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Cytokine modulation of retinoic acid-inducible gene-I (RIG-I) expression in human epidermal keratinocytes
Journal of Dermatological Science (2007) 45, 127—134
www.intl.elsevierhealth.com/journals/jods
Cytokine modulation of retinoic acid-inducible gene-I (RIG-I) expression in human epidermal keratinocytes Hideo Kitamura a, Yasushi Matsuzaki a,*, Kazuyuki Kimura a, Hajime Nakano a, Tadaatsu Imaizumi b, Kei Satoh b, Katsumi Hanada a a
Department of Dermatology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan b Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Received 3 July 2006; received in revised form 2 November 2006; accepted 7 November 2006
KEYWORDS Retinoic acid-inducible gene-I (RIG-I); IFN-g; TNF-a; Keratinocytes; HaCaT; Psoriasis
Summary Background: Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D box family proteins and designated as a putative RNA helicase, which plays various roles in gene expression and cellular functions in response to a variety of RNA viruses. Objective: The present study was designed to investigate the effects of interferon (IFN)-g and tumor necrosis factor (TNF)-a on RIG-I expression in human keratinocytes, and the expression of RIG-I in skin lesions of psoriasis vulgaris, in which IFN-g and TNFa are considered to be involved in its pathogenesis. Methods: The mRNA and protein expression of RIG-I was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Immunohistochemical staining of RIG-I was examined on psoriatic skin section. Results: The levels of RIG-I mRNA and protein were upregulated in HaCaT keratinocytes in the presence of IFN-g or TNF-a. Immunohistochemically, RIG-I was detected in the cytoplasm of the spinous and basal layers of psoriatic skin. Conclusion: Our results suggest that RIG-I might operate not only as a RNA helicase but also as a mediator of the cytokine network in the inflammatory skin diseases, such as psoriasis vulgaris. # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Abbreviations: CARD, caspase recruitment domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCV, hepatitis C virus; IFN, interferon; IL, interleukin; IRF-3, interferon regulatory factor-3; MDA-5, melanoma differentiation-associated gene-5; RIG-I, retinoic acid-inducible gene-I; RT, room temperature; RT-PCR, reverse transcription-polymerase chain reaction; Th, T helper cell; TNF, tumor necrosis factor * Corresponding author. Tel.: +81 172 39 5087; fax: +81 172 37 6060. E-mail address: [email protected] (Y. Matsuzaki). 0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2006.11.003
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1. Introduction Retinoic acid-inducible gene-I (RIG-I) was first identified as a gene induced by retinoic acid in a leukemia cell line [1], and has been classified as a member of the DExH/D box protein family, involved in a wide variety of biological functions related to RNA metabolism such as pre-mRNA splicing, RNA transport, ribosome biogenesis and RNA degradation [2—4]. RIG-I contains a C-terminal region possessing an RNA helicase domain and two caspase recruitment domains (CARDs) at its N-terminus. It is thought that the helicase domain functions as a sensor for double-stranded RNA yielded by viruses, and that the CARDs transmit the information to downstream signals, resulting in the activation of interferon regulatory factor-3 (IRF-3) and nuclear factor-kB (NF-kB) [5,6]. The activated transcription factors stimulate interferon (IFN)-a and -b promoter, and the induced cytokines subsequently function for innate anti-viral defenses [7]. In hepatocytes, hepatitis C virus (HCV) controls the induction of anti-viral defense response by disruption of intracellular signaling processes that confer IRF-3 activation [6]. A recent study has indicated that RIG-I signaling is inhibited by the NS3/4A protease derived from HCV, resulting in HCV persistent infection [8]. On the other hand, MCF-7 human breast cancer cells and T24 urinary bladder carcinoma cells express RIG-I following stimulation by IFN-g in concentration- and time-dependent manners, suggesting that RIG-I may play a role in cellular responses during immune and inflammatory reactions [9]. Chronic inflammatory skin disorders such as psoriasis, allergic contact dermatitis, and atopic dermatitis are characterized by intense infiltration of activated T lymphocytes, which release lymphokines that influence the immune functions of resident skin cells, including keratinocytes [10—13]. Among the T cell-derived cytokines, IFN-g is the most potent activator of proinflammatory functions of keratinocytes. Psoriasis vulgaris is characterized by hyperproliferation and disturbed differentiation of keratinocytes, which might result from accumulation of T-lymphocyte subsets. CD4 + T cells are abundantly present in the upper lesional dermis, whereas CD8 + T cells are in the majority in the lesional epidermis [14]. Studies of the cytokine patterns of infiltrating T cells in the psoriatic lesions have revealed the predominance of T helper cell 1 (Th1) cytokines, e.g., IFN-g and TNF-a, compared with Th2 cytokines, e.g., interleukin (IL)-4, suggesting that psoriasis is an inflammatory disease induced by Th1 lymphocytes [15,16].
H. Kitamura et al. The aim of the present study was to define the effect of various cytokines, especially IFN-g and TNF-a, on the expression of RIG-I in HaCaT cells, an immortalized keratinocyte cell line, and the relevance of RIG-I in keratinocytes present in the epidermis of psoriasis vulgaris.
2. Materials and methods 2.1. Cell culture HaCaT cells, a spontaneously immortalized, nontumorigenic human skin keratinocyte cell line, were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated fatal bovine serum (FBS), 2 mg/ml sodium hydrogen carbonate, 100 mg/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. For experiments with cytokines, 60% confluent HaCaT cells were washed twice with sterile phosphate-buffered saline (PBS) and maintained in MEM with 0.3% FBS prior to addition of recombinant human IFN-g, TNF-a, transforming growth factor (TGF)-b and IL-1b (Roche Diagnostics, Mannheim, Germany). Cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air.
2.2. Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from cultured cells using RNAeasy total RNA isolation kit, as recommended by the manufacturer (Qiagen, Hilden, Germany). Extracted RNA was subjected to reverse transcription using RNA PCR Kit (AMV) Ver. 3.0 (Takara, Kyoto, Japan), according to the instructions provided by the manufacturer. Primers for RIG-I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: RIG-I-F (50 -GCATATTGACTGGACGTGGCA-30 ), RIG-I-R (50 -CAGTCATGGCTGCAGTTCTGTC-30 ), GAPDH-F (50 -CCACCCATGGCAAATTCCATGGCA-30 ), and GAPDH-R (50 -TCTAGACGGCAGGTCAGGTCCACC-30 ). The primers for RIG-I and GAPDH were designed to generate fragments of 644 base pairs (bp) and 598 bp, respectively. The amplification conditions were 94 8C for 1 min; followed by 30 cycles of 94 8C for 1 min, 58 8C for 1 min, and 72 8C for 1 min; and one cycle of 72 8C for 10 min. PCR products were analyzed by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining.
2.3. Western blot analysis Whole cell lysates were purified from HaCaT cells with RIPA lysis buffer (1% nonidet P-40, 0.1% sodium
RIG-I expression in human keratinocytes deoxycholate [SDS] in PBS) with 0.57 mM phenyl methyl sulfonyl fluoride (Sigma—Aldrich, St. Louis, MO, USA), 1 mM sodium orthovanadate (Sigma— Aldrich), and protease inhibitor (Roche Diagnostics). Cytoplasmic and nuclear fractions were separated from HaCaT cells with a NE-PER reagent (Pierce, Rockford, IL, USA) according to the instructions provided by the manufacturer. The concentrations of the extracted protein were quantified by the Bradford method, and 30 mg protein was subjected to electrophoresis in 10% SDS-polyacrylamide gel, and electroblotted to Hybond nitrocellulose membrane (Amersham Biosciences, Chandler, AZ, USA). The membranes were incubated in blocking solution (1% non-fat skim milk in 20 mM Tris [pH 7.4], 500 mM NaCl, and 0.025% Tween 20 [TBS-T]) for 1 h at room temperature (RT), followed by an overnight incubation at 4 8C with rabbit anti-RIG-I antiserum [17] diluted 1:10,000 in blocking solution. The membranes were washed four times in TBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, Amersham Biosciences) in TBS-T for 1 h at RT, and the bands were visualized using ECL-Western blotting detection system (Amersham Biosciences). The membranes were reprobed with an antibody against b-actin (Sigma—Aldrich) to verify equal loading of protein. Western blots were quantitated densitometrically with Scion Image 4.0.2 analysis software.
2.4. Immunofluorescence detection
129 finized and rehydrated using xylene and graded ethanol series. Neutralization of endogenous peroxidase was achieved with 0.3% H2O2 in methanol for 30 min at RT. Sections were coated with nonimmune goat serum overnight at 4 8C, and incubated with anti-RIG-I antiserum (1:2500) for 2 h at RT. Slides were washed with PBS three times, and stained using the avidin—biotin—peroxidase complex method, as recommended by the manufacturer (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA, USA). The skin sections were then lightly counterstained with Mayer’s hematoxylin for microscopic examination. All subjects provided informed consent for all aspects of the investigation according to Declaration of Helsinki Principles.
3. Results 3.1. Cytokine modulation of RIG-I expression in HaCaT cells Previous studies demonstrated that RIG-I protein is expressed in various cells and induced by IFN-g and IL1b [9,17—22]. In this study, we examined the expression of RIG-I in HaCaTcells untreated or treated with TNF-a10 ng/ml, TGF-b 10 ng/ml, IFN-g 10 ng/ml, or IL-1b (5 ng/ml) for 24 h. As shown in Fig. 1, stimulation by IFN-g significantly increased RIG-I protein expression to 6-folds that of unstimulated control HaCaT cells, which showed a faint band. Similarly,
HaCaT cells grown in 4-well chamber slides were incubated with or without 10 ng/ml IFN-g for 24 h, and fixed with 4% paraformaldehyde for 1 h, permeabilized with 0.5% Triton-X, and then treated with 3% bovine serum albumin for blocking. After an overnight incubation at 4 8C, the cells were incubated with rabbit anti-RIG-I antiserum (1:500) for 1 h. After washing with PBS three times, the cells were incubated with rhodamine-conjugated swine anti-rabbit IgG (1:500, Dako, Carpinteria, CA, USA) for 1 h. The cells were rinsed, coverslipped, and examined by fluorescence microscopy.
2.5. Immunohistochemistry Skin biopsies were obtained from six patients with psoriasis vulgaris (age range: 38—76 years) and six non-psoriatic patients as healthy control subjects (age range: 29—70 years). Sections of each tissue sample were stained with hematoxylin and eosin for histopathological evaluation. Sequential sections (7 mm) were cut from each tissue sample embedded in paraffin, and mounted on a glass slide. After an overnight incubation at 37 8C, slides were deparaf-
Fig. 1 Cytokine modulation of RIG-I expression in HaCaT cells. HaCaT cells were stimulated with TNF-a (10 ng/ml), TGF-b (10 ng/ml), IFN-g (10 ng/ml), IL-1b (5 ng/ml), or none for 24 h. Cell lysates were subjected to Western blotting with anti-RIG-I antibody. Displayed data are one representative result of three independent experiments. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
130 TNF-a-induced RIG-I protein expression was also noted at approximately 3-folds that of control HaCaT cells (Fig. 1). In contrast, neither TGF-b nor IL-1b influenced RIG-I expression in HaCaT cells.
3.2. Effect of IFN-g and TNF-a on RIG-I expression in HaCaT cells In the next step, we studied the effect of various concentrations (0.1—100 ng/ml) of IFN-g on RIG-I expression. As shown in Fig. 2A and B, the expression of RIG-I mRNA and protein was induced in IFN-gtreated HaCaT cells in a concentration-dependent manner. Furthermore, to determine the dynamics of RIG-I expression induced by IFN-g, we also examined the time course of RIG-I expression using RT-PCR and Western blot analyses (0—48 h). RIG-I mRNA was expressed at a low level in unstimulated HaCaT cells as demonstrated by RT-PCR analysis (Fig. 2C), gradually increased to a peak level at 12 h after IFN-g stimulation and then somewhat diminished at 24 and 48 h. Similarly, the expression of RIG-I protein clearly increased in a time-dependent manner, and was approximately 6-folds higher than that of unstimulated HaCaTcells, when incubated with IFN-
H. Kitamura et al. g (10 ng/ml) for 24 h (Fig. 2D). These results suggest that RIG-I protein is induced via upregulation of transcription activity by IFN-g. We also characterized TNF-a-induced RIG-I expression in HaCaT cells. The expression of RIG-I mRNA and protein was induced in TNF-a-treated HaCaT cells in a concentration-dependent manner (Fig. 3A and B). The level of RIG-I mRNA increased in a time-dependent manner and reached a maximum at 6 h, earlier peak than IFN-g-stimulated HaCaT, but gradually decreased thereafter (Fig. 3C). On the other hand, the production of RIG-I protein was enhanced at 24 h, and then decreased to basal level at 48 h (Fig. 3D). These results indicate that TNF-a induces a transient increase in RIG-I expression compared with IFN-g.
3.3. RIG-I protein is mainly distributed in the cytoplasm of HaCaT cells To define the subcellular distribution of RIG-I protein, nuclear and cytoplasmic proteins were obtained separately from IFN-g (10 ng/ml) or TNF-a (10 ng/ ml)-treated HaCaT cells after 24 h incubation. The induced increase in RIG-I protein was detected in the
Fig. 2 Time- and concentration-dependent effects of IFN-g on the expression of RIG-I in HaCaT cells. HaCaT cells were incubated in the presence or absence of IFN-g at the indicated concentrations for 24 h. Purified total RNA and cell lysates were subjected to RT-PCR (A) and Western blot analysis (B), respectively. HaCaT cells were incubated in the presence or absence of IFN-g (10 ng/ml) under the same conditions for the indicated time periods. Purified total RNA and cell lysates were subjected to RT-PCR (C) and Western blot analysis (D), respectively. Displayed data are one representative result of three independent experiments in each examination. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
RIG-I expression in human keratinocytes
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Fig. 3 Effects of TNF-a on the expression of RIG-I in HaCaTcells. HaCaTcells were incubated in the presence or absence of TNF-a at the indicated doses for 24 h. Purified total RNA and cell lysates were subjected to RT-PCR (A) and Western blot analysis (B), respectively. HaCaT cells were incubated in the presence or absence of TNF-a (10 ng/ml) under the same conditions for the indicated time periods. Purified total RNA and cell lysates were subjected to RT-PCR (C) and Western blot analysis (D), respectively. Displayed data are one representative result of three independent experiments in each examination. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
Fig. 4 Distribution of RIG-I protein induced by IFN-g or TNF-a in HaCaT cells. (A) HaCaT cells were treated with IFN-g (10 ng/ml) or TNF-a (10 ng/ml) for 24 h, or without stimulation, and nuclear and cytoplasmic fractions were purified, respectively. (B) HaCaT cells were stimulated with IFN-g (10 ng/ml) or none for 24 h and subjected to immunofluorescence staining. Cells were observed under a fluorescent microscope. Scale bars = 10 mm.
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Fig. 5 Immunohistochemical detection of RIG-I in the epidermis of psoriasis. Immunohistochemical staining of RIG-I of normal skin (A) and psoriatic skin (B). Images are representative of six specimens per each group. Scale bars = 100 mm.
cytoplasmic fraction as demonstrated by Western blot analysis, whereas the nuclear fraction did not show RIG-I protein expression (Fig. 4A). This result was confirmed by immunofluorescence staining, which showed cytoplasmic distribution of IFN-g (10 ng/ml)-induced RIG-I protein. These results suggest that the expression of RIG-I protein is significantly enhanced by IFN-g and is distributed in the cytoplasm (Fig. 4B).
3.4. Psoriasis vulgaris keratinocytes overexpress RIG-I protein Biopsy specimens of normal and psoriatic skin were examined for the presence of RIG-I. No staining for RIG-I was noted in the normal skin (Fig. 5A). In contrast, RIG-I was abundantly expressed in keratinocytes of the spinous and basal layers, but not in the cornified layer, in specimens of psoriasis vulgaris (Fig. 5B). Moreover, endothelial cells and fibroblasts in the dermis of psoriasis were positively stained in comparison with the control skin, suggesting that RIG-I might be one of crucial factors for the development of psoriasis.
4. Discussion RIG-I has two tandem motifs with limited homology to CARD and a C-terminal region possessing a DExD/
H box RNA helicase domain, and belongs to the DExH/D box protein family [5]. RIG-I was recently defined to be a double-stranded RNA binding protein that functions independently of Toll-like receptor 3 (TLR3) to signal IFN-production in response to a variety of RNA viruses, including hepatitis C virus (HCV) [5,6]. A recent study has suggested that NS3/ 4A protease derived from HCV, sufficient to support viral RNA replication, disrupts RIG-I downstream signals, resulting in HCV persistent infection, reflecting the evasion of host innate immune defenses [8]. Moreover, the truncated RIG-I without the helicase domain motif significantly induces the activation of IRF-3 and NF-kB even in the absence of any virus or the RNA duplex poly(rI):poly(rC) (poly(I:C)), whereas CARDs-truncated RIG-I induces no activation of downstream signals. These results suggest that the helicase domain suppresses downstream signaling through the CARD domains in nonviral condition [5]. Considering the binding ability of the helicase domain to poly(I:C), RIG-I is an essential component of the signaling pathway upstream of IRF-3 and NF-kB, and probably the direct sensor of viral double-stranded RNA [5,23]. Interestingly, it has been demonstrated that RIG-I is expressed by the induction of inflammatory cytokines in various types of human cells, including endothelial cells, vascular smooth muscle cells, urinary bladder epithelial cells, gingival fibroblasts, and bronchial epithelial cells [9,17—22]. In the present
RIG-I expression in human keratinocytes study, we found that RIG-I protein is predominantly expressed in human keratinocytes treated with IFN-g or TNF-a, whereas neither TGF-b nor IL-1b augmented RIG-I expression. Our results also showed that the induced RIG-I was mainly distributed in the cytoplasm, and RIG-I expression stimulated by IFN-g was higher and more persistent, compared with that induced by TNF-a. However, the rapid induction of RIG-I mRNA was noted at 6 h after TNF-a stimulation, suggesting that IFN-g and TNF-a cooperate to sustain the high expression of RIG-I upon inflammatory reaction or viral infection of the epidermis. IFN-g is a potent cytokine that regulates immune responses by inducing multiple genes in various cell types including keratinocytes. IFN-g exerts its biological activities by binding to specific receptor, IFNg receptor a/b, followed by activation of Janus kinase (JAK) 1/2 and signal transducers and activators of transcription-1 (STAT-1) and induction of various genes [24]. IFN-g and TNF-a belong to Th1 cytokines involved in the development of many inflammatory skin diseases such as psoriasis, characterized by hyperproliferation and disturbed differentiation of keratinocytes [25,26]. In the psoriatic lesions, Th1 cytokines including IFN-g and TNF-a are predominant in comparison with Th2 cytokines such as IL-4, and psoriasis is considered an inflammatory disease induced by Th1 lymphocytes [15,16]. In the present study, we showed that psoriatic keratinocytes on the spinous and basal layers strongly express RIG-I in the cytoplasm, compared with normal keratinocytes of the control specimens, and RIG-I was induced by stimulation IFN-g and TNF-a in cultured human keratinocytes. Furthermore, endothelial cells and fibroblasts in the dermis of psoriatic skin were positively stained for RIG-I, similar to psoriatic keratinocytes. These cells express RIG-I protein by stimulation of IFN-g and IL1b, respectively [20,22]. These results suggest that RIG-I might play an important role in the pathogenesis of psoriasis via induction of various cytokines. Melanoma differentiation-associated gene-5 (MDA-5) was identified as a novel upregulation gene in human melanoma cells induced to terminally differentiate by treatment with IFN-b and mezerein [27]. MDA-5 consists of a CARD and a C-terminal putative RNA helicase domain, and is highly homologous to RIG-I [28]. The level of MDA-5 mRNA is low in normal tissues, whereas its expression is induced in a spectrum of normal and cancer cells by IFN-b [27]. Furthermore, overexpression of MDA-5 by means of a replication incompetent adenoviral vector in cultured melanoma cells resulted in apoptotic cell death, indicating that MDA-5 may contribute to induction of apoptosis during terminal differentiation and IFN treatment [27]. In this study, we
133 identified the distribution of RIG-I in the spinous and basal layers of psoriatic epidermis, however most psoriatic keratinocytes indicate no apoptotic change [29]. Based on these results, the function of RIG-I may be markedly different from MDA-5, although the two proteins have a similar structure. The present study provided the further evidences that the expression of RIG-I induced by IFN-g and TNF-a might play a role in the regulation of proliferation of keratinocytes involved in psoriasis vulgaris. These findings indicate that RIG-I might operate not only as a RNA helicase in viral infection but also as a mediator of the cytokine network during endogenous inflammation.
Acknowledgements We thank Mr. Shigeki Ohashi, Department of Dermatology, Hirosaki University School of Medicine, and Mr. Jyunpei Asano, Second Department of Biochemistry, Hirosaki University School of Medicine, for their kind and helpful advice.
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www.intl.elsevierhealth.com/journals/jods
Cytokine modulation of retinoic acid-inducible gene-I (RIG-I) expression in human epidermal keratinocytes Hideo Kitamura a, Yasushi Matsuzaki a,*, Kazuyuki Kimura a, Hajime Nakano a, Tadaatsu Imaizumi b, Kei Satoh b, Katsumi Hanada a a
Department of Dermatology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan b Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Received 3 July 2006; received in revised form 2 November 2006; accepted 7 November 2006
KEYWORDS Retinoic acid-inducible gene-I (RIG-I); IFN-g; TNF-a; Keratinocytes; HaCaT; Psoriasis
Summary Background: Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D box family proteins and designated as a putative RNA helicase, which plays various roles in gene expression and cellular functions in response to a variety of RNA viruses. Objective: The present study was designed to investigate the effects of interferon (IFN)-g and tumor necrosis factor (TNF)-a on RIG-I expression in human keratinocytes, and the expression of RIG-I in skin lesions of psoriasis vulgaris, in which IFN-g and TNFa are considered to be involved in its pathogenesis. Methods: The mRNA and protein expression of RIG-I was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Immunohistochemical staining of RIG-I was examined on psoriatic skin section. Results: The levels of RIG-I mRNA and protein were upregulated in HaCaT keratinocytes in the presence of IFN-g or TNF-a. Immunohistochemically, RIG-I was detected in the cytoplasm of the spinous and basal layers of psoriatic skin. Conclusion: Our results suggest that RIG-I might operate not only as a RNA helicase but also as a mediator of the cytokine network in the inflammatory skin diseases, such as psoriasis vulgaris. # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Abbreviations: CARD, caspase recruitment domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCV, hepatitis C virus; IFN, interferon; IL, interleukin; IRF-3, interferon regulatory factor-3; MDA-5, melanoma differentiation-associated gene-5; RIG-I, retinoic acid-inducible gene-I; RT, room temperature; RT-PCR, reverse transcription-polymerase chain reaction; Th, T helper cell; TNF, tumor necrosis factor * Corresponding author. Tel.: +81 172 39 5087; fax: +81 172 37 6060. E-mail address: [email protected] (Y. Matsuzaki). 0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2006.11.003
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1. Introduction Retinoic acid-inducible gene-I (RIG-I) was first identified as a gene induced by retinoic acid in a leukemia cell line [1], and has been classified as a member of the DExH/D box protein family, involved in a wide variety of biological functions related to RNA metabolism such as pre-mRNA splicing, RNA transport, ribosome biogenesis and RNA degradation [2—4]. RIG-I contains a C-terminal region possessing an RNA helicase domain and two caspase recruitment domains (CARDs) at its N-terminus. It is thought that the helicase domain functions as a sensor for double-stranded RNA yielded by viruses, and that the CARDs transmit the information to downstream signals, resulting in the activation of interferon regulatory factor-3 (IRF-3) and nuclear factor-kB (NF-kB) [5,6]. The activated transcription factors stimulate interferon (IFN)-a and -b promoter, and the induced cytokines subsequently function for innate anti-viral defenses [7]. In hepatocytes, hepatitis C virus (HCV) controls the induction of anti-viral defense response by disruption of intracellular signaling processes that confer IRF-3 activation [6]. A recent study has indicated that RIG-I signaling is inhibited by the NS3/4A protease derived from HCV, resulting in HCV persistent infection [8]. On the other hand, MCF-7 human breast cancer cells and T24 urinary bladder carcinoma cells express RIG-I following stimulation by IFN-g in concentration- and time-dependent manners, suggesting that RIG-I may play a role in cellular responses during immune and inflammatory reactions [9]. Chronic inflammatory skin disorders such as psoriasis, allergic contact dermatitis, and atopic dermatitis are characterized by intense infiltration of activated T lymphocytes, which release lymphokines that influence the immune functions of resident skin cells, including keratinocytes [10—13]. Among the T cell-derived cytokines, IFN-g is the most potent activator of proinflammatory functions of keratinocytes. Psoriasis vulgaris is characterized by hyperproliferation and disturbed differentiation of keratinocytes, which might result from accumulation of T-lymphocyte subsets. CD4 + T cells are abundantly present in the upper lesional dermis, whereas CD8 + T cells are in the majority in the lesional epidermis [14]. Studies of the cytokine patterns of infiltrating T cells in the psoriatic lesions have revealed the predominance of T helper cell 1 (Th1) cytokines, e.g., IFN-g and TNF-a, compared with Th2 cytokines, e.g., interleukin (IL)-4, suggesting that psoriasis is an inflammatory disease induced by Th1 lymphocytes [15,16].
H. Kitamura et al. The aim of the present study was to define the effect of various cytokines, especially IFN-g and TNF-a, on the expression of RIG-I in HaCaT cells, an immortalized keratinocyte cell line, and the relevance of RIG-I in keratinocytes present in the epidermis of psoriasis vulgaris.
2. Materials and methods 2.1. Cell culture HaCaT cells, a spontaneously immortalized, nontumorigenic human skin keratinocyte cell line, were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated fatal bovine serum (FBS), 2 mg/ml sodium hydrogen carbonate, 100 mg/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. For experiments with cytokines, 60% confluent HaCaT cells were washed twice with sterile phosphate-buffered saline (PBS) and maintained in MEM with 0.3% FBS prior to addition of recombinant human IFN-g, TNF-a, transforming growth factor (TGF)-b and IL-1b (Roche Diagnostics, Mannheim, Germany). Cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air.
2.2. Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from cultured cells using RNAeasy total RNA isolation kit, as recommended by the manufacturer (Qiagen, Hilden, Germany). Extracted RNA was subjected to reverse transcription using RNA PCR Kit (AMV) Ver. 3.0 (Takara, Kyoto, Japan), according to the instructions provided by the manufacturer. Primers for RIG-I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: RIG-I-F (50 -GCATATTGACTGGACGTGGCA-30 ), RIG-I-R (50 -CAGTCATGGCTGCAGTTCTGTC-30 ), GAPDH-F (50 -CCACCCATGGCAAATTCCATGGCA-30 ), and GAPDH-R (50 -TCTAGACGGCAGGTCAGGTCCACC-30 ). The primers for RIG-I and GAPDH were designed to generate fragments of 644 base pairs (bp) and 598 bp, respectively. The amplification conditions were 94 8C for 1 min; followed by 30 cycles of 94 8C for 1 min, 58 8C for 1 min, and 72 8C for 1 min; and one cycle of 72 8C for 10 min. PCR products were analyzed by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining.
2.3. Western blot analysis Whole cell lysates were purified from HaCaT cells with RIPA lysis buffer (1% nonidet P-40, 0.1% sodium
RIG-I expression in human keratinocytes deoxycholate [SDS] in PBS) with 0.57 mM phenyl methyl sulfonyl fluoride (Sigma—Aldrich, St. Louis, MO, USA), 1 mM sodium orthovanadate (Sigma— Aldrich), and protease inhibitor (Roche Diagnostics). Cytoplasmic and nuclear fractions were separated from HaCaT cells with a NE-PER reagent (Pierce, Rockford, IL, USA) according to the instructions provided by the manufacturer. The concentrations of the extracted protein were quantified by the Bradford method, and 30 mg protein was subjected to electrophoresis in 10% SDS-polyacrylamide gel, and electroblotted to Hybond nitrocellulose membrane (Amersham Biosciences, Chandler, AZ, USA). The membranes were incubated in blocking solution (1% non-fat skim milk in 20 mM Tris [pH 7.4], 500 mM NaCl, and 0.025% Tween 20 [TBS-T]) for 1 h at room temperature (RT), followed by an overnight incubation at 4 8C with rabbit anti-RIG-I antiserum [17] diluted 1:10,000 in blocking solution. The membranes were washed four times in TBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, Amersham Biosciences) in TBS-T for 1 h at RT, and the bands were visualized using ECL-Western blotting detection system (Amersham Biosciences). The membranes were reprobed with an antibody against b-actin (Sigma—Aldrich) to verify equal loading of protein. Western blots were quantitated densitometrically with Scion Image 4.0.2 analysis software.
2.4. Immunofluorescence detection
129 finized and rehydrated using xylene and graded ethanol series. Neutralization of endogenous peroxidase was achieved with 0.3% H2O2 in methanol for 30 min at RT. Sections were coated with nonimmune goat serum overnight at 4 8C, and incubated with anti-RIG-I antiserum (1:2500) for 2 h at RT. Slides were washed with PBS three times, and stained using the avidin—biotin—peroxidase complex method, as recommended by the manufacturer (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA, USA). The skin sections were then lightly counterstained with Mayer’s hematoxylin for microscopic examination. All subjects provided informed consent for all aspects of the investigation according to Declaration of Helsinki Principles.
3. Results 3.1. Cytokine modulation of RIG-I expression in HaCaT cells Previous studies demonstrated that RIG-I protein is expressed in various cells and induced by IFN-g and IL1b [9,17—22]. In this study, we examined the expression of RIG-I in HaCaTcells untreated or treated with TNF-a10 ng/ml, TGF-b 10 ng/ml, IFN-g 10 ng/ml, or IL-1b (5 ng/ml) for 24 h. As shown in Fig. 1, stimulation by IFN-g significantly increased RIG-I protein expression to 6-folds that of unstimulated control HaCaT cells, which showed a faint band. Similarly,
HaCaT cells grown in 4-well chamber slides were incubated with or without 10 ng/ml IFN-g for 24 h, and fixed with 4% paraformaldehyde for 1 h, permeabilized with 0.5% Triton-X, and then treated with 3% bovine serum albumin for blocking. After an overnight incubation at 4 8C, the cells were incubated with rabbit anti-RIG-I antiserum (1:500) for 1 h. After washing with PBS three times, the cells were incubated with rhodamine-conjugated swine anti-rabbit IgG (1:500, Dako, Carpinteria, CA, USA) for 1 h. The cells were rinsed, coverslipped, and examined by fluorescence microscopy.
2.5. Immunohistochemistry Skin biopsies were obtained from six patients with psoriasis vulgaris (age range: 38—76 years) and six non-psoriatic patients as healthy control subjects (age range: 29—70 years). Sections of each tissue sample were stained with hematoxylin and eosin for histopathological evaluation. Sequential sections (7 mm) were cut from each tissue sample embedded in paraffin, and mounted on a glass slide. After an overnight incubation at 37 8C, slides were deparaf-
Fig. 1 Cytokine modulation of RIG-I expression in HaCaT cells. HaCaT cells were stimulated with TNF-a (10 ng/ml), TGF-b (10 ng/ml), IFN-g (10 ng/ml), IL-1b (5 ng/ml), or none for 24 h. Cell lysates were subjected to Western blotting with anti-RIG-I antibody. Displayed data are one representative result of three independent experiments. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
130 TNF-a-induced RIG-I protein expression was also noted at approximately 3-folds that of control HaCaT cells (Fig. 1). In contrast, neither TGF-b nor IL-1b influenced RIG-I expression in HaCaT cells.
3.2. Effect of IFN-g and TNF-a on RIG-I expression in HaCaT cells In the next step, we studied the effect of various concentrations (0.1—100 ng/ml) of IFN-g on RIG-I expression. As shown in Fig. 2A and B, the expression of RIG-I mRNA and protein was induced in IFN-gtreated HaCaT cells in a concentration-dependent manner. Furthermore, to determine the dynamics of RIG-I expression induced by IFN-g, we also examined the time course of RIG-I expression using RT-PCR and Western blot analyses (0—48 h). RIG-I mRNA was expressed at a low level in unstimulated HaCaT cells as demonstrated by RT-PCR analysis (Fig. 2C), gradually increased to a peak level at 12 h after IFN-g stimulation and then somewhat diminished at 24 and 48 h. Similarly, the expression of RIG-I protein clearly increased in a time-dependent manner, and was approximately 6-folds higher than that of unstimulated HaCaTcells, when incubated with IFN-
H. Kitamura et al. g (10 ng/ml) for 24 h (Fig. 2D). These results suggest that RIG-I protein is induced via upregulation of transcription activity by IFN-g. We also characterized TNF-a-induced RIG-I expression in HaCaT cells. The expression of RIG-I mRNA and protein was induced in TNF-a-treated HaCaT cells in a concentration-dependent manner (Fig. 3A and B). The level of RIG-I mRNA increased in a time-dependent manner and reached a maximum at 6 h, earlier peak than IFN-g-stimulated HaCaT, but gradually decreased thereafter (Fig. 3C). On the other hand, the production of RIG-I protein was enhanced at 24 h, and then decreased to basal level at 48 h (Fig. 3D). These results indicate that TNF-a induces a transient increase in RIG-I expression compared with IFN-g.
3.3. RIG-I protein is mainly distributed in the cytoplasm of HaCaT cells To define the subcellular distribution of RIG-I protein, nuclear and cytoplasmic proteins were obtained separately from IFN-g (10 ng/ml) or TNF-a (10 ng/ ml)-treated HaCaT cells after 24 h incubation. The induced increase in RIG-I protein was detected in the
Fig. 2 Time- and concentration-dependent effects of IFN-g on the expression of RIG-I in HaCaT cells. HaCaT cells were incubated in the presence or absence of IFN-g at the indicated concentrations for 24 h. Purified total RNA and cell lysates were subjected to RT-PCR (A) and Western blot analysis (B), respectively. HaCaT cells were incubated in the presence or absence of IFN-g (10 ng/ml) under the same conditions for the indicated time periods. Purified total RNA and cell lysates were subjected to RT-PCR (C) and Western blot analysis (D), respectively. Displayed data are one representative result of three independent experiments in each examination. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
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Fig. 3 Effects of TNF-a on the expression of RIG-I in HaCaTcells. HaCaTcells were incubated in the presence or absence of TNF-a at the indicated doses for 24 h. Purified total RNA and cell lysates were subjected to RT-PCR (A) and Western blot analysis (B), respectively. HaCaT cells were incubated in the presence or absence of TNF-a (10 ng/ml) under the same conditions for the indicated time periods. Purified total RNA and cell lysates were subjected to RT-PCR (C) and Western blot analysis (D), respectively. Displayed data are one representative result of three independent experiments in each examination. RIG-I protein levels quantitated by scanning densitometry and corrected for the levels of b-actin in the same sample are shown relative to that of unstimulated HaCaT cells.
Fig. 4 Distribution of RIG-I protein induced by IFN-g or TNF-a in HaCaT cells. (A) HaCaT cells were treated with IFN-g (10 ng/ml) or TNF-a (10 ng/ml) for 24 h, or without stimulation, and nuclear and cytoplasmic fractions were purified, respectively. (B) HaCaT cells were stimulated with IFN-g (10 ng/ml) or none for 24 h and subjected to immunofluorescence staining. Cells were observed under a fluorescent microscope. Scale bars = 10 mm.
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Fig. 5 Immunohistochemical detection of RIG-I in the epidermis of psoriasis. Immunohistochemical staining of RIG-I of normal skin (A) and psoriatic skin (B). Images are representative of six specimens per each group. Scale bars = 100 mm.
cytoplasmic fraction as demonstrated by Western blot analysis, whereas the nuclear fraction did not show RIG-I protein expression (Fig. 4A). This result was confirmed by immunofluorescence staining, which showed cytoplasmic distribution of IFN-g (10 ng/ml)-induced RIG-I protein. These results suggest that the expression of RIG-I protein is significantly enhanced by IFN-g and is distributed in the cytoplasm (Fig. 4B).
3.4. Psoriasis vulgaris keratinocytes overexpress RIG-I protein Biopsy specimens of normal and psoriatic skin were examined for the presence of RIG-I. No staining for RIG-I was noted in the normal skin (Fig. 5A). In contrast, RIG-I was abundantly expressed in keratinocytes of the spinous and basal layers, but not in the cornified layer, in specimens of psoriasis vulgaris (Fig. 5B). Moreover, endothelial cells and fibroblasts in the dermis of psoriasis were positively stained in comparison with the control skin, suggesting that RIG-I might be one of crucial factors for the development of psoriasis.
4. Discussion RIG-I has two tandem motifs with limited homology to CARD and a C-terminal region possessing a DExD/
H box RNA helicase domain, and belongs to the DExH/D box protein family [5]. RIG-I was recently defined to be a double-stranded RNA binding protein that functions independently of Toll-like receptor 3 (TLR3) to signal IFN-production in response to a variety of RNA viruses, including hepatitis C virus (HCV) [5,6]. A recent study has suggested that NS3/ 4A protease derived from HCV, sufficient to support viral RNA replication, disrupts RIG-I downstream signals, resulting in HCV persistent infection, reflecting the evasion of host innate immune defenses [8]. Moreover, the truncated RIG-I without the helicase domain motif significantly induces the activation of IRF-3 and NF-kB even in the absence of any virus or the RNA duplex poly(rI):poly(rC) (poly(I:C)), whereas CARDs-truncated RIG-I induces no activation of downstream signals. These results suggest that the helicase domain suppresses downstream signaling through the CARD domains in nonviral condition [5]. Considering the binding ability of the helicase domain to poly(I:C), RIG-I is an essential component of the signaling pathway upstream of IRF-3 and NF-kB, and probably the direct sensor of viral double-stranded RNA [5,23]. Interestingly, it has been demonstrated that RIG-I is expressed by the induction of inflammatory cytokines in various types of human cells, including endothelial cells, vascular smooth muscle cells, urinary bladder epithelial cells, gingival fibroblasts, and bronchial epithelial cells [9,17—22]. In the present
RIG-I expression in human keratinocytes study, we found that RIG-I protein is predominantly expressed in human keratinocytes treated with IFN-g or TNF-a, whereas neither TGF-b nor IL-1b augmented RIG-I expression. Our results also showed that the induced RIG-I was mainly distributed in the cytoplasm, and RIG-I expression stimulated by IFN-g was higher and more persistent, compared with that induced by TNF-a. However, the rapid induction of RIG-I mRNA was noted at 6 h after TNF-a stimulation, suggesting that IFN-g and TNF-a cooperate to sustain the high expression of RIG-I upon inflammatory reaction or viral infection of the epidermis. IFN-g is a potent cytokine that regulates immune responses by inducing multiple genes in various cell types including keratinocytes. IFN-g exerts its biological activities by binding to specific receptor, IFNg receptor a/b, followed by activation of Janus kinase (JAK) 1/2 and signal transducers and activators of transcription-1 (STAT-1) and induction of various genes [24]. IFN-g and TNF-a belong to Th1 cytokines involved in the development of many inflammatory skin diseases such as psoriasis, characterized by hyperproliferation and disturbed differentiation of keratinocytes [25,26]. In the psoriatic lesions, Th1 cytokines including IFN-g and TNF-a are predominant in comparison with Th2 cytokines such as IL-4, and psoriasis is considered an inflammatory disease induced by Th1 lymphocytes [15,16]. In the present study, we showed that psoriatic keratinocytes on the spinous and basal layers strongly express RIG-I in the cytoplasm, compared with normal keratinocytes of the control specimens, and RIG-I was induced by stimulation IFN-g and TNF-a in cultured human keratinocytes. Furthermore, endothelial cells and fibroblasts in the dermis of psoriatic skin were positively stained for RIG-I, similar to psoriatic keratinocytes. These cells express RIG-I protein by stimulation of IFN-g and IL1b, respectively [20,22]. These results suggest that RIG-I might play an important role in the pathogenesis of psoriasis via induction of various cytokines. Melanoma differentiation-associated gene-5 (MDA-5) was identified as a novel upregulation gene in human melanoma cells induced to terminally differentiate by treatment with IFN-b and mezerein [27]. MDA-5 consists of a CARD and a C-terminal putative RNA helicase domain, and is highly homologous to RIG-I [28]. The level of MDA-5 mRNA is low in normal tissues, whereas its expression is induced in a spectrum of normal and cancer cells by IFN-b [27]. Furthermore, overexpression of MDA-5 by means of a replication incompetent adenoviral vector in cultured melanoma cells resulted in apoptotic cell death, indicating that MDA-5 may contribute to induction of apoptosis during terminal differentiation and IFN treatment [27]. In this study, we
133 identified the distribution of RIG-I in the spinous and basal layers of psoriatic epidermis, however most psoriatic keratinocytes indicate no apoptotic change [29]. Based on these results, the function of RIG-I may be markedly different from MDA-5, although the two proteins have a similar structure. The present study provided the further evidences that the expression of RIG-I induced by IFN-g and TNF-a might play a role in the regulation of proliferation of keratinocytes involved in psoriasis vulgaris. These findings indicate that RIG-I might operate not only as a RNA helicase in viral infection but also as a mediator of the cytokine network during endogenous inflammation.
Acknowledgements We thank Mr. Shigeki Ohashi, Department of Dermatology, Hirosaki University School of Medicine, and Mr. Jyunpei Asano, Second Department of Biochemistry, Hirosaki University School of Medicine, for their kind and helpful advice.
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