Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl]-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)(2)-mediated intracellular signalling events.

In this study, we examined the activation of non-canonical nuclear factor Kappa B (NFκB) signalling in U2OS cells, a cellular metastatic bone cancer model. Whilst Lymphotoxin α1β2 (LTα1β2) stimulated the expected slow, delayed, sustained activation of serine 866/870 p100 phosphorylation and increased cellular expression of p52 NFκB, we found that canonical agonists, Interleukin-1β (IL-1β) and also Tumour necrosis factor-α (TNFα) generated a rapid transient increase in pp100, which was maximal by 15-30 min. This rapid phosphorylation was also observed in other cells types, such as DU145 and HCAECs suggesting the phenomenon is universal. IKKα deletion using CRISPR/Cas9 revealed an IKKα-dependent mechanism for serine 866/870 and additionally serine 872 p100 phosphorylation for both IL-1β and LTα1β2. In contrast, knockdown of IKKβ using siRNA or pharmacological inhibition of IKKβ activity was without effect on p100 phosphorylation. Pre-incubation of cells with the NFκB inducing-kinase (NIK) inhibitor, CW15337, had no effect on IL-1β induced phosphorylation of p100 however, the response to LTα1β2 was virtually abolished. Surprisingly IL-1β also stimulated p52 nuclear translocation as early as 60 min, this response and the concomitant p65 translocation was partially reduced by IKKα deletion. Furthermore, p52 nuclear translocation was unaffected by CW15337. In contrast, the response to LTα1β2 was essentially abolished by both IKKα deletion and CW15337. Taken together, these finding reveal novel forms of NFκB non-canonical signalling stimulated by ligands that activate the canonical NFκB pathway strongly such as IL-1β.

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.

We assessed the effects of over-expressing the dual-specific phosphatase, mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2), in human umbilical vein endothelial cells (HUVECs) on inflammatory protein expression and apoptosis, two key features of endothelial dysfunction in disease.

Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP-4). It plays an important role in macrophage inflammatory responses through the negative regulation of Mitogen activated protein kinase (MAPK) signalling. However, information on the effect of MKP-2 on other aspect of macrophage function is limited.

Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17β-estradiol biosynthesis and its knockdown would be expected to increase 17β-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17β-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17β-estradiol secretion compared to MHP36 cells. As 17β-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17β-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17β-estradiol (100 nm) or 17β-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17β-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17β-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.

Mitogen-activated protein kinase phosphatases (MKPs) play key roles in inflammation and immune mediated diseases. Here we investigated the mechanisms by which MKP-2 modulates central nervous system (CNS) inflammation in experimental autoimmune encephalomyelitis (EAE). Our results show that MKP-2 mRNA levels in the spinal cord and lymphoid organs of EAE mice were increased compared with naive controls, indicating an important role for MKP-2 in EAE development. Indeed, MKP-2-/- mice developed reduced EAE severity, associated with diminished CNS immune cell infiltration, decreased proinflammatory cytokine production and reduced frequency of CD4+ and CD8+ T cells in spleens and lymph nodes. In addition, MKP-2-/- CD11c+ dendritic cells (DCs) had reduced expression of MHC-II and CD40 compared with MKP-2+/+ mice. Subsequent experiments revealed that CD4+ T cells from naïve MKP-2-/- mice had decreased cell proliferation and IL-2 and IL-17 production relative to wild type controls. Furthermore, co-culture experiments showed that bone marrow derived DCs of MKP-2-/- mice had impaired capability in antigen presentation and T cell activation. While MKP-2 also modulates macrophage activation, our study suggests that MKP-2 is essential to the pathogenic response of EAE, and it acts mainly via regulating the important antigen presenting DC function and T cell activation.

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO). Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size. MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group. This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.

Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis.

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2(-/-) mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2(-/-) mice did not, however, demonstrate defective type-1 responses compared with MKP-2(+/+) mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2(+/+), but not MKP-2(-/-), mice was nitric oxide (NO) dependent as infected MKP-2(+/+), but not MKP-2(-/-) mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2(-/-) but not MKP-2(+/+) mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2(-/-) mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.

We have previously shown mitogen-activated protein kinase phosphatase 2 (MKP-2) to be a key regulator of proinflammatory cytokines in macrophages. In the study presented here, we investigated the role of MKP-2 in inflammatory arthritis with a particular focus on neutrophils.

The effects of Leishmania mexicana metacyclic promastigotes upon MAP kinase signalling in mouse bone marrow macrophages and subsequent expression of the disease regulatory proteins iNOS and COX-2 were studied. At a ratio of 5:1, promastigotes caused a marked increase in phosphorylation of the three major MAP kinases, ERK, p38 and JNK. MAP kinase signalling was substantially reduced in TLR-4(-/-) but not TLR-2(-/-) deficient macrophages and completely abolished in double TLR-2/4(-/-) macrophages. A similar outcome was observed using cysteine peptidase B deficient amastigotes. Furthermore, whilst promastigotes had no independent effect on iNOS or COX-2 expression, they prolonged the induction of these proteins stimulated by LPS and enhanced PGE(2) and NO production. Induction of COX-2 and iNOS was also TLR-4 dependent. Blockade of either PGE(2) or NO production with indomethacin or l-NAME reversed promastigote inhibition of LPS induced IL-12 production. Promastigotes also increased macrophage arginase-1 expression and enhanced arginase activity, both of which were substantially reduced in TLR-4 but not TLR-2 deficient macrophages. Surprisingly, arginase inhibition by Nor-NOHA also caused a reversal of promastigote mediated inhibition of macrophage IL-12 production. These data demonstrate for the first time the role of TLR-4 in mediating the effects of L. mexicana promastigotes on MAP kinase activation, up-regulation of COX-2, iNOS as well as arginase-1 expression in macrophages and further shows that PGE(2), NO and arginase activity all contribute substantially to the inhibition of host cell IL-12 production.

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T(H)1 response. By contrast, in the present study footpad infection of MKP-2(-/-) mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2(+/+) mice. Analysis of immune responses following infection demonstrated a reduced T(H)1 response in MKP-2(-/-) mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4(+) and CD8(+) T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2(-/-) mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2(+/+) mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T(H)1/T(H)2 balance was unaltered in MKP-2(-/-) compared with wild-type mice. Although non-stimulated MKP-2(-/-) macrophages were more permissive to L. major growth than macrophages from MKP-2(+/+) mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2(-/-) and MKP-2(+/+) macrophages. Consequently, in the absence of any switch in the T(H)1/T(H)2 balance in MKP-2(-/-) mice, no significant change in disease phenotype was observed.

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 PGE(2) 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.

In this study, we utilised a number of adenoviral constructs in order to examine the role of intermediates of the NF-kappaB pathway in the regulation of inducible nitric oxide synthase (iNOS) induction in rat aortic smooth muscle cells (RASMCs). Lipopolysaccharide (LPS) stimulated a significant increase in iNOS induction and NF-kappaB DNA binding. These parameters were substantially reduced by overexpression of a wild-type Ikappa-Balpha adenoviral construct (Ad.Ikappa-Balpha), confirming a role for NF-kappaB in iNOS induction. Infection with a dominant-negative IKKalpha adenoviral construct (Ad.IKKalpha+/-) did not significantly affect iNOS induction, NF-kappaB DNA binding or Ikappa-Balpha loss. Infection of RASMCs with adenovirus encoding a dominant-negative IKKbeta (Ad.IKKbeta+/-) essentially abolished iNOS induction and activation of the NF-kappaB pathway. Pretreatment of RASMCs with a novel specific inhibitor of IKKbeta, SC-514, significantly reduced iNOS induction, NF-kappaB DNA binding and I-kappaBalpha loss in a concentration-dependent manner. In both RASMCs and human umbilical vein endothelial cells (HUVECs), infection with Ad.IKKbeta+/- also inhibited COX-2 expression in response to LPS. However, Ad.IKKalpha+/- was again without effect. These data suggest that IKKbeta plays a predominant, selective role in the regulation of NF-kappaB-dependent induction of iNOS in RASMCs.