The neuropeptide calcitonin gene-related peptide causes repression of tumor necrosis factor-alpha transcription and suppression of ATF-2 promoter recruitment in Toll-like receptor-stimulated dendritic cells

Abstract

Sensory nerves may dampen inflammatory processes through the release of the neuropeptide calcitonin gene-related peptide (CGRP). CGRP mediates immunosuppressive activities through up-regulation of interleukin-10 or, alternatively, through an interleukin-10-independent pathway that is associated with rapid induction of the transcriptional inducible cAMP early repressor (ICER). In this work, we further investigated the molecular mechanisms of immune modulation by CGRP. Using TLR2-stimulated dendritic cells, we show that inhibition of tumor necrosis factor-alpha production by CGRP is dependent on up-regulation of endogenous ICER. Dendritic cell expression of ICER was selectively induced by CGRP and elevation of cellular cAMP levels but not by numerous pro- and anti-inflammatory cytokines. Treatment of dendritic cells with CGRP did not interfere with the induction of Tnfa gene expression but caused premature repression of TLR2-induced transcriptional activity. ATF-2 was rapidly phosphorylated and recruited to the Tnfa promoter following ligation of TLR2. Concomitant administration of CGRP completely prevented binding of ATF-2 to the Tnfa promoter, whereas recruitment of ICER was markedly elevated. In contrast, CGRP did not influence TLR2-stimulated binding of NF-kappaB p65. Together, these results are consistent with a model suggesting that CGRP causes rapid up-regulation of ICER, which in turn competes with ATF-2 for binding to the Tnfa promoter, leading to repression of gene expression.

FIGURE 1.

Inhibition of dendritic cell TNFα production by CGRP is dependent on ICER. A, BMDC were transfected with control or ICER-specific siRNA. Cells were allowed to recover for 24 or 48 h and stimulated with CGRP for 16 h. Expression of ICER was determined by semiquantitative RT-PCR using titrated amounts of cDNA as template. B, BMDC were transfected with siRNAs together with an expression construct for GFP. After 24 h, cells were treated with culture medium, P3Cys, or a combination of P3Cys and CGRP for 16 h, and expression of TNFα by GFP-positive cells was determined by flow cytometry analysis of fixed and permeabilized cells. The data depicted are representative of three independent experiments yielding comparable results.

FIGURE 2.

ICER is selectively induced by CGRP and elevation of cAMP. BMDC were treated with the indicated mediators, and expression of ICER mRNA was determined by quantitative real-time RT-PCR (n = 3). ***, p < 0.001. Med, medium; Forsk, forskolin.

FIGURE 3.

CGRP causes delayed transcriptional repression of the Tnfa gene. A, BMDC were stimulated for the indicated time periods with P3Cys alone or in combination with CGRP. The transcriptional activity of the Tnfa gene was determined by real-time primary transcript RT-PCR. Induction of Tnfa primary transcripts is given relative to unstimulated cells (n = 3–4). B and C, chromatin samples of BMDC that were untreated or stimulated for 1 h with P3Cys or a combination of P3Cys and CGRP were immunoprecipitated with an antibody against RNA polymerase II (Pol II) or control rabbit IgG. DNA isolated from immunoprecipitates was analyzed by real-time PCR using primers spanning the Tnfa proximal promoter region (B) or a region located ∼5.1 kb upstream of the Tnfa transcriptional start site (C). Values for RNA polymerase II are given as -fold differences relative to the IgG controls (n = 7). D, BMDC were left untreated in medium (M) or were stimulated with P3Cys alone or together with CGRP (C). The addition of CGRP either was concomitant with P3Cys (C+0) or was delayed for 30 (C+30) or 60 (C+60) min. As a control, BMDC were also incubated with P3Cys and inhibitory CGRP (iC), which is an inactive mutant of CGRP lacking the N-terminal seven amino acids (n = 8). *, p < 0.05; ***, p < 0.001.

FIGURE 4.

CGRP inhibits recruitment of ATF-2 to the Tnfa promoter in BMDC. BMDC were untreated or stimulated with P3Cys or a combination of P3Cys and CGRP for 1 h. Chromatin samples of BMDC were immunoprecipitated with antibody against ATF-2, CREB, or NF-κB p65 or with control rabbit IgG. DNA isolated from immunoprecipitates was analyzed by real-time PCR using primers spanning the Tnfa proximal promoter region (A) or a region located ∼5.1 kb upstream of the Tnfa transcriptional start site (B). Values are given as -fold differences relative to the IgG controls (n = 5). *, p < 0.05; **, p < 0.01.

FIGURE 5.

CGRP promotes recruitment of ICER to the Tnfa promoter. BMDC were untreated or stimulated with P3Cys or a combination of P3Cys and CGRP for 1 h. Chromatin samples of BMDC were immunoprecipitated with an affinity-purified polyclonal rabbit antibody against ICER or control rabbit IgG. DNA isolated from immunoprecipitates was analyzed by real-time PCR using primers spanning the Tnfa proximal promoter region (A) or a region located ∼5.1 kb upstream of the Tnfa transcriptional start site (B). Values are given as -fold differences relative to the IgG controls (n = 5). *, p < 0.05; **, p < 0.01.