The Akt1 Isoform Is Required for Optimal IFN-β Transcription through Direct Phosphorylation of β-Catenin

Benjamin N. Gantner, Huali Jin, Feng Qian, Nissim Hay, Bin He and Richard D. Ye
J Immunol September 15, 2012, 189 (6) 3104-3111; DOI: https://doi.org/10.4049/jimmunol.1201669

Abstract

IFN-β is a critical antiviral cytokine that is capable of modulating the systemic immune response. The transcriptional induction of IFN-β is a highly regulated process, involving the activation of pattern recognition receptors and their downstream signaling pathways. The Akt family of serine/threonine kinases includes three isoforms. The specific role for the individual Akt isoforms in pattern recognition and signaling remains unclear. In this article, we report that the TLR3-mediated expression of IFN-β is blunted in cells that lack Akt1. The expression of IFN-β–inducible genes such as CCL5 and CXCL10 was also reduced in Akt1-deficient cells; the induction of TNF-α and CXCL2, whose expression does not rely on IFN-β, was not reduced in the absence of Akt1. Macrophages from Akt1−/− mice displayed deficient clearance of HSV-1 along with reduced IFN-β expression. Our results demonstrate that Akt1 signals through β-catenin by phosphorylation on Ser552, a site that differs from the glycogen synthase kinase 3 β phosphorylation site. Stimulation of a chemically activated version of Akt1, in the absence of other TLR3-dependent signaling, was sufficient for accumulation and phosphorylation of β-catenin at Ser552. Taken together, these results demonstrate that the Akt1 isoform is required for β-catenin–mediated promotion of IFN-β transcription downstream of TLR3 activation.

Introduction

The IFNs are a family of a critical antiviral cytokines whose activity has been known for more than a half century (1). They are now known to exert effects on almost all aspects of the immune response, including immune cell development and effector functions, as well as the signaling pathways that govern pathogen recognition and cellular communication (2). Dysregulation of IFNs can lead to ineffective immunity and autoimmunity (1). A number of cytokines comprise the IFN family, and although the specific roles of each are poorly understood, IFN-β is one of the best studied. A number of unique functions have been attributed to IFN-β, and it is used as a clinical therapeutic in patients with multiple sclerosis (3). Early expression of IFN-β from macrophages has been shown to modify the subsequent inflammatory profile of these cells to produce better, or in some cases worse, outcomes in disease (4).

The transcriptional activation of IFN-β is highly choreographed and well studied, involving multiple signaling pathways and transcription factor complexes (5). Recent studies have demonstrated that the signaling molecules glycogen synthase kinase 3 β (GSK3-β), Akt, and β-catenin are involved in the regulation of IFN-β expression (6–8). GSK3-β activity can restrain the transcription of IFN-β, reportedly because of regulatory effects on c-Jun (6). Another report showed that interfering with Akt activation can block TLR-mediated IFN-β transcription by blunting the activation of the transcription factor IFN response factor 3 (IRF3) (7). It is therefore clear that these pathways are involved in the regulation of IFN-β gene transcription, although the detailed mechanisms are just being delineated (7, 9). Our laboratory has previously described nonredundant roles for specific Akt isoforms in the activation of critical neutrophil functions (10). In this study, we sought to determine whether there were any Akt isoform-specific defects in cytokine responses in macrophages, and found that the Akt1 isoform is required for IFN-β transcription in response to viral-associated stimuli.

The kinase Akt (also known as PKB) is a central player in the signaling pathways that regulate metabolism and cellular transformation. Akt activation proceeds downstream of PI3K activity by bringing Akt into close proximity with its activating kinases. Many pattern recognition receptors, growth factor receptors, and cytokine receptors are able to activate PI3K, and thereby activate Akt. The downstream effects of Akt are quite broad, but one important function is the inhibition of GSK3-β through phosphorylation on Ser9 (11). This results in inhibition of GSK3-β kinase activity, which ordinarily leads to phosphorylation and ubiquitin-mediated destruction of β-catenin. Therefore, Akt activation can promote the transcriptional activity of β-catenin through repression of the GSK3-β–mediated proteosomal degradation. Recently, another mechanism for Akt regulation of β-catenin has been reported. The direct phosphorylation of β-catenin by Akt on Ser552, a site distinct from that targeted by GSK3-β, promotes the transcriptional activity of β-catenin (12). Using Akt1-deficient mice, we demonstrate that this alternate mechanism of β-catenin phosphorylation by the Akt1 isoform is necessary to enhance IFN-β transcription.

Materials and Methods

Reagents

The following reagents were used: polyinosinic:polycytidylic acid [poly(I:C)] LMW (Invivogen), SB-216763 (Cayman Chemical), AP21967, now referred to as A/C heterodimerizer (Clontech), and Verikine Mouse IFN-β ELISA (PBL IFN Source) were used according to manufacturer’s instructions to analyze cell supernatants collected at the indicated time points. Cells were cultured in RPMI 1640 or DMEM made complete with l-glutamine, penicillin-streptomycin, and 10% FBS (Invitrogen). Abs directed against the following Ags were used, with the catalog numbers in parentheses: Akt1 (2967), Akt2 (2964), phospho-Akt (S473) (9271), phospho-IRF3 (S396) (4947), phospho-Erk1/2 (T202/Y204) (9101), phospho-p38 (T180/Y182) (9211), phospho–stress-activated protein kinase/JNK (T183/Y185) (9255), p38 MAPK (9212), and phospho–β-catenin (S552) (9566) from Cell Signaling; phospho-Akt (T308) (124001) from Calbiochem; β-catenin (sc-7963), GAPDH (sc-25778), GFP (sc-8334), inhibitor of κ L chain enhancer (IκBα; sc-371), and histone deacetylase 1 (HDAC1; 2E10) from Millipore. Data were processed using Microsoft Excel (2003) and GraphPad Prism (version 5.0).

Animal and tissue preparation

Animal experiments used C57BL/6 or Akt1−/− mice bred back more than nine generations to C57BL/6, between 6 and 10 wk of age, and were conducted according to protocols approved by the Institutional Animal Care and Use Committee at University of Illinois, Chicago. For in vivo analysis, poly(I:C) in sterile saline was injected into the peritoneal space to a final dose of 5 mg/kg, animals were sacrificed, spleens were excised, and RNA was prepared as described later. For macrophage culture, animals were sacrificed, and femurs and tibias were excised, washed with 70% ethanol and then sterile HBSS. Marrow was aspirated in complete RPMI 1640, single cell suspended, and pelleted. Cells were then cultured in complete RPMI 1640 supplemented with 10% L929 cell (American Type Culture Collection) conditioned complete DMEM for 4 d, lifted, and plated. For signaling analyses, cells were starved in RPMI 1640 without growth factor or serum for 6–8 h before stimulation.

Transfections and reporter assays

HeLa cells (American Type Culture Collection) were cultured in complete DMEM and transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions, using plasmids encoding the following: pEGFP-N1, β-galactosidase, ifnβ-110 luciferase reporter (a kind gift of Dr. T. Maniatis, Harvard University, Cambridge, MA), TLR3 and Toll/IL-1R domain-containing adaptor inducing IFN-β (Trif; kind gifts of Dr. X. Li, Cleveland Clinic Foundation, Cleveland, OH), wild type (WT) and Ser552 mutant β-catenin (kind gifts of Dr. Z. Lu, University of Texas MD Anderson Cancer Center, Houston, TX), myristoylated mutant FK506 binding protein (FKBP; pC4M-F2E, a kind gift of ARIAD Pharmaceuticals), and FKBP12-rapamycin binding domain (FRB)-mouse Akt1 (an in-frame fusion of the mutant FRB fragment from pC4-RHE [ARIAD Pharmaceuticals] to the N terminus of murine Akt1 in pCMV6). The cells were incubated for 18 h. For signaling analyses, cells were starved for a further 24 h in DMEM without serum and stimulated as described prior to lysis. For reporter assays, cells were stimulated, washed with HBSS, and lysed in Reporter Lysis Buffer (Promega). Luciferase expression was assayed using Luciferase Assay Reagent (Promega) and normalized to β-galactosidase expression using the Luminescent β-galactosidase Detection Kit II (Clontech); both signals were detected with a Wallac Victor2 (PerkinElmer). Samples were run in triplicate, and data presented represent at least three independent experiments.

Immunoblotting

Cells were harvested by lysis with SDS-PAGE loading buffer with protease and phosphatase inhibitors (Calbiochem), boiled and sonicated for 15 s, resolved on TGX gradient gels (Biorad) and transferred to nitrocellulose membranes (Bioexpress), blocked, and stained with the indicated Abs. Chemiluminescence detection was performed with Supersignal (Thermo) and imaged using an Imagequant LAS 4000 (GE). Image analysis and quantification was performed using Adobe Photoshop and ImageJ (National Institutes of Health). All data presented represent, and quantifications are derived from, at least three independent experiments.

Nuclear isolations

Macrophages were stimulated and collected at the indicated time points, placed on ice, and washed twice with ice-cold HBSS, scraped in HBSS with protease and phosphatase inhibitors (Calbiochem), and pelleted. Cells were resuspended in cold Nuclear isolation buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, protease and phosphatase inhibitors), incubated on ice for 30 min, IGEPAL CA-630 was added to 0.6%, preparations were vortexed, and lysis was confirmed by microscopy. Nuclei were pelleted, washed three times with nuclear isolation buffer, and prepared for immunoblotting.

RNA isolation and quantitative real-time PCR

Cells were lysed and tissues were harvested and homogenized in TRIzol (Invitrogen), frozen at −80°C, and subsequently processed according to manufacturer’s instructions. RNA grade glycogen (Fermentas) was added to improve RNA precipitations. RNA was immediately reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen) according to instructions provided, using random hexamers (Fermentas) and RNA grade dNTPs (Invitrogen). cDNAs were frozen and subsequently diluted and analyzed using SYBR Advantage qPCR Premix (Clontech), using the default program with dissociation curves on an ABI PRISM 7000 Sequence Detection System, using the following gene-specific primers at 200 nM each: mouse IFN-β: reverse, 5′-CATCTTCTCCGTCATCTCCATAG-3′; forward, 5′-CGTTCCTGCTGTGCTTCTC-3′; mouse TNF-α: reverse, 5′-CACTTGGTGGTTTGCTACGA-3′; forward, 5′-AGTTCTATGGCCCAGACCCT-3′; mouse CXCL2: reverse, 5′-ATCCAGGCTTCCCGGGTGCT-3′; forward, 5′-AAGGAGGAGCCTGGGCTGCT-3′; mouse CXCL10: reverse, 5′-GCTCTCTGCTGTCCATCCAT-3′; forward, 5′-CCCACGTGTTGAGATCATTG-3′; mouse CCL5: reverse, 5′-TCCTTCGAGTGACAAACACG-3′; forward, 5′-ACCTCTCCCTAGAGCTGCCT-3′; mouse GAPDH: reverse, CTTGCTCAGTGTCCTTGCTG-3′; forward, 5′-TGCGACTTCAACAGCAACTC-3′. Triplicate samples were analyzed for each data point; data presented are representative of at least three independent experiments.

HSV-1 infection

Macrophages were infected with HSV-1(F) at 0.1 PFU/cell and harvested at the indicated time points for mRNA (as described earlier) or viral plaque assays. Cells were subjected to freeze–thaw three times to liberate virus; viral titers were quantified by plating on Vero cells and assaying plaque formation. Plaque assay data shown are representative of two independent experiments.

Results

Akt1 is required for optimal transcription of the IFN-β gene in response to poly(I:C) and viral infection

To determine the role for specific Akt isoforms in promoting transcription, we studied mice with a targeted deletion of the Akt1 gene for viral-associated stimulation of IFN-β transcription. Macrophages from Akt1−/− mice were prepared and tested for their response to TLR3 stimulation with the synthetic dsRNA mimetic, poly(I:C). After 2 h of stimulation, IFN-β mRNA production was assayed by quantitative PCR. Macrophages from C57BL/6 (B6) were used as controls. Whereas the B6 macrophages responded robustly to poly(I:C) stimulation, the Akt1−/− cells expressed significantly less IFN-β transcript (p < 0.005; Fig. 1A). Assays of IFN-β protein expression also showed a similar defect in the Akt1−/− cells (p < 0.002), which remained at background levels after 3 h of stimulation (Fig. 1B). We observed similar results from in vivo analysis after i.p. injection of 5 mg/kg poly(I:C). Four hours after injection, we observed a 36 ± 13-fold increase in IFN-β transcript in the spleens of B6 animals, whereas Akt1−/− spleens demonstrated no change from baseline (p < 0.02; data not shown). IFN-β stimulates secondary transcriptional responses including the expression of genes coding for CCL5 (also known as RANTES) and CXCL10 (also known as IP-10) (13). As expected, both genes were expressed in B6 macrophages in response to poly(I:C) stimulation, but their induction was abrogated in the absence of Akt1 (Fig. 1C, 1D). We next determined whether genes unrelated to IFN-β were similarly affected, so we analyzed the mRNA response of TNF-α (Fig. 1E) and CXCL2 (also known as MIP-2; Fig. 1F). Akt1−/− cells displayed no reductions in the response of these IFN-β–independent genes; in fact, a small increase in mRNA production was observed. This may be explained by the report from Androulidaki et al. (14) that Akt1 may restrain certain types of proinflammatory gene transcription via expression of a microRNA. Taken together, these data demonstrate that there is a significant role for the Akt1 isoform in the transcription of the IFN-β gene and its downstream effects, but that it is unlikely to promote general pathways responsible for the induction of other inflammatory cytokines.

FIGURE 1.
FIGURE 1.

Defective IFN-β and IFN-β–dependent transcriptional responses in the absence of Akt1. (A) IFN-β transcripts detected in WT (B6) and Akt1−/− macrophages before and after poly(I:C) stimulation (2 h at 10 μg/ml). IFN-β mRNA was assayed using qPCR, and values were normalized to GAPDH mRNA. (B) IFN-β protein level was assessed by ELISA after 3 h of stimulation. Expression of CCL5 (C), CXCL10 (D), TNF-α (E), and CCL2 (F) mRNA from WT and Akt1−/− macrophages was conducted as in (A) before and after poly(I:C) stimulation. WT and Akt1−/− macrophages were infected with HSV-1 for 3 h, and mRNA was assessed as described earlier (G); at the indicated time points, HSV-1 viral burden was evaluated by viral plaque assay (H). All data are presented as means ± SD of three samples for a single experiment that is representative of three independent experiments, except (H), which is representative of two independent experiments.

We next analyzed the role of Akt1 in response to viral infection. HSV-1 is a large DNA virus known to infect mucosal epithelial cells and establish latent infection within neuronal tissues. HSV-1 infection stimulates IFN-β transcription through TLR3 stimulation and through the RIG-I–like receptors (RLRs) (15). Two recent studies highlight the importance of IFN-β in host defense by demonstrating that HSV-1 targets this cytokine by encoding separate virulence factors to interfere with RLRs, as well as TANK-binding kinase 1 (TBK1), which activates IRF3, a critical transcription factor for IFN-β (15, 16). Additional studies have recently demonstrated the importance of TLR3 and its signaling adaptor, Trif, by showing that patients with defects in these genes are at greater risk for HSV-1 encephalitis, a potentially fatal manifestation of viral infection (17). To examine a potential role for Akt1 in response to HSV-1 infection, we challenged Akt1−/− macrophages with HSV-1. Three hours after viral infection, the IFN-β transcript level was determined. As shown in Fig. 1G, Akt1−/− macrophages exhibited a reduced IFN-β response to HSV-1 challenge compared with B6 control cells. Consistent with this, Akt1−/− cells were less effective in controlling HSV-1 replication than B6 macrophages, as determined by viral plaque assays (Fig. 1H). Because HSV-1 is known to stimulate multiple pathways leading to IFN-β transcription, it is possible that more than one of these pathways for viral recognition were affected by the absence of Akt1.

Loss of Akt1 affected poly(I:C)-mediated Akt phosphorylation, but not IRF3 or NF-κB signaling pathways

The regulation of Akt is complex. Activation of the kinase proceeds downstream from PI3K, which acts on phosphoinositol lipids. These modified lipids recruit Akt to the membrane where it is subsequently phosphorylated on its catalytic site at Thr308 by phosphoinositide-dependent kinase-1 and on a regulatory site in the C terminus at Ser473 by mammalian target of rapamycin complex 2 (11). Akt kinase activity has been shown to play a role in TLR3 signaling (7, 18, 19). We therefore wished to determine whether the genetic loss of Akt1 alone was sufficient to affect Akt signaling. Macrophages were prepared from B6 and Akt1−/− mice, stimulated with poly(I:C), and characterized for phosphorylation on Akt using an anti–phospho-Akt Ab that does not discriminate between different Akt isoforms. In B6 cells, phosphorylation of Akt at Ser473 increased within 10 min of stimulation and was maintained for at least 60 min. By contrast, Akt phosphorylation in the Akt1−/− cells was significantly diminished (Fig. 2). We obtained similar results for phosphorylation of Akt on Thr308 (data not shown). Because macrophages express both Akt1 and Akt2, we analyzed the expression of both kinases. As expected, no Akt1 was detected in cells from Akt1−/− mice. However, the absence of Akt1 did not change Akt2 protein expression (Fig. 2). These data demonstrate that Akt1 plays a nonredundant role in response to TLR3 stimulation, and that its loss results in abrogation of normal Akt signaling.

FIGURE 2.
FIGURE 2.

Macrophages from Akt1−/− mice demonstrate defective phosphorylation on Akt in response to poly(I:C) stimulation. Macrophages prepared from Akt1−/− and B6 animals were treated with poly(I:C) for the indicated time points, protein was resolved by SDS-PAGE followed by immunoblotting for phosphorylated and total Akt species. Quantification of band intensities is presented in lower panel as means ± SDs of at least three independent experiments; values were normalized to GAPDH protein levels for each sample (*p < 0.05).

We next wished to evaluate other signaling pathways relevant to IFN-β transcription where Akt has been reported to play a role. IRF3 is a critical transcription factor involved in the induction of IFN-β. Phosphorylation of IRF3 at Ser396, which may be catalyzed by the kinase TBK1, is sufficient to drive IRF3 to the nucleus to promote transcription of IFN-β (20). Because Akt signaling has been shown to promote the activation of TBK1 (7), we sought to examine whether Akt1 is important for IRF3 phosphorylation in response to poly(I:C) stimulation. In B6 macrophages, we consistently detected increases in phospho-IRF3 Ser396 levels within 30 min of poly(I:C) stimulation. In Akt1−/− cells, we detected a trend of decreased IRF3 phosphorylation, but it did not reach statistical significance (Fig. 3). The NF-κB pathway is activated by many stimuli including those that induce IFN-β expression. A variety of signaling events converge on the activation of the IκB kinase (IKK) kinase complex, which phosphorylates IκB, and thereby targets it for ubiquitin-mediated degradation. We assayed IκBα protein levels and observed decreases in protein abundance within 30 min of poly(I:C) stimulation. However, lysates from the B6 and Akt1−/− macrophages displayed no differences in IκBα protein abundance at any of the time points analyzed (Fig. 3). We also evaluated the MAPK signaling pathways, which are known to play important roles in activating transcription of IFN-β (5). No decreases in the phosphorylation of p38 MAPK or JNK MAPK were observed in Akt1−/− cells (data not shown). This led us to exclude a role for these pathways in the Akt1-mediated IFN-β transcription.

FIGURE 3.
FIGURE 3.

Akt1−/− macrophages demonstrate normal IRF3 phosphorylation and IκBα degradation in response to poly(I:C) stimulation. Macrophages prepared from Akt1−/− and B6 animals were treated as in Fig. 2 and immunoblotted for phosphorylated IRF3 and total IκBα. Quantification of band intensities is presented in bottom panels as means ± SDs of at least three independent experiments; values were normalized to GAPDH protein levels for each sample.

Akt1 activates β-catenin signaling induced by poly(I:C)

β-Catenin acts as a transcriptional activator through its accumulation in cells and translocation to the nucleus, where it promotes transcription. Yang et al. (8) demonstrated that viral and LPS stimulation of IFN-β is associated with β-catenin accumulation and phosphorylation at Ser552. Because Ser552 of β-catenin has been reported to be phosphorylated by Akt (12), we wished to determine whether β-catenin levels were impacted by the loss of Akt1. Macrophages were prepared from B6 or Akt1−/− mice and stimulated with poly(I:C). At the indicated time points, nuclei were isolated and protein was resolved on polyacrylamide gel and immunoblotted for β-catenin, GAPDH (a cytosolic protein), and HDAC1 (a nuclear protein). Quantification for β-catenin at 60 min after poly(I:C) stimulation revealed increased β-catenin protein levels in the nucleus of B6-derived macrophages. In comparison, significantly less β-catenin was found in the nuclei of Akt1−/− macrophages (Fig. 4A).

FIGURE 4.
FIGURE 4.

Macrophages from Akt1−/− mice demonstrate defects in β-catenin accumulation and phosphorylation. (A) Macrophages prepared from Akt1−/− and B6 animals were treated with poly(I:C) for the indicated time points. Cells were separated into nuclear and cytoplasmic fractions, and protein was resolved by SDS-PAGE and immunoblotted for total β-catenin, nuclear, and cytoplasmic markers. Quantification for the 60-min time point was normalized to HDAC1 protein levels. (B) Macrophages were treated for the indicated time points with poly(I:C), whole-cell lysates were resolved by SDS-PAGE and immunoblotted for total β-catenin and β-catenin phosphorylated on Ser552. Quantifications were derived as in Fig. 2 (*p < 0.05, **p < 0.002).

We next investigated phosphorylation of β-catenin in these cells. Ser552 has been reported to be a direct target of Akt, and its phosphorylation can promote transcriptional activity (12). We therefore wished to determine whether phosphorylation on Ser552 required Akt1. Macrophages from B6 and Akt1−/− mice were stimulated for the indicated time points with poly(I:C), and total cell lysates were resolved by PAGE and probed for β-catenin, as well as phosphorylated β-catenin at Ser552. As expected from the nuclear blots, total β-catenin increased in B6 cells at 30 and 60 min, but Akt1−/− demonstrated significantly less β-catenin protein at these time points. B6 cells also demonstrated increased accumulation of phospho–β-catenin on Ser552 after stimulation, whereas there was significantly less of the phosphorylated species in the Akt1−/− cells (Fig. 4B). Taken together, these findings demonstrate that on poly(I:C) treatment, β-catenin accumulates and translocates to the nuclei, and this correlates with phosphorylation on Ser552. These events require the Akt1 isoform.

Inhibition of GSK3-β enhances IFN-β transcription but cannot rescue the loss of Akt1

As discussed earlier, Akt can potentially regulate the β-catenin pathway through two different, but nonmutually exclusive mechanisms. Akt can inhibit the kinase GSK3-β through phosphorylation, which should result in the accumulation of β-catenin protein. This may alone be sufficient to enhance IFN-β transcription. However, Akt can also phosphorylate Ser552 directly. We therefore used SB-216763, a chemical inhibitor of GSK3-β kinase activity, to discriminate between these two possibilities for their impact on IFN-β induction. Macrophages from B6 or Akt1−/− mice were prepared and treated with or without SB-216763 before stimulation with poly(I:C) for 2 h. IFN-β mRNA, as assayed by qPCR, increased with poly(I:C) treatment as expected. Pretreatment with SB-216763 significantly increased the IFN-β mRNA response (Fig. 5A). These results are consistent with the report that inhibition of GSK3-β enhances LPS-stimulated IFN-β expression (9). The Akt1−/− macrophages failed to respond to poly(I:C). In contrast with B6 macrophages, pretreatment with the GSK3-β inhibitor failed to promote any increase in the IFN-β mRNA in response to poly(I:C) (Fig. 5A). The failure of GSK3-β inhibition to rescue the Akt1−/− phenotype demonstrates a nonredundant role for Akt1 that goes beyond inhibition of GSK3-β.

FIGURE 5.
FIGURE 5.

Inhibition of GSK3-β potentiates IFN-β transcription but cannot rescue transcription in Akt1−/− macrophages. (A) Macrophages from B6 or Akt1−/− mice were pretreated with or without 10 μM SB-216763 for 30 min to inhibit GSK3-β kinase activity. Cells were subsequently treated with or without poly(I:C) for 2 h, and IFN-β mRNA was assayed by quantitative PCR and normalized to GAPDH mRNA. Data were processed and presented as in Fig. 1. (B) Cells pretreated with inhibitor or controls, as in (A), were stimulated with poly(I:C) for 15 min, and protein was resolved by SDS-PAGE and immunoblotted for total and phospho–β-catenin species. Data are representative of three independent experiments.

We next determined the effect of GSK3-β inhibition on β-catenin protein accumulation and phosphorylation. As predicted, immunoblot analysis of cells treated with SB-216763 as described earlier demonstrated that both B6 and Akt1−/− macrophages contained increased concentrations of β-catenin protein, regardless of poly(I:C) stimulation. However, only B6 macrophages demonstrated increased Ser552 phosphorylation on β-catenin, and only on stimulation with poly(I:C) (Fig. 5B). Akt1−/− cells failed to demonstrate any increase in Ser552 phosphorylation regardless of inhibitor treatment or poly(I:C) stimulation (Fig. 5B). These data support the hypothesis that increases in β-catenin levels can enhance the transcription of IFN-β mRNA in macrophages, but that Akt1 is required for IFN-β induction through phosphorylation of Ser552 on β-catenin.

Ser552 of β-catenin is required for enhanced transcription of IFN-β

Because β-catenin levels correlate with IFN-β mRNA responses in macrophages, we next determined whether ectopic expression of β-catenin could enhance IFN-β transcription. To accomplish this, we reconstituted the TLR3 pathway in HeLa cells and analyzed the effects of increased levels of β-catenin on reporter gene activity. Cells were transfected with the expression constructs for TLR3, Trif, ifnβ-110 luciferase reporter, and β-galactosidase (a readout of transfection efficiency). Either the WT or a Ser552 to Ala (S552A) mutant of β-catenin was transfected into the HeLa cells, and their expression was confirmed (Fig. 6B). An empty vector control was introduced in place of the β-catenin constructs. Treatment of the “empty vector” transfected cells with poly(I:C) induced a significant increase in luciferase reporter activity, demonstrating that the TLR3 signaling pathway could be reconstituted in the transfected cells. Cells transfected with the WT β-catenin construct showed a further increase in luciferase reporter activity on stimulation with poly(I:C), compared with the “empty vector” controls. In contrast, cells transfected with the S552A mutant of β-catenin demonstrated no further increase in luciferase activity over WT transfected cells (Fig. 6A). These data demonstrate that β-catenin contributes causally to transcription of IFN-β mRNA, and that phosphorylation on Ser552 is critical for this effect in the reconstituted cells.

FIGURE 6.
FIGURE 6.

β-Catenin can enhance IFN-β promoter activity, and this requires Ser552. (A) HeLa cells were reconstituted with components of the TLR3 signaling pathway, IFN-β–110 luciferase reporter, and either empty vector, WT, or mutant β-catenin expression constructs. Cells were stimulated with poly(I:C) for 4 h, and luciferase reporter expression was detected as described. Data presented are normalized to ectopic β-galactosidase expression and shown as means ± SDs of three samples from a single experiment that is representative of three independent experiments. (B) Endogenous and ectopically expressed β-catenin protein levels were detected by SDS-PAGE and immunoblotting.

Activation of Akt1 is sufficient for Ser552 phosphorylation on β-catenin

We demonstrated earlier that Akt1 is required for phosphorylation of β-catenin on Ser552, and the importance of this site in promotion of IFN-β transcription. We next investigated whether Akt1 kinase activity alone is sufficient to drive phosphorylation on Ser552 and β-catenin accumulation. To accomplish this, we generated a chemically inducible version of Akt1 (FRB-Akt1), whose kinase function could be activated rapidly by addition of a drug to the cells. FRB-Akt1 is composed of mouse Akt1 with an N-terminal fusion to FRB, the rapamycin-binding domain of mammalian target of rapamycin. This FRB domain was mutated to allow binding to an analog of rapamycin, AP21967, with a bulky substitution at the C16 methoxy group that prevents binding or inhibition of endogenous mammalian target of rapamycin activity (21). It was previously demonstrated that such a construct could be recruited to a myristoylated FKBP protein (myrFKBP), and that this alone could drive Akt activation (22). In this system, AP21967 drives Akt1 to the plasma membrane, leading to robust phosphorylation of Akt1 on both Ser473 and Thr308, and subsequent phosphorylation of the Akt target protein, GSK3-β (Fig. 7B and data not shown). Using this approach, we next determined whether chemically inducible Akt1 activity could influence the transcriptional activity of IFN-β. Cells transfected with components of the TLR3 signaling pathway as described earlier were additionally transfected with the FRB-Akt and myrFKBP constructs. As earlier, poly(I:C) treatment significantly enhanced the IFN-β transcriptional reporter activity. In identically transfected cells, simultaneous stimulation with both poly(I:C) and AP21967, to chemically activate the FRB-Akt1 construct, significantly enhanced the reporter activity beyond that of poly(I:C) alone (Fig. 7A). Activation of FRB-Akt1 in this system alone, however, is not sufficient to drive reporter activity beyond the level of untreated cells (data not shown). These data demonstrate that increasing Akt1 signaling can enhance the transcriptional activity of the IFN-β promoter. In addition, it demonstrates that signaling downstream of our chemically inducible Akt1 construct can functionally interact with the endogenous signaling pathways governing transcriptional activation of IFN-β.

FIGURE 7.
FIGURE 7.

Akt1 kinase activity alone is sufficient for phosphorylation on Ser552 and accumulation of β-catenin protein. (A) HeLa reporter assays were performed as in Fig. 6 with a chemically inducible version of Akt1. Identically transfected cells were stimulated with buffer, poly(I:C) alone (20 μg/ml), or poly(I:C) and the activating drug, AP-21967 (100 nM), for 4 h. Cells were subsequently analyzed for luciferase activity, and data were processed and are presented as in Fig. 6. (B) Cells transfected as in (A) were stimulated with the poly(I:C) (20 μg/ml) or AP-21967 (100 nM) for the indicated time points. Protein was resolved by SDS-PAGE and immunoblotted with the indicated Abs. Quantifications were normalized to GAPDH protein expression, and are presented as in Fig. 2.

We next examined the sufficiency of Akt1 signaling for the activation of β-catenin. Cells were transfected, as described earlier, for the luciferase reporter assays. We either activated TLR3 signaling by stimulating with poly(I:C) or activated Akt1 alone by stimulating with AP21967, using the same doses as earlier for luciferase reporter assays. Cells were analyzed by immunoblotting at the indicated time points. Stimulation with poly(I:C) led to rapid and sustained phosphorylation of Akt on Ser473 (Fig. 7B). Akt1 was also phosphorylated on Thr308 (data not shown). Chemical activation of Akt1 alone led to similar phosphorylation on Akt (Fig. 7B and data not shown). Stimulation of either TLR3 or FRB-Akt1 alone led to similar accumulation of β-catenin and its phosphorylation on Ser552 (Fig. 7B). Taken together, these data demonstrate that Akt1 is sufficient to drive the phosphorylation of Ser552 on β-catenin, and that this signaling contributes to the enhanced transcription of IFN-β (Fig. 8).

Discussion

The signaling pathways that control the expression of IFN-β are critical to antiviral immunity, and understanding them may aid in therapeutic manipulation of the immune response (2). Our results demonstrate that the Akt1 isoform is critical in regulating this cytokine in response to TLR3 and viral stimulation, despite normal expression of the Akt2 protein. We demonstrate that loss of Akt1 leads to a significant reduction of the IFN-β mRNA response to TLR3 activation, both in vitro and in vivo. The defect in transcription results in decreases in IFN-β protein and reduced transcription of IFN-β–dependent genes. A similar defect in IFN-β expression is seen in response to infection of the knockout macrophages with HSV-1, and these cells are less able to control viral replication. Despite these defects and consistent with prior reports, we observe no defects in the transcription of proinflammatory cytokines such as TNF-α. These findings suggest that Akt1 regulates selected functions downstream of TLR3 signaling and prompted further investigation of the mechanisms that control IFN-β expression.

It has been previously reported, using genetic and biochemical approaches, that IFN-β induction involves TBK1 phosphorylation of IRF3, a critical transcription factor that binds the enhancer of IFN-β (23, 24). Interestingly, pathogens such as HSV-1 have developed evasive strategies to target this pathway, thereby facilitating productive infection (15). In addition, IRF3 is also regulated by IKKε through phosphorylation. Although a PI3K-dependent component has been implicated in the activation of IRF3 (25), it was not until recently that a role for Akt in IFN-β induction began to be recognized. β-catenin, one of the substrates for Akt phosphorylation, is closely involved in IFN-β induction. A β-catenin–dependent pathway is responsible for intracellular nucleic acid sensor-mediated IFN-β production, through mechanisms that include direct binding of β-catenin to the C-terminal domain of IRF3 (8). Based on published data and results from this study, the Akt1 isoform is primarily responsible for the two functions of Akt in regulating β-catenin signaling (Fig. 8). One is the inhibition of GSK3-β by phosphorylation on Ser9 (11). β-Catenin promotes the constitutive targeting of β-catenin for proteosomal degradation (26), hence serving as a negative regulator of IFN-β production (9). By suppressing GSK3-β, Akt increases β-catenin protein levels. We have shown that in Akt1−/− macrophages, Akt1 isoform constitutes a significant part of activated Akt and is required for β-catenin accumulation in the nucleus. A second and more recently uncovered function is the direct phosphorylation of β-catenin by Akt on Ser552, which leads to β-catenin nuclear translocation and enhances its transcriptional activity (8, 12). We have shown that Akt1 deficiency abrogates Ser552 phosphorylation on β-catenin, and this defect persists even after stabilization of β-catenin protein level by inhibition of GSK3-β (Fig. 5). Based on these findings, we conclude that suppression of GSK3-β and direct phosphorylation of β-catenin at Ser552 are two independent functions of Akt1 (Fig. 8), both required for IFN-β induction. We have shown that ectopic expression of WT β-catenin can enhance the activity of the IFN-β promoter on TLR3 stimulation, and this function is dependent on Ser552 because a mutant β-catenin bearing an alanine at this site fails to enhance promoter activity (Fig. 6). Finally, we show that in cells that recapitulate TLR3 signaling, the activation of a chemically inducible version of Akt1 alone is sufficient to drive accumulation and Ser552 phosphorylation of β-catenin similar to TLR3 stimulation. Taken together, these data demonstrate that viral-associated stimulation of IFN-β is enhanced by activation of Akt1, and the activation of Akt1 is necessary and sufficient to drive direct phosphorylation and the transcriptional activity of β-catenin.

FIGURE 8.
FIGURE 8.

A working model for Akt1-dependent enhancement of IFN-β transcription. Stimulation of TLR3 by poly(I:C) activates Akt1, which directly phosphorylates on two downstream targets: phosphorylation of β-catenin at Ser552 enhances its transcriptional activity, whereas phosphorylation of GSK3-β results in increased β-catenin protein levels. Combined, Akt1 is able to stimulate β-catenin–mediated transcriptional activity, which, together with other signaling pathways, enhances IFN-β gene induction.

Results from this study provide direct evidence that Akt1 is sufficient for β-catenin phosphorylation at Ser552. However, even though a chemically active Akt1 is sufficient for stimulation of β-catenin phosphorylation and its transcriptional activity, it is insufficient to drive the induction of IFN-β, which requires other signaling events such as IRF3 phosphorylation. Although we have observed a consistent decrease in the TBK1-mediated phosphorylation of IRF3 in the absence of Akt1, it was not statistically significant (Fig. 3). This may be explained by the fact that IRF3 phosphorylation is regulated independently, by kinases such as TBK1 and IKKε; the Akt2 isoform may also be able to perform this function. In addition, we saw no defects in IκBα degradation or activation of the MAPK signaling pathways. These findings support a selective function of the Akt1 isoform in the regulation of IFN-β induction.

Akt1 and Akt2 are closely related isoforms of Akt, and both are highly expressed in macrophages and other phagocytes (10). Our observation that Akt1 deficiency alters TLR3-dependent induction of IFN-β, despite normal expression of Akt2, demonstrates that Akt1 has a nonredundant function in host defense. Several possibilities exist that may explain the observations. One is that Akt1 functions differently from Akt2 in macrophages and is the only Akt isoform required for IFN-β induction. Supporting this possibility, we have shown in our reconstitution assay that a chemically active Akt1 is sufficient for β-catenin phosphorylation at Ser552. Akt isoform-specific signaling may be accomplished through selective interactions with phosphatases, as has been shown with the PHLPP family that differentially regulates Akt isoforms (27). The other possibility is that both Akt1 and Akt2 are required for IFN-β induction because they each regulate a distinct signaling pathway. Genetic approaches will be required to test this possibility in Akt2−/− mice. Finally, it is also possible that both isoforms of Akt are required to maintain a level of signals for the activation of downstream pathways. In recent years, Akt isoform-dependent functions have been reported, and a study of these cases may help to understand how Akt1 regulates IFN-β production. In platelets, all three Akt isoforms are expressed, and genetic deletion of any of these results in significant defects in platelet activation (28–30). The absence of Akt2 leads to a type 2 diabetic phenotype (31, 32), whereas deficiency in Akt1 results in growth retardation and increased apoptosis (33). Of particular interest, Akt2, but not Akt1, is primarily responsible for the bactericidal functions of neutrophils including cell migration, granule contents release, and superoxide production (10). The defects may be attributed to preferential distribution of Akt2 to plasma membrane on activation, whereas the Akt1 remains in the cytosolic compartment (10). Given that TLR3 signaling occurs in the intracellular compartment, it is possible that Akt1 localization dictates its functions, such as a TLR3-dependent induction of IFN-β. These findings suggest that specific targeting of one Akt isoform may be of therapeutic value in terms of inhibiting or activating the respective pathways.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. T. Maniatis for the ifnβ-110 reporter construct, Dr. X. Li for the TLR3 and Trif expression plasmids, and Dr. Z. Lu for the β-catenin constructs. We also thank Matthew Perryman for technical assistance.

Footnotes

  • This work was supported by National Institutes of Health Grants R01 AI033503 and R56 AI040176. B.N.G. was supported in part by National Institutes of Health Grant T32 HL007829.

  • Abbreviations used in this article:

    FKBP
    FK506 binding protein
    FRB
    FKBP12-rapamycin binding domain
    GSK3-β
    glycogen synthase kinase 3 β
    HDAC1
    histone deacetylase 1
    IκBα
    inhibitor of κ L chain enhancer
    IKK
    IκB kinase
    IRF3
    IFN response factor 3
    poly(I:C)
    polyinosinic:polycytidylic acid
    RLR
    RIG-I–like receptor
    TBK1
    TANK-binding kinase 1
    Trif
    Toll/IL-1R domain-containing adaptor inducing IFN-β
    WT
    wild type.

  • Received June 19, 2012.
  • Accepted July 5, 2012.

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