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IFN-ßbeta;-induced SOCS-1 negatively regulates CD40 gene expression in macrophages and microglia

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA

¹Correspondence: Department of Cell Biology, 1918 University Blvd., MCLM 395, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA. E-mail: tika{at}uab.edu

SPECIFIC AIMS

The aberrant expression of CD40 is involved in human diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer’s disease. CD40 expression is induced by a variety of stimuli, including IFN- and lipopolysaccharide (LPS). This study was designed to study the molecular basis by which IFN-ßbeta;, a cytokine with immunomodulatory properties, regulates CD40 gene expression in macrophages and microglia, and the negative regulatory function of IFN-ßbeta;-induced suppressor of cytokine signaling-1 (SOCS-1).

PRINCIPAL FINDINGS

1. IFN-ßbeta;-induced STAT-1 activation and GAS elements in the CD40 promoter are critical for IFN-ßbeta;-induced CD40 gene expression
The molecular basis of IFN-ßbeta; induced CD40 gene expression has yet to be addressed. We initiated experiments to examine the kinetics of IFN-ßbeta;-induced CD40 gene transcription in macrophages (RAW264.7 cells). CD40 mRNA expression was detected 1 h after addition of IFN-ßbeta;, peaked at 4 h and then returned to basal levels at 12 h (Fig. 1 A). IFN-ßbeta;-induced CD40 protein expression followed in a time-dependent manner in RAW264.7 cells (Fig. 1B ). STAT-1^Tyr701 and STAT-1^Ser-727 were strongly phosphorylated after 0.5–2 h of IFN-ßbeta; treatment and STAT-2^Tyr690 was detected at 0.5 h after IFN-ßbeta; treatment (Fig. 1C ). These results indicate that IFN-ßbeta; activates the STAT-1/STAT-2 signaling pathway.

View larger version (39K): [in this window] [in a new window]   Figure 1. IFN-ßbeta;-induced STAT-1 activation and GAS elements in CD40 promoter are critical for IFN-ßbeta;-induced CD40 gene expression. A) RAW264.7 cells were treated with medium or IFN-ßbeta; (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RNase protection assay (RPA) for CD40 and GAPDH mRNA. B) RAW264.7 cells were treated in the absence or presence of IFN-ßbeta; for up to 72 h and then stained with either PE-conjugated anti-CD40 or PE-conjugated isotype-matched control antibody (Ab). Cells were subjected to FACS analysis. C) RAW264.7 cells were incubated in the absence or presence of IFN-ßbeta; (100 U/ml) for up to 4 h. Protein lysates were prepared and subjected to immunoblotting with antiphospho-STAT-1Tyr701, antiphospho-STAT-1Ser727, antiphospho-STAT-2Tyr690, stripped, and reprobed with anti-STAT-1, STAT-2, and antiactin as loading controls. D) WT and GAS mutant constructs of the human CD40 promoter. E) RAW264.7 cells were transiently transfected with 0.2 µg of indicated constructs, treated with medium or IFN-ßbeta; (100 U/ml) for 12 h, and then analyzed for luciferase activity. F) WT or STAT-1-/- primary murine microglia were treated with IFN-ßbeta; for 4 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. Representative of 3 experiments.

Within the CD40 promoter, there are three GAS elements and four NF-B elements. To define the cis-acting elements necessary for IFN-ßbeta;-induced CD40 promoter activity, GAS mutant constructs of the human CD40 promoter (Fig. 1D ) were tested. Mutation of the dGAS and mGAS elements leads to a 65% inhibition of IFN-ßbeta;-induced CD40 promoter activity, whereas mutation of the pGAS element did not inhibit IFN-ßbeta; activation of the CD40 promoter (Fig. 1E ). Mutation of the four NF-B sites had no influence on IFN-ßbeta;-induced CD40 promoter activity (data not shown). These results suggest that two of the three GAS elements (dGAS and mGAS) and the transcription factor STAT-1, but not the NF-B elements, play an important role in IFN-ßbeta;-induced CD40 promoter activity. To determine the importance of STAT-1 activation in IFN-ßbeta;-induced CD40 gene expression, primary microglia from STAT-1 deficient mice were examined. In these cells, IFN-ßbeta;-induced CD40 mRNA expression was attenuated compared to wild-type (WT) microglia (Fig. 1F ). These experiments indicate that STAT-1 plays a critical role in IFN-ßbeta;-induced CD40 gene expression.

2. IFN-ßbeta; induces CD40 expression in primary macrophages and microglia
In primary human macrophage cultures, low constitutive expression of CD40 mRNA and protein was observed, which was enhanced by IFN-ßbeta; at the mRNA levels. In primary murine microglia, CD40 mRNA was strongly inducible by IFN-ßbeta; in a time-dependent manner (1–8 h) and followed by expression of CD40 protein (data not shown). These data demonstrate that IFN-ßbeta; induction of CD40 expression occurs in primary macrophages and microglia.

3. IFN-ßbeta; induces SOCS-1 expression in macrophages, and ectopic expression of SOCS-1 inhibits recruitment of STAT-1 and RNA Pol II to the CD40 promoter and modification of H3 and H4 histones in response to IFN-ßbeta;
Negative regulation of signal transduction pathways is necessary for an appropriate cellular and physiological response to cytokine stimulation. SOCS-1 is one of eight cytokine-inducible inhibitors of cytokine signaling. Constitutive expression of SOCS-1 mRNA was undetectable; however, SOCS-1 mRNA was quickly induced between 1–2 h after addition of IFN-ßbeta; (Fig. 2 A), and SOCS-1 protein expression was detected between 2–8 h of IFN-ßbeta; treatment (Fig. 2B ). Activation of the murine SOCS-1 promoter was tested in RAW264.7 cells; IFN-ßbeta; activates SOCS-1 promoter activity (Fig. 2C ).

View larger version (37K): [in this window] [in a new window]   Figure 2. IFN-ßbeta; Induces SOCS-1 expression in macrophages, and ectopic expression of SOCS-1 inhibits recruitment of STAT-1 and RNA Pol II to the CD40 promoter, and modification of H3 and H4 histones in response to IFN-ßbeta;. A) RAW264.7 cells were incubated in the absence or presence of IFN-ßbeta; (100 U/ml) for up to 12 h, then total RNA was isolated and analyzed by RPA for SOCS/1 and GAPDH mRNA. B) RAW264.7 cells were incubated in the absence or presence of IFN-ßbeta; (100 U/ml) for up to 24 h. Protein lysates were subjected to immunoblotting with anti-SOCS-1 antibody, stripped and reprobed with anti-actin as a loading control. C) RAW264.7 cells were transiently transfected with 0.2 µg of the murine SOCS-1 promoter construct, treated with medium or IFN-ßbeta; (100 U/ml) for 8 h, and analyzed for luciferase activity. D) Protein lysates were prepared from RAW264.7 and RAW-SOCS-1 cells and subjected to immunoblotting with anti-SOCS-1 antibody for confirmation of ectopic SOCS-1 expression, stripped and reprobed with anti-actin as a loading control (E) RAW264.7 and RAW-SOCS-1 cells were treated with IFN-ßbeta; (100 U/ml) for up to 24 h, and CD40, IRF-1 and GAPDH mRNA was analyzed. F) RAW264.7 and RAW-SOCS-1 cells were incubated in the absence or presence of IFN-ßbeta; (100 U/ml) for up to 8 h, then the cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against STAT-1, histone acetylation (Ac-H3, Ac-H4), RNA Pol II, phospho-Pol IISer5, or normal rabbit IgG.

To study regulation of IFN-ßbeta; signaling by SOCS-1, RAW264.7 cells overexpressing SOCS-1 (RAW-SOCS-1) were utilized. SOCS-1 protein expression was confirmed by immunobloting (Fig. 2D ). IFN-ßbeta;-induced CD40 mRNA expression was suppressed at all time points in RAW-SOCS-1 cells compared with WT RAW264.7 cells. Similar results were observed for IFN-ßbeta;-induced IRF-1 mRNA expression (Fig. 2E ).

To monitor transcription factor binding in vivo, RAW264.7 cells were incubated in the absence or presence of IFN-ßbeta; for up to 8 h, and chromatin immunoprecipitation (ChIP) assays were performed (Fig. 2F ). STAT-1 was weakly associated with the CD40 promoter in untreated cells, and increased recruitment of STAT-1 was observed 0.5 to 1 h after IFN-ßbeta; treatment. IFN-ßbeta;-induced STAT-1 recruitment was repressed in RAW-SOCS-1 cells. These results indicate that the STAT-1 transcription factor is recruited to the CD40 promoter upon IFN-ßbeta; treatment, and this recruitment is inhibited by SOCS-1 overexpression. The acetylation of histones H3 and H4 increased after IFN-ßbeta; treatment, and were inhibited by SOCS-1 in RAW-SOCS-1 cells (Fig. 2F ). Increased recruitment of Pol II was observed at 0.5 h after IFN-ßbeta; addition, and peaked at 1 h. Ser-5 phosphorylation of Pol II CTD was increased 0.5 h after IFN-ßbeta; stimulation, reached maximal levels at 2 h, and then diminished over time. Recruitment of Pol II and phosphorylation of Pol II^Ser-5 were inhibited in RAW-SOCS-1 cells (Fig. 2F ). These results suggest that IFN-ßbeta;-induced recruitment of Pol II and phosphorylation of Pol II^Ser-5 on the CD40 promoter is concurrent with activation of the CD40 gene and that overexpression of SOCS-1 blocks the IFN-ßbeta;-induced general gene transcription machinery.

CONCLUSIONS AND SIGNIFICANCE

IFN-ßbeta; is a pleiotropic cytokine with numerous immunoregulatory effects on cells of the innate and adaptive immune systems. Many effects of IFN-ßbeta; are immunosuppressive in nature, such as inhibition of class II MHC expression, suppression of matrix metalloproteinase-9 (MMP-9) expression, inhibition of interleukin (IL)-12, and induction of IL-10. In this study, we demonstrate that IFN-ßbeta; induces the expression of the costimulatory molecule CD40, which is critical for efficient antigen presentation to T cells, thereby leading to T cell activation. However, IFN-ßbeta; also induces expression of the SOCS-1 protein, which functions in a negative regulatory feedback loop to inhibit IFN-ßbeta; signaling, and ultimately CD40 expression (Fig. 3 ). IFN-ßbeta; induction of CD40 occurs at the transcriptional concentration and involves recruitment of the transcription factor STAT-1 as well as RNA Pol II to the CD40 promoter in vivo in a stepwise and coordinated order. IFN-ßbeta; also induced permissive modifications of histones H3 and H4 that were concurrent with activation of the CD40 gene. SOCS-1 is a critical regulator of the Janus-activated kinase (JAK)-STAT signaling pathway. IFN-ßbeta; induces SOCS-1 expression and SOCS-1 negatively regulates IFN-ßbeta;-induced CD40 gene expression. Overexpression of SOCS-1 attenuated IFN-ßbeta;-induced CD40 gene expression. Additionally, IFN-ßbeta;-induced STAT-1 phosphorylation and recruitment to the CD40 promoter was abolished by SOCS-1 overexpression, as was RNA Pol II recruitment, and histone H3 and H4 permissive modifications.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of proposed model of IFN-ßbeta;-induced CD40 gene expression. IFN-ßbeta; activates JAK-STAT-1 pathway (30 min to 2 h); STAT-1 then dimerizes, translocates into the nucleus, and binds to GAS elements in the CD40 promoter. Concurrent with STAT-1 recruitment, IFN-ßbeta; leads to modifications in H3 and H4 and recruitment of RNA Pol II. IFN-ßbeta; also transiently induces the expression of SOCS-1 (mRNA between 1 to 4 h; protein between 1 to 8 h), which binds to activated JAK1 and inhibits its catalytic activity, subsequently inhibiting STAT-1 activation. Inhibition of STAT-1 activation by SOCS-1 results in a reduction of IFN-ßbeta;-induced CD40 gene expression.

IFN-ßbeta; is currently used for therapeutic treatment of patients with MS, although efficacy is attenuated over time. The beneficial effects of IFN-ßbeta; are thought to be mediated by induction of IL-10 expression, suppression of MMP-9 expression, inhibition of vascular cell adhesion molecule-1 expression, improving the integrity of the blood-brain-barrier, and dampening T cell and macrophage inflammatory responses. Our results suggest that SOCS-1 induction by IFN-ßbeta; will ultimately lead to suppression of IFN-ßbeta; signaling, which in the context of MS may contribute to the loss of effectiveness of IFN-ßbeta; in this particular disease. A more detailed analysis of the effect of SOCS-1 on other IFN-ßbeta; regulated genes in T cells, endothelial cells, astrocytes, and macrophages/microglia will provide important information on the role of SOCS-1 in cells involved in CNS inflammatory responses.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5493fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5493fjev1 20/7/985    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qin, H. Articles by Benveniste, E. N. Search for Related Content PubMed PubMed Citation Articles by Qin, H. Articles by Benveniste, E. N.