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In this study we used adenovirus infection to overexpress the dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2), in human umbilical vein endothelial cells and examined inflammatory protein expression and apoptosis, two key features of endothelial dysfunction in disease. We generated an adenoviral version of MKP-2 (Adv.MKP-2) and infected HUVECs for 40 h. TNF! stimulated MAP kinase phosphorylation and protein expression was measured by Western blotting. Cellular apoptosis was assayed by FACS. Infection with Adv.MKP-2 selectively abolished TNF!-mediated JNK activation and had little effect upon ERK or p38 MAP kinase. Adv.MKP-2 abrogated COX-2 expression whilst induction of the endothelial cell adhesion molecules ICAM and VCAM, two NF"B-dependent proteins, were not affected. However, when ICAM and VCAM expression was partly reduced by blockage of the NF"B pathway Adv.MKP-2 was able to reverse this inhibition. This correlated with enhanced TNF!-induced I"B! loss, a marker of NF"B activation. TNF! in combination with NF"B blockade also increased HUVEC apoptosis; this was significantly reversed by Adv.MKP-2. Protein markers of cellular damage and apoptosis, H2AX phosphorylation and caspase-3 cleavage, were also reversed by MKP-2 overexpression.

In this study we examined the potential for PAR(2) and TNFalpha to synergise at the level of MAP kinase signalling in PAR(2) expressing NCTC2544 cells. However, to our surprise we found that activation of PAR(2) by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFalpha stimulated IkappaBalpha loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR(4), however inhibition was reversed by pre-incubation with the novel PAR(2) antagonist K14585, suggesting that the effect is specific for PAR(2) activation. SLIGKV-OH was found to be more potent in inhibiting TNFalpha-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G(q/11) inhibitor YM25480 also reversed PAR(2) mediated inhibition. Activation of PAR(2) was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR(2) or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G(q/11), in mediating inhibition of TNFalpha-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.

The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.

MAP Kinase Phosphatase-2 (MKP-2) is a dual specific nuclear phosphatase which is selective for both ERK and JNK, MAP kinases implicated in the regulation of apoptosis in response to genotoxic stress. Here we report the conditional expression of MKP-2 in human embryonic kidney cells 293. We demonstrate that Flag-WT-MKP-2 is able to rescue cells from apoptotic commitment when subjected to UV-C or cisplatin treatment. We establish that upon stimulation all three major MAP kinase families (ERK, JNK and p38 MAP kinases) are activated. However, MKP-2 is surprisingly only able to deactivate JNK in vivo. Furthermore, whilst pre-treatment of cells with either the JNK inhibitor SP600125, or the MEK-1 inhibitor PD98059, also reverses UV-C and cisplatin-induced apoptosis, the anti-apoptotic effect of MKP-2 overexpression is not additive with SP600125 but is with PD098059, suggesting that MKP-2 is involved in specifically terminating JNK activity and not ERK. The inability of MKP-2 to dephosphorylate ERK in vivo is also not due to the inability of Flag-MKP-2 to bind both ERK and JNK; phosphorylated forms of each kinase are co-precipitated with both WT and CI-MKP-2. Immunofluorescence studies however demonstrate that ERK is exclusively cytosolic in origin and not translocated to the nucleus following UV-C and cisplatin treatment whilst JNK is principally nuclear. These studies demonstrate the in vivo specificity of MKP-2 for JNK and not ERK and show that nuclear-targeted JNK is involved in genotoxic stress-induced apoptosis.

MAP kinase phosphatase-2 (MKP-2) is a member of a family of dual specificity phosphatases (DSPs) that function in both the cytosol and nucleus to inactivate the MAP kinases. The mechanism that controls the subcellular distribution of these proteins is currently unclear. In this study, we have used site-directed mutagenesis to remove two novel nuclear localization sequences, NLS-1 and -2, either alone or in combination (DNLS). Loss of NLS-1 or NLS-2 alone did not alter the nuclear targeting of MKP-2 but mutation of both resulted in MKP-2 being retained within the cytosol. Furthermore, whilst expression of WT-MKP-2, NLS-1 or NLS-2 reduced both sorbitol- or UV-stimulated nuclear c-Jun N-terminal kinase (JNK) activity in HEK293 cells, this effect was absent in cells expressing DNLS-MKP-2. Similarly, transient transfection of WT-MKP-2, NLS-1 or NLS-2, but not DNLS-MKP-2 was able to substantially reduce agonist-stimulated ANF reporter activity in rat cardiac myocytes. Taken together, these results indicate that whilst both novel NLS participate in the nuclear localization of MKP-2, the expression of either sequence is sufficient to retain nuclear targeting.

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., Author Summary In cells of the immune system are switch-on enzymes called kinases which regulate responses to infectious agents such as Leishmania. However, in the same cells there are switch-off enzymes known as phosphatases which function to turn off the kinases once they have done their work. A lot of studies have focussed on the role of kinases but not phosphatases in response to infection; we therefore generated a novel mouse in which the gene for one of these phosphatases, called MKP-2, has been deleted. We found that in the absence of this phosphatase unexpected things happened. The profile of inflammatory proteins, produced by a special cell of the immune system, called a macrophage, that functions to regulate infection by Leishmania, changed in ways which meant the macrophage could either fight infection very effectively or very weakly. In actual fact, we found that the macrophages with no MKP-2 fought off Leishmania poorly and mice deficient in MKP-2 had a modified immune response favouring the growth of the parasite. This is the first study to give critical insight into the role of MKP-2 in fighting Leishmania infection and demonstrates very well the importance of this class of enzyme in pathogen biology.