Inhibition of NF-κB by a TAT-NEMO–binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis

Abstract

Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-B (NF-B) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-B in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selec- IntroductionThe introduction of biologically active peptides into human polymorphonuclear neutrophils (PMNs) may help in clarifying intracellular signal transduction and ultimately could have therapeutic implications. However, all peptide delivery methods available thus far are inefficient. Previously, peptide transduction domains (PTDs) were identified that shuttle even large proteins in excess of 100 000 Da into mammalian cells in vitro and in vivo. 1 These "Trojan-horse" peptides include the homeodomain of Antennapedia (a Drosophila transcription factor), a short amino acid sequence of HIV-1, and the herpes simplex virus 1 (HSV-1) structural protein VP22. [2][3][4] We explored the use of an 11-amino acid sequence (amino acids 47-57) from the HIV TAT protein (HIV-TAT) in PMNs to target nuclear factor-B (NF-B).NF-B is a transcription factor controlling gene expression during inflammation, immunity, cell proliferation, stress response, and apoptosis. 5-8 NF-B is activated by many agents including cytokines, viral infection, UV radiation, and free radicals. 9 In unstimulated cells, NF-B is sequestered in the cytoplasm by tightly bound inhibitors (IB␣, IB␤, IB⑀). The inhibitors are phosphorylated and rapidly degraded, allowing NF-B to translocate into the nucleus and activate target genes. IB␣ is phosphorylated on serine residues by the multicomponent IB kinase (IKK) containing 2 catalytic subunits (IKK␣ and IKK␤) and one regulatory subunit (IKK␥). [10][11][12][13] In contrast to other cell types, the role of NF-B in PMNs is incompletely characterized due to rapid NF-B degradation by proteolytic enzymes, difficulties with PMN transfection, and the lack of specific NF-B inhibitors. Previous studies with pharmacologic compounds, such as pyrrolidine dithiocarbamate (PDTC), SN50, and gliotoxin, have suggested that NF-B is involved in regulating PMN apoptosis. [14][15][16] However, the specificity of these agents has been questioned. [17][18][19] We used a highly specific small peptide to block the interaction of IKK␥ with the IB kinase complex (IKK). 20 We generated a linear 2-domain peptide containing the NEMO-binding domain (NBD) to specifically block NF-B and the TAT-PTD to shuttle NBD into PMNs. Materials and methods MaterialsGranulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 8 (IL-8) were obtained from R&D Systems (Wiesbaden-Nordenstedt, Germany). Lipopolysaccharide (LPS), tumor necrosis factor ␣ (TNF-␣), dexamethasone (DEX), Ficoll-Hypaque, and pr...

Febrile temperatures control antineutrophil cytoplasmic autoantibody–induced neutrophil activation via inhibition of phosphatidylinositol 3‐kinase/Akt

“…Further investigations are needed to address these differential effects of heat. We reported earlier that NF-B controls neutrophil survival, and that moderate heat in vitro abrogates the NF-B-dependent signals in neutrophils after challenge with TNF␣ and lipopolysaccharide (LPS) (17,18,29). We observed the same NF-B inhibition in neutrophils isolated from mice that were exposed to 40.5°C for 30 minutes (18), suggesting that these effects also occur under in vivo conditions.…”

Amelioration of acute inflammation by systemic administration of a cell‐permeable peptide inhibitor of NF‐κB activation

“…Other investigators have reported that NBD peptide can also inhibit NF-B activation and NF-B-dependent gene expression in endothelial cells (21) and neutrophils (22). Investigators in our group and others have recently shown that continuous administration of the NBD peptide can block osteoclastogenesis and reverse inflammatory bone loss (14,23).…”

NF-κB and the regulation of hematopoiesis

“…11 More recently, inhibition of IKK/Nemo interaction with Nemo-binding domain (NBD) peptide, a potent inhibitor of the classical NF-kB pathway, was shown to increase constitutive cell death which was also observed following induction with LPS. 12 Figure 2 NF-kB in granulocyte and osteoclast differentiation. (a) In the bone marrow, granulocytes undergo differentiation from a promyelocyte stage (PM) to a metamyelocyte (MY) stage and finally into bone marrow polymorphonuclear neutrophilic granulocytes (bm-PMNs).…”