This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 27/4/806    most recent 01.ATV.0000258867.79411.96v1 Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harvey, E. J. Articles by Ramji, D. P. Search for Related Content PubMed PubMed Citation Articles by Harvey, E. J. Articles by Ramji, D. P. Pubmed/NCBI databases Substance via MeSH

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:806.)
© 2007 American Heart Association, Inc.

Vascular Biology

Critical Role for Casein Kinase 2 and Phosphoinositide-3-Kinase in the Interferon-–Induced Expression of Monocyte Chemoattractant Protein-1 and Other Key Genes Implicated in Atherosclerosis

Elizabeth J. Harvey; Na Li; Dipak P. Ramji

From the Cardiff School of Biosciences, Cardiff University, UK.

Correspondence to Dipak P. Ramji, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK. E-mail Ramji{at}cardiff.ac.uk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— The interferon- (IFN-)–mediated regulation of macrophage gene expression is of crucial importance in the pathogenesis of atherosclerosis. The mechanisms underlying the actions of IFN- signaling in macrophages were investigated using monocyte chemoattractant protein (MCP)-1 as a model gene.

Methods and Results— The IFN-–induced expression of MCP-1 in macrophages was attenuated by inhibitors of phosphoinositide-3-kinase (PI3K), casein kinase 2 (CK2), and Janus kinase (JAK)-2. AKT was the downstream target for PI3K action. Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that signal transducer and activator of transcription (STAT)-1 interacted with IFN- responsive elements in the MCP-1 gene promoter. The IFN-–induced activity of the MCP-1 gene promoter and an artificial promoter containing STAT1 responsive elements was inhibited by expression of dominant negative forms of JAK-1 and -2, STAT1, CK2, and AKT. The action of CK2 and AKT on STAT1 activation was mediated, at least in part, through the regulation of serine 727 phosphorylation. Analysis of a number of other genes regulated by this cytokine and implicated in atherosclerosis revealed a gene-specific action for PI3K/AKT in IFN- signaling.

Conclusions— These studies provide novel insights into the role of PI3K/AKT and CK2 in IFN- signaling relevant to changes in macrophage gene expression during atherosclerosis.

The mechanisms underlying the IFN-–regulated expression of monocyte chemoattractant protein-1 and other key genes implicated in atherosclerosis were investigated. We show critical roles for CK2 and PI3K in addition to the JAK/STAT pathway in IFN-–signaling in macrophages, which have implications in the development of atherosclerosis.

Key Words: atherosclerosis • chemokines • gene expression • IFN-gamma • macrophage • monocyte chemoattractant protein-1 • signal transduction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Atherosclerosis is a form of chronic inflammation regulated by the action of cytokines.¹ The cytokine interferon (IFN)-, which is expressed at high levels in atherosclerotic lesions, plays a crucial role in the pathogenesis of this disease and regulates the function and properties of all the cell types present in the vessel wall.² For example, IFN- is the major cytokine involved in the induced expression of MCP-1 and other chemokines seen during atherogenesis.^1,2 Such chemokines play a crucial role in the disease by controlling the recruitment of immune cells to the site of inflammation, plaque stability, cellular proliferation, and foam cell formation.^3–4 An atheroprotective effect has been demonstrated for deficiency in a number of chemokines and chemokine receptors, including MCP-1.^3–4

Analysis of the mechanisms underlying IFN-–inducible gene transcription has identified a key role for the JAK–STAT pathway.⁵ Several recent studies have, however, suggested the existence of alternative pathways for IFN- action.⁶ Our current understanding of such alternative pathways is relatively poor, and further studies are required both to improve our knowledge of the molecular basis of IFN- action and for the development of novel therapeutic strategies to combat atherosclerosis. Our previous studies on the IFN-–mediated suppression of lipoprotein lipase gene transcription revealed a role for transcription factors Sp1 and Sp3 along with the PI3K pathway in the response.^7–8 In addition, we have recently identified a novel role for CK2 in the IFN-–stimulated expression of the inducible cAMP early repressor (ICER).⁹ The inhibition of CK2 has been found to prevent the progression of another inflammatory disorder, glomerulonephritis,¹⁰ in a rat model of the disease, the progression of which may be regulated through similar mechanisms to atherosclerosis. The purpose of this study was to investigate the role of CK2 and PI3K in IFN- signaling in macrophages using MCP-1 and other markers of atherosclerosis as model genes.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
Cell lines (mouse macrophage J774.2, human monocytic U937, and human hepatoma Hep3B) were from the European Collection of Animal Cell Cultures. LY294002 and AG490, wortmannin, and SH-6 were purchased from Calbiochem, Sigma, and Alexis Biochemicals, respectively. Antibodies were from Santa-Cruz (STAT1 p89/94, Sp1 X, MCP-1), Sigma (ß-actin), and Upstate Biotechnology (STAT1 pSer727, STAT1 pTyr701). Recombinant IFN- and the chromatin immunoprecipitation (ChIP) kit were from Peprotech and Active Motif, respectively.

Cell Culture
Human monocyte-derived macrophages (isolated from buffy coats as reported previously^9,11–12) and the cell lines were maintained in Dulbecco modified Eagle’s medium (DMEM) (J744.2, Hep3B) or RPMI 1640 (U937, primary cultures) supplemented with heat-inactivated (56°C, 30 minutes) fetal calf serum [5% (v/v) for primary cultures and 10% (v/v) for all other cells], 100U/mL penicillin, and 100 µg/mL streptomycin. The cultures were maintained at 37°C in a humidified atmosphere containing 5% (v/v) CO₂ in air. Treatment with IFN- (1000 U/mL) or the different pharmacological inhibitors was performed as previously described.^9,12

Transient Transfection and Reporter Gene Assays
Hep3B cells were transfected using the polyethylenimine (PEI) transfection reagent. Plasmid DNA (5 to 10 µg) in 5% (wt/vol) glucose was mixed with PEI solution (pH 7.2) and the resulting complex diluted with DMEM before being added to cells. These were then incubated with the complex for 24 hours at 37°C, washed twice with culture medium, and treated with the mediators. The luciferase activity in cell extracts was determined using a commercially available kit (Promega) and normalized to the total protein concentration, which was measured using the Micro BCA protein assay reagent kit (Pierce). The plasmid constructs used in this research were dominant negative (DN)-STAT1(Y701F), DN-STAT1(S727A) and 3xly6e from Dr J.E. Darnell (The Rockefeller University); DN-JAK1 and DN-JAK2 from Dr D. E. Levy (New York University); DN-PKB(AAA) from Dr B. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel); DN CK2 from Drs E. M. Chambaz and C. Cochet (INSERM, Grenoble), and MCP[213]Luc from Dr R.M. Ransohoff (Cleveland Clinic Foundation).

Reverse Transcription–Polymerase Chain Reaction
Semiquantitative RT-PCR on total cellular RNA was carried out as described before,^9,11–12 except for variations in the annealing temperature and total number of cycles (please see supplemental materials, available online at http://atvb.ahajournals.org, for conditions and primer sequences). The products were size-fractionated by agarose gel electrophoresis and analyzed using a Syngene gel documentation system (GRI).

Western Blot Analysis, Electrophoretic Mobility Shift Assays, and ChIP
Whole cell extracts were prepared in buffers containing phosphatase and protease inhibitors and used for Western blot analysis as previously described.^9,12 EMSA was carried out essentially as previously reported.^9,11–12 The sequences of the oligonucleotides used were: 5'-GGCACCCTGCCTGACTCCACCCCCCTGGCTTA-3' and 5'-GTTGTAAGCCAGGGGGGTGGAGTCAGGCAGGG-3' (MCP-GC); 5'-GGCTTCCACTTCCTGGAAACACCCGAGGG-3' and 5'-GAGCCCTCGGGTGTTTCCAGGAAGTGGAA-3' (MCP-GAS) (please see supplemental materials for sequences of oligonucleotides that were used as competitors). ChIP was carried out using reagents supplied in the ChIP-IT kit according to manufacturer’s instructions. PCR was carried out using the primers: 5'-CCCATTTGCTCATTTGGTCT-3' and 5'-CTTATTGAAAGCGGGCAGAG-3' (MCP-1 promoter); and 5'-ATGGTTGCCACTGGGGATCT-3' and 5'-TGCCAAAGCCTAGGGG- AAGA-3' (negative control primers against non-promoter genomic DNA; supplied by the manufacturer). The products were separated by agarose gel electrophoresis.

Statistical Analyses of Data
Semiquantitative measurements of the signals in Western blots or RT-PCR were performed by densitometric analysis using the GeneTools software (GRI). Statistical comparisons between data were carried out using the Student t test with P<0.05 considered as statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The IFN-–Induced Expression of MCP-1 Is Suppressed by Inhibition of CK2 and PI3K
Our previous studies have shown that the murine J774.2 cell line is a useful model system to investigate the mechanisms underlying IFN-–regulated macrophage gene expression with demonstrated conservation with primary cultures.^7–9,11 This cellular system was used initially to delineate the potential role of PI3K and CK2 in the action of IFN- on MCP-1 expression. IFN- induced MCP-1 mRNA and protein expression in a time-dependent manner, with maximal levels at around 3 hours after stimulation, and this was accompanied by the secretion of the protein into the medium (data not shown). Apigenin, a widely used CK2 inhibitor,^9–10,13attenuated the IFN-–induced MCP-1 expression (Figure 1A). Similarly, this response was attenuated by 2 inhibitors of the PI3K pathway, LY294002 and wortmannin (Figure 1B). A similar profile was seen when the action of LY294002 and apigenin on MCP-1 protein levels was investigated by Western blot analysis (Figure 1C; multiple products represent various glycosylation forms of the protein). To rule out the possibility that the results obtained were peculiar to the J774.2 cell line, representative experiments were repeated on primary cultures of human monocyte-derived macrophages. Both apigenin and LY294002 inhibited the expression of MCP-1 mRNA obtained in the presence of IFN- (Figure 1D). A similar pattern was seen in human THP-1 macrophages, the human hepatoma Hep3B cell line, human umbilical vein endothelial cells, and the human endothelial EA.hy926 cell line (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 1. The IFN-–induced expression of MCP-1 is attenuated by inhibitors of CK2 and PI3K. J774.2 macrophages (A-C) or primary human monocyte-derived macrophages (D) were treated for 3 hours with vehicle (–) or IFN- /vehicle (+) in the absence or the presence of apigenin (10 µmol/L in A and 20 µmol/L in C and D), LY294002 (5 and 20 µmol/L in B, 20 µmol/L in C and D), or wortmannin (5 and 20 µmol/L). For A, B, and D, total cellular RNA was isolated and subjected to RT-PCR using primers against MCP-1 or ß-actin. The products were size-fractionated by agarose gel electrophoresis. –RT shows the outcome of a reaction in which the reverse transcriptase step was omitted (RNA from cells treated with vehicle alone was used). For C, equal amounts of protein were subjected to Western blot analysis using antisera against MCP-1 and ß-actin as indicated. The results shown are representative of at least 3 independent experiments (statistical analysis of the relative expression levels, as determined by densitometric scanning, from all experiments showed that the attenuation of IFN-–induced MCP-1 mRNA or protein expression by the inhibitors was statistically significant; P<0.01).

AKT Acts as a Downstream Effector of PI3K in IFN-–Induced Expression of MCP-1
Because AKT (also called protein kinase B) is a key downstream effector of PI3K action,¹⁴ its role in the regulation of MCP-1 expression was investigated. The AKT inhibitor SH-6 attenuated the IFN-–induced expression of MCP-1 (P<0.001) (Figure 2A). Such an effect on MCP-1 expression was specific to SH-6 and not seen with rapamycin, an inhibitor of another downstream component of PI3K signaling¹⁴ (data not shown). Consistent with an inhibitory action of SH-6, expression of a DN form of AKT (mutated at residues 179, 308, and 473) attenuated the IFN-–mediated activation of the proximal human MCP-1 gene promoter (213 bp upstream of the transcription start site linked to the luciferase reporter gene)¹⁵ in Hep3B cells (Figure 2B). These cells were employed for all transfection experiments because of difficulties in transfecting J774.2 and primary macrophages with exogenous DNA, and because the induction of MCP-1 expression by IFN- and its attenuation by inhibitors of CK2 and LY294002 was similar in macrophages and Hep3B cells (data not shown). Further experiments showed that IFN- induced the activity of AKT, and this was attenuated by LY294002 and the JAK2 inhibitor AG490 but not by apigenin (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2. AKT acts as a downstream effector of PI3K in the regulation of MCP-1 expression by IFN-. A, J774.2 macrophages were treated for 3 hours with either vehicle alone (–) or IFN- /vehicle (+) in the absence or the presence of SH-6 (20 µmol/L) as indicated. RT-PCR was then carried out using primers against MCP-1 and ß-actin as shown. The products were size-fractionated by agarose gel electrophoresis. –RT shows the outcome of a reaction in which the reverse-transcriptase step was omitted (RNA from cells treated with vehicle alone was used). Statistical analysis of relative expression, as determined by densitometric scanning, from 3 independent experiments showed that the inhibition of IFN-–induced MCP-1 expression by SH-6 was significant (P<0.001). B, Hep3B cells were cotransfected with pcDNA3 or an expression vector encoding DN-AKT, and MCP[213]Luc. After transfection, the cells were incubated in the presence or absence of IFN-. For each plasmid, the relative luciferase activity in cells incubated in the absence of IFN- has been arbitrarily assigned as 1, with those from cells in the presence of this cytokine expressed relative to this as fold induction. The results are mean±SD from 5 independent experiments carried out in triplicate. Asterisks indicates significant inhibition of the IFN-–induced reporter gene activity seen in cells transfected with the pcDNA3 plasmid (P<0.001).

JAK-STAT Signaling Is Also Required for the IFN- Induced Expression of MCP-1 in Macrophages: Role of CK2 and AKT in the Regulation of STAT1 Activity
Previous analysis of the MCP-1 promoter in astrocytoma revealed a functional interaction between an IFN-–activated site (GAS), binding to STAT1, and a GC-element, which interacts with Sp1.¹⁵ Disruption of GAS abrogated induction by IFN- whereas mutations in the GC box reduced promoter activity without affecting fold induction.¹⁵ As STAT1 binding was essential for IFN- inducibility, it was possible that the action of CK2 and/or AKT on MCP-1 expression was mediated through regulation of STAT1 activity. As previous studies were performed on astrocytomas, it was necessary to confirm DNA-protein interactions in other cellular systems such as macrophages before the action of AKT and CK2 could be investigated. EMSA with the GC element from the murine MCP-1 promoter showed 3 DNA-protein complexes, the intensity of which was similar in untreated and IFN-–stimulated cells (Figure 3A). In contrast, the GAS element produced 2 DNA-protein complexes, the binding of which was increased when the cells were incubated with IFN- for 30 minutes (Figure 3B). The specificity of the DNA–protein complexes was confirmed by competition EMSA (Figure 3A and 3B); DNA binding was competed by inclusion of an excess of unlabeled oligonucleotides containing the corresponding sequence from the MCP-1 promoter (M in Figure 3A and 3B) or those containing the binding site for the relevant transcription factor (Sp1 or STAT1) but not by those containing an unrelated sequence (AP1 or NFB binding site).

View larger version (30K): [in this window] [in a new window]   Figure 3. The MCP-1 promoter interacts with transcription factors Sp1 and STAT1. A and B, J774.2 macrophages were either left untreated (–) or stimulated with IFN- [3 hours in A (+); indicated period of time in B]. EMSA were then performed using whole cell extracts and radiolabeled MCP-GC (A) or MCP-GAS (B) probes. Competition assays were carried out using an excess of unlabeled probe (M) or those containing binding sites for Sp1, AP1, STAT1, and NFB as indicated. DNA-protein complexes are shown by a vertical line labeled C. C, U937 monocytes, differentiated into macrophages with phorbol-12-myristate-13 acetate for 24 hours, were incubated for 30 minutes in the presence or absence of IFN-. ChIP analysis was then performed using antibodies specific for Sp1 and STAT1 along with a negative control IgG (NA). PCR primers were specific to the MCP-1 promoter containing the STAT1 and Sp1 binding sites (MCP-1 prom) or a region of nonpromoter genomic DNA (control) (IN represents diluted input DNA). Products were resolved by agarose gel electrophoresis (image). Densitometric analysis was performed and the relative levels of Sp1 and STAT1 binding in comparison to NA (assigned as 0) are shown as mean±SD in the graph (**P<0.01).

DNA-protein interactions at the MCP-1 promoter in vivo have not yet been analyzed. ChIP assays using STAT1 and Sp1 antibodies were therefore performed. As shown in Figure 3C, the IFN-–induced binding of STAT1, but not Sp1, to the MCP-1 promoter also occurred in vivo. U937 macrophages, in which the IFN- inducibility of MCP-1 expression was similar to J774.2 cells (data not shown), were used for ChIP assays because they were recommended by the manufacturer.

Having established that STAT1 from macrophages binds to the GAS element in the MCP-1 promoter and its binding is induced by IFN- in vivo, the role of key components of the JAK-STAT pathway was investigated. Initial experiments showed that the JAK2 inhibitor AG490 attenuated the IFN-–induced expression of MCP-1 (supplemental IA, available online at http://atvb.ahajournals.org). Further studies used DN constructs against JAK1, JAK2, and STAT1^16–17 with a luciferase vector containing the proximal MCP-1 promoter. As shown in supplemental Figure IB, a marked inhibition of IFN-–induced reporter gene activity was obtained by expression of DN-JAK1 and -JAK2. DN-STAT1, mutated at tyrosine 701 or serine 727, also produced a statistically significant inhibition albeit to a lesser extent than DN-JAKs (supplemental Figure IB). Such an inhibition was specific to DN STAT1 and not seen with DN forms of C/EBPß or NF-B (data not shown).

To assess the role of CK2 and AKT in the regulation of STAT1, the action of DN-CK2 and DN-AKT on the activity of a luciferase-based plasmid containing 3 copies of a STAT1 binding site (3xly6e) was analyzed. Similar to AKT (Figure 2), the positive action of the DN-CK2 plasmid was confirmed by its ability to attenuate the IFN-–induced expression of MCP-1 mRNA in transfected cells (data not shown). As expected, DN-JAK1, -JAK2, and -STAT1 (Tyr701 or Ser727) inhibited the IFN-–induced activity of the 3xly6e plasmid (Figure 4). More importantly, a statistically significant inhibition of this activity was seen with DN-CK2 and -AKT (Figure 4), thereby implicating both these kinases in transcriptional activation by IFN- via STAT1. This could potentially be attributable to an effect on the phosphorylation of STAT1, which was investigated in more detail.

View larger version (13K): [in this window] [in a new window]   Figure 4. CK2 and AKT modulate STAT1 activity. Hep3B cells were cotransfected with the STAT1-responsive 3xly6e plasmid along with pcDNA3, DN-STAT1-Y701F (STAT-Y), DN-STAT1-S727A (STAT-S), DN-JAK1, DN-JAK2, pSG5, DN-CK2, or DN-AKT as indicated. The cells were then cultured for 6 hours in the presence or absence of IFN-. The relative luciferase activity in cells transfected with pSG5 (control plasmid for DN-CK2) or pcDNA3 (control plasmid for all other DN constructs) and incubated in the absence of IFN- has been arbitrarily assigned as 1 with those in the presence of the cytokine represented to this as fold induction (mean±SD). Asterisks indicate significant inhibition of the IFN-–induced activity seen in cells transfected with the control plasmids (*P<0.05; **P<0.01; ***P<0.001).

Figure 5A shows that STAT1 was phosphorylated on tyrosine 701 after stimulation of cells with IFN- for 5 minutes and at subsequent time points. Phosphorylation of STAT1 at serine 727 was also seen after 5 minutes, reaching high levels after 30 minutes and at subsequent time points (Figure 5A). In contrast, there was little change in the total amount of the STAT1 protein (Figure 5A). The IFN-–induced phosphorylation of STAT1 at tyrosine 701 was inhibited by AG490 but not by apigenin or LY294002 (Figure 5B). In contrast, all 3 agents inhibited the phosphorylation of STAT1 at serine 727 (Figure 5B), thereby suggesting that CK2 and AKT inhibit the full activation of STAT1.

View larger version (33K): [in this window] [in a new window]   Figure 5. CK2 and AKT affect the activation of STAT1 through phosphorylation of serine 727. J774.2 macrophages were treated with IFN- for the time shown (A) or incubated for 3 hours with vehicle (–) or IFN-/vehicle in the absence or the presence of LY294002 (LY; 20 µmol/L), apigenin (Api; 20 µmol/L), or AG490 (AG; 50 µmol/L) (B). Western blot analysis was performed using antibodies specific for phosphorylated forms of STAT1 (pSTAT-Tyr701 or pSTAT-Ser727) or total STAT1 (pSTAT p84/91). The images shown are representative of at least 3 independent experiments. The graph shows relative expression (mean±SD), as determined by densitometric scanning, from 3 experiments. The value from cells treated with IFN- /vehicle has been arbitrarily assigned as 1 with that from the others represented to this value (**P<0.01; ***P<0.001).

Gene-Specific Action of PI3K/AKT in IFN- Signaling
Having established that CK2 and PI3K/AKT were required for IFN-–induced MCP-1 expression and affected the phosphorylation of STAT1 at serine 727, we wondered whether they played a general role in the regulation of genes that require JAK-STAT signaling or if there was a gene-specific mode of action. This possibility was initially investigated by RT-PCR for several genes: the chemokines IFN-inducible T cell chemoattractant (ITAC), monokine induced by IFN- (Mig), IFN-inducible protein-10 (IP-10); and suppressor of cytokine signaling (SOCS-1; the IFN- inducibility of which was absent in STAT1-deficient macrophages).⁶ MCP-1 and ICER⁹ were included in some experiments for comparative purposes. In the case of ICER, a number of alternatively spliced transcripts exist, all of which are functionally indistinguishable and induced by IFN-.⁹ These are collectively referred to as ICER in this study. As shown in Figure 6, the JAK2 inhibitor AG490 attenuated the IFN- induced expression of MCP-1 (P<0.01), SOCS-1 (P<0.001), ITAC (P<0.05), Mig (P<0.001), and IP-10 (P<0.05) but not ICER. On the other hand, the IFN- inducibility of all the genes analyzed was attenuated by apigenin (data not shown; P<0.01 for MCP-1 and <0.001 for ICER, SOCS-1, ITAC, Mig, and IP-10), thereby suggesting a more general role for CK2 in IFN- signaling. In contrast, inhibition of PI3K attenuated the IFN-–induced expression of MCP-1, ITAC (P<0.01), Mig (P<0.001), and IP-10 (P<0.05) but not SOCS-1. These data suggest a potential gene-specific effect of PI3K/AKT in IFN- signaling, which was investigated further by the use of the Oligo GEArray Mouse Atherosclerosis Microarray from SuperArray. This contains 113 oligonucleotides specific to genes related to the pathogenesis of atherosclerosis as well as control genes. Analysis of the data from 3 independent experiments showed that from the 66 genes listed, the expression of 50 was induced in response to IFN- and 3 suppressed while 6 were unaffected (supplemental Table I). Of the 50 IFN-–inducible genes, the upregulation of 39 was inhibited by LY294002 (supplemental Table I). From these, the results for selected genes was confirmed by RT-PCR (supplemental Figure II) showing a good correlation between the data from microarray analysis and RT-PCR.

View larger version (35K): [in this window] [in a new window]   Figure 6. The role of the JAK–STAT and PI3K pathways in the regulation of macrophage gene expression by IFN-. J774.2 macrophages were treated for 3 hours with vehicle (–) or IFN-/vehicle (+) in the absence or the presence of AG490 [50 µmol/L (A; top panel) or 50 µmol/L and 100 µmol/L (A; bottom panel)] or LY294002 (5 µmol/L and 20 µmol/L) (B). Total cellular RNA was subjected to RT-PCR using primers against the indicated genes, and the products were size-fractionated by agarose gel electrophoresis. The images shown are representative of at least 3 independent experiments. Statistical analysis of data from densitometric scanning showed significant inhibition by AG490 of IFN-–induced expression of MCP-1 (P<0.01), SOCS-1 (P<0.001), ITAC (P<0.05), Mig (P<0.001), and IP-10 (P<0.05), and by LY294002 of MCP-1 (P<0.01), ITAC (P<0.01), Mig (P<0.001), and IP-10 (P<0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The findings presented in this study provide novel insights into the role of CK2 and PI3K/AKT along with the JAK–STAT pathway in the IFN-–mediated regulation of MCP-1 and macrophage gene expression relevant to atherosclerosis. Firstly, we show that CK2 plays a key role in IFN- signaling by regulating the phosphorylation of serine 727 in STAT1. Consistent with this finding, we have previously shown that although CK2 activity is present constitutively, this is increased markedly by IFN-.⁹ Secondly, PI3K/AKT also modulates the expression of several genes regulated by IFN- along with the phosphorylation of STAT1 at serine 727. However, not all IFN-–regulated genes requiring the JAK–STAT pathway need PI3K for this response. Overall, these studies along with our previous research^8–9 suggest the existence of at least 3 differential pathways for the IFN-–mediated regulation of macrophage gene expression (supplemental Figure III). Future studies should investigate the roles of CK2, AKT, and other components of these pathways in animal models of atherosclerosis.

Although PI3K/AKT has been implicated in the IFN-–induced expression of some genes,^18–20 our studies reveal a key role in the regulation of a large number of genes, particularly those implicated in atherosclerosis, and identify a gene-specific action. On the basis of our understanding of other receptor-associated tyrosine kinases,^14,21 PI3K is likely to be activated through JAKs after activation of the IFN- receptor by the cytokine. The stimulation of PI3K activity then leads to the subsequent activation of the downstream effector AKT. CK2 has been shown to phosphorylate AKT in Jurkat cells.²² However, this does not appear to be the case in macrophages because apigenin has no effect on IFN- induced activity of AKT (data not shown).

The inhibition of CK2 and PI3K/AKT signaling decreased the IFN-–stimulated activation of a GAS-regulated promoter and the phosphorylation of STAT1 at serine 727 (Figures 4 and 5). The exact kinase(s) responsible for this serine phosphorylation has not yet been identified and it is thus possible that AKT and/or CK2 may act directly on STAT1 or alternatively in the activation of downstream effector kinase(s). The precise role of STAT1 serine 727 phosphorylation is not fully understood but is known to be necessary for full promoter activation by this transcription factor.^5–6 Gene targeted mice expressing a mutant STAT1 in which serine 727 has been substituted by alanine have increased mortality following infection with Listeria monocytes and reduced induction of IFN-–stimulated genes in macrophages, thereby suggesting a crucial role for this modification in a full-fledged innate immunity in response to the cytokine.²³ In addition, STAT1 phosphorylation at serine 727 has previously been suggested to mediate interactions with a number of coactivator proteins.^6,24 Differential recruitment of such coactivator proteins may be one reason for the selective action of PI3K/AKT in IFN- signaling mediated through the JAK–STAT pathway. Alternatively, the nature of other proteins that interact with the promoter region (eg, Sp1 in MCP-1) and the modulation of their action by IFN- may be responsible for such a selective action. Analysis of the promoter regions of IFN-–inducible genes that are not affected by inhibition of PI3K/AKT will be necessary to resolve this issue.

In conclusion, IFN- is a key regulator of the inflammatory response and regulates the expression of a large number of genes implicated in atherosclerosis. Although the role of the JAK–STAT pathway in IFN- signaling is well established, little is understood about the role and contribution of alternative pathways in the action of this cytokine. Our studies provide novel insights into the role of CK2 and PI3K/AKT in IFN- signaling.

	Acknowledgments

 
Sources of Funding

This work was supported by the British Heart Foundation.

Disclosures

None.

	Footnotes

 
Original received September 15, 2006; final version accepted January 9, 2007.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.[Free Full Text]
2. Harvey EJ, Ramji DP. Interferon- and atherosclerosis: pro- or anti-atherogenic? Cardiovasc Res. 2005; 67: 11–20.[Abstract/Free Full Text]
3. Sheikine YA, Hansson GK. Chemokines as potential therapeutic targets in atherosclerosis. Curr Drug Targets. 2006; 7: 13–27.[CrossRef][Medline] [Order article via Infotrieve]
4. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95: 858–866.[Abstract/Free Full Text]
5. Stark GR, Kerr IM, Williams BRG, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem. 1998; 67: 227–264.[CrossRef][Medline] [Order article via Infotrieve]
6. Ramana CV, Gil MP, Schreiber RD, Stark GR. Stat-1-dependent and –independent pathways in IFN--dependent signaling. Trends Immunol. 2002; 23: 96–101.[CrossRef][Medline] [Order article via Infotrieve]
7. Tengku-Muhammad TS, Hughes TR, Cryer A, Ramji DP. Involvement of both the tyrosine kinase and the phosphatidylinositol-3' kinase signal transduction pathways in the regulation of lipoprotein lipase expression in J774.2 macrophages by cytokines and lipopolysaccharide. Cytokine. 1999; 11: 463–468.[CrossRef][Medline] [Order article via Infotrieve]
8. Hughes TR, Tengku-Muhammad TS, Irvine SA, Ramji DP. A novel role of Sp1 and Sp3 in the interferon--mediated suppression of macrophage lipoprotein lipase gene transcription. J Biol Chem. 2002; 277: 11097–11106.[Abstract/Free Full Text]
9. Mead JR, Hughes TR, Irvine SA, Singh NN, Ramji DP. Interferon- stimulates the expression of the inducible cAMP early repressor in macrophages through the activation of casein kinase 2. A potentially novel pathway for interferon- -mediated inhibition of gene transcription. J Biol Chem. 2003; 278: 17741–17751.[Abstract/Free Full Text]
10. Yamada M, Katsuma S, Adachi T, Hirasawa A, Shiojima S, Kadowaki T, Okuno Y, Koshimizu TA, Fujii S, Sekiya Y, Miyamoto Y, Tamura M, Yumura W, Nihei H, Kobayashi M, Tsujimoto G. Inhibition of protein kinase CK2 prevents the progression of glomerulonephritis. Proc Natl Acad Sci U S A. 2005; 102: 7736–7741.[Abstract/Free Full Text]
11. Irvine SA, Foka P, Rogers SA, Mead JR, Ramji DP. A critical role for the Sp1-binding sites in the transforming growth factor-ß-mediated inhibition of lipoprotein lipase gene expression in macrophages. Nucleic Acids Res. 2005; 33: 1423–1434.[Abstract/Free Full Text]
12. Singh NN, Ramji DP. Transforming growth factor-ß-induced expression of the apolipoprotein E gene requires c-Jun N-terminal kinase, p38 kinase, and casein kinase 2. Arterioscler Thromb Vasc Biol. 2006; 26: 1323–1329.[Abstract/Free Full Text]
13. Shen J, Channavajhala P, Seldin DC, Sonenshein GE. Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IB. J Immunol. 2001; 167: 4919–4925.[Abstract/Free Full Text]
14. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002; 296: 1655–1657.[Abstract/Free Full Text]
15. Zhou ZHL, Chaturvedi P, Han YL, Aras S, Li YS, Kolattukudy PE, Ping D, Boss JM, Ransohoff RM. IFN- induction of the human monocyte chomoattractant protein (hMCP)-1 gene in astrocytoma cells: functional interaction between an IFN--activated site and a GC-rich element. J Immunol. 1998; 160: 3908–3916.[Abstract/Free Full Text]
16. Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB, Wang J. Mechanism of STAT3 activation by insulin-like growth factor 1 receptor. J Biol Chem. 2000; 275: 15099–15105.[Abstract/Free Full Text]
17. Bromberg JF, Fan Z, Brown J, Mendelsohn J, Darnell JE Jr. Epidermal growth factor-induced growth inhibition requires Stat1 activation. Cell Growth Differen. 1998; 9: 505–512.[Abstract]
18. Navarro A, Anand-Apte B, Tanabe Y, Feldman G, Larner AC. A PI-3 kinase-dependent, Stat1-independent signaling pathway regulates interferon-stimulated monocyte adhesion. J Leukoc Biol. 2003; 73: 540–545.[Abstract/Free Full Text]
19. Nguyen H, Ramana CV, Bayes J, Stark GR. Roles of phosphatidylinositol 3-kinase in interferon--dependent phosphorylation of STAT1 on serine 727 and activation of gene expression. J Biol Chem. 2001; 276: 33361–33368.[Abstract/Free Full Text]
20. Venkatesan BA, Mahimsinathan L, Ghosh-Choudhury N, Gorin Y, Bhandari B, Valente AJ, Abboud HE, Choudhury GG. PI3 kinase-dependent Akt kinase and PKC independently regulate interferon- -induced STAT1 serine phosphorylation to induce monocyte chemotactic protein-1 expression. Cellular Signal. 2006; 18: 508–518.
21. Okugawa S, Ota Y, Kitazawa T, Nakayama K, Yanagimoto S, Tsuka K, Kawada M, Kimura S. Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages. Am J Physiol Cell Physiol. 2003; 285: C399–C408.[Abstract/Free Full Text]
22. Di Maira G, Salvi M, Arrigoni G, Martin O, Sarno S, Brustolon F, Pinna LA, Ruzzene M. Protein kinase CK2 phosphorylates and upregulates Akt/PKB. Cell Death Differ. 2005; 12: 668–677.[CrossRef][Medline] [Order article via Infotrieve]
23. Varinou L, Ramsauer K, Karaghiosoff M, Kolbe T, Pfeffer K, Muller M, and Decker T. Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN--dependent innate immunity. Immunity. 2003; 19: 793–802.[CrossRef][Medline] [Order article via Infotrieve]
24. Zhang JJ, Zhao Y, Chait BT, Lathem WW, Ritzi M, Knippers R, Darnell JE Jr. Ser 727-dependent recruitment of MCM5 by Stat1 in IFN--induced transcriptional activation. EMBO J. 1998; 17: 6963–6971.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				H. Luo, Y. Li, J.-J. Mu, J. Zhang, T. Tonaka, Y. Hamamori, S. Y. Jung, Y. Wang, and J. Qin Regulation of Intra-S Phase Checkpoint by Ionizing Radiation (IR)-dependent and IR-independent Phosphorylation of SMC3 J. Biol. Chem., July 11, 2008; 283(28): 19176 - 19183. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 27/4/806    most recent 01.ATV.0000258867.79411.96v1 Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harvey, E. J. Articles by Ramji, D. P. Search for Related Content PubMed PubMed Citation Articles by Harvey, E. J. Articles by Ramji, D. P. Pubmed/NCBI databases Substance via MeSH