Abstract
Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.
Highlights
We investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-␣ (TNF-␣) and interleukin (IL)12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs
We investigated the mechanisms that underlie the differential regulation of TNF-␣ and IL-12 production using macrophages deficient in MK2, one of the signaling molecules, which has been described to be downstream of p38 [21]
Because IL-12 is a key proinflammatory cytokine thought to be involved in controlling malaria infection and in disease pathogenesis [9], we studied mechanisms that underlie the observed marked increase in IL-12 production by MK2Ϫ/Ϫ macrophages stimulated with GPIs
Summary
Materials—SB203580, U0126, and PD98059 were from Calbiochem. Actinomycin D was from Sigma. We the TNF-␣ transcript was maximal at 4 h, with a decreased analyzed the production of TNF-␣ and IL-12 by cells pretreated level at 8 h (Fig. 3B) These data could be explained by difwith the ERK and p38 MAPK inhibitors and stimulated with the ferent kinetics of TNF-␣ mRNA synthesis or, more likely, by other TLR ligands, LPS, Pam3CSK4, and CpG To analyze whether increased NF-B binding to the gene promoter is responsible for the enhanced mRNA synthesis by MK2Ϫ/Ϫ macrophages stimulated with GPIs compared with WT cells, we performed EMSA using TNF-␣ and IL-12p40 promoter-specific DNA probes for NF-B binding. Inhibition of p38 activity and ERK activation weakened the binding in both WT and MK2Ϫ/Ϫ macrophages, and the results agree with the TNF-␣ mRNA and protein levels in these cells. This is consistent with the requirement of NF-B p50 homodimer forming a complex with
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