TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five
intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene
expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR
adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information
on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing
activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4
ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase
2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on
Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated
factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor
SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38
MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of
MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream
regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen
