The activating receptor NKG2D and its ligands RAE-1 play an important role in the NK, γδ+, and CD8+ T cell-mediated immune response to tumors. Expression levels of RAE-1 on target cells have to be tightly controlled to allow immune cell activation against tumors but to avoid destruction of healthy tissues. In this study, we report that cell surface expression of RAE-1ε is greatly enhanced on cells lacking JunB, a subunit of the transcription complex AP-1. Furthermore, tissue-specific junB knockout mice respond to 12-O-tetradecanoyl-phorbol-13-acetate, a potent AP-1 activator, with markedly increased and sustained epidermal RAE-1ε expression. Accordingly, junB-deficient cells are efficiently killed via NKG2D by NK cells and induce IFN-γ production. Our data indicate that the transcription factor AP-1, which is involved in tumorigenesis and cellular stress responses, regulates RAE-1ε. Thus, up-regulated RAE-1ε expression due to low levels of JunB could alert immune cells to tumors and stressed cells.

Effective immune responses against infections and tumors require a close collaboration between the innate and the adaptive immune system. In the innate immune system, NK cells, which are cytolytic and cytokine-producing effector cells, serve as a first line of immune defense (1, 2). Their activation is determined by a delicate balance between inhibitory receptors, most of which are specific for self-MHC class I, and activating receptors.

The activating receptor NKG2D is expressed on all NK cells, on NKT cells, γδ+ T cells, and in mice on activated CD8+ T cells. It was shown that, in mice, the protein family that comprises the retinoic acid early inducible gene-1 (RAE-1α-ε), H60, and MULT-1 (murine UL16-binding protein-like transcript-1) are recognized by NKG2D (3, 4). rae-1α, -β, and -γ were initially described as three separate genes in 129 mice that were induced by retinoic acid treatment of the F9 teratocarcinoma (5), and subsequently RAE-1δ and ε were identified to be ligands for mouse NKG2D (mNKG2D)3 (3, 6). In humans, the UL16-binding proteins (ULBPs) and the MHC class I chain-related gene (MIC) families bind to NKG2D (7, 8). Activation of the NKG2D receptor is partially controlled by inhibitory receptors in vitro (9). In vivo, however, ectopic expression of NKG2D ligands on tumor cells leads to NK cell-mediated tumor rejection even in the presence of self-MHC class I (10, 11). Therefore, expression levels of NKG2D ligands must be tightly controlled to allow immune responses to tumors and viruses, yet to avoid harmful damage of healthy tissues.

Indeed, NKG2D-ligand expression is largely absent from most healthy tissues (3). Increased expression of NKG2D ligands is detected during certain autoimmune diseases, e.g., in the prediabetic pancreas islets of NOD mice (12) and on synoviocytes of rheumatoid arthritis patients (13). It was shown that rae-1δ and -ε expression was up-regulated on peritoneal macrophages from C57BL/6 mice after treatment with certain TLR agonists, whereas rae-1α, -β, and -γ were regulated on macrophages from BALB/c mice (14). Recently, Gasser et al. (15) reported regulation of mouse and human NKG2D ligands by a DNA damage pathway. Furthermore, NKG2D ligands are expressed on tumor cell lines and to varying degrees during cutaneous malignancy in mice (6). Taken together, up-regulated expression of NKG2D ligands distinguishes abnormal and stressed cells.

One hallmark of stressed cells is aberrant activity of transcription factors, particularly of the AP-1 complex. The AP-1 transcription factor is a dimeric complex that consists of members of the Jun (JunB, c-Jun, JunD), Fos, and activating transcription factor protein families (16, 17). It was shown that AP-1 regulates gene expression in response to extracellular stimuli including growth factors and cytokines, stress signals, oncogenic stimuli, and bacterial and viral infections (18). Alterations in AP-1 activity result in changes of target gene expression and subsequent complex biological processes, such as cell proliferation, differentiation, and apoptosis. For the AP-1 member JunB, both positive and negative effects on target gene expression were reported depending on its interacting partners, the promoter context and cell type. There is growing evidence that altered expression of the JunB subunit causes aberrant AP-1 total activity, which is often associated with pathophysiological responses. For example, deregulated expression of AP-1 subunits was observed during certain autoimmune diseases, including rheumatoid arthritis (19), and seems to be associated with psoriasis (20).

With regard to human cancer, both enhanced or decreased expression levels of JunB were described (16, 17). The importance for JunB to control differentiation and proliferation was confirmed in genetically modified mice, which express low levels of JunB in the myeloid lineage (21). These mice develop myeloid proliferative disease, which resembles early human chronic myeloid leukemia. In addition, in these mice, immunological defects with regard to Th2 differentiation were described (22).

Because much evidence exists that JunB and other AP-1 members regulate gene expression during cell differentiation, transformation, and cellular stress responses, we investigated whether AP-1 affects the expression of NKG2D ligands. In this study, we define a pivotal role for JunB in the regulation of RAE-1ε expression in vitro and in vivo and its impact on NK cell activation.

For detection of NKG2D ligands by flow cytometry, cell lines were stained with a mNKG2D-fusion protein (mNKG2D-FP) as described in Ref.3 . A human NKG2D-FP, which does not recognize mNKG2D ligands, was used as an irrelevant chimeric protein. The following Abs were used: anti-RAE-1ε, clone 205001 (R&D Systems); rat IgG2a, clone 54447 (R&D Systems); and goat anti-rat Ig-PE (BD Biosciences).

Wild-type (wt) and junB−/− mouse embryonic fibroblasts (MEFs) were isolated from a E9.5 mouse embryo of mixed genetic background (C57BL/6 × 129/Sv) as described previously (23). Midgestation junB−/− or heterozygous junB+/− embryos were the source for the infection of primary endothelial cells by the N-TKmT retrovirus. JunB+/− mice were backcrossed to C57BL/6 for at least six generations. To rescue expression of JunB, junB−/− MEFs cells were transduced with the retroviral vector pMX-pie (containing an internal ribosome entry site (IRES)-GFP element) encoding JunB or the empty vector (kindly provided by T. Kitamura, University of Tokyo, Tokyo, Japan) and selected with puromycin (1 μg/ml). Experiments were performed using cells 3 wk after puromycin selection. Approximately 95% of selected cells expressed GFP from the JunB-IRES-GFP expression vector.

For quantitative real-time PCR, labeled probes (Applied Biosystems) and primers were used as described previously (14). Total RNA was treated with DNaseI, and first-strand cDNAs were synthesized using oligo(dT)15 primers. mRNA levels were normalized to mRNA levels of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT).

Mice with floxed-junB alleles (24) were crossed with Collagen1α2-Cre (Col1α2-Cre) mice (25) to create Col1α2/junBΔ/− mice. Efficient recombination in the epidermis was verified by RT-PCR and Southern blot analysis (L. Florin, M. Schorpp-Kistner, and P. Angel, unpublished observations). Col1α2-Cre and junB floxed mice were backcrossed into C57BL/6 background for at least three generations, and 12- to 13-wk-old female mice were used. One day after shaving, 100 μl of 10 nmol/μl 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in acetone or solvent alone was applied on back skin three times in an interval of 48 h. Six or 24 h after the last treatment, total RNA was isolated from the epidermis. Heterozygous Col1α2/junBΔ/+ mice were used as controls. The procedures for performing animal experiments were approved by Regierungspräsidium Karlsruhe (Karlsruhe, Germany).

NK cells were isolated from C57BL/6 splenocytes using mAb-DX5-coated magnetic beads (Miltenyi Biotec), expanded for 7 days with 1700 U/ml human IL-2, and used as effectors in a standard 5-h 51Cr release cytotoxicity assay (3). For blocking experiments, the anti-mNKG2D mAb (R&D Systems; clone 191004) or an isotype control (R&D Systems; clone 141945) was included in the cytotoxicity assay at a concentration of 10 μg/ml.

A total of 2.5 × 105 NK cells was cultured with equal numbers of target cells in the presence of 1700 U/ml human IL-2 for 24 h. Concentrations of mouse IFN-γ in the supernatants were determined by ELISA (BD Biosciences).

To investigate the importance of the JunB protein in transcriptional regulation of RAE-1, we analyzed NKG2D-ligand expression in genetically modified embryonic cell lines deficient in JunB (Fig. 1). NKG2D-ligands were expressed at comparably low levels in wt MEFs and in heterozygous junB+/− endothelioma cells. By contrast, the expression of mNKG2D-ligands was greatly enhanced in MEFs and endothelioma cells deficient in JunB. Together, these data indicate that NKG2D-ligands are up-regulated in the absence of JunB.

FIGURE 1.

Expression of NKG2D ligands on mouse embryonic cell lines. Cells were stained with a mNKG2D-FP (filled histogram) or an irrelevant fusion protein (hNKG2D-FP; gray solid line) and analyzed by flow cytometry. Histograms of wt or junB−− MEFs (left panels) and junB+/− or junB−− endothelioma cells (right panels) are depicted. One representative of at least four independently performed experiments is shown. MFI, Mean fluorescence intensity.

FIGURE 1.

Expression of NKG2D ligands on mouse embryonic cell lines. Cells were stained with a mNKG2D-FP (filled histogram) or an irrelevant fusion protein (hNKG2D-FP; gray solid line) and analyzed by flow cytometry. Histograms of wt or junB−− MEFs (left panels) and junB+/− or junB−− endothelioma cells (right panels) are depicted. One representative of at least four independently performed experiments is shown. MFI, Mean fluorescence intensity.

Close modal

Because mNKG2D binds to several different ligands, we determined which NKG2D-ligand was modulated by JunB by using real-time PCR and flow cytometric analysis (Fig. 2). A 20-fold enhancement of rae-1ε mRNA level was observed in junB−/− MEFs as compared with wt controls (Fig. 2,A), whereas rae-1δ was not detectable. Accordingly, MEFs and endothelioma cells deficient in JunB stained at high levels with an Ab specific for RAE-1ε (Fig. 2 B and data not shown), confirming the enhanced rae-1ε mRNA expression at the protein level. JunB−/− MEFs also showed enhanced mRNA expression of MULT-1 as compared with wt, whereas expression levels of H60 were not detectable (data not shown). No positive staining was detected with the Ab CX1, which recognizes RAE-1α, -β, and -γ (data not shown).

FIGURE 2.

RAE-1ε is up-regulated on junB−− MEFs. A, mRNA expression levels of rae-1δ and -ε were determined by quantitative real-time PCR. ND, Not detectable. SDs of triplicates of one experiment are indicated. B, Cell surface expression of RAE-1ε on MEFs was determined using a specific Ab (filled histogram) or an isotype control (gray solid line). C, wt MEFs (filled histogram), junB−− MEFs transduced with either vector control (solid line), and a vector encoding JunB (dashed line) were stained with an anti-RAE-1ε mAb. Staining with an isotype control mAb is shown as dotted line. Cells were gated on GFP-positive cells. Experiments shown in A and B were performed at least four times with similar results. In C, one representative of two independently performed experiments is shown.

FIGURE 2.

RAE-1ε is up-regulated on junB−− MEFs. A, mRNA expression levels of rae-1δ and -ε were determined by quantitative real-time PCR. ND, Not detectable. SDs of triplicates of one experiment are indicated. B, Cell surface expression of RAE-1ε on MEFs was determined using a specific Ab (filled histogram) or an isotype control (gray solid line). C, wt MEFs (filled histogram), junB−− MEFs transduced with either vector control (solid line), and a vector encoding JunB (dashed line) were stained with an anti-RAE-1ε mAb. Staining with an isotype control mAb is shown as dotted line. Cells were gated on GFP-positive cells. Experiments shown in A and B were performed at least four times with similar results. In C, one representative of two independently performed experiments is shown.

Close modal

Next, we investigated whether the enhanced expression of RAE-1ε can be reversed, at least to some extent, in junB−/− MEFs by ectopic expression of JunB (Fig. 2 C). Upon retroviral transduction of junB−/− MEFs with an expression vector encoding JunB, cell surface expression of RAE-1ε was reduced in comparison to junB−/− MEFs transduced with the empty vector control. Quantitative Western blotting of these cell populations expressing GFP from the IRES-GFP expression vector revealed that the protein levels of JunB reintroduced in junB−/− MEFs, corresponded to endogenous levels expressed in MEFs derived from wt mice (data not shown).

These data show that ectopic expression of JunB can at least partially revert high expression levels of rae-1ε in junB−/− MEFs. We cannot exclude that, in the absence of JunB also other pathways are indirectly implicated.

Negative regulation of RAE-1ε by JunB might be mediated by binding of JunB to an AP-1 recognition sequence in the rae-1ε promoter, as previously suggested for JunB-dependent negative regulation of kgf and gm-csf (26). Thus, we performed a search for AP-1 binding sites within the potential rae-1ε promoter (NT_039491.3), and found a total of 11 putative AP-1 binding sites within the promoter region up to 6 kb upstream of the transcriptional start of rae-1ε. Two AP-1 binding sites represent 8-mer Jun/activating transcription factor sequences, whereas nine of them belong to the class of 7-mer Fos/Jun recognition sequences. From −4.0 kb to −4.5 kb a cluster of four Fos/Jun consensus AP-1 sites were identified, which suggest that rae-1ε could potentially be a direct target gene of AP-1.

It is also possible that repression by JunB is indirect, because it was recently shown that AP-1 can participate in more global mechanisms including chromatin remodeling (27). Taken together, the loss of JunB could regulate expression of RAE-1ε directly and/or indirectly.

To determine whether the absence of JunB also affects RAE-1ε expression in vivo, we analyzed tissue-specific junB knockout mice, to circumvent the lethality of junB−/− embryos (28). In Col1α2/junBΔ/− mice, JunB expression is absent in the dermis, epidermis, and lymphocytes, with no obvious phenotype in skin development and homeostasis (L. Florin, M. Schorpp-Kistner, and P. Angel, unpublished observations). Because under homeostatic conditions AP-1 basal activity is low in the epidermis of adult wt mice, the phorbol ester TPA–which is a very potent mitogenic activator of AP-1 leading to skin inflammation, epidermal hyperproliferation, and multistage carcinogenesis–was applied (29). Six hours after the last TPA treatment, a 2- to 3-fold induction of rae-1ε transcription was observed in the epidermis of control mice, which drops to levels similar to those before TPA treatment at 24 h (Fig. 3).

FIGURE 3.

Up-regulation of rae-1ε in the epidermis of Col1α2/junBΔ/− mice after treatment with TPA. Control mice (Col1α2/junBΔ/+) or Col1α2/junBΔ/− mice were treated three times with TPA or solvent control every second day. Total RNA of tissue samples was collected before and 6 h or 24 h after the last treatment, and rae-1ε expression was determined by real-time PCR. One representative of two independently performed experiments is shown, and SDs of triplicates of one real-time PCR are indicated. The real-time PCR was repeated three times with similar results.

FIGURE 3.

Up-regulation of rae-1ε in the epidermis of Col1α2/junBΔ/− mice after treatment with TPA. Control mice (Col1α2/junBΔ/+) or Col1α2/junBΔ/− mice were treated three times with TPA or solvent control every second day. Total RNA of tissue samples was collected before and 6 h or 24 h after the last treatment, and rae-1ε expression was determined by real-time PCR. One representative of two independently performed experiments is shown, and SDs of triplicates of one real-time PCR are indicated. The real-time PCR was repeated three times with similar results.

Close modal

In the epidermis of Col1α2/junBΔ/− mice, basal level expression of rae-1ε was similarly low, when compared with control mice, but rae-1ε up-regulation 6 h after TPA treatment was considerably higher (7–8 times). Moreover, expression of rae-1ε remained at high levels for at least 24 h. These data demonstrate that JunB is also a negative regulator of rae-1ε in vivo, allowing an increased and sustained induction of expression in response to TPA.

So far, our data demonstrate greatly enhanced levels of RAE-1ε on junB-deficient cells. Next, we asked whether the up-regulation of RAE-1ε on junB−/− MEFs leads to NK cell activation (Fig. 4). Cytotoxicity assays revealed that junB−/− MEFs are more efficiently killed by NK cells as compared with wt MEFs (Fig. 4 A). Addition of a neutralizing anti-mNKG2D mAb substantially blocked NK cell killing of junB−/− MEFs, which demonstrates that the enhanced killing is mediated by an interaction between NKG2D and its ligands.

FIGURE 4.

JunB deficiency sensitizes MEFs for lysis by NK cells and induces IFN-γ production. A, Wt or junB−− MEFs were used as targets in a standard 51Cr NK cell-mediated cytotoxicity assay. The anti-mNKG2D mAb (clone 191004) or an isotype control (clone 141945) was included in the cytotoxicity assay. B, NK cells were cultured in the absence (control) or presence of the indicated target cell lines, and IFN-γ release was determined by specific ELISA. One representative experiment of at least three independently performed experiments is shown. SDs of triplicates of one experiment are indicated.

FIGURE 4.

JunB deficiency sensitizes MEFs for lysis by NK cells and induces IFN-γ production. A, Wt or junB−− MEFs were used as targets in a standard 51Cr NK cell-mediated cytotoxicity assay. The anti-mNKG2D mAb (clone 191004) or an isotype control (clone 141945) was included in the cytotoxicity assay. B, NK cells were cultured in the absence (control) or presence of the indicated target cell lines, and IFN-γ release was determined by specific ELISA. One representative experiment of at least three independently performed experiments is shown. SDs of triplicates of one experiment are indicated.

Close modal

Furthermore, upon cocultivation with junB−/− MEFs, NK cells produced elevated levels of IFN-γ as compared with the coculture with wt MEFs (Fig. 4 B). Thus, the absence of JunB sensitizes target cells to NKG2D-mediated lysis and activates the production of IFN-γ.

The AP-1 subunits JunB and c-Jun are involved in cell proliferation, differentiation, apoptosis, and regulation of cytokines, thereby acting in the mesenchymal/epidermal cell cross talk (16, 17). Our data (Fig. 3) indicate that in the absence of JunB, epidermal cells react with an enhanced and sustained expression of rae-1ε in response to the mitogen and tumor promoter TPA. It was reported recently that rae-1 expression is also detected in carcinomas and papillomas, which developed several weeks after treatment with the tumor initiator dimethylbenz(a)anthracene combined with the tumor promoter TPA (6). In these studies, 24 h after TPA treatment, rae-1 expression was only minimally enhanced, which is consistent with our data on control mice. In Col1α2/junBΔ/[]− mice, however, we already observed enhanced levels of rae-1ε at 6 h, which remained at high levels at least for 24 h post-TPA treatment, demonstrating that JunB negatively regulates the expression of rae-1ε. It is interesting to note that, on one hand, expression levels of c-Jun and JunB are regulated by TPA with different kinetics during the course of skin carcinogenesis (18, 29). On the other hand, expression of rae-1ε varies considerably among individual papilloma and carcinoma isolates (6). Based on our findings implying antagonistic regulation of RAE-1ε by JunB and c-Jun in mutant cells and mice, it is tempting to speculate that these differences in RAE-1ε expression levels are, at least in part, due to differences in the molar ratio between c-Jun and JunB in a given tumor. Therefore, in the future, it will be important to simultaneously determine the levels of RAE-1, c-Jun, and JunB in papilloma, squamous cell carcinoma, and other tumors.

Our results (Fig. 4) demonstrate that killing of junB−/− target cells by NK cells is greatly enhanced. In addition, we also show that high levels of IFN-γ, a cytokine-mediating immunosurveillance against certain tumors, are induced in response to junB−− MEFs. In this context, it was demonstrated that γδ+ T cells, which express NKG2D, are crucial for immunosurveillance to cutaneous malignancies characterized by high expression of rae-1 (6). We assume that NKG2D-expressing immune cells are activated by an enhanced expression of RAE-1ε in junB−− epidermis in vivo.

JunB is involved in inflammatory processes and negatively regulates cell proliferation in certain cell types (16, 17, 20). Accordingly, synovial fibroblasts from rheumatoid arthritis patients (19) or keratinocytes from psoriasis patients (20) express low levels of JunB (19). Interestingly, synoviocytes from rheumatoid arthritis patients, which proliferate at high levels, also express elevated amounts of the human NKG2D-ligand MICA (13). It will be important to investigate whether the expression of human NKG2D ligands also depends on AP-1, particularly its negative regulation by JunB.

Altered activity of AP-1, due to deregulated expression of AP-1 subunits, is associated with several pathophysiological processes, all characterized by uncontrolled cell proliferation and differentiation (16, 17). Based on our data, loss of JunB in premalignant cells could alarm the innate immune system by up-regulation of a ligand for the activating receptor NKG2D, whereas enhanced levels of JunB, as reported at least for advanced stages skin cancer (29), may counteract efficient expression of RAE-1ε, thereby leading to tumor evasion of the immune system. Alternatively, aberrant expression of AP-1 subunits, including JunB, might also cause harmful immune cell activation during chronic inflammation and autoimmune disease.

We thank Drs. Axel Szabowski, Jochen Hess, Oliver Pein, and Björn Textor for helpful discussions, and Dr. Carola Schellack, Dr. Elisabeth Suri-Payer, and Lewis Lanier for critically reading the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Boehringer Ingelheim Fonds and the German-Israeli Foundation for Scientific Research and Development.

3

Abbreviations used in this paper: mNKG2D, mouse NKG2D; wt, wild type; MEF, mouse embryonic fibroblast; IRES, internal ribosome entry site; RAE-1, retinoic acid early inducible gene-1; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.

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