Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides

Abstract

The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

FIGURE 1.

Inhibition of inflammation-induced but not basal cytokine production by NBD peptides. PBMC were preincubated with the indicated concentrations of NBD peptides (A and B) or PHA-408 (E and F) for 2 h prior to stimulation with LPS (A and E) or vehicle (B and F) for 18 h. RASF were preincubated with the indicated concentrations of NBD peptides (C and D) for 2 h prior to stimulation with IL-1β (C) or vehicle (D) for 18 h. Filled and open columns represent mutant (Mut) NBD peptides and WT NBD peptides, respectively. Data are expressed as percentage of LPS- or IL-1β-stimulated cells or untreated cells (Basal). The data represent the mean ± S.D. of at least three independent determinations from one representative experiment. The experiments were performed at least twice with similar results. **, p < 0.005, significant differences compared with LPS or IL-1β-stimulated cells. NBD peptides used were IKK-2 11-mer.

FIGURE 2.

Inhibition of IL-1β-induced IKK-2 phosphorylation and activity in RASF by NBD peptides. RASF were stimulated for 10 min with 1 ng/ml IL-1β following cell treatment for 1 h with NBD peptides (A and B) or PHA-408 (D, closed circles) or were stimulated for 10 min with 1 ng/ml IL-1β preceding the treatment of cell lysates for 1 h with NBD peptides (C) or PHA-408 (D, open circles). Cell lysates from the same experiment (A and B) or separate experiments (C and D) were used for Western blot analysis (A) or kinase activity (B–D). The data (B–D) represent the mean ± S.D. of at least three independent determinations. The experiments were repeated at least twice with similar results. *, p < 0.05, significant differences compared with IL-1β-stimulated cells. NS, nonstimulation. NBD peptides used were IKK-2 11-mer. Mut, mutant.

FIGURE 3.

Lack of inhibitory effects of NBD peptides on NF-κB signaling intermediates upstream of the IKK complex and MAPK pathway. RASF were treated with the indicated concentrations of NBD peptides (A and C) or PHA-408 (A) or IRAK-4 inhibitor (B) for 30 min, followed by stimulation with 1 ng/ml IL-1β for 10 min. Cell lysates were analyzed by Western blot. Tubulin was used as a control for protein loading. NS, nonstimulation. NBD peptides used were IKK-2 11-mer. Mut, mutant.

FIGURE 4.

Differential potency of NBD-containing peptides. Purified recombinant GST-NEMO-(2–200) (2 nm) was incubated with purified recombinant C terminus FLAG-tagged full-length IKK-2 (2 nm). The ability of WT or mutant (W739A and W741A) NBD peptides to disrupt NEMO-IKK-2 binding was measured by Alphascreen. The data represent the mean ±S.E. of four independent experiments.

FIGURE 5.

Disruption of TRAF-6-dependent NEMO-IKK-2 interactions in cell-free system by NBD peptides. A, HeLa cell extracts were prepared and treated with 0, 0.25, 0.5, 1, or 2 mm ATP with or without 60 nm exogenous TRAF6. B and C, HeLa cell extracts were treated with 0.1 or 1% DMSO (vehicle), PHA-408, WT, or mutant NBD peptides (0.1 or 1% DMSO, final concentration) in the absence or presence of 2 mm ATP and 60 nm exogenous TRAF6 and were analyzed by Western blot. The experiments were repeated three times with similar results. HSP27 was used as a control for protein loading. NBD peptides used were IKK-2 11-mer.

FIGURE 6.

Release of IKK-2 from an IKK complex in RASF by NBD peptides. A, RASF were pretreated with 150 μm of WT or mutant (Mut) NBD peptides for 30 min prior to a 10-min treatment with 1 ng/ml IL-1β. The IKK complex was immunoprecipitated (IP) with anti-NEMO mAbs, and the immunoprecipitated material (top) as well as the original cell lysates (Inputs, bottom) were subjected to Western blot analysis. RASF lysates were also subjected to native PAGE and Western blot analysis using anti-NEMO mAbs (B, top). The band corresponding to the IKK complex was excised, and the proteins were extracted in 2× reducing SDS sample loading buffer, resolved using SDS-PAGE, and subjected to Western blot analysis using anti-IKK-1/2 Abs (B, middle) or anti-NEMO mAbs (B, bottom). C, RASF cells were preincubated with the indicated concentrations of WT or mutant NBD peptides for 30 min before stimulation with IL-1β for 10 min, and samples were analyzed by Western blot. Immunoreactive bands were visualized using the Li-Cor Odyssey Infrared Imaging System. The experiments were repeated 2–4 times with similar results. NS, nonstimulation. NBD peptides used were IKK-2 11-mer.

FIGURE 7.

Disruption of NEMO-IKK-2 interactions in RASF by NBD peptides. RASF cells were incubated with the indicated concentrations of WT or mutant (mut) NBD peptides for 30 min before stimulation with 1 ng/ml IL-1β for 15 min. NEMO-IKK2 interactions were visualized at ×20 magnification (A) using PLA as detailed under “Experimental Procedures.” The signal was also quantified from an average of nine fields, which was then divided by the numbers of cells in each field, thus giving a signal average/cell (B). The experiments were repeated three times with similar results. The data represent the mean ± S.E. **, p < 0.005, significant differences compared with IL-1β-stimulated cells. NBD peptides used were IKK-2 11-mer.