In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.

No abstract

Mitogen-activated protein kinase phosphatase-2 (MKP-2The amplitude and duration of MAP kinase signaling within a specific subcellular compartment are key features in the integration of extracellular stimuli and their effects on cellular (1). Three main MAP kinase groups, the ERKs, JNK, and p38 MAP kinases, are involved in regulating functions such as proliferation, apoptosis, and differentiation in response to growth factors, peptide hormones, stress, and infection (2). Perturbations in MAP kinase signaling are features of several different types of diseases including several types of cancers (3), diabetes (4), atherosclerosis (5, 6), and immune disorders.The kinetics of MAP kinase activation are strictly controlled principally by the mitogen-activated protein kinase phosphatases (MKPs), 3 a family of at least 10 dual specific phosphatases (DUSPs) that function to terminate MAP kinase signaling within a defined subcellular location (7). They share a common C-terminal catalytic domain and an N-terminal non-catalytic domain containing the MAP kinase interaction motif (8). Each isoform has unique yet overlapping features including substrate specificity, subcellular distribution, and factors regulating induction. For example, MKP-1 is a nuclear DUSP of the type 1 class and selective for all three major MAP kinases in vitro, whereas MKP-3, a type II DUSP, is a cytosolic phosphatase selective solely for ERK over the other kinases (7). Due to effects upon MAP kinase signaling, pertubations in the MKPs have been implicated principally in cancer (9). However, more recently, a role has been established in inflammation (10) and some cardiovascular disorders (11).One poorly studied MKP is MKP-2 (12). This DUSP (DUSP-4) is a member of the type 1 family and has been shown to be induced in response to a number of stimuli including phorbol esters and growth hormones (12-14). Nuclear targeting is regulated by two distinct nuclear targeting sequences (15). Substrate specificity for ERK and JNK was originally demonstrated in vitro (16); however, selective inhibition of JNK has been implicated in cellular studies (17,18). Although MKP-2 has been recently regarded as a surrogate for the more well described MKP-1, recent cellular studies demonstrate a role in protection against apoptosis (17) and in senescence (19). However, there is still a lack of information describing the function of DUSP-4 in different cell types, in particular regarding substrate selectivity in vivo.We have recently developed a DUSP-4 deletion mouse model and demonstrated a novel immunological phenotype in vivo (20). Using embryonic fibroblasts from DUSP-4 deletion mice, we now examine the effect of deletion upon MAP kinase signaling and growth parameters. We find that despite very moderate increases in ERK and JNK activity, MKP-2 deletion has profound effects upon cellular proliferation. In particular, we identify a role for MKP-2 in G 2 /M phase transition and demonstrate that MKP-2 plays a role in cell survival in response to the apo-* This work was supporte...

Highlights► TLR-4 activation by Leishmania mexicana promastigotes and CPB-deficient amastigotes. ► Prolonged, TLR-4 dependent iNOS and COX-2 expression by L. mexicana promastigotes. ► Enhanced TLR-4 dependent-arginase-1 expression. ► Regulation of IL-12 induction by an arginase-1 dependent mechanism.

The MKPs (mitogen-activated protein kinase phosphatases) are a family of at least ten DUSPs (dual-specificity phosphatases) which function to terminate the activity of the MAPKs (mitogen-activated protein kinases). Several members have already been demonstrated to have distinct roles in immune function, cancer, fetal development and metabolic disorders. One DUSP of renewed interest is the inducible nuclear phosphatase MKP-2, which dephosphorylates both ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) in vitro. Recently, the understanding of MKP-2 function has been advanced due to the development of mouse knockout models, which has resulted in the discovery of novel roles for MKP-2 in the regulation of sepsis, infection and cell-cycle progression that are distinct from those of other DUSPs. However, many functions for MKP-2 still await to be characterized.