Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.     

Enteral arginine modulates inhibition of AP-1/c-Jun by SP600125 in the postischemic gut

We previously demonstrated that enteral arginine increased c-Jun/activator protein-1 (AP-1) DNA-binding activity and iNOS expression in a rodent model of mesenteric ischemia/reperfusion (I/R). The objective of this study was to specifically investigate the role of AP-1 in arginine's deleterious effect on the postischemic gut. We hypothesized that AP-1 inhibition would mitigate the effects of arginine. Using a rodent model of mesenteric I/R we demonstrated that gut neutrophil infiltration, activity of c-Jun/AP-1, as well as iNOS expression were increased by I/R and further increased by arginine while lessened by inhibition of c-Jun using the pharmacologic c-Jun N-terminal kinase inhibitor, SP600125. Similar results were demonstrated using a cell culture model of oxidant stress in IEC-6 cells. Importantly, effects of SP600125 were comparable to those of c-Jun silencing. Lastly, the specific iNOS inhibitor, 1400W, had no effect on either AP-1 or c-Jun. In conclusion, SP600125 attenuated the activity of c-Jun/AP-1, iNOS expression, and neutrophil infiltration induced by arginine following mesenteric I/R. Our data suggest that AP-1 inhibition mitigates the injurious inflammatory effects of arginine in the postischemic gut. Further investigation into the pathologic role of enteral arginine in the postischemic gut is warranted.     

Leishmania parasites act as a Trojan horse that paralyzes the translation system of host macrophages

GP63 is an abundant GPI-anchored surface metalloprotease of Leishmania. Jaramillo et al. (2011) show that GP63 manipulates the translation system of host macrophages by cleaving mTOR, which leads to 4E-BP1 dephosphorylation. This study pioneers the observation that Leishmania parasites metabolically paralyze their host cells using an elegant translation shutoff mechanism.     

Differentiation-associated genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in KYSE450 cells

A group of potential differentiation-associated genes had been identified by microarray analysis as c-Jun/AP-1 target genes essential for epithelial differentiation program. Our previous study showed that c-Jun/AP-1 could bind and activate these gene promoters in vivo using chromatin immunoprecipitation. To further understand how the mitogen-activated protein kinase signaling pathways regulate AP-1 activity and expression of c-Jun target genes, our strategy was based on the use of 12-o-tetradecanoylophorbol-13-acetate (TPA) and pharmacological reagents to induce or block c-Jun expression. The mRNA and protein expression of these genes increased in response to TPA-induced c-Jun/AP-1 expression. Inhibitors of JNK (SP600125) and PKC (GF109203X) mainly blocked expression and phosphorylation of c-Jun, while inhibition of MEK-ERK activity with PD98059 (an inhibitor of MEK) had little effect. Expression of involucrin and keratin 4 in response to TPA was attenuated by pretreatments with GF109203X and SP600125, but not PD98059, suggesting involvement of PKC and JNK in this response. Taken together, these results suggested that differentiation-associated genes were regulated by TPA-induced c-Jun/AP-1 mainly via a PKC/JNK pathway in esophageal cancer cell line KYSE450.     

Leishmania infection: surfaces and immunity

Infections with Leishmania parasites are initiated by bites from infected sandflies; the injected promastigotes are attacked by phagocytic cells but succeed in entering cells of the macrophage family and surviving in them. The secrets of the success of the extracellular form in penetrating the host cell and of the intracellular form in surviving in a potentially hostile environment are yet to be unraveled. The infectivity of the extracellular promastigote is related to the expression on its surface of molecules that interact with the surface of the host cell. One of these molecules is the promastigote surface protease, or gp63, which is also a dominant surface antigen; this enzyme is thought to be involved in binding to the macrophage via the cell receptors for mannose and fucose and for the third component of complement. Another important surface component is the lipophosphoglycan, consisting of a series of phosphorylated disaccharides linked to a novel lipid anchor in the membrane. This is also released from the parasite surface and was earlier identified as a highly immunogenic antigen excreted into culture medium. It can activate complement and may in this way promote attachment of the parasite to the macrophage. Other surface structures include the acid phosphatase, a glyco-inositol phospholipid, another glycolipid, and membrane proteins of 80 and 17 kilodaltons. All of these may play a role in attachment of the promastigote to the macrophage host cell, as well as in the survival of the amastigote within the macrophage, perhaps by inhibiting the activities of destructive enzymes. The roles in infectivity of these components of the Leishmania surfaces and their interactions with the various receptors on macrophages are discussed. The immune responses induced by these and other parasite antigens during infections in humans and experimental animals are also described briefly, especially those responses that may contribute to protection from infection, or to diagnosis and epidemiology.     

Mitogen activated protein kinase-dependent activation of c-Jun and c-Fos is required for neuronal differentiation but not for growth and stress response in PC12 cells

MAPK-dependent activation of AP-1 protein c-Jun is involved in PC12 cell differentiation and apoptosis. However, the role of other AP-1 proteins and their connection to MAPKs during growth, differentiation and apoptosis has remained elusive. Here we studied the activation of AP-1 proteins in response to ERK, JNK, and p38 signaling upon NGF, EGF and anisomycin exposures. All treatments caused different kinetics and strength of MAPK and AP-1 activities. NGF induced persistent ERK and AP-1 activities, whereas upon EGF and anisomycin exposures, their activities were only weakly and transiently induced. The sustained AP-1 activity was associated with concomitant c-Fos and c-Jun expression and phoshorylation, which were JNK and ERK dependent. While inhibition of the ERK, JNK, and p38 activities partially prevented AP-1 activity and suppressed differentiation, none of them was required for anisomycin-induced apoptosis. The importance of c-Fos and c-Jun as mediators of differentiation was demonstrated by the findings that the corresponding siRNAs suppressed NGF-induced neurite outgrowth. However, the capacity of c-Fos to promote differentiation required cooperation with Jun proteins. In contrast, Fra-2 expression was not required for the differentiation response. Together, the results show that sustained c-Jun and c-Fos activities mediate MAPK signaling and are essential for differentiation of PC12 cells.     

Mammalian antimicrobial peptide influences control of cutaneous Leishmania infection

Cathelicidin-type antimicrobial peptides (CAMP) are important mediators of innate immunity against microbial pathogens acting through direct interaction with and disruption of microbial membranes and indirectly through modulation of host cell migration and activation. Using a mouse knock-out model in CAMP we studied the role of this host peptide in control of dissemination of cutaneous infection by the parasitic protozoan Leishmania. The presence of pronounced host inflammatory infiltration in lesions and lymph nodes of infected animals was CAMP-dependent. Lack of CAMP expression was associated with higher levels of IL-10 receptor expression in bone marrow, splenic and lymph node macrophages as well as higher anti-inflammatory IL-10 production by bone marrow macrophages and spleen cells but reduced production of the pro-inflammatory cytokines IL-12 and IFN-γ by lymph nodes. Unlike wild-type mice, local lesions were exacerbated and parasites were found largely disseminated in CAMP knockouts. Infection of CAMP knockouts with parasite mutants lacking the surface metalloprotease virulence determinant resulted in more robust disseminated infection than in control animals suggesting that CAMP activity is negatively regulated by parasite surface proteolytic activity. This correlated with the ability of the protease to degrade CAMP in vitro and co-localization of CAMP with parasites within macrophages. Our results highlight the interplay of antimicrobial peptides and Leishmania that influence the host immune response and the outcome of infection.     

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effect cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.