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Stat signaling cascade and induces p53-dependent antiviral protection
BBRC Biochemical and Biophysical Research Communications 329 (2005) 1139–1146 www.elsevier.com/locate/ybbrc
Intracellular interferon triggers Jak/Stat signaling cascade and induces p53-dependent antiviral protection q Masaharu Shin-Ya a, Hideyo Hirai a, Etsuko Satoh a, Tsunao Kishida a,b, Hidetsugu Asada a, Fumiko Aoki a, Masako Tsukamoto a, Jiro Imanishi a, Osam Mazda a,* a
Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan b Louis Pasteur Center for Medical Research, Kyoto 606-8225, Japan Received 5 February 2005
Abstract Intracellular interferons (IFNs) exert biological functions similar to those of extracellular IFNs, but the signal transduction pathway triggered by the intracellular ligands has not been fully revealed. We investigated the signaling cascade by sequence-specific knockdown of signaling molecules by means of the RNA interference. Truncated IFN-b gene was constructed so that the N-terminal secretory signal sequence was deleted (SD.IFN-b). Cells transfected with this construct showed phosphorylation and activation of the STAT1 without any detectable secretion of the cytokine. The MHC class I expression was significantly augmented, while the augmentation was suppressed by short interfering RNA duplexes specific for JAK1, TYK2, and IFN-a/b receptor (IFNAR) 1 and 2c chains. The SD.IFN-b also induced p53 and phosphorylation of p53 at Ser15. Specific silencing of p53 abrogated the antiviral effect of SD.IFN-b, suggesting that the tumor suppressor is critically involved in antiviral defense mediated by intracellular IFN. 2005 Elsevier Inc. All rights reserved. Keywords: Interferon; Autocrine; p53; RNA interference; STAT1
Cytokines are secreted from producer cells, subsequently binding to specific receptors on the surface of the target cells, in which intracellular signal transmission cascade is triggered to induce biological responses. Cumulative evidences indicate that biological functions of some cytokines also exert inside the producer cells without being secreted out from the cells. These include IFN-c [1–3], v-sis [4], interleukin-3 (IL-3) [5], IFN-a [6,7], and IL-6 [8]. Because these cytokines act in an autocrine manner, the signals triggered by intracellular q Abbreviations: SD.hIFN-b, signal sequence-deleted hIFN-b; IFN, interferon; mAb, monoclonal antibody; siRNA, small interfering RNA; RNAi, RNA interference. * Corresponding author. Fax: +81 75 251 5331. E-mail address: [email protected] (O. Mazda).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.088
ligands may have important biological implications in the cytokine action in autocrine cells. However, the signaling pathway triggered by intracellular ligand has not been fully elucidated at molecular level. In the case of extracellular IFN-b, the engagement of IFN-a/b receptor (composed of the IFNAR1 and IFNAR2c chains) on the cell surface triggers activation of JAK1 and TYK2, leading to phosphorylation and activation of the STAT1 transcriptional factor, which in turn induces expression of IFN-responsive genes through activation of the IFN-stimulated response element (ISRE)-containing promoters (reviewed in [9]). Intracellular IFN-a [6,7] as well as IFN-c [2,3] were shown to induce phosphorylation and activation of the STAT1, but it has not been demonstrated whether the conventional JAK/STAT pathways participate in
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the intracellular ligand-mediated signal transmission. Moreover, it remains controversial whether the intracellular ligand engages IFNAR1 and IFNAR2c receptor molecules to trigger the signaling cascade. Conventional experimental technologies including immunocoprecipitation of ligand–receptor complex can hardly demonstrate intracellular ligand–receptor interaction. Using the RNAi technology, we investigated whether each component of the IFNAR-JAK1/TYK2-STAT pathway was a prerequisite for the signal transduction cascade triggered by the extracellular and intracellular IFN-b. We found that the IFNAR1 and 2c chains as well as the JAK1, TYK2, and STAT1 molecules were critically involved in the signal transmission pathway. Moreover, we also demonstrated that the intracellular IFN signal results in p53 induction that plays essential roles in antiviral protection achieved by the intracellular cytokine. Materials and methods Plasmid vectors. The pGEG.hIFN-b contains the full-length human IFN-b (hIFN-b) cDNA in which a ClaI restriction site was introduced at the junction between the nucleotide sequences corresponding to the N-terminal secretory signal peptide and the mature hIFN-b in such a manner that the encoding amino acid sequence was
identical to that of wild-type IFN-b (Fig. 1A, upper figure). The signal sequence was deleted to generate pGEG.SD.hIFN-b (Fig. 1A, lower figure). Control plasmids, pGEG.GL3 and pGEG.EGFP, were described previously [10]. Cells, transfection, and neutralizing antibodies. A human embryonic kidney fibroblast cell line, 293, was transfected with 30 lg plasmids by electroporation, while a human EwingÕs sarcoma cell line, A4573, was transfected with 2 lg plasmids by poly-amidoamine dendrimer (Superfect; Qiagen, Hilden, Germany) [11,12]. The neutralizing antihIFN-b mAb was kindly provided by Dr. N. Naruse (Toray, Tokyo, Japan). Western blotting analysis. Cells were extracted in lysis buffer (1% NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 20 mM Tris–HCl, pH 7.4, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail) and subjected to SDS–PAGE. Samples were transferred onto PVDF membranes (Millipore, Bedford, MA, USA) and probed with specific antibodies against phospho-STAT1 (Tyr 701) (BD Pharmingen, San Diego, CA, USA), p53 (BD Pharmingen), and phospho-p53 (Ser15) (Cell Signaling Technology, Beverly, MA, USA). The blots were developed with ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA). Reporter assay. STAT1 transactivation was measured as follows: cells were co-transfected with (i) 2 lg hIFN-b, SD.hIFN-b, or control plasmid, (ii) 0.5 lg pISRE-TA-Luc (a reporter plasmid containing five repeats of the ISRE sequence upstream of the luciferase gene) (Mercury Pathway Profiling Luciferase System; BD Clontech), and (iii) 0.5 lg pb-actin.b (an internal control plasmid containing the b-galactosidase (b-gal) gene driven by the b-actin promoter). After culture, the cells were extracted, and the luciferase and b-gal activities were measured. The luciferase/b-gal activity ratio was regarded as the relative ISRE activity as described [10].
Fig. 1. Intracellular IFN triggered the JAK/STAT signal transmission cascade. (A) Schematic representation of the hIFN-b gene constructs. (B,C) A4573 cells were transfected with the indicated plasmids and cultured with/without rhIFN-b (added every 24 h) or anti-hIFN-b mAb. After 72 h of culture, cells were stained with anti-MHC class I antibody followed by FACS analysis. Mean fluorescence intensities (MFI) (B) and histograms (C) are shown. Bars, ±SE. (D) A4573 cells were transfected with the indicated plasmids or treated with rhIFN-b. Cells were extracted at various time points and Western blotting analysis was performed using anti-phospho STAT1 (pY701) mAb as a probe. (E) A4573 cells were co-transfected with a mixture of pISRE-TA-Luc, pb-actin.b, and the indicated plasmids, and cultured with/without rhIFN-b (added every 24 h). One to three days later, cells were lysed and the extracts were subjected to Luc and b-gal assays. The ratios of Luc to b-gal activities are plotted. Bars, ±SE. ##P < 0.001.
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Table 1 Sequences of siRNA duplexes used in this study Forward IFNAR1(451) IFNAR1(591) IFNAR1(741) IFNAR1(1204) IFNAR2(502) IFNAR2(601)
0
Reverse 0
5 -AGCUCAGAUUGGUCCUCCAdTdT-3 5 0 -ACUCUUCAGGUGUAGAAGAdTdT-3 0 5 0 -AGACCACAGUUGAAAAUGAdTdT-3 0 5 0 -CACUUCAAAUGCUGAGAGAdTdT-3 0 5 0 -CCACAUUAAUGUGGUGGUGdTdT-3 0 5 0 -GAAGCAUAAACCCGAAAUAdTdT-3 0
RNA interference. Small interfering RNA (siRNA) duplexes targeting JAK1 (Darmacon, Lafayette, CO, USA), TYK2 (Darmacon), and p53 (Cell Signaling Technology) were purchased from the sources shown. Four siRNA duplexes specific for IFNAR1 as well as 2 siRNA duplexes for IFNAR2c were synthesized (Table 1). The siRNAs were delivered into the 293 and A4573 cells by electroporation and RNAiFect (Qiagen), respectively. Vesicular stomatitis virus infection. Cells were plated at a density of 4.0 · 104 cells/well in 96-well flat-bottomed microtiter plates. After incubation for 24 h under the standard conditions, cells were infected with vesicular stomatitis virus (VSV) at a multiplicity of infection (MOI) of 10 and cultured for a further 24 h. Cell viability was estimated by dye exclusion method [13]. Serial dilutions of each supernatant were added to FL cells in 96-well plates, and 24 h later the cytopathic effect (CPE) was observed under phase-contrast microscopy. VSV in the supernatants was titrated according to the method of Kaerber [14]. RT-PCR analyses. Total RNA was reverse-transcribed using an Omniscript RT Kit (Qiagen). The resultant cDNA was subjected to PCR amplification using primers for 2 0 –5 0 -oligoadenylate synthetase (2 0 –5 0 OAS) (5 0 -tggctgaattacccatgctt-3 0 and 5 0 -tggacaagggatgtgaaaat-3 0 ), MxA (5 0 -gcatcccaccctctattact-3 0 and 5 0 -tgtcttcagttcctttgtcc-3 0 ), PKR (5 0 -ttggctcaggtggatttgg-3 0 and 5 0 -ggcttttcttccacacagtc-3 0 ), and b-actin (5 0 -cttctacaatgagctgcgtg-3 0 and 5 0 -tcatgaggtagtcagtcagg-3 0 ) genes. The resultant products were separated by gel electrophoresis through a 3% agarose gel and stained with ethidium bromide. Statistical analysis. Data in each experiment are expressed as means ± SE of triplicate samples. Statistical analyses were performed using StudentÕs t test. All the experiments were repeated more than three times and reproducible results were obtained.
Results Intracellular IFN-b showed similar biological activities to those of extracellular rhIFN-b protein To stimulate cells with intracellular IFN-b, the cells were transfected with the SD.hIFN-b gene which was engineered so that the gene product was not secreted out from the transfectants (Fig. 1A, lower figure). To stimulate cells with extracellular IFN-b, recombinant human IFN-b (rhIFN-b) protein was added to culture supernatant of cells. Cells transfected with the fulllength IFN-b gene (Fig. 1A, upper figure) receive both the intracellular and extracellular IFN stimuli. Biological activities as well as signaling pathways elicited by intracellular and/or extracellular IFNs were comparatively examined.
5 0 -UGGAGGACCAAUCUGAGCUdTdT-3 0 5 0 -UCUUCUACACCUGAAGAGUdTdT-3 0 5 0 -UCAUUUUCAACUGUGGUCUdTdT-3 0 5 0 -UAUUUCGGGUUUAUGCUUCdTdT-3 0 5 0 -CACCACCACAUUAAUGUGGdTdT-3 0 5 0 -UCUCUCAGCAUUUGAAGUGdTdT-3 0
When A4573 or 293 cells were transfected with the full-length hIFN-b gene, both cell lines showed drastic secretion of hIFN-b, and the cytokine concentrations in the supernatant reached 3200 ± 860 IU/ml (293) and 16,000 ± 1300 IU/ml (A4573) on day 3 (data not shown). Based on these results, the rhIFN-b protein was added to 293 and A4573 cells at concentrations of 5000 and 20,000 IU/ml, respectively, in the following experiments. In contrast, cells transfected with the SD.hIFN-b construct did not secrete the cytokine at a detectable level (<5 IU/ml) (data not shown). A4573 cells were transfected with SD.hIFN-b gene or control plasmid (pGEG.GL3), and after the transfection cells were cultured with/without rhIFN-b protein. Flowcytometric analysis was performed to evaluate the class I MHC expression on the cell surface. Figs. 1B and C show the mean fluorescence intensities (MFI) and the histograms, respectively. In the absence of hIFN-b gene transfection and rhIFN-b stimulation, the cells expressed MHC class I at an intermediate level as indicated by flowcytometric analysis of the cells transfected with control plasmid (pGEG.GL3) (the MFI was 120 ± 0.38). The expression level was drastically elevated when the cells were treated with rhIFN-b protein (pGEG.GL3 (+rhIFN-b); the MFI was 825 ± 1.29). Interestingly, SD.hIFN-b gene transfection also elevated the MHC class I expression on the cell surface (pGEG.SD.hIFN-b (rhIFN-b); the MFI was 735 ± 2.62). The augmentation of MHC class I expression was not significantly affected by the addition of an excess amount of anti-hIFN-b neutralizing mAb (anti-IFN-b mAb at this concentration completely blocked rhIFN-b activity at 20,000 IU/ml, data not shown), suggesting that the results were not caused by a trace amount of extracellular hIFN-b potentially secreted from the SD.hIFN-b gene-transfected cells (Fig. 1B, bar second to the right; the MFI was 800 ± 2.82). The cells given both the SD.IFN-b gene transfer and rhIFN-b protein showed the highest expression of MHC class I (pGEG.SD.IFN-b (+rIFN-b); the MFI was 1161 ± 11.2). The biological activities of intracellular and extracellular IFN-b signals were also tested by examining another function of IFN, i.e., antiviral properties. A4573 cells were transfected with SD.hIFN-b gene and/or trea-
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ted with rhIFN-b protein, followed by infection with a lethal dose of the vascular stomatitis virus (VSV). Although control cells were killed by the virus, either the SD.hIFN-b gene transfection or rhIFN-b treatment rendered the A4573 cells highly resistant to the viral infection (data not shown).
bar). The SD.hIFN-b activity was also significantly blocked by specific knockdown of JAK1 or TYK2 signaling molecules (Figs. 3A, left three bars, and D). These results strongly suggest that intracellular IFN exerts its biological function through the IFNAR1 and 2c, JAK1 and TYK2 signaling molecules that play indispensable roles in the signaling cascade.
Intracellular IFN-b exerted its biological activities through the JAK/STAT signaling pathway
p53 induction by intracellular IFN-b-induced signal
It has been established that the extracellular IFN-bmediated signal is associated with activation of intracellular signaling molecules, JAK1, TYK2, and STAT1 (reviewed in [15]). We found that SD.hIFN-b induced tyrosine phosphorylation (Fig. 1D) and the transactivation function (Fig. 1E) of STAT1, suggesting that the intracellular IFN-b triggered JAK/STAT pathway. Next, we examined whether the JAK/STAT signaling cascade is essentially involved in the biological activities of intracellular IFN-b. The SD.hIFN-b-mediated up-regulation of MHC class I was markedly blocked by the IFNAR1-specific and IFNAR2-specific siRNA duplexes (Table 1 and Fig. 2A, left three bars) that reduced the expression of the corresponding receptor molecules (Fig. 2D), while nonspecific siRNA did not show any detectable suppressive effect (Fig. 2A, rightmost
It was documented that extracellular IFN induces the p53 tumor suppressor [16]. We investigated expression and phosphorylation status of p53 in 293 cells after extracellular and intracellular IFN stimulation. Immunoblotting analyses demonstrated that p53 protein was accumulated in SD.hIFN-b or hIFN-b gene-transfected cells (Fig. 4B, upper panel), while p53 was not significantly induced after the treatment with rhIFN-b protein (Fig. 4A, upper panel). SD.hIFN-b and hIFN-b gene transfer also induced phosphorylation of p53 at Ser15 (Fig. 4B, middle panel), a site involved in p53–MDM2 interaction [17], while rhIFN-b protein had very faint, if any, effect on the phosphorylation status of the tumor suppressor protein (Fig. 4A, middle panel). We also examined whether the p53 induced by intracellular IFN-b was involved in antivirus defense. When 293 cells
Fig. 2. The IFNAR-mediated pathway was essential in the signal transmission triggered by intracellular IFN-b. (A–C) A4573 cells were transduced with the indicated siRNAs (days 4 and 1) prior to transfection with pGEG.SD.hIFN-b (A,B) or pGEG.hIFN-b plasmids (C). Cells were then cultured in the presence (B) or absence (A,C) of rhIFN-b (added every 24 h). Seventy-two hours later, FACS analyses were performed. Bars, ±SE. (D) Cells were transduced with the indicated siRNAs (days 4 and 1), and on day 0 Western blotting analyses were performed using the indicated antibodies as probes. ##P < 0.001.
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Fig. 3. The JAK1 and TYK2 adaptor molecules were also essential in the intracellular IFN-b-mediated signal transduction. (A–C) A4573 cells were transduced with the indicated siRNAs (days 4 and 1) prior to transfection with pGEG.SD.hIFN-b (A,B) or pGEG.hIFN-b plasmids (C). Cells were then cultured in the presence (B) or absence (A,C) of rhIFN-b (added every 24 h). Seventy-two hours later, FACS analyses were performed. Bars, ±SE. (D) Cells were transduced with the indicated siRNAs (days 4 and 1), and on day 0 Western blotting analyses were performed using the indicated antibodies as probes. **P < 0.01; ##P < 0.001.
were infected with VSV, robust amplification of the virus was detected in the cells (Fig. 4C, leftmost bar). The virus titer was reduced markedly in cells transfected with the SD.hIFN-b gene (Fig. 4C, bar second to the right). The antivirus activity was significantly blocked by p53-specific siRNA duplex (Fig. 4C, rightmost bar), which effectively silenced the tumor suppressor molecule (Fig. 4E, rightmost lane). The SD.hIFN-b-mediated suppression of virus replication could potentially be due to apoptotic cell death that could be induced by p53 [18]. To assess this possibility, we measured the viability of cells after VSV infection. As shown in Fig. 4D, SD.hIFN-b-transfected cells survived the virus challenge (bar second to the right), which drastically killed the cells transfected with control plasmid (leftmost bar). Genetic silencing of p53 significantly cancelled the SD.hIFN-b-induced resistance to the lethal VSV infection (Fig. 4D, rightmost bar). These results strongly suggest that the SD.hIFN-b-induced suppression of viral replication was not due to cell death. We also assessed whether p53 is involved in expression of IFN-inducible genes with antiviral activities. The 2 0 -5 0 OAS, MxA, and PKR are induced after transfection with SD.hIFN-b gene or rhIFN-b treatment, regardless of the presence or absence of p53-specific siR-
NA (Fig. 4F). It was shown that p53 is not a prerequisite for the induction of these antiviral proteins.
Discussion In the present study, RNAi experiments successfully delineated the signal transduction molecules that crucially participated in the intracellular IFN-b-mediated signal transmission cascade. Although intracellular IFN-a [6,7] as well as IFN-c [2,3] have been shown to induce phosphorylation and activation of the STAT1, it remained to be elucidated whether the conventional JAK/STAT pathway is crucially involved in the signaling cascade triggered by intracellular ligand. Activation of a molecule does not always mean that the molecule plays an indispensable role in signal transmission. Indeed, for an example, TYK2 is activated by extracellular IL-6 [19], IL-10 [20] and thrombopoietin [21], but this kinase is not a prerequisite for the biological activities of these cytokines as revealed by experiments using TYK2 gene-deficient mice [21,22]. The present study indicated that the IFNAR1, IFNAR2c, JAK1, and TYK2 molecules are all essential and nonredundant for the signal transmission triggered by intracellular IFN-b.
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Fig. 4. Intracellular IFN-mediated signal prompted p53 induction that plays an essential role in antiviral activity. (A,B) 293 cells were treated with rhIFN-b protein (A) or transfected with the indicated plasmids (B). After culturing for the indicated periods, cell extracts were subjected to Western blotting analysis using the indicated antibodies as probes. (C,D) 293 cells were transduced with scramble or p53-specific siRNAs (days 4 and 1) followed by transfection with the indicated plasmids (day 0). Twenty-four hours later, cells were infected with VSV at MOI of 10. On day 2, the virus titer in the culture supernatant was evaluated (C), while cell viability was measured by dye exclusion (D). Bars, ±SE. *P < 0.05; ##P < 0.001. (E,F) 293 cells were transduced with the indicated siRNA (days 4 and 1) and/or plasmids (day 0) as in (C). After culturing for 48 h with or without rhIFN-b, cell lysates were subjected to Western blotting analyses using anti-p53 or anti-b-actin antibodies as probes (E), while RNA was extracted from the cells and subjected to RT-PCR analyses using the indicated primers (F).
It has been controversial whether intracellular ligands are capable of binding to corresponding receptor molecules, because cytokine receptors are basically distributed on the cell surface exposing their ligand-binding domain to the extracellular space. In the case of IFNc, it was proposed that the intracellular ligand may specifically bind to the cytoplasmic domain, instead of the extracellular domain, of the IFN-c receptor a chain through the C-terminus basic region of the IFN-c [3]. However, IFN-b does not share sequence homology with IFN-c at the C-terminus region, and intracellular IFN-b-IFNAR binding has not been reported. It remains to be revealed at which intracellular compartment the intracellular IFN-b interacts with its receptor. Both the receptor and ligand molecules are type I cell surface/ secretory molecules that may be synthesized in the rough endoplasmic reticulum and transported to the plasma membrane through the Golgi apparatus [23]. Inside the IFN producer cells, the binding of intracellular ligand to the receptor molecule may take place during these traffic processes, before distribution of the receptor
molecule to cell surface, although this concept needs to be examined. JAK1, TYK2, and STAT1 are ubiquitously expressed and associated with various cytokine receptors. Genetic disruption experiments demonstrated that JAK1 [24] as well as STAT1 [25,26] are required for signal transduction triggered by interaction between extracellular IFN-a and cell surface IFNAR [27], while TYK2 controls cell surface expression and proteosomal degradation of IFNAR1 chain rather than directly contributing to the signaling event [28]. In our experimental settings, activity of intracellular IFN was also suppressed by TYK2-specific siRNA duplexes (Fig. 3A, second bar). Therefore, TYK2 may also contribute to stability and/or transport of IFNAR inside the IFNproducing cells. The STAT proteins are a family of latent transcription factors expressed in many cell types and regulate a wide range of biological functions, including cell growth, differentiation, survival, development, and cell death [9,29]. Recently, Townsend et al. reported that
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STAT1 induced accumulation of p53 by negatively modulating transcription of the mdm2 gene. STAT1 also activates p53 function not only by inhibiting the MDM2–p53 interaction but also by binding directly to p53 [30,31]. The linkage between extracellular IFN-a/b signaling and p53 was reported by Takaoka et al. [16] who revealed that transcription of p53 is up-regulated by extracellular IFN stimulus. In our system, intracellular IFN signaling induced accumulation of p53 as well as phosphorylation of p53 at Ser15 more significantly than did extracellular IFN (Figs. 4A and B). The phosphorylation of p53 at Ser15 inhibits the interaction between p53 and MDM2 proteins, leading to the accumulation and activation of p53 [17]. This may explain the mechanism by which p53 was accumulated by intracellular IFN signaling. The present study suggests an intriguing biological significance of the intracellular ligand-mediated signal, i.e., an antiviral defense system. Cells produce IFN in response to viral infection. In the IFN-producing cells, intracellular IFN may prevent viral amplification, while extracellular IFN secreted from the producers acts on bystander cells and renders them resistant to the virus infection. The IFNAR-JAK/STAT-mediated signals and p53 induction may contribute to this innate defense mechanism.
Acknowledgments We thank Dr. N. Naruse (Toray, Tokyo, Japan), Dr. T. Sudo (Basic Research Laboratories, Toray), Mochida Pharmaceutical (Tokyo, Japan), and Otsuka Pharmaceutical (Tokushima, Japan) for kindly providing the anti-hIFN-b mAb, hIFN-b cDNA, rhIFN-b, and antibodies specific for hIFNAR1 and hIFNAR2, respectively. Drs. Y. Sokawa, T. Iida, and H. Nakanishi (Kyoto Prefectural University of Medicine) provided helpful support. We also thank Dr. W.T.V. Germeraad (University Hospital Maastricht, Maastricht, the Netherlands) for critical reading of the manuscript.
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Intracellular interferon triggers Jak/Stat signaling cascade and induces p53-dependent antiviral protection q Masaharu Shin-Ya a, Hideyo Hirai a, Etsuko Satoh a, Tsunao Kishida a,b, Hidetsugu Asada a, Fumiko Aoki a, Masako Tsukamoto a, Jiro Imanishi a, Osam Mazda a,* a
Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan b Louis Pasteur Center for Medical Research, Kyoto 606-8225, Japan Received 5 February 2005
Abstract Intracellular interferons (IFNs) exert biological functions similar to those of extracellular IFNs, but the signal transduction pathway triggered by the intracellular ligands has not been fully revealed. We investigated the signaling cascade by sequence-specific knockdown of signaling molecules by means of the RNA interference. Truncated IFN-b gene was constructed so that the N-terminal secretory signal sequence was deleted (SD.IFN-b). Cells transfected with this construct showed phosphorylation and activation of the STAT1 without any detectable secretion of the cytokine. The MHC class I expression was significantly augmented, while the augmentation was suppressed by short interfering RNA duplexes specific for JAK1, TYK2, and IFN-a/b receptor (IFNAR) 1 and 2c chains. The SD.IFN-b also induced p53 and phosphorylation of p53 at Ser15. Specific silencing of p53 abrogated the antiviral effect of SD.IFN-b, suggesting that the tumor suppressor is critically involved in antiviral defense mediated by intracellular IFN. 2005 Elsevier Inc. All rights reserved. Keywords: Interferon; Autocrine; p53; RNA interference; STAT1
Cytokines are secreted from producer cells, subsequently binding to specific receptors on the surface of the target cells, in which intracellular signal transmission cascade is triggered to induce biological responses. Cumulative evidences indicate that biological functions of some cytokines also exert inside the producer cells without being secreted out from the cells. These include IFN-c [1–3], v-sis [4], interleukin-3 (IL-3) [5], IFN-a [6,7], and IL-6 [8]. Because these cytokines act in an autocrine manner, the signals triggered by intracellular q Abbreviations: SD.hIFN-b, signal sequence-deleted hIFN-b; IFN, interferon; mAb, monoclonal antibody; siRNA, small interfering RNA; RNAi, RNA interference. * Corresponding author. Fax: +81 75 251 5331. E-mail address: [email protected] (O. Mazda).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.088
ligands may have important biological implications in the cytokine action in autocrine cells. However, the signaling pathway triggered by intracellular ligand has not been fully elucidated at molecular level. In the case of extracellular IFN-b, the engagement of IFN-a/b receptor (composed of the IFNAR1 and IFNAR2c chains) on the cell surface triggers activation of JAK1 and TYK2, leading to phosphorylation and activation of the STAT1 transcriptional factor, which in turn induces expression of IFN-responsive genes through activation of the IFN-stimulated response element (ISRE)-containing promoters (reviewed in [9]). Intracellular IFN-a [6,7] as well as IFN-c [2,3] were shown to induce phosphorylation and activation of the STAT1, but it has not been demonstrated whether the conventional JAK/STAT pathways participate in
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the intracellular ligand-mediated signal transmission. Moreover, it remains controversial whether the intracellular ligand engages IFNAR1 and IFNAR2c receptor molecules to trigger the signaling cascade. Conventional experimental technologies including immunocoprecipitation of ligand–receptor complex can hardly demonstrate intracellular ligand–receptor interaction. Using the RNAi technology, we investigated whether each component of the IFNAR-JAK1/TYK2-STAT pathway was a prerequisite for the signal transduction cascade triggered by the extracellular and intracellular IFN-b. We found that the IFNAR1 and 2c chains as well as the JAK1, TYK2, and STAT1 molecules were critically involved in the signal transmission pathway. Moreover, we also demonstrated that the intracellular IFN signal results in p53 induction that plays essential roles in antiviral protection achieved by the intracellular cytokine. Materials and methods Plasmid vectors. The pGEG.hIFN-b contains the full-length human IFN-b (hIFN-b) cDNA in which a ClaI restriction site was introduced at the junction between the nucleotide sequences corresponding to the N-terminal secretory signal peptide and the mature hIFN-b in such a manner that the encoding amino acid sequence was
identical to that of wild-type IFN-b (Fig. 1A, upper figure). The signal sequence was deleted to generate pGEG.SD.hIFN-b (Fig. 1A, lower figure). Control plasmids, pGEG.GL3 and pGEG.EGFP, were described previously [10]. Cells, transfection, and neutralizing antibodies. A human embryonic kidney fibroblast cell line, 293, was transfected with 30 lg plasmids by electroporation, while a human EwingÕs sarcoma cell line, A4573, was transfected with 2 lg plasmids by poly-amidoamine dendrimer (Superfect; Qiagen, Hilden, Germany) [11,12]. The neutralizing antihIFN-b mAb was kindly provided by Dr. N. Naruse (Toray, Tokyo, Japan). Western blotting analysis. Cells were extracted in lysis buffer (1% NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 20 mM Tris–HCl, pH 7.4, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail) and subjected to SDS–PAGE. Samples were transferred onto PVDF membranes (Millipore, Bedford, MA, USA) and probed with specific antibodies against phospho-STAT1 (Tyr 701) (BD Pharmingen, San Diego, CA, USA), p53 (BD Pharmingen), and phospho-p53 (Ser15) (Cell Signaling Technology, Beverly, MA, USA). The blots were developed with ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA). Reporter assay. STAT1 transactivation was measured as follows: cells were co-transfected with (i) 2 lg hIFN-b, SD.hIFN-b, or control plasmid, (ii) 0.5 lg pISRE-TA-Luc (a reporter plasmid containing five repeats of the ISRE sequence upstream of the luciferase gene) (Mercury Pathway Profiling Luciferase System; BD Clontech), and (iii) 0.5 lg pb-actin.b (an internal control plasmid containing the b-galactosidase (b-gal) gene driven by the b-actin promoter). After culture, the cells were extracted, and the luciferase and b-gal activities were measured. The luciferase/b-gal activity ratio was regarded as the relative ISRE activity as described [10].
Fig. 1. Intracellular IFN triggered the JAK/STAT signal transmission cascade. (A) Schematic representation of the hIFN-b gene constructs. (B,C) A4573 cells were transfected with the indicated plasmids and cultured with/without rhIFN-b (added every 24 h) or anti-hIFN-b mAb. After 72 h of culture, cells were stained with anti-MHC class I antibody followed by FACS analysis. Mean fluorescence intensities (MFI) (B) and histograms (C) are shown. Bars, ±SE. (D) A4573 cells were transfected with the indicated plasmids or treated with rhIFN-b. Cells were extracted at various time points and Western blotting analysis was performed using anti-phospho STAT1 (pY701) mAb as a probe. (E) A4573 cells were co-transfected with a mixture of pISRE-TA-Luc, pb-actin.b, and the indicated plasmids, and cultured with/without rhIFN-b (added every 24 h). One to three days later, cells were lysed and the extracts were subjected to Luc and b-gal assays. The ratios of Luc to b-gal activities are plotted. Bars, ±SE. ##P < 0.001.
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Table 1 Sequences of siRNA duplexes used in this study Forward IFNAR1(451) IFNAR1(591) IFNAR1(741) IFNAR1(1204) IFNAR2(502) IFNAR2(601)
0
Reverse 0
5 -AGCUCAGAUUGGUCCUCCAdTdT-3 5 0 -ACUCUUCAGGUGUAGAAGAdTdT-3 0 5 0 -AGACCACAGUUGAAAAUGAdTdT-3 0 5 0 -CACUUCAAAUGCUGAGAGAdTdT-3 0 5 0 -CCACAUUAAUGUGGUGGUGdTdT-3 0 5 0 -GAAGCAUAAACCCGAAAUAdTdT-3 0
RNA interference. Small interfering RNA (siRNA) duplexes targeting JAK1 (Darmacon, Lafayette, CO, USA), TYK2 (Darmacon), and p53 (Cell Signaling Technology) were purchased from the sources shown. Four siRNA duplexes specific for IFNAR1 as well as 2 siRNA duplexes for IFNAR2c were synthesized (Table 1). The siRNAs were delivered into the 293 and A4573 cells by electroporation and RNAiFect (Qiagen), respectively. Vesicular stomatitis virus infection. Cells were plated at a density of 4.0 · 104 cells/well in 96-well flat-bottomed microtiter plates. After incubation for 24 h under the standard conditions, cells were infected with vesicular stomatitis virus (VSV) at a multiplicity of infection (MOI) of 10 and cultured for a further 24 h. Cell viability was estimated by dye exclusion method [13]. Serial dilutions of each supernatant were added to FL cells in 96-well plates, and 24 h later the cytopathic effect (CPE) was observed under phase-contrast microscopy. VSV in the supernatants was titrated according to the method of Kaerber [14]. RT-PCR analyses. Total RNA was reverse-transcribed using an Omniscript RT Kit (Qiagen). The resultant cDNA was subjected to PCR amplification using primers for 2 0 –5 0 -oligoadenylate synthetase (2 0 –5 0 OAS) (5 0 -tggctgaattacccatgctt-3 0 and 5 0 -tggacaagggatgtgaaaat-3 0 ), MxA (5 0 -gcatcccaccctctattact-3 0 and 5 0 -tgtcttcagttcctttgtcc-3 0 ), PKR (5 0 -ttggctcaggtggatttgg-3 0 and 5 0 -ggcttttcttccacacagtc-3 0 ), and b-actin (5 0 -cttctacaatgagctgcgtg-3 0 and 5 0 -tcatgaggtagtcagtcagg-3 0 ) genes. The resultant products were separated by gel electrophoresis through a 3% agarose gel and stained with ethidium bromide. Statistical analysis. Data in each experiment are expressed as means ± SE of triplicate samples. Statistical analyses were performed using StudentÕs t test. All the experiments were repeated more than three times and reproducible results were obtained.
Results Intracellular IFN-b showed similar biological activities to those of extracellular rhIFN-b protein To stimulate cells with intracellular IFN-b, the cells were transfected with the SD.hIFN-b gene which was engineered so that the gene product was not secreted out from the transfectants (Fig. 1A, lower figure). To stimulate cells with extracellular IFN-b, recombinant human IFN-b (rhIFN-b) protein was added to culture supernatant of cells. Cells transfected with the fulllength IFN-b gene (Fig. 1A, upper figure) receive both the intracellular and extracellular IFN stimuli. Biological activities as well as signaling pathways elicited by intracellular and/or extracellular IFNs were comparatively examined.
5 0 -UGGAGGACCAAUCUGAGCUdTdT-3 0 5 0 -UCUUCUACACCUGAAGAGUdTdT-3 0 5 0 -UCAUUUUCAACUGUGGUCUdTdT-3 0 5 0 -UAUUUCGGGUUUAUGCUUCdTdT-3 0 5 0 -CACCACCACAUUAAUGUGGdTdT-3 0 5 0 -UCUCUCAGCAUUUGAAGUGdTdT-3 0
When A4573 or 293 cells were transfected with the full-length hIFN-b gene, both cell lines showed drastic secretion of hIFN-b, and the cytokine concentrations in the supernatant reached 3200 ± 860 IU/ml (293) and 16,000 ± 1300 IU/ml (A4573) on day 3 (data not shown). Based on these results, the rhIFN-b protein was added to 293 and A4573 cells at concentrations of 5000 and 20,000 IU/ml, respectively, in the following experiments. In contrast, cells transfected with the SD.hIFN-b construct did not secrete the cytokine at a detectable level (<5 IU/ml) (data not shown). A4573 cells were transfected with SD.hIFN-b gene or control plasmid (pGEG.GL3), and after the transfection cells were cultured with/without rhIFN-b protein. Flowcytometric analysis was performed to evaluate the class I MHC expression on the cell surface. Figs. 1B and C show the mean fluorescence intensities (MFI) and the histograms, respectively. In the absence of hIFN-b gene transfection and rhIFN-b stimulation, the cells expressed MHC class I at an intermediate level as indicated by flowcytometric analysis of the cells transfected with control plasmid (pGEG.GL3) (the MFI was 120 ± 0.38). The expression level was drastically elevated when the cells were treated with rhIFN-b protein (pGEG.GL3 (+rhIFN-b); the MFI was 825 ± 1.29). Interestingly, SD.hIFN-b gene transfection also elevated the MHC class I expression on the cell surface (pGEG.SD.hIFN-b (rhIFN-b); the MFI was 735 ± 2.62). The augmentation of MHC class I expression was not significantly affected by the addition of an excess amount of anti-hIFN-b neutralizing mAb (anti-IFN-b mAb at this concentration completely blocked rhIFN-b activity at 20,000 IU/ml, data not shown), suggesting that the results were not caused by a trace amount of extracellular hIFN-b potentially secreted from the SD.hIFN-b gene-transfected cells (Fig. 1B, bar second to the right; the MFI was 800 ± 2.82). The cells given both the SD.IFN-b gene transfer and rhIFN-b protein showed the highest expression of MHC class I (pGEG.SD.IFN-b (+rIFN-b); the MFI was 1161 ± 11.2). The biological activities of intracellular and extracellular IFN-b signals were also tested by examining another function of IFN, i.e., antiviral properties. A4573 cells were transfected with SD.hIFN-b gene and/or trea-
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ted with rhIFN-b protein, followed by infection with a lethal dose of the vascular stomatitis virus (VSV). Although control cells were killed by the virus, either the SD.hIFN-b gene transfection or rhIFN-b treatment rendered the A4573 cells highly resistant to the viral infection (data not shown).
bar). The SD.hIFN-b activity was also significantly blocked by specific knockdown of JAK1 or TYK2 signaling molecules (Figs. 3A, left three bars, and D). These results strongly suggest that intracellular IFN exerts its biological function through the IFNAR1 and 2c, JAK1 and TYK2 signaling molecules that play indispensable roles in the signaling cascade.
Intracellular IFN-b exerted its biological activities through the JAK/STAT signaling pathway
p53 induction by intracellular IFN-b-induced signal
It has been established that the extracellular IFN-bmediated signal is associated with activation of intracellular signaling molecules, JAK1, TYK2, and STAT1 (reviewed in [15]). We found that SD.hIFN-b induced tyrosine phosphorylation (Fig. 1D) and the transactivation function (Fig. 1E) of STAT1, suggesting that the intracellular IFN-b triggered JAK/STAT pathway. Next, we examined whether the JAK/STAT signaling cascade is essentially involved in the biological activities of intracellular IFN-b. The SD.hIFN-b-mediated up-regulation of MHC class I was markedly blocked by the IFNAR1-specific and IFNAR2-specific siRNA duplexes (Table 1 and Fig. 2A, left three bars) that reduced the expression of the corresponding receptor molecules (Fig. 2D), while nonspecific siRNA did not show any detectable suppressive effect (Fig. 2A, rightmost
It was documented that extracellular IFN induces the p53 tumor suppressor [16]. We investigated expression and phosphorylation status of p53 in 293 cells after extracellular and intracellular IFN stimulation. Immunoblotting analyses demonstrated that p53 protein was accumulated in SD.hIFN-b or hIFN-b gene-transfected cells (Fig. 4B, upper panel), while p53 was not significantly induced after the treatment with rhIFN-b protein (Fig. 4A, upper panel). SD.hIFN-b and hIFN-b gene transfer also induced phosphorylation of p53 at Ser15 (Fig. 4B, middle panel), a site involved in p53–MDM2 interaction [17], while rhIFN-b protein had very faint, if any, effect on the phosphorylation status of the tumor suppressor protein (Fig. 4A, middle panel). We also examined whether the p53 induced by intracellular IFN-b was involved in antivirus defense. When 293 cells
Fig. 2. The IFNAR-mediated pathway was essential in the signal transmission triggered by intracellular IFN-b. (A–C) A4573 cells were transduced with the indicated siRNAs (days 4 and 1) prior to transfection with pGEG.SD.hIFN-b (A,B) or pGEG.hIFN-b plasmids (C). Cells were then cultured in the presence (B) or absence (A,C) of rhIFN-b (added every 24 h). Seventy-two hours later, FACS analyses were performed. Bars, ±SE. (D) Cells were transduced with the indicated siRNAs (days 4 and 1), and on day 0 Western blotting analyses were performed using the indicated antibodies as probes. ##P < 0.001.
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Fig. 3. The JAK1 and TYK2 adaptor molecules were also essential in the intracellular IFN-b-mediated signal transduction. (A–C) A4573 cells were transduced with the indicated siRNAs (days 4 and 1) prior to transfection with pGEG.SD.hIFN-b (A,B) or pGEG.hIFN-b plasmids (C). Cells were then cultured in the presence (B) or absence (A,C) of rhIFN-b (added every 24 h). Seventy-two hours later, FACS analyses were performed. Bars, ±SE. (D) Cells were transduced with the indicated siRNAs (days 4 and 1), and on day 0 Western blotting analyses were performed using the indicated antibodies as probes. **P < 0.01; ##P < 0.001.
were infected with VSV, robust amplification of the virus was detected in the cells (Fig. 4C, leftmost bar). The virus titer was reduced markedly in cells transfected with the SD.hIFN-b gene (Fig. 4C, bar second to the right). The antivirus activity was significantly blocked by p53-specific siRNA duplex (Fig. 4C, rightmost bar), which effectively silenced the tumor suppressor molecule (Fig. 4E, rightmost lane). The SD.hIFN-b-mediated suppression of virus replication could potentially be due to apoptotic cell death that could be induced by p53 [18]. To assess this possibility, we measured the viability of cells after VSV infection. As shown in Fig. 4D, SD.hIFN-b-transfected cells survived the virus challenge (bar second to the right), which drastically killed the cells transfected with control plasmid (leftmost bar). Genetic silencing of p53 significantly cancelled the SD.hIFN-b-induced resistance to the lethal VSV infection (Fig. 4D, rightmost bar). These results strongly suggest that the SD.hIFN-b-induced suppression of viral replication was not due to cell death. We also assessed whether p53 is involved in expression of IFN-inducible genes with antiviral activities. The 2 0 -5 0 OAS, MxA, and PKR are induced after transfection with SD.hIFN-b gene or rhIFN-b treatment, regardless of the presence or absence of p53-specific siR-
NA (Fig. 4F). It was shown that p53 is not a prerequisite for the induction of these antiviral proteins.
Discussion In the present study, RNAi experiments successfully delineated the signal transduction molecules that crucially participated in the intracellular IFN-b-mediated signal transmission cascade. Although intracellular IFN-a [6,7] as well as IFN-c [2,3] have been shown to induce phosphorylation and activation of the STAT1, it remained to be elucidated whether the conventional JAK/STAT pathway is crucially involved in the signaling cascade triggered by intracellular ligand. Activation of a molecule does not always mean that the molecule plays an indispensable role in signal transmission. Indeed, for an example, TYK2 is activated by extracellular IL-6 [19], IL-10 [20] and thrombopoietin [21], but this kinase is not a prerequisite for the biological activities of these cytokines as revealed by experiments using TYK2 gene-deficient mice [21,22]. The present study indicated that the IFNAR1, IFNAR2c, JAK1, and TYK2 molecules are all essential and nonredundant for the signal transmission triggered by intracellular IFN-b.
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Fig. 4. Intracellular IFN-mediated signal prompted p53 induction that plays an essential role in antiviral activity. (A,B) 293 cells were treated with rhIFN-b protein (A) or transfected with the indicated plasmids (B). After culturing for the indicated periods, cell extracts were subjected to Western blotting analysis using the indicated antibodies as probes. (C,D) 293 cells were transduced with scramble or p53-specific siRNAs (days 4 and 1) followed by transfection with the indicated plasmids (day 0). Twenty-four hours later, cells were infected with VSV at MOI of 10. On day 2, the virus titer in the culture supernatant was evaluated (C), while cell viability was measured by dye exclusion (D). Bars, ±SE. *P < 0.05; ##P < 0.001. (E,F) 293 cells were transduced with the indicated siRNA (days 4 and 1) and/or plasmids (day 0) as in (C). After culturing for 48 h with or without rhIFN-b, cell lysates were subjected to Western blotting analyses using anti-p53 or anti-b-actin antibodies as probes (E), while RNA was extracted from the cells and subjected to RT-PCR analyses using the indicated primers (F).
It has been controversial whether intracellular ligands are capable of binding to corresponding receptor molecules, because cytokine receptors are basically distributed on the cell surface exposing their ligand-binding domain to the extracellular space. In the case of IFNc, it was proposed that the intracellular ligand may specifically bind to the cytoplasmic domain, instead of the extracellular domain, of the IFN-c receptor a chain through the C-terminus basic region of the IFN-c [3]. However, IFN-b does not share sequence homology with IFN-c at the C-terminus region, and intracellular IFN-b-IFNAR binding has not been reported. It remains to be revealed at which intracellular compartment the intracellular IFN-b interacts with its receptor. Both the receptor and ligand molecules are type I cell surface/ secretory molecules that may be synthesized in the rough endoplasmic reticulum and transported to the plasma membrane through the Golgi apparatus [23]. Inside the IFN producer cells, the binding of intracellular ligand to the receptor molecule may take place during these traffic processes, before distribution of the receptor
molecule to cell surface, although this concept needs to be examined. JAK1, TYK2, and STAT1 are ubiquitously expressed and associated with various cytokine receptors. Genetic disruption experiments demonstrated that JAK1 [24] as well as STAT1 [25,26] are required for signal transduction triggered by interaction between extracellular IFN-a and cell surface IFNAR [27], while TYK2 controls cell surface expression and proteosomal degradation of IFNAR1 chain rather than directly contributing to the signaling event [28]. In our experimental settings, activity of intracellular IFN was also suppressed by TYK2-specific siRNA duplexes (Fig. 3A, second bar). Therefore, TYK2 may also contribute to stability and/or transport of IFNAR inside the IFNproducing cells. The STAT proteins are a family of latent transcription factors expressed in many cell types and regulate a wide range of biological functions, including cell growth, differentiation, survival, development, and cell death [9,29]. Recently, Townsend et al. reported that
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STAT1 induced accumulation of p53 by negatively modulating transcription of the mdm2 gene. STAT1 also activates p53 function not only by inhibiting the MDM2–p53 interaction but also by binding directly to p53 [30,31]. The linkage between extracellular IFN-a/b signaling and p53 was reported by Takaoka et al. [16] who revealed that transcription of p53 is up-regulated by extracellular IFN stimulus. In our system, intracellular IFN signaling induced accumulation of p53 as well as phosphorylation of p53 at Ser15 more significantly than did extracellular IFN (Figs. 4A and B). The phosphorylation of p53 at Ser15 inhibits the interaction between p53 and MDM2 proteins, leading to the accumulation and activation of p53 [17]. This may explain the mechanism by which p53 was accumulated by intracellular IFN signaling. The present study suggests an intriguing biological significance of the intracellular ligand-mediated signal, i.e., an antiviral defense system. Cells produce IFN in response to viral infection. In the IFN-producing cells, intracellular IFN may prevent viral amplification, while extracellular IFN secreted from the producers acts on bystander cells and renders them resistant to the virus infection. The IFNAR-JAK/STAT-mediated signals and p53 induction may contribute to this innate defense mechanism.
Acknowledgments We thank Dr. N. Naruse (Toray, Tokyo, Japan), Dr. T. Sudo (Basic Research Laboratories, Toray), Mochida Pharmaceutical (Tokyo, Japan), and Otsuka Pharmaceutical (Tokushima, Japan) for kindly providing the anti-hIFN-b mAb, hIFN-b cDNA, rhIFN-b, and antibodies specific for hIFNAR1 and hIFNAR2, respectively. Drs. Y. Sokawa, T. Iida, and H. Nakanishi (Kyoto Prefectural University of Medicine) provided helpful support. We also thank Dr. W.T.V. Germeraad (University Hospital Maastricht, Maastricht, the Netherlands) for critical reading of the manuscript.
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