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. 2013 Oct;33(10):588-96.
doi: 10.1089/jir.2012.0065. Epub 2013 May 25.

Serum response factor indirectly regulates type I interferon-signaling in macrophages

Lan Xie  1 , Amy L Sullivan, Jana G Collier, Christopher K Glass
Affiliations

Serum response factor indirectly regulates type I interferon-signaling in macrophages

Lan Xie et al. J Interferon Cytokine Res. 2013 Oct.

Abstract

Serum response factor (SRF) is required for diverse aspects of development and homeostasis, but potential roles in the regulation of inflammation and immunity have not been systematically investigated. Here, we demonstrate that SRF is unexpectedly required for optimal responses of elicited peritoneal macrophages to type I interferons. Knockdown of SRF expression in these cells impairs induction of numerous interferon (IFN)-stimulated genes (ISGs) in response to zymosan, LPS, and poly I:C. This effect is primarily due to a defect in the ability of induced type I interferons to mediate secondary activation of ISGs. SRF does not appear to be required for expression of established components of the type I interferon signaling pathway, with IFN-β-dependent phosphorylation of STAT1 and STAT2 normally occurring in SRF-depleted macrophages. Collectively, these findings suggest that SRF can indirectly modulate type I interferon-signaling, without interfering with the classic JAK/STAT/ISGF3 pathway.

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Figures

FIG. 1.
FIG. 1.
Genome wide analysis of the role of serum response factor (SRF) in transcription regulation in macrophages. (A) Logos of sequence motifs over-represented in the promoters of genes induced>2-fold in elicited macrophages following zymosan treatment for 1h. Relative height of letters indicates the frequency of occurrence at each position. A binding site for SRF is the third most highly enriched sequence motif in the promoters of this set of genes. (B) Srf knockdown efficiency was confirmed by Q-PCR. Thioglycollate-elicited macrophages were transfected with control or SRF siRNAs, as indicated, and the levels of Srf mRNA are shown, normalized to Gapdh expression. (C) Gene Spring representation of the gene expression profiles of control or SRF siRNA-transfected primary macrophages following the treatments indicated. The genes shown are those that were induced more than 3-fold after 6 h of zymosan treatment and were reduced by more than 33% with SRF knockdown at either 1 or 6 h of zymosan treatment. (D) Gene ontology (GO) analysis for biological process annotations of zymosan-induced genes (6 h) that were downregulated with SRF knockdown. (E) Highly enriched sequence motifs in the promoters of genes induced by zymosan (6 h) that were downregulated following SRF knockdown.
FIG. 2.
FIG. 2.
Expression profiles of SRF-dependent interferon-stimulated genes (ISGs). (A, B, and C) mRNA expression profiles of representative SRF-dependent ISGs (Ifit2, Ifit3, Isg15) were analyzed by Q-PCR. Thioglycollate-elicited macrophages were transfected with control or SRF siRNAs, as indicated, and treated with (A) zymosan (1 mg/mL), (B) LPS (100 ng/mL), and (C) polyI:C (20 μg/mL) for 0, 1, 3, and 6 h, respectively. Results shown are representative of 3 independent experiments. Statistical significance (denoted by asterisks) was determined by performing student's t-tests on the indicated mRNA levels after 6 h of zymosan stimulation with control or SRF-specific siRNA treatment in the 3 experiments *P<0.05 versus ctrl; **P<0.01 versus ctrl. Error bars represent the standard deviations of the duplicate PCR reactions used in the representative experiment shown.
FIG. 3.
FIG. 3.
SRF regulation of ISGs is specific for type I interferons. (A and B) Expression profiles of representative type I interferon-dependent genes (Ifit2, Ifit3, and Isg15) were analyzed by Q-PCR. Thioglycollate-elicited macrophages were transfected with control siRNA or siRNA targeted against SRF, as indicated, and left either untreated or treated with (A) IFN-β (1000 Unit/mL) and (B) IFN-α (1000 Unit/mL) for 1, 3, or 6 h. (C) Expression profiles of representative type II interferon-dependent genes (Irf1, Oas2, and Stat1) were analyzed by Q-PCR. siRNA transfections were carried out in primary macrophages as described in A and B, but the cells were treated with IFN-γ (20 μg/mL) for 0, 1, 3, or 6 h. Results shown are representative of 3 independent experiments. Statistical significance (denoted by asterisks) was determined as described in Figure 2. *P<0.05 versus ctrl; **P<0.01 versus ctrl.
FIG. 4.
FIG. 4.
SRF affects ISGs independent of protein synthesis and mRNA stability. (A and B) Ifit2 expression levels were analyzed by Q-PCR. Thioglycollate-elicited macrophages were transfected with control or SRF siRNAs, as indicated, and left either untreated or treated with (A) IFN-β (1000 Unit/mL) or (B) polyI:C (20 μg/mL) for 6 h in the absence or presence of cycloheximide (10 μg/mL). (C) Ifit3 expression levels were analyzed by Q-PCR in primary macrophages using the same siRNA knockdown procedure as described in A and B. Cells were untreated or treated with IFN-β (1000 Unit/mL) for 6 h, Act-D (1 μg/mL) was then added, and cells were harvested 0, 2, 4, and 6 h after Act-D treatment, as indicated. Results are expressed as the mRNA levels observed relative to the appropriate control or SRF siRNA-transfected samples induced with IFN-β without subsequent Act-D treatment (0 h). Results shown are the average relative expressions observed in 3 independent experiments.
FIG. 5.
FIG. 5.
SRF affects ISGs without affecting the expression, phosphorylation, and nuclear translocation of ISGF3 signaling molecules. (A) Western blots showing consistent levels of the indicated proteins with or without SRF knockdown, before or after treatment with 1000 Unit/mL of IFN-β for 30 min. Lysates were probed with anti-GAPDH antibodies as a loading control. (B) Nuclear and cytoplasmic extracts from primary macrophages transfected with control or SRF siRNAs and treated with IFN-β for 0 or 30 min, as indicated, were immunoblotted for the indicated proteins. Extracts were probed with anti-lamin and anti-GAPDH antibodies to show the integrity of the nuclear and cytoplasmic extracts, respectively, and to serve as loading controls. Results shown are representative of 3 independent experiments.
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