Regulation of NF-κB Circuitry by a Component of the Nucleosome Remodeling and Deacetylase Complex Controls Inflammatory Response Homeostasis

Suresh B Pakala,Tri M Bui-Nguyen,Sirigiri Divijendra Natha Reddy,Da-Qiang Li,Shaohua Peng,Suresh K Rayala,Richard R Behringer,Rakesh Kumar

doi:10.1074/jbc.m110.139469

Abstract

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-kappaB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-kappaB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1.HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1(-/-) mice were high compared with wild-type mice. Accordingly, MTA1(-/-) mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-kappaB circuitry, in the regulation of inflammatory responses.

Highlights

LPS-stimulated macrophages impairs NF-␬B signaling and control of nucleosome remodeling coregulators and comexpression of inflammatory molecules
MTA1 Is an Inflammation-inducible Gene—During an investigation involving treatment of primary murine peritoneal macrophages with Escherichia coli LPS, we discovered unexpectedly that LPS stimulation of macrophages results in a substantial induction of MTA1 mRNA but not MTA2 mRNA or MTA3 mRNA as measured by quantitative realtime PCR (q-PCR) (Fig. 1A)
The results suggest that MTA1 may be an inflammation-inducible gene

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Cell Culture—Antibodies against MTA1 (A300280A), MTA2 (A300 –395A), MTA3 (A300 –160A), and RNA polymerase II (pol II) (A300 – 653A) were purchased from Bethyl Laboratories (Montgomery, TX); and HDAC2 (catalog no. sc-9959), NF-␬B p65 (p65) (catalog no. sc-372), phosphoNF-␬B p65 (catalog no. sc-33020), NF-␬B p50 (catalog no. sc-7178), phospho-NF-␬B p50 (catalog no. sc-33022-R), and NF-␬B p65 (catalog no. 286-H) X (catalog no. sc-7151 X) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Q-PCR was performed with the gene-specific primers listed in supplemental Table 1. Mutations in the NF-␬B consensus sequence of murine Mta[1] promoter were created by using the site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) using the primers listed in supplemental Table 2. Immunoprecipitation and Immunoblot Analysis—Cell lysates were prepared in radioimmune precipitation assay lysis buffer, and Western blot analysis was performed as described previously (20). 1 mg of cell lysates was incubated with MTA1 or pol II or HDAC2 or p65 antibody and agarose beads with constant rotation followed by extensive washing (20 mM HEPES (pH 7.6, 150 mM KCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 8% glycerol). With the DNA isolated at the end of the ChIP analysis, PCR was performed using the primers mentioned in supplemental Table 3. Statistical analysis of the data were performed by using Student’s t test

RESULTS AND DISCUSSION

EXPRESSION OF CONCERN

ADDITIONS AND CORRECTIONS