NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway

Abstract

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-kappaB pathway. It has been suggested through indirect studies that NF-kappaB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK-/- mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense.

FIG. 1.

NIK interacts with Nod2. (A) MDP induces p100 processing in a Nod2-dependent manner. HEK 293 cells were transfected with 100 ng of p100 expression construct in combination with different amounts of Myc-tagged Nod2 expression plasmid. Cells were stimulated with 20 μg/ml MDP for 24 h, and p100 processing was detected with anti-NF-κB2 monoclonal antibody, which recognizes both p100 and p52. Myc-tagged Nod2 protein was detected by anti-Myc immunoblotting. The film was scanned, and the band intensity of p52 was calibrated as described in Materials and Methods. (B and C) Interaction of NIK with Nod2. HEK 293 cells were cotransfected with Myc-tagged Nod2 (B) or truncated Myc-tagged Nod2ΔCARD (C) and with FLAG-tagged NIK, RIP, RICK, and p38 expression plasmids. The cell lysates were immunoprecipitated with rabbit polyclonal anti-FLAG antibody overnight. The resulting immune complexes were fractionated by SDS-PAGE, transferred to membranes, and subsequently probed with monoclonal anti-Myc antibody. The lysates (10% of immunoprecipitation input) derived from each transfection were also loaded in gels as control and immunoblotted using anti-FLAG and anti-Myc antibodies.

FIG. 2.

Essential roles of Nod2 and NIK in mediating responses to MDP and LPS. (A) Nod2 mediates MDP-induced IκBα degradation and p38 phosphorylation. Nod2^+/+ and Nod2^−/− BMDM were stimulated with MDP (20 μg/ml) or LPS (10 ng/ml) for different times. Cell lysates were prepared, and IκBα degradation and p38 phosphorylation were detected by Western blotting using specific antibodies. NIK (B and C) and Nod2 (D) are required for synergistic production of p52 by MDP and LPS. BMDM from aly/+ and aly/aly mice lacking functional NIK (B) or BMDM from NIK^+/+ and NIK^−/− mice (C) and also BMDM from WT and Nod2^−/− mice (D) were stimulated by various concentrations of LPS in the presence or absence of MDP (20 μg/ml) for 24 h, and processing of p100 was analyzed by Western blotting using anti-NF-κB2 antibody recognizing both p100 and p52. The films were scanned, and the band density for p52 was normalized to the tubulin constitutive control.

FIG. 3.

NIK regulates LPS- and CpG-induced p52 nuclear translocation. BMDM were stimulated with LPS (100 ng/ml) and CpG (0.2 μM) for the indicated time points. Cells were lysed, and nuclear fractions were subjected to Western blot analysis with the indicated antibody.

FIG. 4.

Impaired BLC gene induction in NIK^−/− cells. BMDM were stimulated for 24 h with LPS (0.02 to 0.4 ng/ml) and 20 μg/ml MDP. Gene induction (mRNA) was determined by real-time PCR. The relative expression of genes was normalized to the level of 18s RNA. Data are presented as means ± standard deviations of triplicate wells. **, P < 0.01 for cytokine concentrations from knockout mice compared with data from wild-type mice. Results are representative of two independent experiments.

FIG. 5.

Diminished BLC secretion in NIK^−/− cells. BMDM were stimulated for 24 h (LPS, 0.04 ng/ml; MDP, 20 μg/ml) and the production of cytokine and chemokine were determined by specific ELISA. Data are presented as means ± standard deviations of triplicate wells. *, P < 0.05, and **, P < 0.01, for cytokine concentrations from knockout mice compared with data from wild-type mice. Results are representative of three independent experiments.

FIG. 6.

Helicobacter infection induced BLC gene activation and chemokine secretion in a NIK-dependent manner. (A) BMDM were infected with H. felis at the indicated multiplicity of infection (MOI) for 24 h, and induction of cytokines was detected with specific ELISA kits. Data are presented as means ± standard deviations of triplicate wells. Results are representative of three independent experiments. (B) BMDM were infected with H. felis at the indicated multiplicity of infection (MOI) for 24 h. Relative gene expression was detected by real-time PCR. Data are presented as means ± standard deviations of triplicate wells. **, P < 0.01 for cytokine concentrations from knockout mice compared with data from wild-type mice. Results are representative of three independent experiments.