MEK-dependent and -independent signaling events downstream of Tpl-2

Comparison of Tpl-2 small molecule inhibitor (SMI) versus MEK SMI activities in LPS-treated human monocytes will reveal a subset of genes that require Tpl-2 but are independent of MEK. Tpl-2 is a highly conserved (94% human versus mouse) serine-threonine kinase expressed in cells important to the inflammatory response, including monocytes, macrophages, dendritic cells, and B/T cells. The role of Tpl-2 in monocytes and macrophages has been especially well-studied. It has been shown in these cells that Tpl-2 is required for the expression of cytokines in response to Toll-like receptor ligands, including LPS. In resting cells, Tpl-2 forms a complex with p105 and ABIN-2. Upon stimulation, this complex dissociates. Dissociated p105 is processed to p50, which impacts transcription. Dissociated Tpl-2 phosphorylates MEK, which, in turn, phosphorylates ERK. ERK activates the transcription factor AP-1 and its downstream gene targets. An open question in the field is whether Tpl-2 acts solely through MEK to drive gene expression, or does Tpl-2 have any MEK-independent targets. Answering this question is important both for understanding basic Tpl-2 biology as well as its role in disease. Based on published data, Tpl-2 is important for inflammatory cytokine production, and in animal models where these cytokines contribute to disease (septic shock, IBD), blocking Tpl-2 ameliorates disease symptoms. We have a highly selective SMI of Tpl-2 that effectively blocks cytokine production in purified human monocytes. This SMI can block production of the same cytokines that a MEK inhibitor blocks, which is expected given that Tpl-2 lies upstream of MEK. However, the MEK SMI only partially inhibits certain cytokines, while the Tpl-2 SMI fully blocks them, suggesting there are additional factors downstream of Tpl-2 that are not MEK-dependent. Human monocytes will be purified from buffy coats (total of 5 donors) using a negative selection kit (Miltenyi 130-091-153) and plated at 1 x 10^6/ml in RPMI + 10% FBS + 100 ng/ml rhM-CSF. The next day, media will be aspirated and substituted with low-serum media (RPMI + 0.5% FBS). Tpl-2 and MEK inhibitors will be added to a subset of the plates at concentrations of 2.8 uM and 0.31 uM, respectively (the amounts required to fully inhibit the expression of several cytokines based on our cell-based assays), and allowed to be taken up by the cells for 1 hr. Then, 10 ng/ml of LPS will be added to a subset of the plates to activate the cells. After 6 hrs, RNA will be harvested from all of the plates. Supernatants will be saved and analyzed by ELISA to confirm cell activity/cytokine expression.