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Glycobiology Advance Access originally published online on April 20, 2007
Glycobiology 2007 17(7):12C-15C; doi:10.1093/glycob/cwm045
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Double-stranded RNA induces galectin-9 in vascular endothelial cells: involvement of TLR3, PI3K, and IRF3 pathway

Tadaatsu Imaizumi1,2, Hidemi Yoshida2, Nozomu Nishi6, Hiroshi Sashinami3, Takanori Nakamura6, Mitsuomi Hirashima7, Chikara Ohyama4, Ken Itoh5 and Kei Satoh2

2 Department of Vascular Biology
3 Department of Microbiology and Immunology
4 Department of Urology
5 Center for Advanced Medical Research, Hirosaki University School of Medicine, 5-Zaifucho, Hirosaki 036-8562, Japan
6 Department of Endocrinology
7 Department of Immunology and Immunopathology, Kagawa University School of Medicine, Kagawa 761-0793, Japan

1 To whom correspondence should be addressed; E-mail: timaizum{at}cc.hirosaki-u.ac.jp

Received on January 11, 2007; revised on April 1, 2006; accepted on April 13, 2007


   Abstract
Top
 Abstract
Introduction
 Results and discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Galectin-9 is a member of the galectin family, which induces various biological reactions such as chemotaxis of eosinophils and apoptosis of T cells. We previously reported that polyinosinic–polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA), induces the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs). In the present study, we addressed the possible involvement of two potential receptors for dsRNA, Toll-like receptor (TLR) 3 and retinoic acid-inducible gene-I (RIG-I), in the expression of galectin-9 in HUVECs. Poly IC-induced galectin-9 expression was almost completely suppressed by RNA interference (RNAi) against TLR3, but not against RIG-I. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited the induction of galectin-9 by poly IC. RNAi against interferon regulatory factor 3 (IRF3) also inhibited poly IC-induced galectin-9 expression. We conclude that TLR3, PI3K, and IRF3 are involved in the poly IC-induced galectin-9 expression in HUVECs.

Key words: dsRNA / galectin-9 / vascular endothelial cells / TLR3 / RIG-I / PI3K / IRF3


   Introduction
Top
 Abstract
 Introduction
Results and discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Galectins are a family of ß-galactoside-binding animal lectins with conserved carbohydrate-binding domains (Hirabayashi and Kasai 1993Go), and galectin-9 has multiple immunomodulatory functions. Galectin-9 induces the chemotaxis of eosinophils (Matsumoto et al. 1998Go; Hirashima 1999Go, 2000Go), inhibits the function of Th1 cells via binding to T cell immunoglobulin (Ig)- and mucin-domain-containing molecule-3 (TIM-3) (Zhu et al. 2005Go), and promotes maturation of human monocyte-derived dendritic cells (Dai et al. 2006). Vascular endothelial cells play an important role in the initiation of inflammatory and immune reactions, and we found that the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs) was stimulated by interferon (IFN)-{gamma} (Imaizumi, Kumagai et al. 2002Go). The IFN-{gamma}-induced galectin-9 expression was inhibited by 15-deoxy-{Delta}12,14-prostaglandin J2, which also exerts regulatory effects on inflammatory responses (Imaizumi et al. 2003Go).

Polyinosinic–polycytidylic acid (poly IC) is an authentic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics viral infection of the cells. In endothelial cells, poly IC induces the expression of various genes such as interleukin-6, vascular adhesion molecule-1, epithelial neutrophil activatin peptide-78, and retinoic acid-inducible gene-I (RIG-I) (Offerman et al. 1995; Imaizumi et al. 2005Go). We previously demonstrated that poly IC also induces the expression of galectin-9 in HUVECs (Ishikawa et al. 2004Go). However, receptors or signaling molecules involved in the poly IC-induced expression of galectin-9 have not been identified. Toll-like receptors (TLRs) recognize the molecular pattern of pathogens, which are important in innate immunity. TLR3 recognizes dsRNA, and binding of dsRNA to TLR3 activates the signaling to induce anti-viral responses (Alexopoulou et al. 2001Go). RIG-I, a putative RNA helicase (Imaizumi, Aratani et al. 2002Go), may also bind dsRNA and activate anti-viral responses independently of TLR3 (Yoneyama et al. 2005Go).

The present study was undertaken to examine whether TLR3 or RIG-I may be involved in the poly IC-induced expression of galectin-9 in HUVECs and whether other TLR ligands affect the galectin-9 expression. Poly IC is known to activate multiple signaling systems, including phosphatidylinositol 3-kinase (PI3K) and interferon regulatory factor 3 (IRF3) (Sarkar et al. 2004Go; Guillot et al. 2005Go; Yoneyama et al. 2005Go), and we also addressed the possible involvement of PI3K and IRF3 in poly IC-induced galectin-9 expression.


   Results and discussion
Top
 Abstract
 Introduction
 Results and discussion
Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
dsRNA elicits various cellular responses similar to those provoked by viral infection. Vascular endothelial cells serve as one of the important components of immune and inflammatory reactions (McIntyre et al. 1997Go) and express various cytokines and adhesion molecules in response to dsRNA (Offerman et al. 1995Go; Imaizumi et al. 2005Go). Cellular recognition of dsRNA and subsequent activation of anti-viral signaling are important for host defense against RNA viruses. TLR3 is localized on the endosome membrane and serves as a receptor for dsRNA, whereas RIG-I is a cytoplasmic molecule (Imaizumi et al. 2004Go) and is suggested to function as another receptor for dsRNA (Yoneyama et al. 2005Go). Both TLR3 and RIG-I independently turn on the anti-viral responses via activation of IRF3 (Yoneyama et al. 2005Go).

In the present study, we first examined the effect of short-interfering RNA (siRNA) against TLR3 or RIG-I on the poly IC-induced expression of galectin-9. In accordance with a previous report (Ishikawa et al. 2004Go), poly IC induced galectin-9 in HUVECs (Figure 1A). Poly IC also enhanced the expression of messenger ribonucleic acid (mRNA) for TLR3 and RIG-I. Transfection of the cells with siRNA against TLR3 or RIG-I markedly inhibited the expression of target mRNA, and the poly IC-induced galectin-9 expression was inhibited by knockdown of TLR3, but not of RIG-I (Figure 1A and B). Thus, TLR3 may be involved in the poly IC-induced galectin-9 expression in HUVECs. Expression of galectin-1 was not altered by poly IC treatment or transfection with the siRNAs.


Figure 1
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  		Fig. 1. Expression of galectin-9 in HUVECs treated with poly IC. (A) HUVECs were transfected with siRNA against TLR3, RIG-I, or non-silencing control. After 24 h of transfection, the cells were treated with 20 µg/mL poly IC for 8 h. Total RNA was extracted and single-strand cDNA was synthesized. Specific cDNAs for galectin-9, galectin-1, TLR3, RIG-I, and GAPDH were amplified by PCR. (B) siRNA against TLR3 or non-silencing control was transfected into HUVECs as in (A). After incubation for 24 h, the cells were treated with 20 µg/mL poly IC for an additional 24 h. Lysates were subjected to western blot analysis for galectin-9 and actin. (C) HUVECs were transfected with siRNA against IRF3 or non-silencing control. After 24 h of transfection, the cells were treated with 20 µg/mL poly IC for 8 h. Total RNA was extracted and RT–PCR for galectin-9, IRF3, and GAPDH were performed as in (A). (D) siRNA against IRF3 or non-silencing control was transfected into HUVECs as in (C). After incubation for 24 h, the cells were treated with 20 µg/mL poly IC for an additional 24 h. Western blot analyses for galectin-9 and actin were performed as in (B). (E) HUVECs were pretreated with 20 µM LY294002 for 1 h, and treated with 20 µg/mL poly IC for 8 h. The expression of galectin-9 mRNA was examined by RT–PCR as in Figure 1. (F) The cells were pretreated with LY294002 as above, and treated with 20 µg/mL poly IC for 24 h. Western blot analysis for galectin-9 and actin was performed. (G) The signaling pathway leading to the poly IC-induced galectin-9 expression.

 
Various signaling systems, including NF-{kappa}B, p38 MAPK, IRF3, and PI3K, are known to be involved in the gene expression induced by poly IC. We previously found that neither NF-{kappa}B nor p38 MAPK is involved in the endothelial expression of galectin-9 induced by poly IC (Ishikawa et al. 2004Go). In the present study, we examined whether IRF3 is involved in the galectin-9 induction by poly IC. Transfection of IRF3 siRNA markedly inhibited the IRF3 expression and poly IC-induced galectin-9 expression (Figure 1C and D). IRF3 may be involved in the galectin-9 expression mediated through TLR3. PI3K catalyzes the production of PI(3,4,5)P3, which is followed by phosphorylation of downstream kinases. A recent study showed that the PI3K signaling pathway is activated via TLR3 (Sarkar et al. 2004Go), and that LY294002, an inhibitor of PI3K, reduces the production of CC chemokine ligand 5 (CCL5) in response to poly IC (Guillot et al. 2005Go). In the present study, pretreatment of the cells with LY294002 inhibited the poly IC-induced galectin-9 expression (Figure 1E and F). PI3K is known to activate the IRF3 pathway but suppress the NF-{kappa}B pathway activated via TLR3 in glial cells (Zhao et al. 2006Go). Taken together, these results may imply that poly IC induces galectin-9 expression in HUVECs via TLR3, PI3K, and IRF3. Our results summarized in Figure 1G.

Next, we examined whether galectin-9 expression is regulated via other TLRs. We used Escherichia coli lipopolysaccharide (LPS), heat-killed Listeria monocytogenes (HKLM), loxoribine (LOX) or CpG oligonucleotide as ligands for TLR4, TLR2, TLR7, or TLR9, respectively. The effect of E. coli LPS on the expression of galectin-9 is summarized in Figure 2A–C. Treatment of HUVECs with LPS induced galectin-9 mRNA, and this was inhibited by knockdown of TLR4; however, LPS did not induce the expression of galectin-9 protein. IFN-{gamma}, used as a positive control, significantly induced the galectin-9 protein expression. This result suggests that certain post-transcriptional mechanisms may be necessary for the LPS-induced galectin-9 expression in HUVECs. HKLM and CpG oligonucleotide induced the expression of CCL5 mRNA, but not galectin-9 in HUVECs. LOX did not induce the galectin-9 expression in HUVECs, although it induced interleukin-8 (IL-8) mRNA. TLRs recognize the molecular pattern of the pathogens, and TLR3 recognizes dsRNA and may play an important role in the innate immunity against viral infection. TLR3 may be involved in galectin-9 expression; and this result suggests a certain role of galectin-9 in anti-viral responses in endothelial cells.


Figure 2
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  		Fig. 2. (A) HUVECs were treated with 10 µg/mL E. coli LPS for up to 24 h, and RT–PCR analysis for galectin-9 and GAPDH was performed. (B) siRNA against TLR4 or non-silencing control was transfected into HUVECs. After 24 h of transfection, the cells were treated with 10 µg/mL LPS for 16 h. Total RNA was extracted and RT–PCRs for galectin-9, TLR4, and GAPDH were performed as in (A). (C) HUVECs were treated with 10 µg/mL LPS or 10 ng/mL IFN-{gamma} for up to 24 h. Western blot analysis for galectin-9 and actin was performed. (D) HUVECs were treated with HKLM (at a multiplicity of 10:1), 500 µM LOX, or 2.5 µM CpG oligonucletide for 16 h, and expression of mRNA for galectin-9, CCL5, IL-8, and GAPDH was examined by RT–PCR analysis. Galectin-9 cDNA was used for a positive control of the reaction.

 
In conclusion, the TLR3, PI3K, and IRF3 pathway may be involved in poly IC-induced galectin-9 expression in HUVECs.


   Materials and methods
Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
Conflict of interest statement
 Acknowledgments
 References
 
Reagents
HUVECs were purchased from Cambrex (Walksville, MD). Humedia EB-2 and its supplements were from Kurabo (Osaka, Japan). Poly IC, LPS from E. coli, and anti-actin IgG were from Sigma (St Louis, MO). TLR agonists, LOX and CpG oligonucleotide M362, were from InvivoGen (San Diego, CA). Recombinant human [r(h)] IFN-{gamma} was from Roche (Mannheim, Germany). LY294002, an inhibitor of PI3K, was from Cell Signaling Technology (Beverly, MA). Lipofectamine 2000, oligo(dT)12-18 and M-MuLV reverse transcriptase were from Invitrogen (Carlsbad, CA). SiRNAs (#SI102655156 for TLR3, #SI102657403 for RIG-I, #SI03117359 for IRF3, and #SI03095645 for TLR4), an RNeasy total RNA isolation kit and Taq DNA polymerase were purchased from Qiagen (Hilden, Germany). Oligonucleotide primers were from Fasmac (Kanagawa, Japan). Immobilon polyvinylidene difluoride (PVDF) membrane was from Millipore Japan (Tokyo, Japan). A chemiluminescent substrate Supersignal west femto was from Pierce (Rockford, IL).

Cell culture
HUVECs were cultured as described (Ishikawa et al. 2004Go). Briefly, HUVECs were cultured using HuMedia EB-2 supplemented with 2% fetal bovine serum, 10 ng/mL epidermal growth factor, 1 µg/mL hydrocortisone, 5 ng/mL basic fibroblast growth factor, and 10 µg/mL heparin. The cells of the first to the fifth passage were used for experiments. HUVECs were transfected with siRNA using Lipofectamine 2000 according to the manufacturer's instructions. Briefly, the cells were maintained in the medium without antibiotics for 24 h, and then incubated with transfection mixture for 24 h. HUVECs were treated with poly IC, LPS, HKLM, LOX, CpG oligonucleotide M362, or r(h) IFN-{gamma}. LY294002 was added to HUVECs 30 min before the application of poly IC.

Reverse transcription–polymerase chain reaction (RT–PCR)
RT–PCR analysis for the expression of galectin-9, galectin-1, TLR3, and RIG-I was performed as described previously (Imaizumi et al. 2002Go; Ishikawa et al. 2004Go). Total RNA was extracted from HUVECs using an RNeasy total RNA isolation kit. Single-stranded complementary deoxyribonucleic acid (cDNA) for a PCR template was synthesized from 1 µg of total RNA using primer oligo(dT)12-18 and M-MuLV reverse transcriptase. Specific primers for galectin-9, galectin-1, TLR3, RIG-I, IRF3, TLR4, CCL5, IL-8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows:

galectin-9-F (5'-GAGATGGCCTTCAGCAGTTCC-3'),
galectin-9-R (5'-CGCCTATCTCTGCACATGGGT-3'),
galectin-1-F (5'-TGGTCGCCAGCAACCTGAATCTCA-3'),
galectin-1-R (5'-TAGTTGATGGGCCTCCAGGTTGAGG-3'),
TLR3-F (5'-CTCAGAAGATTACCAGCCGCC-3'),
TLR3-R (5'-CCATTATGAGACAGATCTAATG-3'),
RIG-I-F (5'-GCATATTGACTGGACGTGGCA-3'),
RIG-I-R (5'-CAGTCATGGCTGCAGTTCTGTC-3'),
IRF3-F (5'-TACGTGAGGCATGTGCTGA-3'),
IRF3-R (5'-AGTGGGTGGCTGTTGGAAAT-3'),
TLR4-F (5'-TGCAATGGATCAAGGACCAGAGGC-3'),
TLR4-R (5'-GTGCTGGGACACCACAACAATCACC-3'),
CCL5-F (5'-CTACTCGGGAGGCTAAGGCAGGAA-3'),
CCL5-R (5'-GAGGGGTTGAGACGGCGGAAGC-3'),
IL-8-F (5'-AGGAGTGCTAAAGAACTTCGA-3'),
IL-8-R (5'-TGAATTCTCAGCCCTCTTCAA-3'),
GAPDH-F (5'-CCACCCATGGCAAATTCCATGGCA-3'),
GAPDH-R (5'-TCTAGACGGCAGGTCAGGTCCACC-3').

The reaction condition for galectin-9 was 1x (94 °C, 1 min), 26x (94 °C, 1 min; 62 °C, 1 min; 72 °C, 1 min), and 1x (72 °C, 10 min). The reaction condition for galectin-1, RIG-I, IRF3, IL-8, and GAPDH was similar to that for galectin-9 except that amplification was repeated for 30 cycles and the annealing temperature was 55 °C. For the analysis of TLR3, TLR4, and CCL5, amplification was performed for 35 cycles with the annealing temperature at 58 °C. The products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide. There are three isoforms of galectin-9 depending on the length of the linker peptide, short, medium and long types; and the size of the amplified product for galectin-9 was 978 bp, which corresponds to the medium type. The size of the products for TLR3, galectin-1, RIG-I, IRF3, TLR4, CCL5, IL-8, and GAPDH was 287, 346, 644, 425, 449, 318, 219, and 696 bp, respectively.

Western blotting
Western blot analysis for galactin-9 and actin was performed as described previously (Imaizumi et al. 2002Go; Ishikawa et al. 2004Go). Briefly, cells were washed with 20 mM cold phosphate-buffered saline, pH 7.4, and lysed with Laemmli's reducing sample buffer. The lysates were subjected to electrophoresis on a 4–20% gradient polyacrylamide gel, and the proteins were transferred to an Immobilon PVDF membrane. The membrane was incubated with rabbit anti-galectin-9 IgG (0.2 µg/mL) or anti-actin IgG, and immunodetection was performed using anti-rabbit IgG labeled with horseradish peroxidase and a Supersignal west femto chemiluminescent substrate.


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Conflict of interest statement
Acknowledgments
 References
 
None declared.


   Acknowledgments
Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
References
 
The authors thank Kumiko Munakata, Michiko Nakata, and Miho Ono for their help. Part of this study was supported by grants from the Japan Society for the Promotiom of Science (#17590773) and from the Aomori Bank for medical research in Hirosaki University.


   Abbreviations
 
CCL5, CC chemokine ligand 5; cDNA, complementary deoxyribonucleic acid; dsRNA, double-stranded RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HKLM, heat-killed Listeria monocytogenes; HUVECs, human umbilical vein endothelial cells; IFN, interferon; IL-8, interleukin-8; IRF3, interferon regulatory factor 3; LOX, loxoribin; LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; PI3K, phosphatidylinositol 3-kinase; poly IC, polyinosinic–polycytidylic acid; PVDF, polyvinylidene difluoride; r(h) IFN-{gamma}, recombinant human interferon-{gamma}; RIG-I, retinoic acid-inducible gene-I; RNAi, RNA interference; siRNA, short-interfering RNA; TLR, Toll-like receptor.


   References
Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
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