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Genes to Cells (2009) 14, 511-521. doi:10.1111/j.1365-2443.2009.01287.x
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Intracellular galectin-9 activates inflammatory cytokines in monocytes

 Ai Matsuura1, Junichi Tsukada2, Takamitsu Mizobe1, Takehiro Higashi2, Fumihiko Mouri1, Rena Tanikawa1, Akira Yamauchi3, Mitsuomi Hirashima4 and Yoshiya Tanaka1

1 First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
2 Cancer Chemotherapy Center, University of Occupational and Environmental Health, Kitakyushu, Japan
3 Department of Cell Regulation, Faculty of Medicine, Kagawa University, Kagawa, Japan
4 Department of Immunology and Immunopathology, Faculty of Medicine, Kagawa University, Kagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Whether galectin-9 plays a role in inflammatory responses remains elusive. The present study was designed to determine the role of intracellular galectin-9 in activation of inflammatory cytokine genes in human monocytes. Galectin-9 expression vector pBKCMV3-G9 was transiently co-transfected into THP-1 monocytic cells along with luciferase reporters carrying gene promoters of IL-1{alpha} (IL1A), IL-1β (IL1B) and IFN{gamma}. Transient transfection studies showed that galectin-9 over-expression activated all three gene promoters, suggesting that intracellular galectin-9 induces inflammatory cytokine genes in monocytes. Galectin-9 over-expression also activated NF-IL6 (C/EBP β) and AP-1, but not NF-{kappa}B. In contrast, extracellular galectin-9 is not involved in regulation of inflammatory cytokines. Immunoprecipitation/Western blotting, using anti-galectin-9 Ab and anti-NF-IL6 Ab, showed physical association of intracellular galectin-9 with NF-IL6. RT-PCR confirmed that galectin-9 over-expression increased IL-1{alpha} and IL-1β mRNA levels in THP-1 cells. The interaction of galectin-9 with NF-IL6 was enhanced following LPS treatment in THP-1 cells. Intracellular galectin-9 synergized with LPS to activate NF-IL6. Nuclear translocation of galectin-9 was also observed in THP-1 cells treated with LPS. Our results indicate that galectin-9 is a LPS-responsive factor, and further demonstrate that intracellular galectin-9 transactivates inflammatory cytokine genes in monocytes through direct physical interaction with NF-IL6.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Galectins are a protein family of animal lectins that modulate various extracellular (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellular (by interacting with cytoplasmic and nuclear proteins) signaling pathways. To date, 15 galectins have been cloned in mammals (Barondes et al. 1994a). Galectins exhibit affinity for β-galactosides, which share certain conserved sequence elements (Barondes et al. 1994b), and play modulatory roles in diverse biological processes such as cell adhesion, cell proliferation (Perillo et al. 1998; Asakura et al. 2002; Nishi et al. 2003; Zick et al. 2004), cell apoptosis (Perillo et al. 1995; Kashio et al. 2003), chemoattraction and immunomodulation of inflammation (Liu 2000; Rabinovich et al. 2002; Almkvist & Karlsson 2004). Galectins can act as inflammatory mediators, and are involved in the recruitment of polymorphonuclear leukocytes from the blood stream and cross-link of polymorphonuclear leukocytes with the endothelium (Almkvist & Karlsson 2004).

Members of the galectin family are structurally classified into three groups. Galectin-1, 2, 7, 10 and 13 are prototype galectins, whereas galectin-3 is a chimera type. In contrast, galectin-4, 8, 9 and 12 belong to the tandem repeat type subfamily, which is characterized structurally by the presence of two distinct carbohydrate recognition domains, with different sugar binding specificities, joined by a linker peptide. Galectin-9 exhibits various biological activities such as chemoattraction, cell aggregation, induction of superoxide production and prolongation of cell survival of eosinophils (Matsumoto et al. 2002). In addition, galectin-9 can function as a proapoptotic factor in a variety of cells, including activated T lymphocytes (Kashio et al. 2003), thymocytes and tumor cells (Kageshita et al. 2002). Galectin-9 is also a potential prognostic factor as it exhibits antimetastatic properties in breast cancer (Irie et al. 2005). Galectin-9 is expressed in activated T cells, mouse embryonic kidney, and tissues of patients with Hodgkin's disease, monocytes/macrophages, Jurkat, THP-1, and RPMI-8866 cells (Spitzenberger et al. 2001). In mice, this factor is widely distributed, including the liver, small intestine, thymus, kidney, spleen and lung. Phorbol 12-myriastate 13-acetate up-regulates galectin-9 production in Jurkat cells (Chabot et al. 2002). Furthermore, matrix metalloproteinase and protein kinase C are involved in the release of galectin-9 from Jurkat cells (Chabot et al. 2002). Human endothelial cells treated with interferon (IFN)-{gamma} exhibit increased production of galectin-9 (Imaizumi et al. 2002). Lipopolysaccharide (LPS) enhances the expression levels of galectin-9 mRNA and protein in a time-dependent manner (Kasamatsu et al. 2005). The present study was designed to determine the significance of galectin-9 in the inflammatory processes. The results demonstrated that intracellular galectin-9 functions as a transcriptional activator of inflammatory cytokine genes in monocytes/macrophages.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Galectin-9 induces expression of IL-1{alpha} and IL-1β mRNAs in THP-1 monocytic cells

Galectin-9 is expressed in various types of cells such as T-cells, fibroblasts, endothelial cells, mast cells, macrophages, astrocytes and eosinophils. In the present study, mRNA expression of inflammatory cytokines such as IL-1{alpha} and IL-1β in THP-1 cells transiently transfected with galectin-9 expression vectors was examined by using RT-PCR. Three kinds of galectin-9 expression vectors, pBKCMV3-G9(S), pBKCMV3-G9(M) and pBKCMV3-G9(L) were used. Three isoforms of galectin-9 that differed only in the length of their linker peptide region have been identified (Hirashima 2000). The short-sized isoform of galectin-9, galectin-9(S) was found to have a linker peptide region of 14 amino acids, whereas the medium and the long-sized isoforms of galectin-9, galectin-9(M) and galectin-9(L) have a linker peptide region of 26 and 58 amino acids, respectively. The expression vectors for galectin-9(S), galectin-9(M) and galectin-9(L) were pBKCMV3-G9(S), pBKCMV3-G9(M) and pBKCMV3-G9(L), respectively. THP-1 cells that did not over-express galectin-9 showed no significant expression of IL-1{alpha} or IL-1β mRNA (Fig. 1A,B). Transient transfection of either pBKCMV3-G9(S), pBKCMV3-G9(M) or pBKCMV3-G9(L) into THP-1 cells induced both IL-1{alpha} and IL-1β mRNA expression (Fig. 1A,B). These results indicate that galectin-9 enhances the mRNA expression of IL-1{alpha} and IL-1β.


Figure 1
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  		Figure 1  Expression of IL-1{alpha} and IL-1β mRNAs in THP-1 cells transfected with galectin-9 expression vectors. Three micro grams of pBKCMV3-G9(S), pBKCMV3-G9(M) or pBKCMV3-G9(L) were transiently transfected into THP-1 cells, and 24 h after transfection, expression of IL-1{alpha} (A) and IL-1β (B) mRNAs was examined by RT-PCR. RT-PCR was carried out as described in Experimental Procedures section. As a positive control, THP-1 cells were treated with LPS. Representative results of three experiments with similar findings.

 
Effects of galectin-9 on IL1A, IL1B and IFN{gamma} promoter activities in THP-1 cells

To examine the effects of galectin-9 on the transcriptional regulation of inflammatory cytokine genes, pBKCMV3-G9(S), pBKCMV3-G9(M) or pBKCMV3-G9(L) was co-transfected into THP-1 cells along with pGL3IL1{alpha} reporter for IL1A gene promoter. Over-expression of each of the three isoforms of galectin-9 resulted in similar enhancement of IL1A promoter activity (Fig. 2). Furthermore, transfection of pBKCMV3-G9(S) dose-dependently induced promoter activities of IL1B and IFN{gamma} as well as IL1A (Fig. 3A–C). Transfection of pBKCMV3-G9(S) at 1.5 µg resulted in approximately threefold, eightfold and eightfold increase in activities of IL1A, IL1B and IFN{gamma} promoters. In contrast, no activation of IL1B promoter was observed, when galectin-8 expression vector pBKCMV3–G8 was used instead of pBKCMV3-G9 (Fig. 3D).


Figure 2
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  		Figure 2  Effects of galectin-9(S), (M) and (L) on IL1A promoter activity in THP-1 cells. An amount of 1.5 µg of pBKCMV3-G9(S), pBKCMV3-G9(M) or pBKCMV3-G9(L) were transiently transfected into THP-1 cells, together with 1.0 µg of IL1A promoter reporter. The total amount of transfected DNA was kept constant (3.0 µg) by adding control vector. Data are mean ± SD of triplicate samples. Luciferase assays were carried out as described in Experimental Procedures. Data were normalized by internal control Renilla luciferase activity.

 

Figure 3
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  		Figure 3  Galectin-9 activates cytokine gene promoters in THP-1 cells. pBKCMV3-G9(S) (0.5–1.5 µg) was transiently transfected together with 1.0 µg of cytokine promoter reporters [(A) IL1A, (B) IL1B, (C) IFN{gamma}] into THP-1 cells. The total amount of transfected DNA was kept constant (3.0 µg) by adding control vector. Data are mean ± SD of triplicate samples. Luciferase assays were carried out as described in Experimental Procedures. Data were normalized by internal control Renilla luciferase activity.

 
In contrast to the data obtained from transient transfection studies using pBKCMV3-G9(S), incubation of THP-1 cells carrying IL1A promoter reporter pGL3IL1{alpha} with exogenous galectin-9 protein, hG9NC (null) 100 nM for 24 h resulted in inhibition of IL1A promoter activity. Furthermore, the galectin-9-induced inhibition of IL1A promoter activity was prevented by lactose (30 mM), but not sucrose (30 mM) (Fig. 4). The most biological effects of extracellular galectins are mediated by their carbohydrate-binding activity. These data indicate that unlike intracellular galectin-9, extracellular galectin-9 is not involved in activation of inflammatory cytokine gene promoters.


Figure 4
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  		Figure 4  Exogenous galectin-9 protein inhibits IL1A promoter activity in THP-1 cells. pGL3IL1{alpha} (1.0 µg) was transfected into THP-1 cells. The cells were incubated with or without 30 mM lactose or 30 mM sucrose for 24 h, followed by stimulation with 100 nM galectin-9 protein (hG9NC (null)) for 24 h. Cell were collected and assayed for IL1A promoter activity. The total amount of transfected DNA was kept constant (3 µg) by the addition of control vector. Data are mean ± SD of triplicate samples. Luciferase assays were carried out as described in Experimental Procedures. Data were normalized by internal control Renilla luciferase activity.

 
Effects of galectin-9 on transcription factors NF-IL6, NF-{kappa}B and AP-1 in THP-1 cells

Induction of genes of inflammatory cytokines such as IL1A and IL1B and IFN{gamma} involves binding of several transcription factors such as NF-{kappa}B, NF-IL6 and AP-1 to their target sites within the promoter lesions. NF-IL6 transactivates IL1B gene promoter through its binding to two different sites, –91 to –83 and –41 to –33 (Natsuka et al. 1992; Kominato et al. 1995). Based on our finding that intracellular galectin-9 induces inflammatory cytokines in monocytes, we co-transfected pG2mfNF{kappa}B, pG3mfNF6 x 3 and pAP1(1)-Luc, along with pBKCMV3-G9(S), into THP-1 cells. As shown in Fig. 5A,B, galectin-9 enhanced the activities of both AP-1 and NF-IL6, suggesting functional cooperation between galectin-9 and the bZIP family members. In contrast, galectin-9 failed to induce NF-{kappa}B activity, although LPS significantly induced the latter in THP-1 cells (Fig. 5C).


Figure 5
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  		Figure 5  Effects of galectin-9 on various transcriptional factors in THP-1 cells. pBKCMV3-G9(S) (0.5–1.5 µg) was transiently transfected into THP-1 cells together with 1.0 µg of pG3mfNF6 x 3 (A), pAP1(1)-Luc (B), pG2mfNF{kappa}B (C), pGL3TNF{alpha} (D) and PGL3HTmNF-IL6 (E). The total amount of transfected DNA was kept constant (3.0 µg) by the addition of control vector. Data are mean ± SD of triplicate samples. Luciferase assays were carried out as described in Experimental Procedures. Data were normalized by internal control Renilla luciferase activity. As a positive control, THP-1 cells were treated with LPS (C and D).

 
To confirm the lack of activation of NF-{kappa}B by galectin-9, we transfected pGL3TNF{alpha}, a luciferase reporter harboring TNF-{alpha} promoter region located from –199 to +82, into THP-1 cells, together with pBKCMV3-G9(S). TNF-{alpha} promoter element –199 to +82 possesses a binding site for NF-{kappa}B, but not NF-IL6 or AP-1 (Natsuka et al. 1992; Geist et al. 1997). pGL3TNF{alpha} containing the NF-{kappa}B site was activated by LPS, but not by galectin-9 expression (Fig. 5D). Furthermore, to determine whether –91 to –83 NF-IL6 site within the IL1B promoter plays a pivotal role in the transactivation of the IL1B promoter by galectin-9, a mutated luciferase reporter, pGL3HTmNF-IL6 was used. HTmNF-IL6 was identical to the wild-type HT, but contained nucleotide substitutions within the –91 to –83 NF-IL6 binding site. As a result, mutation of the –91 to –83 NF-IL6 binding site completely prevented activation by galectin-9 (Fig. 5E).

Physical association of intracellular galectin-9 with NF-IL6

The finding that galectin-9 induced NF-IL6 activity in THP-1 cells led us to examine the physical association of galectin-9 with NF-IL6. In these experiments, pcNFIL6 and/or pBKCMV3-G9(S) were transfected into HEK293T cells and 24 h after transfection, the cell lysates were immunoprecipitated with anti-galectin-9 Ab followed by WB using anti-NF-IL6 Ab. No expression of galectin-9 was detected in HEK293T transfected with a mock vector (Fig. 6A). Anti-galectin-9 Ab immunoprecipitation contained NF-IL6, only when both of pcNFIL6 and pBKCMV-G9(S) were transfected into HEK293T cells, suggesting the association of NF-IL6 with galectin-9 in the absence of DNA (Fig. 6A). In addition, pBKCMV3-G9(S) transfection into HEK293T cells also induced NF-IL6 activity (data not shown). Moreover, our IP/WB data using anti-gelectin-9 Ab showed a single galectin-9 protein band only when pBKCMV3-G9(S) was transfected into HEK293T cells, indicating the specificity of anti-galectin-9 Ab used in the present study to galectin-9 protein (Fig. 6B).


Figure 6
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  		Figure 6  Galectin-9 physically interacts with NF-IL6 in HEK293T cells. (A) Cells lysates prepared from HEK293T cells transfected with pcNFIL6 and/or pBKCMV3-G9(S) were immunoprecipitated with anti-galectin-9 Ab and then blotted with anti-NF-IL6 Ab. Lane 1; Mock transfection, lane 2; pBKCMV3-G9(S) transfection, lane 3; pcNFIL6 transfection, lane 4; co-transfection of both pBKCMV3-G9(S) and pcNFIL6. IP-WB assay were carried out as described in Experimental Procedures section. Representative results of three experiments with similar findings. (B) Cells lysates prepared from HEK293T cells transfected with pBKCMV3-G9(S) or a mock vector. IP-WB assay were carried out as described in Experimental Procedures. Representative results of three experiments with similar findings.

 
Galectin-9 is an LPS-responsive factor
LPS functions as a mediator of various inflammatory responses to activate cytokine genes such as IL-1{alpha}, IL-1β and TNF-{alpha} in monocytes/macrophages and is involved in the pathogenesis of various inflammatory diseases. In the present study, confocal microscopy identified galectin-9 in the cytoplasm of untreated THP-1 monocytic cells (Fig. 7A). Galectin-9 protein expression was further confirmed in WB using anti-galectin-9 Ab (data not shown). In addition, nuclear translocation of galectin-9 was observed 1 h after LPS stimulation of THP-1 cells (Fig. 7A,B), and reached a maximum at 6 h after LPS treatment (Fig. 7A,B). Next, we investigated physical interaction of NF-IL6 with galectin-9 in THP-1 cells. As no co-immunoprecipitation of NF-IL6 with galectin-9 was detected in THP-1 cells which did not over-express galectin-9 (Fig. 7C), pBKCMV3-G9(S) was transiently transfected into THP-1 cells. In THP-1 cells carrying pBKCMV3-G9(S), co-immunoprecipitaion of NF-IL6 with galectin-9 was observed. Treatment of THP-1 cells with LPS further enhanced co-immunoprecipitaion of NF-IL6 with galectin-9 (Fig. 7C). In addition, when THP-1 cells carrying pBKCMV3-G9(S) were treated with LPS, galectin-9 synergized with LPS to activate NF-IL6 in THP-1 cells (Fig. 7D). These data clearly demonstrated cooperativity of galectin-9 with NF-IL6.


Figure 7
Figure 7
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  		Figure 7  Galectin-9 is an LPS-responsive factor, which is translocated into the nucleus and is physically associated with NF-IL6 following treatment of THP-1 cells with LPS. (A) THP-1 monocytic cells were stimulated with LPS (1 µg/mL) or left untreated. Confocal microscopy analysis for galectin-9 was carried out as described in Experimental Procedures. Magnification, 63x and 1.4 oil DIC for all panels (scale bar = 10 µm). (B) Nuclear translocation of galectin-9 was observed after stimulation of LPS by fluorescence microscopy with DAPI stain (blue). (C) THP-1 cells were transfected with pBKCMV3-G9(S) (1.5 µg). 24 h after transfection, cells were treated with LPS for 6 h, or left untreated. IP-WB assay were carried out as described in Experimental Procedures. Representative results of three experiments with similar findings. (D) pBKCMV3-G9(S) (1.0 µg) was transiently transfected together with 1.0 µg of pG3mfNF6 x 3 into THP-1 cells. Cells were stimulated with LPS (0.5 µg/mL) or left untreated. The total amounts of transfected DNA was kept constant (3.0 µg) by adding control vector. Data are mean ± SD of triplicate samples. Luciferase assays were carried out as described in Experimental Procedures section. Data were normalized by internal control Renilla luciferase activity.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Galectin-9 was first described in Hodgkin's lymphoma as a tumor antigen. Galectin-9 exhibits various biological functions such as cell aggregation, chemoattraction and apoptosis. A wide variety of cells including monocytes/macrophages express galectin-9. Galectin-9 expression has been reported to be down-regulated during monocytic differentiation of HL60 cells (Kashio et al. 2003). In contrast, a recent study demonstrated that LPS enhances expression of galectin-9 protein and mRNA in the periodontal ligament cells (Kasamatsu et al. 2005). In our study, the results of confocal microscopy indicated evident expression of galectin-9 protein in untreated THP-1 monocytes. Moreover, it is interesting to note that stimulation of THP-1 monocytic cells with LPS induced nuclear translocation of galectin-9 within 1 h, that is, galectin-9 is an LPS-responsive protein in monocytes (Fig. 7). We also demonstrated that intracellular galectin-9 in THP-1 monocytes induced the expression of both IL-1{alpha} and IL-1β mRNAs (Fig. 1A,B) and transactivation of inflammatory cytokine gene promoters, such as IL1A, IL1B and IFN{gamma} (Fig. 3A–C). In contrast, extracellular gelectin-9 failed to activate the IL1A promoter in the same cells. Interestingly, our transient transfection data further showed that intracellular galectin-9 induced the activation of transcription factors such as NF-IL6 and AP-1 (Fig. 5A,B). No activation of NF-{kappa}B by intracellular galectin-9 was observed in THP-1 cells (Fig. 5C). These results strongly suggest that induction of inflammatory cytokine gene promoters is mediated through activation of leucin zipper transcription factors such as NF-IL6 and AP-1 by intracellular galectin-9. In fact, NF-IL6 and AP-1 have been demonstrated to be involved in transactivation of the inflammatory cytokine genes (Akira & Kishimoto 1992). Furthermore, our IP/WB using HEK293T cells and THP-1 cells showed co-immunoprecipitation of NF-IL6 with galectin-9, indicating direct physical association of NF-IL6 with galectin-9. Thus, the interaction between intracellular galectin-9 and leucin zipper transcription factors such as NF-IL6 and AP-1 appears to result in activation of inflammatory cytokine genes. Moreover, since six different members of C/EBPs have been identified, our data may suggest the possibility that galectin-9 cooperates with the other C/EBP family proteins. Considered together, the above results emphasize the role of nuclear galectin-9 in inflammatory gene-regulation.

NF-IL6 (C/EBP β), a member of the basic leucine zipper (bZIP) family of transcription factors was initially identified as a nuclear factor that binds to the IL-1 responsive elements (Akira et al. 1990). NF-IL6 also plays a role in regulation of the genes encoding several acute-phase protein genes, albumin, c-fos, and adipocyte specific proteins (Poli et al. 1990; Isshiki et al. 1991). Furthermore, various genes involved in inflammatory and immune responses, including IL-8, granulocyte/colony-stimulating factor, IL-1 and immunoglobulin genes, have been demonstrated to be activated by NF-IL6 (Akira & Kishimoto 1992). NF-IL6 is present ubiquitously in cells in an inactive protein form that support transcription following posttranslational modification and/or physical association with other transcription factors. The role of NF-IL6 as an LPS-responsive transcription factor in a pre-existing inactive form in unstimulated monocytes/macrophages has been established (Shirakawa et al. 1993).

In addition, NF-IL6 is heterodimerized with members of the C/EBP family and the AP-1 family through their bZIP regions and is also associated with several transcription factors outside the bZIP family members such as NF-{kappa}B (Stein et al. 1993), Tax (Tsukada et al. 1997), PU.1 (Yang et al. 2000), heat shock factor 1 (Xie et al. 2002), calcium channel blocker (CCB) (Eickelberg et al. 1999), chicken ovalbumin upstream promoter transcriptional factor (COUP-TF) (Schwartz et al. 2000) and glucocorticoid receptor (GR) (Nishio et al. 1993). Our previous study reported that NF-IL6 vigorously activates IL-1β core promoter via protein-tethered transactivation mediated by PU.1 (Yang et al. 2000). In the present study, we further demonstrated that intracellular galectin-9 transactivated inflammatory cytokine genes through physical association with NF-IL6 in monocytes. In contrast, extracellular galectin-9 has been reported to induce apoptosis not only of T cells but also of B cells (BALL-1), monocytes (THP-1), and myelocytes (HL-60), when these cells were treated with 1 µM galectin-9 for 24 h (Kashio et al. 2003), indicating that extracellular galectin-9 can function as a proapoptotic factor for various immune cells. Our transient transfection studies also showed partial inhibition of IL1A gene promoter activity in THP-1 cells incubated with exogenous galectin-9 (Fig. 4). These results demonstrate distinct functions for extracellular and intracellular gelectin-9.

There are only a few reports regarding intracellular functions of galectins. Nuclear export of phosphorylated galectin-3 prevents apoptosis through the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways (Takenaka et al. 2004). Galectin-3 is involved in RNA processing and cell cycle-regulation through activation of transcription factors when translocated to the nucleus. The nuclear localization of galectin-3, a pre-mRNA splicing factor is associated with proliferation of normal cells. Galectin-3 located in nuclei of papillary cancer cells up-regulates transcriptional activity of a thyroid-specific transcription factor TTF-1 via interaction between with TTF-1 homeodomain (Paron et al. 2003). Cyclin D1 promoter is also enhanced through enhancement/stabilization of CRE-associated complex formation by gelectin-3 (Lin et al. 2002). In contrast, galectin-4 is considered to be involved in p27-mediated activation of the myelin basic protein promoter via its physical interaction with p27 (Wei et al. 2007).

Monocytes are instrumental in mediating immediate immune and inflammatory responses. Various important inflammatory and immuno-regulatory cytokines are expressed by activated monocytes/macrophages. These cells can respond almost instantly to a variety of stimuli including LPS, IL-1 and other cytokines, and undergo a rapid transformation from resting monocytes to activated ones. The inflammatory cytokine genes in resting monocytes/macrophages are also silent, but rapidly transcribed in competent cells on stimulation. Production of inflammatory cytokines is tightly regulated in monocytes/macrophages. (Auron & Webb 1994). In this regard, it is noteworthy that galectin-9 is a LPS-responsive factor, which can form a complex with NF-IL6 to transactivate inflammatory cytokine gene promoters in monocytes/macrophages. In conclusion, the present study demonstrated a novel function for gelectin-9; transactivation of inflammatory cytokine genes in monocytes through physical interaction with leucin zipper transcription factors such as NF-IL6.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell cultures

Human THP-1 monocytic cell line (JCRB0112) was purchased from Health Science Research Resource Bank (Osaka, Japan), and human endothelial kidney 293T cells (HEK293T cells) were used in the present study. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 0.5% of penicillin and streptomycin in a humidified incubator under 5% CO2 at 37 °C. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. Cells were split at one-third of dilution every 3 or 4 days to avoid over-crowding and were further split at 1 : 2 on the day before transfection.

Plasmids

Human IL-1β gene (IL1B) promoter region (HT sequence) located between positions –131 to +12 (pGL3HT), IFN-{gamma} promoter sequence from –572 to +75 (pGL3IFNG), human IL-1{alpha} gene (IL1A) promoter fragment from –1437 to +725 (pGL3IL1{alpha}), TNF{alpha} promoter region located from –199 to +82 (pGL3TNF{alpha}) were inserted into pGL3-basic luciferase reporter (Promega, Madison, WI), respectively (Mizobe et al. 2007). Mutations of the –91 to –83 upstream NF-IL6 binding site within the IL1B promoter were the same as those reported previously (Kominato et al. 1995). HTmNF-IL6 (the core sequence 5'-gTGTtAAgT-3') was identical to the wild-type HT, but contained nucleotide substitutions within the –91 to –83 NF-IL6 binding site. pG2mfNF{kappa}B; NF-{kappa}B luciferase reporter and pG3mfNF6 x 3; NF-IL6 luciferase reporter were described previously (Mizobe et al. 2007). pAP1(1)-Luc designed for monitoring induction of activator protein (AP)-1 and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) signal transduction pathway was purchased from Panomics (Redwood City, CA).

The expression vectors for galectin-9(S), galectin-9(M) and galectin-9(L) were pBKCMV3-G9(S), pBKCMV3-G9(M) and pBKCMV3-G9(L), respectively. Galectin-9 is a tandem-repeat type and more susceptible to proteolysis than other galectins due to the presence of a relatively long linker peptide. We found protease-resistant galectin-9 by modification of its linker peptide, hG9NC (null). The NF-IL6 (C/EBP β) expression vector pcNFIL6 was described previously (Tsukada et al. 1997). pBKCMV3-G8(M) was a galectin-8 expression vector.

Confocal and fluorescence microscopy

THP-1 cells (1 x 105) were collected and treated with 4% formaldehyde (Sigma Chemical Co., St. Louis, MO) in FACS medium for 15 min. and then with 0.1% saponin (Sigma) in FACS medium. The cells were incubated with a specific antibody (Ab) against galectin-9 (GalPharma, Kagawa, Japan) for 30 min at 4 °C. Subsequently, the cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG Ab at saturating concentrations in FACS medium. We performed confocal analysis of galectin-9 using an inverted laser scan microscope (model LSM5-pascal, Carl Zeiss Microscope System, Germany). For detection of DNA/nuclei using 4',6-diamidino-2-phenylindole (DAPI), section were incubated for 2 min in a 300-nM solution of DAPI dilactate in PBS. Cells were overlaid and observed by fluorescence microscopy (Axiovert 135 inverted microscope; Zeiss, oberkochen, Germany) for enhanced green fluorescent protein (eGFP)-galectin-9 (green) and DAPI nuclear staining (blue). Green and blue channel images were merged using Axio-Vision software (Zeiss).

Transfections and luciferase assays

THP-1 cells were transfected by the DEAE-dextran methods as described previously (Shirakawa et al. 1993; Tsukada et al. 1997). This technique was used because, unlike electroporation, it did not induce endogenous IL1A and IL1B. Cells (2 x 106 cells per plate) were transfected with 3 µg of plasmids. Transfection of plasmids into HEK-293T cells was carried out using a transfection kit; Transfast (Promega) according to the protocol recommended by the manufacturer. At 24 h after transfection, cells were lysed with Passive Lysis Buffer (Promega). The cell lysates were used for a dual-luciferase reporter assay system (Promega). Samples were normalized to Renilla luciferase activity as an internal control for transfection efficiency.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNAs of THP-1 cells were extracted by Isogen RNA extraction kit (Nippon Gene, Tokyo). Total RNA (0.5 µg) was used along with a reverse transcriptase RNA PCR kit; Access RT-PCR System (Promega) according to the instructions provided by the supplier. An aliquot of the PCR mixture was subjected to electrophoresis in 3% agarose gel. The primers used were human IL-1{alpha} sense, 5'-GTCTCTGAATCAGAAATCCTTCTATC-3'; human IL-1{alpha} anti-sense, 5'-CATGTCAAATTTCACTGCTT CATCC-3'; human IL-1β sense, 5'-CAGAGAGTCCTGTG CTGAAT-3'; human IL-1β anti-sense, 5'-GTAGGAGAGGTC AGAGAGGC-3' (Herbein et al. 1994), β-actin sense, 5'-TCATGA AGTGTGACGTTGACATCCGT-3'; and β-actin anti-sense, 5'-CCTAGAAGCATTTGCGGTGCAAGATG-3'.

Protein extraction, immunoprecipitation (IP) and Western blotting (WB)

Cells pellet was lysed with lysis buffer (in mM, 150 NaCl, 50 Tris–HCl, 2 sodium orthovanadate, and 5 sodium pyrophosphate) with protease inhibitor mixture (complete mini) and 1% Triton X-100. The same amount of 2 x SDS (sodium dodecylsulfate) buffer (125 mM Tris–HCl, 4% SDS, 20% glycerol, and 0.02% bromophenol blue) with 2-mercaptoethanol was added to each sample, then boiled for 3 min. Cells lysates were incubated with anti-galectin-9 Ab and 50 µL of Protein G Micro Beads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 0.5 h on ice. Anti-NF-IL6 Ab (Santa Cruz Biotechnology Inc.) was used for WB. IP and WB were carried out as described previously (Mizobe et al. 2007).

Statistical analysis

All data were expressed as mean ± SD. Differences between groups were examined for statistical significance using the Student's t-test. A P value < 0.05 denoted the presence of a statistically significant difference.


   Acknowledgements
 
This work was supported in part by a Research Grant-In-Aid for Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan and University of Occupational and Environmental Health, Japan.


   Footnotes
 
Communicated by: Shigeo Koyasu

* Correspondence: jtsukada{at}med.uoeh-u.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 20 January 2009





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