Inhibition of Tumor Progression Locus 2 Protein Kinase Decreases Lipopolysaccharide-Induced Tumor Necrosis Factor .ALPHA. Production Due to the Inhibition of the Tip-Associated Protein Induction in RAW264.7 Cells

Abstract

The activation of mitogen-activated protein kinases (MAPKs) is critically involved in inflammatory events through mediation of the production of various inflammatory cytokines. The Tpl2 (tumor progression locus 2)-MEK (MAPK/ERK kinase)-ERK (extracellular signal-regulated kinase) signaling pathway plays an essential role in the production of tumor necrosis factor a (TNFa) in macrophages stimulated with lipopolysaccharide (LPS). Here, we studied the molecular mechanisms of Tpl2-mediated TNFa production using a potent Tpl2 kinase inhibitor, 1,7-naphtyridine-3-carbonitrile, and LPS-stimulated RAW264.7 cells. This inhibitor was effective in suppressing the in vitro Tpl2 kinase activity, and caused a significant reduction in TNFa production via specific suppression of the phosphorylation of MEK and ERK but not that of p38 and c-Jun N-terminal kinase (JNK). A p38 inhibitor, SB203580, also inhibited the TNFa production dose-dependently. Although the TNFa mRNA level was not altered by either inhibitor, the Tpl2 inhibitor increased the nuclear TNFa mRNA level, while decreasing that in the cytoplasm. Tip-associated protein (TAP), a key molecule in the nucleocytoplasmic transport of TNFa mRNA, was up-regulated by LPS, but this increase was impaired by the Tpl2 inhibitor. In all cases, SB203580 was without effect in the presence of LPS. These results suggest that the LPS-induced TNFa production via the Tpl2-MEK-ERK signaling pathway is regulated by changing the TAP level at the nucleocytoplasmic transport level. These results improve understanding of TNFa regulatory mechanisms and might provide a new therapeutic strategy against inflammatory diseases.

Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

“…Initial experiments suggested that TPL-2/ERK might regulate TNF production at a post-transcriptional level by promoting the export of Tnf mRNA from the nucleus [29]. In apparent support of this hypothesis, a recent study using TPL-2 and MEK inhibitors showed that TPL-2/ERK controls, at the post-transcriptional level, the expression of the nuclear mRNA export receptor Tip-associated protein (TAP) [68]. However, pre-TNF protein levels in LPS-stimulated primary macrophages are minimally affected by pharmacological MEK inhibition or TPL-2 deficiency, indicating that defective intracellular TNF mRNA transport is unlikely to explain the failure of Tpl2 −/− macrophages to produce TNF [67].…”

Pharmacological Inhibition of TPL2/MAP3K8 Blocks Human Cytotoxic T Lymphocyte Effector Functions

“…Since the human and murine Tpl2 gene products are 93% identical at the amino acid level [22], we utilized a commercially available small molecule inhibitor of Tpl2 function (Tpl2 inhibitor) to test the role of Tpl2 in regulating cytokine secretion in CD8 + T cells from both species. This inhibitor is cell permeable, reversible, and blocks Tpl2 activation in an ATP-dependent manner [23], [24]. Further, this Tpl2 inhibitor has been shown to block TNF-α secretion from murine bone marrow derived macrophages, as well as human PBMCs responding to LPS stimulation [24]–[26].…”

“…This inhibitor is cell permeable, reversible, and blocks Tpl2 activation in an ATP-dependent manner [23], [24]. Further, this Tpl2 inhibitor has been shown to block TNF-α secretion from murine bone marrow derived macrophages, as well as human PBMCs responding to LPS stimulation [24]–[26]. Thus, the commercially available Tpl2 inhibitor can block Tpl2 activation in both mouse and human innate immune cells.…”

“…Many of these inhibitors were developed to target protein kinases since the discovery of their ability to block not only the relevant kinase activity but also the production of pro-inflammatory cytokines such as IL-1β and TNF-α[32]. Recently, Tpl2 has been identified as a ‘druggable’ target due to its role in activating innate immune cells to produce TNF-α in mice and human [23], [24], [33]. Therapeutic use of Tpl2 inhibitors has been suggested to impede prolonged and uncontrolled inflammation in diseases such as rheumatoid arthritis and inflammatory bowel disease based on preliminary studies in mice [26], [34]–[36].…”

The role of tumor progression locus 2 protein kinase in glial inflammatory response

“…; Hirata et al . ). The kinase has also been shown to regulate secretion of several other cytokines and chemokines in different cell systems (Vougioukalaki et al .…”