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International Immunology Advance Access originally published online on May 19, 2008
International Immunology 2008 20(7):901-909; doi:10.1093/intimm/dxn048
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© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The NF-{kappa}B, p38 MAPK and STAT1 pathways differentially regulate the dsRNA-mediated innate immune responses of epidermal keratinocytes

Xiuju Dai, Koji Sayama, Mikiko Tohyama, Yuji Shirakata, Lujun Yang, Satoshi Hirakawa, Sho Tokumaru and Koji Hashimoto

Department of Dermatology, Ehime University Graduate School of Medicine, Toon-city, Ehime 791-0295, Japan

Correspondence to: K. Sayama; E-mail: sayama{at}m.ehime-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The epidermis is the primary boundary between the body and the environment, and it serves as the first line of defense against microbial pathogens. Production of chemokines and cytokines is an important step in the initiation of innate immune responses to viral infections. Epidermal keratinocytes produce IFN-{alpha}, -β and macrophage inflammatory protein (MIP)-1{alpha} in response to double-stranded RNA (dsRNA) or viral infections. We showed that human keratinocytes produced cytokines [tumor necrosis factor (TNF)-{alpha}, IL-1β and IL-15] and chemokines [MIP-1β, RANTES and liver and activation-regulated chemokine (LARC)] in response to dsRNA, with activation of the nuclear factor {kappa}B (NF-{kappa}B), p38 mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription 1 (STAT1) pathways. To study the roles of these pathways in their production, we transfected keratinocytes with adenoviral vectors (Ax) carrying a dominant-negative form of inhibitor {kappa}B {alpha} (I{kappa}B{alpha}) (I{kappa}B{alpha}M), a dominant-negative mutant form of STAT1 (STAT1F) or suppressors of cytokine signaling 1 (SOCS1). Transfection with AxI{kappa}B{alpha}M or addition of a p38 inhibitor (SB203580) significantly decreased the dsRNA-mediated production of TNF-{alpha}, IL-1β and MIP-1{alpha}, but not of IFN-β, IL-15, MIP-1β, RANTES or LARC. Transfection with AxSTAT1F or AxSOCS1 inhibited the dsRNA-mediated production of TNF-{alpha}, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC, but not IFN-β or IL-1β. In conclusion, the NF-{kappa}B, p38 MAPK and STAT1 pathways differentially regulate dsRNA-mediated innate immune responses in epidermal keratinocytes.

Keywords: chemokines, cytokine, innate immunity, IRF3, SOCS1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The skin is the primary interface between the body and the environment and serves as the first line of defense against microbial pathogens. Epidermal keratinocytes, the main constituent of the epidermis, actively participate in innate immune responses by producing cytokines, chemokines (1–3) and anti-microbial peptides (4). In addition, human keratinocytes can be targets for viruses, such as herpes simplex virus (HSV) (5), human papillomavirus (6) and varicella-zoster virus (7). HSV- or varicella-zoster virus-infected keratinocytes are known to produce cytokines and chemokines (5, 8). Keratinocytes in virus-infected skin lesions produce macrophage inflammatory protein (MIP)-1{alpha} (7), suggesting that these cells play a role in the virus-mediated innate immune reaction of the skin. However, the regulatory mechanisms behind virus-mediated immune reaction of keratinocytes remain unclear.

Toll-like receptors (TLRs) play important roles in innate and adaptive immunity by recognizing microbial pathogens. The intracellular signals of the TLRs have been classified into myeloid differentiation primary response gene (88)-dependent and Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta-dependent pathways (9). TLR3 is a receptor for virus-associated double-stranded RNA (dsRNA) and activates nuclear factor {kappa}B (NF-{kappa}B), mitogen-activated protein kinases (MAPKs) and IFN regulatory factor 3 (IRF3) in an effort to control the viral infection (9, 10). In addition to TLR3, the RNA helicase retinoic acid-inducible gene (RIG-1) and melanoma differentiation-associated gene 5 (MDA5) have also been implicated in viral dsRNA recognition (11, 12). In vitro studies suggest that both RIG-1 and MDA5 detect RNA viruses and polyinosine–polycytidylic acid (polyI:C), a synthetic dsRNA analogue (11). Previously, we showed that the synthetic dsRNA, polyI:C, induced production of IFNs and MIP-1{alpha} in cultured human keratinocytes (8, 13). In airway epithelial cells, polyI:C induced the expression of various chemokines, including MIP-1{alpha}, MIP-1β, RANTES, 10 kDa IFN-{gamma}-inducible protein (IP-10), IL-8 and liver and activation-regulated chemokine (LARC), as well as the expression of several cytokines, including IL-6, tumor necrosis factor (TNF)-{alpha}, IL-1β and IL-15 (14). However, production of these cytokines and chemokines in keratinocytes with dsRNA has not been studied. TNF-{alpha} and IL-1β regulate the early phase of inflammation in viral infections (15, 16), while IL-15 contributes to innate immunity by regulating the function of NK cells (17) and Langerhans cells (18). Local over-expression of IL-15 in the epidermis protects mice from cutaneous HSV infection by enhancing their anti-viral immune responses (19). Thus, it is important to study the regulatory mechanisms of the production of cytokines and chemokines in keratinocytes during viral infection to understand the defense mechanisms of the skin.

The production of most cytokines and chemokines is regulated primarily at the level of transcription, through activation of specific sets of transcription factors controlled by NF-{kappa}B, MAPKs and IRFs (13, 16). The signal transducers and activators of transcription (STAT) family plays an important role in cytokine production (20). Upon viral infection, type I IFN is rapidly induced and activates the transcription factor complex ISGF3, consisting of STAT1, STAT2 and IRF9. ISGF3 binds to cis-elements, termed IFN-stimulated response elements which usually reside within the promoters of IFN-inducible genes, such as TLR3 (8) and IRF7 (21). Some IFN-inducible genes can also be activated by IRF3 in virus-infected cells, where it forms a complex with CBP/p300 co-activators to bind to IRSE sites in the promoters of IFN-inducible genes, such as ISG15 and IP-10 (20, 21). The transcriptional regulation of these genes is dependent on the cellular context and stimulation (22). We reported previously that blockade of STAT1 suppressed the IFN- or polyI:C-induced expression of TLR3 and IRF7 in keratinocytes (13). However, details of the polyI:C-mediated production of other chemokines and cytokines in epidermal keratinocytes and the regulatory mechanisms involved remain unclear. Here, we report that dsRNA-induced production of chemokines and cytokines in epidermal keratinocytes was differentially regulated by NF-{kappa}B, p38 MAPK and STAT1 pathways.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Reagents
PolyI:C (Amersham Biosciences Corp., Piscataway, NJ, USA) was dissolved in deionized distilled water at a concentration of 2 mg ml–1 and stored at –70°C. SB203580 (Calbiochem–Novabiochem International Co., San Diego, CA, USA) was dissolved in dimethyl sulfoxide at a concentration of 2 mM and stored at –20°C. Anti-IRF3 antibody, anti-β-actin, anti-RIG-I and anti-MDA5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mAb to TLR3 was obtained from Alexis Biochemicals (San Diego, CA, USA). The antibodies for inhibitor {kappa}B {alpha} (I{kappa}B{alpha}), phospho-I{kappa}B{alpha}, p38, phospho-p38, extracellular signal-regulated kinase (ERK), phospho-ERK, c-Jun N-terminal kinase (JNK), phospho-JNK, phospho-STAT1, phospho-STAT2 and phospho-STAT3 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Cell culture and polyI:C stimulation
Normal human keratinocytes were cultured in MCDB153 medium, supplemented with insulin (5 µg ml–1), hydrocortisone (5 x 10–7 M), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), bovine hypothalamic extract (50 µg ml–1) and Ca2+ (0.03 mM), as described previously (23). Cells that had been passaged four times were used in the experiments, and subconfluent keratinocyte cultures were treated with 100 ng ml–1 of polyI:C for a predetermined period.

This study was performed according to the principles set forth in the Declaration of Helsinki. All procedures involving human subjects received prior approval from the ethical committee of Ehime University School of Medicine. All subjects provided written informed consent to participation in the study.

Adenovirus construction and transfection
AxI{kappa}B{alpha}M, AxSOCS1 and AxSTAT1F were prepared as described previously (13). Cultured normal human keratinocytes were transfected with AxI{kappa}B{alpha}M, AxSOCS1 or AxSTAT1F and then stimulated as described previously (13). AxLacZ was used as a control.

Immunofluorescence microscopy
Keratinocytes on chamber slides were fixed for 5 min in methanol:acetone (1:1) and washed with PBS. The cells were then treated with anti-IRF3 antibody overnight at 4°C. After washing with PBS, the cells were incubated with fluorescein-labeled goat anti-rabbit IgG for 30 min at 37°C and then washed four times. The stained cells were visualized at a magnification of x40 under a confocal microscope (LSM 510; Carl Zeiss, Jena, Germany).

Reverse Transcription–PCR
Total RNA from the cultured cells was isolated at the indicated time points using Isogen (Nippon Gene, Tokyo, Japan). Reverse transcription (RT)–PCR was performed using RT–PCR High Plus (Toyobo, Osaka, Japan), according to the manufacturer's protocol. The primer pairs used are listed in Table 1. The products were visualized on 2% agarose gels containing ethidium bromide and were then sequenced to confirm the accuracy of amplification.


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  		Table 1. The primer pairs used for RT–PCR

 
ELISA
Culture supernatants were collected after treatment with polyI:C and stored at –70°C until use. ELISA kits for TNF-{alpha} and MIP-1{alpha} were purchased from Endogen (Auburn, MA, USA). ELISAs were performed according to the manufacturer's protocol. The optical density at 450 nm was measured with an Immuno Mini NJ-2300 microplate reader (Nalgene Nunc International K.K., Tokyo, Japan). All assays were performed in triplicate.

Protein preparation and western blotting
The cells were harvested by transferring them into an extraction buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM s–HCl (pH 7.4) and protease inhibitors. Equal amounts of protein were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes. The analysis was performed using a Vistra ECF Kit (Amersham Biosciences K.K., Tokyo, Japan) and a FluoroImager (Molecular Dynamics Inc., Sunnyvale, CA, USA).

Statistical analysis
At least three independent studies were performed and yielded similar results. The results of one representative experiment are shown in each of the figures. Quantitative data are expressed as means ± standard deviations. Statistical significance was determined using the paired Student's t-test. Differences were deemed statistically significant at P < 0.05. The levels of statistical significance are indicated in the figures as follows: *P < 0.05, **P < 0.01 and ***P < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
PolyI:C enhanced mRNA expression of cytokines and chemokines in normal human keratinocytes
The presence of dsRNA during viral infection induces numerous inflammatory mediators in a variety of cell types (16, 22). As an initial step, we analyzed mRNA induction by polyI:C in cultured normal human keratinocytes. The levels of mRNA expression of inflammatory cytokines, including IFN-β, TNF-{alpha}, IL-1β and IL-15, and chemokines, including MIP-1{alpha}, MIP-1β, RANTES and LARC, were enhanced by polyI:C (Fig. 1a). The mRNA level of IL-18 was unchanged (data not shown).


Figure 1
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  		Fig. 1. PolyI:C enhanced mRNA expression of cytokines and chemokines in keratinocytes. (a) Keratinocytes were stimulated with polyI:C (100 ng ml–1) for the indicated times, and RT–PCR was performed to detect the transcription of GAPDH, IFN-β, TNF-{alpha}, IL-1β, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC. (b) Keratinocytes were stimulated with polyI:C for the indicated times, and the levels of TLR3, RIG-1 and MDA5 proteins were detected using western blotting.

 
The expression of the dsRNA detectors (24) TLR3, RIG-I and MDA5 in cultured human keratinocytes was also investigated using western blotting. As shown in Fig. 1(b), unstimulated cells (time, 0 h) expressed TLR3, but not RIG-I or MDA5. On stimulation with polyI:C, the level of TLR3 protein increased rapidly, and significant RIG-I and MDA5 expression was induced after 12 h of stimulation. The induction of RIG-I by polyI:C confirmed previous reports in human astrocytoma cells (25) and fibroblasts (26).

NF-{kappa}B regulated polyI:C-induced TNF-{alpha}, IL-1β and MIP-1{alpha} production
As NF-{kappa}B regulates the production of many inflammatory cytokines (16), we examined its involvement in the polyI:C-induced production of cytokines and chemokines in keratinocytes. I{kappa}B{alpha} was rapidly phosphorylated by polyI:C (Fig. 2a), as reported previously (13). Next, the keratinocytes were transfected with an adenoviral vector (Ax) carrying a dominant-negative mutant form of I{kappa}B{alpha} (I{kappa}B{alpha}M) to block NF-{kappa}B signaling (13). Transfection with I{kappa}B{alpha}M significantly suppressed the mRNA induction of TNF-{alpha}, IL-1β and MIP-1{alpha} by polyI:C, while the levels of mRNA expression of other genes were unaffected (Fig. 2b). ELISAs for TNF-{alpha} and MIP-1{alpha} confirmed the inhibitory effect of I{kappa}B{alpha}M on their production (Fig. 2c). Thus, NF-{kappa}B signaling appeared to be involved in the induction of TNF-{alpha}, IL-1β and MIP-1{alpha} by polyI:C in keratinocytes.


Figure 2
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  		Fig. 2. NF-{kappa}B is involved in the polyI:C-induced expression of TNF-{alpha}, IL-1β and MIP-1{alpha} in keratinocytes. (a) Keratinocytes were treated with polyI:C for the indicated times, cell extracts were prepared and I{kappa}B{alpha} and phospho-I{kappa}B{alpha} expression were detected by western blotting. (b) Keratinocytes were infected with AxLacZ or AxI{kappa}B{alpha}M for 24 h, and the cultures were stimulated with polyI:C for the indicated times. RT–PCR was performed to determine the mRNA expression of various cytokines and chemokines. (c) Keratinocytes were infected with AxLacZ or AxI{kappa}B{alpha}M and then stimulated with polyI:C for 30 h. The supernatants were collected, and the expression of TNF-{alpha} and MIP-1{alpha} was analyzed by ELISA.

 
p38 MAPK regulated polyI:C-induced TNF-{alpha}, IL-1β and MIP-1{alpha} production
The MAPK system is activated by infection with several viruses (16, 22). It has previously been shown in epithelial cells that dsRNA activated p38 MAPK, which is required for the induction of TNF-{alpha} and IL-1β (27). Considering the important role MAPK signaling plays in host defense, we examined the activation of three major MAPK subfamilies, p38, ERK1/2 and JNK, in keratinocytes upon polyI:C stimulation. As shown in Fig. 2(a), after adding polyI:C, phospho-p38 was increased significantly, whereas the changes in phospho-ERK and phospho-JNK were not remarkable. To determine whether p38 is involved in polyI:C-induced cytokine and chemokine production in keratinocytes, p38 activity was inhibited using SB203580. Treatment with SB203580 resulted in a partial, but statistically significant, reduction in polyI:C-induced TNF-{alpha}, IL-1β and MIP-1{alpha} mRNA expression (Fig. 3B). This result was confirmed by ELISA (Fig. 3c). These observations suggest that polyI:C-induced production of TNF-{alpha}, IL-1β and MIP-1{alpha} involves p38 signaling.


Figure 3
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  		Fig. 3. Activation of the p38 pathway is required for polyI:C-induced TNF-{alpha}, IL-1β and MIP-1{alpha} expression in keratinocytes. (a) Keratinocytes were treated with polyI:C for the indicated times, cell extracts were prepared and the levels of p38, phospho-p38, ERK, phospho-ERK, JNK and phospho-JNK were determined by western blotting. The intensity of the protein band was quantified using NIH Image (Molecular Dynamics), and the relative increases in phospho-p38, phospho-ERK and phospho-JNK compared with the total protein are shown with the data. (b) Cultures were processed with dimethyl sulfoxide or SB202380 (20 µM) for 30 min prior to treatment with polyI:C for the indicated times. RNA was prepared, and RT–PCR was performed to detect the mRNA expression of several cytokines and chemokines. (c) Cultures were pre-treated with dimethyl sulfoxide or SB202380 for 30 min prior to stimulation with polyI:C for 30 h, and the supernatants were collected. TNF-{alpha} and MIP-1{alpha} production were examined by ELISA.

 
Expression of suppressors of cytokine signaling 1 suppressed the activation of STATs and reduced polyI:C-induced TNF-{alpha}, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC production
The JAK–STAT pathway is involved in immune regulation via IFNs (13), which are induced by viral infection and dsRNA. The suppressors of cytokine signaling (SOCS)/cytokine-inducible SH2-containing protein family, which is induced by STAT activation, negatively regulates the JAK–STAT pathway by inhibiting STAT activation (28). Previously, we showed that the STAT1–SOCS1 pathway regulates innate immunity in keratinocytes (13). In this study, we investigated whether the JAK–STAT pathway was involved in the production of cytokines and chemokines in polyI:C-treated keratinocytes. As in our previous report (13), we detected significant phosphorylation of STAT1, STAT2 and STAT3 upon polyI:C stimulation (Fig. 4a), which was blocked by SOCS1 (Fig. 4a), while the total protein levels of STATs were unchanged (data not shown). Furthermore, SOCS1 inhibited the mRNA induction of most of the genes, including the cytokines TNF-{alpha} and IL-15 and the chemokines MIP-1{alpha}, MIP-1β, RANTES and LARC (Fig. 4b). The data for TNF-{alpha} and MIP-1{alpha} were confirmed at the protein level by ELISA (Fig. 4c).


Figure 4
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  		Fig. 4. Over-expression of SOCS1 suppressed the induction of TNF-{alpha}, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC and blocked the nuclear translocation of IRF3 in polyI:C-treated keratinocytes. (a) Keratinocyte cultures were transfected with AxLacZ or AxSOCS1 for 24 h, stimulated with polyI:C for 4 h and the extracts were subjected to western blotting to detect phospho-STAT1, phospho-STAT2 and phospho-STAT3. (b) Keratinocytes were transfected with AxLacZ or AxSOCS1 for 24 h prior to treatment with polyI:C for the indicated time periods, and the transcription of several cytokines and chemokines was assayed by RT–PCR. (c) Keratinocyte cultures were transfected with AxLacZ or AxSOCS1 for 24 h. PolyI:C was added to the cultures, and the supernatants were collected after 30 h. ELISA was performed to evaluate the levels of TNF-{alpha} and MIP-1{alpha}. (d) Keratinocytes were transfected with AxLacZ or AxSOCS1 for 24 h prior to treatment with polyI:C for the indicated times. The cells were then fixed, and immunofluorescence was used to detect the nuclear translocation of IRF3.

 
Expression of SOCS1 blocked the polyI:C-induced nuclear translocation of IRF3
It has been suggested that the action of SOCS is not confined to JAK/STAT signaling (29). We found that SOCS1 not only suppressed STAT activation (Fig. 4a) but also blocked the polyI:C-induced nuclear translocation of IRF3 (Fig. 4d), which is upstream of the JAK–STAT pathway during dsRNA signaling. These observations suggested that SOCS1 plays a central role in regulating the immune response in polyI:C-treated keratinocytes.

STAT1 regulated polyI:C-induced TNF-{alpha}, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC production
A specific role of STAT1 activation in MIP-1{alpha} production in dsRNA signaling has been demonstrated (13). We examined whether STAT1 activation was essential for polyI:C-induced cytokine and chemokine expression. An Ax carrying a dominant-negative mutant form of STAT1 (STAT1F), which specifically blocks STAT1 activation with no effect on other STATs (13), was transfected into keratinocytes. These keratinocytes were then treated with polyI:C. STAT1F regulated the mRNA expression of polyI:C-induced cytokines and chemokines in a pattern similar to SOCS1 (Fig. 5a). Moreover, STAT1F decreased the production of TNF-{alpha} and MIP-1{alpha} (Fig. 5b). As STAT1F did not influence the dsRNA-induced activation of IRF3 (data not shown), the similarities between the effects of STAT1F and those of SOCS1 on polyI:C-triggered inflammation suggest that STAT1 contributed to the response to dsRNA in keratinocytes.


Figure 5
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  		Fig. 5. STAT1F inhibited the induction of TNF-{alpha}, IL-15, MIP-1{alpha}, MIP-1β, RANTES and LARC by polyI:C in keratinocytes. (a) Keratinocytes were transfected with AxLacZ or AxSOCS1 for 24 h prior to treatment with polyI:C for the indicated time periods, and the transcription of several cytokines and chemokines was measured by RT–PCR. (b) Keratinocytes were infected with AxLacZ or AxSTAT1F and then stimulated with polyI:C for 30 h. The supernatants were collected, and the levels of TNF-{alpha} and MIP-1{alpha} were analyzed by ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
We found that human keratinocytes produced TNF-{alpha}, IL-1β, IL-15, MIP-1β, RANTES and LARC following exposure to dsRNA, in addition to MIP-1{alpha} and IFN-β. Figure 6 summarizes the regulatory mechanisms of the polyI:C-induced production of cytokines and chemokines. Specific sets of transcription factors required for cytokine and chemokine production vary among cell types (16, 22). In airway epithelial cells, the p38 MAPK pathway alone regulates the dsRNA-induced production of TNF-{alpha} and IL-1β (27). In contrast, in keratinocytes, both p38 MAPK and NF-{kappa}B are required for the production of TNF-{alpha} and IL-1β. This may be explained by the observation that the p38 MAPK pathway is required for the efficient activation of NF-{kappa}B in response to viral infection (30) and other environmental stresses (31). In airway epithelial cells (14) and NIH 3T3 cells (32), both the IRF3 pathway and NF-{kappa}B signal are required for dsRNA-induced expression of RANTES. On the other hand, p38 MAPK regulates viral infection-induced RANTES in bronchial epithelial cells (33) and in microglia (30), but not in fibroblasts (32). In keratinocytes, STAT1, but not the NF-{kappa}B or p38 pathways, is required for RANTES production. Although all the NF-{kappa}B, p38 MAPK and STAT1 pathways regulate polyI:C-induced MIP-1{alpha} production in keratinocytes, NF-{kappa}B signaling is not essential for MIP-1{alpha} production in airway epithelial cells (14). Moreover, dsRNA-mediated LARC production is regulated by NF-{kappa}B activation in airway epithelial cells (14), but dsRNA-activated STAT1 signal is essential for LARC production in human keratinocytes. Although dsRNA elicits innate immune reactions in a variety of organs, the regulatory mechanisms to produce cytokines and chemokines differ among them. We also noted that the IFN-β, TNF-{alpha} and LARC genes were induced much earlier compared with other cytokines or chemokines, in response to polyI:C stimulation. In addition, inhibition of the STAT1 signal significantly suppressed the late phase, but not the early phase, of TNF-{alpha} and LARC production. This kinetic regulation of TNF-{alpha} and LARC demonstrates that different transcription factors contribute to the early and late phases of expression of these genes. In particular, the early phase of TNF-{alpha} and LARC induction might occur in response to direct dsRNA receptor-mediated TNF-{alpha} and LARC transcription, which might be activated by NF-{kappa}B and p38, although neither blocking the NF-{kappa}B signal nor inhibiting p38 resulted in a remarkable decrease in LARC production. As opposed to early and direct TNF-{alpha} and LARC induction, the late phase of the production of these genes is indirect and is most probably mediated by dsRNA-induced IFN-β, which activates STAT1 and amplifies the dsRNA signaling (13).


Figure 6
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  		Fig. 6. Regulation of dsRNA-mediated cytokines and chemokines by different signaling pathways in keratinocytes. After infection with virus or treatment with dsRNA, NF-{kappa}B, p38 MAPK and IRF3 are activated, resulting in IFN-β production. The de novo-expressed IFN-β stimulates activation of the JAK–STAT pathway. The activation of NF-{kappa}B, p38 MAPK and JAK–STAT pathways differentially regulates dsRNA-mediated production of cytokines and chemokines.

 
TLR3, RIG-I and MDA5 have been implicated in the recognition of dsRNA and the subsequent induction of anti-viral responses (24). In the endosome, the viral dsRNA and its mimic polyI:C are recognized by TLR3, whereas RIG-I and MDA5 have been identified as cytosomal dsRNA detectors. Moreover, RIG-I and MDA5 also activate NF-{kappa}B and IRF3 and stimulate the subsequent production of type I IFNs and pro-inflammatory cytokines when stably expressed in cells (24, 34). Despite this knowledge, the exact contributions of TLR3, RIG-I and MDA5 to dsRNA-mediated signal transduction and cytokine production have yet to be clarified. In human astrocytoma cells, polyI:C-induced RANTES up-regulation is significantly inhibited by siRNA for RIG-I (25), and over-expression of RIG-I in gingival fibroblasts enhances the production of IL-1β and IL-8 induced by polyI:C (26). However, the knockdown of RIG-I and MDA5 through siRNA transfection failed to inhibit polyI:C-mediated RANTES, IP-10 and IL-8 production in airway epithelial cells (35). In this study, we demonstrated that cultured normal human keratinocytes express substantial TLR3, but not MDA5 and RIG-I, suggesting that the primary reactions triggered by polyI:C are most probably mediated by TLR3, while RIG-I and MDA5, both of which are significantly induced by polyI:C, might contribute to the amplification of dsRNA signaling in keratinocytes. Further research is required to elucidate the respective functions of TLR3, RIG-I and MDA5 in dsRNA signaling and anti-viral responses in human keratinocytes.

Our finding that SOCS1 blocked polyI:C-induced IRF3 nuclear translocation was unexpected. The mechanism of IRF3 activation by polyI:C is probably dependent on the kinases I kappa B kinase {varepsilon} and TRAF-associated NF-{kappa}B activator (TANK)-binding kinase 1 (36). Although Kinjyo et al. (29) have suggested that SOCS1 suppressed TRAF6-dependent IKK activation when over-expressed, no studies have described the effect of SOCS in the region directly upstream of IRF3. Further research is required to clarify the molecular mechanism by which SOCS1 inhibited IRF3 activation.

Induction of type I IFNs is one of the earliest events in viral infections, in fact preceding the generation of a specific immune response (37). The promoter of the human IFN-β gene is complex, with several partially overlapping positive and negative regulatory elements (38). Three families of transcription factors, IRF3 (39), NF-{kappa}B and AP-1 (38, 40), all of which are activated in response to dsRNA or viral infection, have been shown to participate in the induction of IFNs. Activation of the IFN-β promoter requires the coordinated action of several transcription factors (41); however, not all these transcription factors may be necessary for dsRNA-mediated IFN-β induction if one of them is present in excess (42). In the current study, we found that SOCS1 over-expression blocked the activation of IRF3, but that it had no effect on IFN-β expression; furthermore, both the introduction of AxI{kappa}B{alpha}M and treatment with a p38 MAPK inhibitor failed to block IFN-β induction. It has been shown that individual cis-elements, in the absence of the others, can drive dsRNA-induced transcription of transfected reporter genes, indicating that each element is capable of communicating with the basal transcription machinery and with the relevant co-activators (38), and this may apply to the complex promoter of the IFN-β gene in polyI:C-treated keratinocytes. Compared with the regulation of the other cytokines and chemokines, the contribution of the many overlapping signaling mechanisms in IFN-β transcription suggests that IFN-β is a key element in viral infection.

Overall, our findings provide insight into the precise roles of NF-{kappa}B, p38 and STAT1 in the virus-provoked innate immune responses of epidermal keratinocytes.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
This study was supported by Integrated Center for Science (INCS), Ehime University; Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.S. (19390297) and K.H. (18390314). And this work was also supported by Health and Labour Sciences Research Grants from the ministry of Health, Labour and Welfare of Japan to K.H. (H19-nanchi-ippan-004).


   Acknowledgements
 
We thank Teruko Tsuda and Eriko Tan for technical assistance.


   Abbreviations
 
Ax, adenovirus vector
dsRNA, double-stranded RNA
ERK, extracellular signal-regulated kinase
HSV, herpes simplex virus
I{kappa}B, inhibitor {kappa}B
I{kappa}B{alpha}M, dominant-negative mutant form of I{kappa}B{alpha}
IRF, IFN regulatory factor
JNK, c-Jun N-terminal kinase
LARC, liver and activation-regulated chemokine
MAPK, mitogen-activated protein kinase
MDA5, melanoma differentiation-associated gene 5
MIP, macrophage inflammatory protein
NF-{kappa}B, nuclear factor {kappa}B
polyI:C, polyinosine–polycytidylic acid
RIG-1, RNA helicase retinoic acid-inducible gene
RT, reverse transcription
SOCS, suppressors of cytokine signaling
STAT, signal transducers and activators of transcription
STAT1F, dominant-negative mutant form of STAT1
TLR, Toll-like receptor
TNF, tumor necrosis factor

   Notes
 
Transmitting editor: E. Vivier

Received 19 December 2007, accepted 22 April 2008.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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 References
 

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