Abstract
Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression.
Highlights
The transcription factor nuclear factor kappa B (NF-kB) regulates the expression of a number of immunological mediators, including the chemokines, cytokines, adhesion molecules and enzymes that produce secondary inflammatory mediators such as nitric oxide synthase [1]
We have demonstrated the activation of NF-kB transcriptional repressor homodimer ( p50/p50) in L. amazonensisinfected macrophages and the downregulation of inflammatory mediators such as nitric oxide synthase (iNOS) expression in infected macrophages associated with the p50/p50 formation [13]
To verify whether the nuclear levels of p50 were augmented in infected macrophages, immunofluorescence assay demonstrated the nuclear translocation of p50 after the infection of macrophages with L. amazonensis for 1 h and 5 h
Summary
The transcription factor nuclear factor kappa B (NF-kB) regulates the expression of a number of immunological mediators, including the chemokines, cytokines, adhesion molecules and enzymes that produce secondary inflammatory mediators such as nitric oxide synthase (iNOS) [1]. The NF-kB family comprises five different members containing the Rel homology domain that may originate homo- or heterodimers, NF-kB1 (p105/p50), NF-kB2 (p100/p52), RelA (p65), RelB and c-Rel [2]. NF-kB1 and NF-kB2 are synthesized as the large p105 and p100 precursors, which are post-translationally processed to the DNA-binding subunits p50 and p52, respectively [3]. The canonical activation of NF-kB involves the phosphorylation of IkB (NF-kB inhibitor) by the IkB kinase (IKK) complex, leading to proteasome-mediate IkB degradation and nuclear translocation of NF-kB dimer RelA–p50 [4]. NF-kB is frequently activated during infections and.
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