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. 2004 Jul;24(14):6488-500.
doi: 10.1128/MCB.24.14.6488-6500.2004.

The IkappaB kinase complex and NF-kappaB act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of AP-1

Daniel Krappmann  1 , Elmar Wegener, Yoshiaki Sunami, Meral Esen, Andreas Thiel, Benjamin Mordmuller, Claus Scheidereit
Affiliations

The IkappaB kinase complex and NF-kappaB act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of AP-1

Daniel Krappmann et al. Mol Cell Biol. 2004 Jul.

Abstract

Toll-like receptors (TLRs) recognize conserved products of microbial pathogens to initiate the innate immune response. TLR4 signaling is triggered upon binding of lipopolysaccharides (LPS) from gram-negative bacteria. Using comparative gene expression profiling, we demonstrate a master regulatory role of IkappaB kinase (IKK)/NF-kappaB signaling for immediate-early gene induction after LPS engagement in precursor B cells. IKK/NF-kappaB signaling controls a large panel of gene products associated with signaling and transcriptional activation and repression. Intriguingly, the induction of AP-1 activity by LPS in precursor B cells and primary dendritic cells fully depends on the IKK/NF-kappaB pathway, which promotes expression of several AP-1 family members, including JunB, JunD, and B-ATF. In pre-B cells, AP-1 augments induction of a subset of primary NF-kappaB targets, as shown for chemokine receptor 7 (CCR7) and immunoglobulin kappa light chain. Thus, our data illustrate that NF-kappaB orchestrates immediate-early effects of LPS signaling and controls secondary AP-1 activation to mount an appropriate biological response.

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Figures

FIG. 1.
FIG. 1.
Analysis of LPS and IKKγ-dependent gene regulation by Northern blotting. 70Z/3, 1.3E2, or 1.3E2 IKKγ/PKCθ-expressing cells were stimulated for 2 h with PMA (P), LPS (L), LPS in the presence of CHX (C), or CHX alone. As a control, whole-cell extracts were prepared and analyzed by EMSA for NF-κB DNA binding activity (upper left panel). In parallel, total RNA was extracted and subjected to Northern blotting with cDNA probes for the indicated genes. Membranes were repetitively used after stripping, and equal loading was verified by using a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe.
FIG. 2.
FIG. 2.
LPS and NF-κB-dependent target gene profiling in murine pre-B cells. Either mock-infected (control retrovirus) cells or a clone of 70Z/3 cells infected with IκBαΔN-expressing retrovirus (IκBαΔN retrovirus) were left untreated or stimulated with LPS for 90 min before total RNA was extracted and subjected to microarray analysis. Shown are the baseline signals (first column), the signal intensity after LPS stimulation (second column), the induction (fold) (third column), and the change call comparing LPS to baseline signals (fourth column) for 70Z/3 control (left) or IκBαΔN (right). A graphical representation of the severalfold inductions for each gene in 70Z/3CV cells (black) and 70Z/3 IκBΔN cells (gray) is shown. Genes were sorted as shown in Table 1. Genes in gray boxes are refractory to IκBαΔN-mediated LPS induction. Note that basal CD40 expression is highly reduced in IκBαΔN-expressing cells. The deceptive increase in 70Z/3 IκBαΔN cells after LPS treatment results from the very low expression level and could not be verified by Northern blotting (data not shown).
FIG. 3.
FIG. 3.
Analysis of LPS-mediated NF-κB-independent gene regulation in 70Z/3 cells. (A and B) CV- or IκBΔNV-infected 70Z/3 cells (a pool or two independent clones, as indicated were treated with LPS for the times indicated, and RNA was prepared and subjected to Northern blotting as indicated. LPS-mediated induction of IκBɛ, PIM1, MKP-7, MIP-1α, CHOP10, PAC1, and GAPDH was analyzed.
FIG. 4.
FIG. 4.
NF-κB is required for LPS-induced AP-1 activation in pre-B cells and dendritic cells. (A) Untreated 70Z/3, CV- and IκBΔN-infected 70Z/3, or 1.3E2 cells were stimulated with LPS for 12 h, and AP-1 DNA binding activity was determined by EMSA. (B) Human primary dendritic cells were mock transfected or transfected with IκBαΔN expression vector and afterwards stimulated with LPS for 1 and 24 h. AP-1 DNA binding activity was analyzed by EMSA. IκBαΔN expression resulted in the complete loss of NF-κB DNA binding activity in dendritic cells (compare results to reference ; data not shown). n.s., nonspecific.
FIG. 5.
FIG. 5.
IKK/NF-κB activation regulates LPS induction of AP-1 family members in pre-B cells. (A) 70Z/3 or 1.3E2 cells or 1.3E2IKKγ/PKCθ-expressing cells were stimulated as described in the Fig. 1 legend and analyzed for expression of Jun/Fos/ATF/MAF family members as indicated. (B and C) 70Z/3 cells were stimulated with LPS or PMA (B) or UV light (C) for the times indicated, and whole-cell extracts were analyzed for NF-κB and AP-1 DNA binding and JunB, JunD, and p65 expression levels by Western blotting. The migrations of two AP-1 DNA complexes that have a differential mobility are indicated (C1 and C2). (D) AP-1 DNA binding activity after UV light (3 h) or LPS (8 h) stimulation was subjected to supershifting analysis with anti-JunB, anti-JunD, or anti-cFos antibody. Mobilities of AP-1 C1, AP-1 C2, and supershifted complexes (ss) are indicated. (E) 70Z/3 cells were stimulated with LPS or UV light for 4 h in the absence or presence of CHX. NF-κB and AP-1 DNA binding activity were analyzed by EMSA. n.s., nonspecific.
FIG. 6.
FIG. 6.
AP-1 activation in response to LPS correlates with NF-κB-mediated up-regulation of JunB, JunD, and B-ATF. (A) 70Z/3 cells infected with CV, Flag-A-Fos virus (AFosV), or Flag-IκBαΔNV(IκBΔNV) and 1.3E2 cells were stimulated for 0, 2, 4, and 8 h with LPS, and extracts were analyzed for NF-κB and AP-1 DNA binding activity by EMSA and for JunB, JunD, JNK1, ERK1/2, IκBα, Flag-IκBαΔN, and FlagA-Fos expression by Western blotting. (B) Cells were treated as described above (A), and total RNA was extracted and analyzed for JunB, JunD, B-ATF, and GAPDH expression by Northern blotting. (C) The different cell clones were stimulated with UV light, and extracts were prepared after 0, 1.5, 3, and 6 h and analyzed for NF-κB and AP-1 activity by EMSA.
FIG. 7.
FIG. 7.
NF-κB and AP-1 cooperate in transcriptional and biological responses to LPS. (A) 70Z/3CV-, Flag-A-FosV-, and IκBαΔNV-infected cells were stimulated with LPS for 0, 2, 4, and 8 h, and total RNA was prepared and analyzed for mRNA expression of the indicated genes by Northern blotting. (B) Cells were treated as described above (A), and DNA binding activity of Oct-1 and Oct-2 was determined by EMSA. OCT-2 and Igκ light chain expression was analyzed by Western blotting (lower panels). (C) Either parental 70Z/3 cells or control (pLXSN) infected and A-Fos- and IκBαΔN-infected 70Z/3 cells were treated for 20 h with LPS (1 μg/ml) or IFN-γ, and sIgM expression was examined by flow cytometry.
FIG. 8.
FIG. 8.
Schematic representation of primary LPS response genes in 70Z/3 pre-B cells. Primary transcriptional response genes can be grouped according to the requirement of IKK and/or NF-κB for gene induction. Whereas the largest group of genes requires classical IKK and NF-κB signaling (center), induction of a single gene is completely independent of IKK and NF-κB activation (left). Induction of a few genes depends on IKK activation but unknown transcription factors, indicating that the IKK complex may regulate an alternative NF-κB independent pathway(s). Depicted are some representative genes.
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