutic strategy for airway inflammation. Investigation of the role of MAP3K8 in regulation of the inflammatory response was conducted using epithelial cell lines. A MAP3K8 gene knockdown experiment using small interfering RNA (siRNA) was carried out to establish the effect of MAP3K8 on the inflammatory response following IL-1β stimulation. In addition the effect of MAP3K8 gene knockdown on the therapeutic outcome of dexamethasone (Dex) was investigated. ELISAs were used to establish whether knockdown of MAP3K8 resulted in inhibition of IL-1β-induced IL-6, IL-8 and RANTES. The effect of treatment with Dex in combination with MAP3K8 gene silencing on ERK and MEK phosphorylation was determined by Western Blotting. The impact of MAP3K8 gene silencing on global gene expression was also assessed using Affymetrix Human Gene 1.1ST arrays. Differential expression and network analyses were performed on the data generated. A significant rise in MAP3K8 gene expression was found 2 hours after IL-1β stimulation in both A549 and normal human bronchial epithelial (NHBE) cells. In addition, siRNA against MAP3K8 resulted in approximately 40% inhibition of IL-6, IL-8 and RANTES expression after IL-1β stimulation. Furthermore, MAP3K8 gene silencing enhanced by approximately 40% the decrease in IL-8 and RANTES release seen at different concentrations of Dex. The combination of MAP3K8 gene silencing and Dex also resulted in a greater inhibition of phosphorylated ERK compared to that seen with Dex alone. Data from global gene expression arrays revealed that the MAP3K8 regulated inflammatory response was predominantly involved in the ERK/MAPK and SAPK/JNK pathways but not the p38 MAPK pathway. Among the genes regulated by MAP3K8, it is interesting to note that IL-6, MMP-1, MMP-3, MMP-12 and CCL20 have previously been reported to be linked with airway diseases, such as COPD and asthma. Furthermore, MAP3K8 regulated the AP-1 transcription factor up-regulation of IL-1β-induced IL-6 and MMP-1 through the ERK1/2 and JNK signaling pathways. MAP3K8 however appears to regulate NF-kB activation through IKKα/β activation independent of the MAPK pathway regulated by itself. In conclusion, MAP3K8 plays a key role in the inflammatory cytokine response post IL-1β stimulation. MAP3K8 gene silencing by siRNA not only suppresses these key inflammatory cytokines but also enhances the therapeutic effect of steroids. MAP3K8 inhibitors might therefore provide a new therapeutic strategy for airway inflammation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo