IκBζ Is a Transcriptional Key Regulator of CCL2/MCP-1

Abstract

CCL2, also referred to as MCP-1, is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive CCL2 secretion has been linked to many inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that IκBζ, an atypical IκB family member and transcriptional coactivator required for the selective expression of a subset of NF-κB target genes, is a key activator of the Ccl2 gene. IκBζ-deficient macrophages exhibited impaired secretion of CCL2 when challenged with diverse inflammatory stimuli, such as LPS or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of IκBζ. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that IκBζ is directly recruited to the proximal promoter region of the Ccl2 gene and is required for transcription-enhancing histone H3 at lysine-4 trimethylation. Finally, IκBζ-deficient mice showed significantly impaired CCL2 secretion and monocyte infiltration in an experimental model of peritonitis. Together, these findings suggest a distinguished role of IκBζ in mediating the targeted recruitment of monocytes in response to local inflammatory events.

The Atypical Inhibitor of NF-κB, IκBζ, Controls Macrophage Interleukin-10 Expression

“…MEFs and Raw264.7 cells were cultured in DMEM/high glucose, 10% FCS, and MycoZapPlusCL (Lonza). Raw264.7/TetOn-IB cells were described previously (15) and cultured in the presence of 1 mg/ml neomycin (G418, Lonza). Cells were seeded at a density of 10 5 cells/cm 2 24 h before the experiment.…”

“…Expression plasmids for Nfkb1 (p50) and IB have been described previously (15,17). For reporter gene assays, the following luciferase promoter constructs were used: pGL2basic-Il10 and truncation mutants (28), pGL3basic-Lcn2 (29), and pGL3basic-Il6 and pGL3basic-Elam1 (16).…”

“…The atypical IB protein IB has been recently implicated in differential NF-B target gene expression in macrophages (15,16) even though its physiological function remains largely unknown. The IB-encoding Nfkbiz gene is rapidly induced as a primary NF-B response gene by various inflammatory stimuli and, through association with the NF-B subunit p50, is thought to exert its transcription-enhancing activity on secondary response genes mainly at the level of chromatin remodeling (12,17,18).…”

“…Recently, we showed that in macrophages, expression of CCL2 strictly depends on the presence of IB and, consequently, that expression of CCL2 is abolished in macrophages from IB-deficient mice (15). Furthermore, several other proinflammatory gene products, including IL-6, IL12p40, IL-17, IFN␥, and GM-CSF, have been found to be regulated by IB (16, 19 -23).…”

“…Furthermore, several other proinflammatory gene products, including IL-6, IL12p40, IL-17, IFN␥, and GM-CSF, have been found to be regulated by IB (16, 19 -23). Intriguingly, however, despite impaired expression of these pro-inflammatory cytokines, Nfkbiz Ϫ/Ϫ mice display a pro-inflammatory phenotype characterized by periocular inflammation, inflammatory skin alterations, and an M1 hyperpolarized macrophage state (15,16,24,25). In view of the phenotype of Nfkbiz Ϫ/Ϫ mice, we therefore investigated a potential role of IB for the regulation of IL-10 as an essential anti-inflammatory cytokine.…”