European Journal of Pharmacology 742 (2014) 42–46
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
CDr10b inhibits the expression of cyclooxygenase-2 and inducible nitric oxide synthase induced by lipopolysaccharide Gyo-Jeong Gu a,1, Se-Jin Lim b,1, Sang-il Ahn a, Sung-Chan Lee c, Young-Tae Chang d,e, Tae Hyun Choi f, Byoung Soo Kim f, Yong-Bin Eom b, Na Kyung Lee b, Hyung-Sun Youn a,b,n a
Departments of Medical Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea c Aptabio Therapeutics Inc., Kyunggi-do, Yong-in city 446-908, Republic of Korea d Department of Chemistry and MedChem Program, Life Sciences Institute, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore e Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium (SBIC), 11 Biopolis Way, #02-02 Helios, Agency for Science, Technology and Research (AnSTAR), Biopolis, Singapore 138667, Singapore f Department of Imaging, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2014 Received in revised form 19 August 2014 Accepted 24 August 2014 Available online 6 September 2014
The pathophysiological processes of inflammation can lead to a host of diseases, such as periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer. The dysregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activation play important roles in the development of certain inflammatory diseases. Here, we investigated the effects of CDr10b which is originally developed for a microglia staining probe on inflammation, by modulating NF-κB activation and iNOS and COX-2 expression induced by lipopolysaccharide (LPS) in murine macrophages. The CDr10b suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. All the results suggest that CDr10b is a promising novel agent for the treatment of inflammatory diseases. & 2014 Elsevier B.V. All rights reserved.
Keywords: Inflammation Nuclear factor-κB Cyclooxygenase-2 Inducible nitric oxide synthase CDr10b
1. Introduction Toll-like receptors (TLRs) play an important role in recognition of conserved pathogen-associated molecular patterns (PAMPs) derived from various microbial pathogens including viruses, bacteria, protozoa and fungi, and in the subsequent initiation of innate immune responses (Akira and Takeda, 2004; Medzhitov et al., 1997). The activation of TLRs by agonists can induce inflammatory responses that are key etiological conditions for the development of many chronic inflammatory diseases. Inflammation, which is mediated by multiple molecular mechanisms, refers to the pathological and physiological processes known to be involved in numerous diseases. Two of the most important enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2), have important roles in the development of certain inflammatory diseases (Moncada, 1999; Turini and DuBois, 2002).
n Corresponding author at : Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University, Chungnam, Asan-Si 336-745, Republic of Korea. Tel.: þ 82 41 530 3086; fax: þ 82 41 530 3085. E-mail address: [email protected] (H.-S. Youn). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2014.08.036 0014-2999/& 2014 Elsevier B.V. All rights reserved.
NOS, which catalyzes the conversion of L-arginine to nitric oxide, is classified into several isoforms, based on the cell type or the location and manner of expression, such as endothelial NOS (eNOS), neuronal NOS (nNOS), and a macrophage or inducible NOS (iNOS) (Murakami and Ohigashi, 2007). The eNOS and nNOS are constitutively active enzymes in vascular endothelial cells and neuronal tissues. On the other hand, iNOS enzymes are induced under both normal and pathological conditions in a variety of cell types, including macrophages, keratinocytes, hepatocytes, astrocytes, and microglial cells. Since dysregulated iNOS expression might be intimately involved in the development of certain inflammatory diseases, iNOS is a very important therapeutic target in the development of anti-inflammatory drugs (Vallance, 2003). Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids, is the molecular target for antiinflammatory remedies (Rouzer and Marnett, 2009). The COX consists of at least two isoforms: COX-1 and COX-2. The COX-1 enzyme is constitutively expressed in many normal tissues. In contrast, although COX-2 is undetectable in most of the normal mammalian tissues, it is an inducible enzyme expressed in them in response to pathophysiological stimuli including ultraviolet light, dioxin, and lipopolysaccharide (LPS) (Murakami and Ohigashi, 2007). It is reported that the expression of COX-2 is regulated at
G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
43
Fig. 1. (A) The structure of CDr10b. (B) Excitation/emission spectra of CDr10b.
transcriptional and post-transcriptional levels by pro-inflammatory agents, cytokines, growth factors, and tumor-promoters (Alvarez et al., 2005; Antman et al., 2005). Previously, we reported the development of a Compound of Designation red 10 binding (CDr10b) (Fig. 1) for the isolation and imaging of live microglia, when investigating their roles in neuroinflammatory diseases (Leong et al., 2014). Herein, we report immunomodulatory activity of CDr10b in the context of NF-κB activation induced by LPS (TLR4 agonist) in murine macrophages. The CDr10b suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. These results suggest that CDr10b can be developed as a potent anti-inflammatory drug.
2. Materials and methods
2.4. Plasmids The NF-κB (2x)-luciferase reporter construct was provided by Frank Mercurio (Signal Pharmaceuticals, San Diego, CA, USA). The heat shock protein 70 (HSP70)-β-galactosidase reporter plasmid was purchased from Robert Modlin (University of California, Los Angeles, CA, USA). The COX-2 and iNOS luciferase reporter constructs were obtained from Dr. Daniel Hwang (University of California, Davis, CA, USA). A wild type of MyD88 was provided by J. Tschopp (University of Lausanne, Lausanne, Switzerland). The wild-type IKKβ was obtained from M. Karin (University of California, San Diego, CA). The wild type of p65 was obtained from J. Ye (Pennington Biomedical Research Center, Baton Rouge, LA). All DNA constructs were prepared in large scale for transfection using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA, USA).
2.1. Synthetic procedure of CDr10b CDr10b was synthesized by following the established procedure (Leong et al., 2014). The 8-(4-Aminophenyl)-4,4-difluoro-1, 3-dimethyl-4-bora-3a,4a-diaza-s-indacene (aminophenyl bodipy) (100 mg, 320 mmole) and 3-ethoxy-4-methoxybenzaldehyde (86 mg, 480 mmole) were dissolved with acetonitrile (50 ml). Then, 3 eq. of pyrrolidine and acetic acid was added to the reaction mixture. The reaction mixture was heated to 60 1C for 3 h. After completion of the reaction, NaHCO3 saturated solution (500 ml) and chloroacetyl chloride 200 ml were added to the reaction mixture and stirred vigorously for 5 min. The crude compound was purified using column chromatography with MeOH/DCM eluent system (0–5% of MeOH), and 148 mg of product was obtained. The spectroscopic properties and analytical data were identical as the reference. 2.2. Reagents LPS was obtained from List Biological Laboratories (San Jose, CA, USA), and it was dissolved in endotoxin-free water. All other reagents were purchased from Sigma-Aldrich, unless otherwise indicated. 2.3. Cell culture RAW 264.7 cells (a murine monocytic cell line; ATCC TIB71) and 293T human embryonic kidney cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 units/ml Penicillin (Invitrogen, Carlsbad, CA, USA), and 100 μg/ml Streptomycin (Invitrogen). Cells were maintained at 37 1C in a 5% CO2/95% air environment.
2.5. Transfection and reporter gene luciferase assay The assays were performed as previously described (Youn et al., 2005, 2006b). RAW264.7 cells were co-transfected with a luciferase plasmid and a plasmid containing heat shock protein (HSP)70-βgalactosidase as an internal control, using SuperFect transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions and then pretreated with 20 or 50 μM CDr10b for 1 h and then treated with LPS (10 ng/ml) for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined using the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The luciferase activity was normalized by β-galactosidase activity.
2.6. Immunoblotting Immunoblotting was performed as previously described (Youn et al., 2006a, 2006c). Equal amounts of cell extracts were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked to prevent nonspecific binding of antibodies in phosphate-buffered saline containing 0.1% Tween-20 and 3% nonfat dry milk. The immunoblotting was performed with the indicated antibodies, and the secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences, Arlington Heights, IL, USA). The reactive bands were visualized with the enhanced chemiluminescence Western blot detection reagents (Intron, Seongnam, Gyeonggi-do, South Korea).
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2.7. Cell viability test
2.8. Data analysis
The cell viability was assessed using MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] based colorimetric assay. RAW264.7 cells were treated with CDr10b (20, 50, 100 μM) for 4 h. Twenty microliters of the CellTiter 96 AQueous One Solution Reagent was added directly to culture wells. The plate was incubated at 37 1C for 4 h in a humidified, 5% CO2 atmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader.
The data were obtained from triplicate experiments. The values are expressed as the mean 7standard error of the mean (S.E.M.).
3. Results 3.1. CDr10b inhibits LPS-induced NF-κB activation To evaluate the cytotoxic nature of CDr10b in RAW 264.7 cells, the MTS viability assay was used. It was apparent that CDr10b was not cytotoxic at the tested concentrations of 20–100 μM. Therefore, the cellular toxicity could be excluded from the possible reason for any observed changes in the subsequent study. Next, we determined the effect of CDr10b on LPS-induced NF-κB activation, by using the NF-κB luciferase reporter assay. The CDr10b inhibited NF-κB activation which was induced by LPS (Fig. 2). The NF-κB is activated in response to stimuli of various pathogens (Hacker and Karin, 2006). Therefore, the suppression of NF-κB activation is considered as an important strategy for antiinflammation and anti-cancer therapies. 3.2. CDr10b suppresses LPS-induced iNOS expression
Fig. 2. CDr10b inhibits LPS-induced NF-κB activation. NF-κB luciferase assay in RAW264.7 cells. Values are expressed as mean7 S.E.M. (n¼ 3). nn denotes significantly different from LPS alone, Po 0.01. Veh, vehicle.
The next experiment assessed the capability of CDr10b to regulate iNOS expression, which is one of the target genes regulated through NF-κB activation in macrophages. The CDr10b suppressed the iNOS expression that was induced by LPS in RAW264.7 cells, as determined by iNOS-luciferase reporter assay (Fig. 3A). The CDr10b also suppressed the iNOS protein that was induced by LPS, as determined by iNOS immunoblotting assay
Fig. 3. CDr10b inhibits LPS-induced iNOS expression. (A) iNOS luciferase assay in RAW264.7 cells. Values represent the mean 7S.E.M. (n¼3). nn Significantly different from LPS alone, P o 0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates were analyzed for iNOS and β-actin protein by immunoblots. Veh, vehicle.
Fig. 4. CDr10b inhibits LPS-induced COX-2 expression. (A) COX-2 luciferase assay in RAW264.7 cells. Values represent the mean7 S.E.M. (n ¼3). nn Significantly different from LPS alone, Po 0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates were analyzed for COX-2 and β-actin protein by immunoblots. Veh, vehicle.
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Fig. 5. CDr10b suppresses MyD88-dependent signaling pathway of TLR4. (A–C) 293T cells were transfected with NF-κB luciferase reporter plasmid and the expression plasmid of MyD88 (A), IKKβ (B) or p65 (C). Cells were further treated with 20 or 50 μM CDr10b for 18 h. Relative luciferase activity was normalized with β-gal activity. Values represent mean7 S.E.M. (n¼ 3). þdenotes a result that is significantly different from MyD88 alone, Po 0.01 ( þ þ) (A). # denotes a result that is significantly different from IKKβ alone, P o 0.01 (##) (B). n denotes a result that is significantly different from p65 alone, Po 0.01 (nn) (C). Veh, vehicle.
(Fig. 3B). All of the results suggested that CDr10b inhibited iNOS expression induced by LPS. 3.3. CDr10b suppresses LPS-induced COX-2 expression Next, we determined whether CDr10b could regulate COX-2 expression, which is another target gene regulated through NF-κB activation. The CDr10b suppressed the COX-2 expression that was induced by LPS in RAW264.7 cells, as determined by COX-2luciferase reporter assay (Fig. 4A). The CDr10b also suppressed the COX-2 protein that was induced by LPS, as determined by COX-2 immunoblotting assay (Fig. 4B). 3.4. CDr10b suppresses MyD88-dependent signaling pathway of TLRs To further investigate the regulation of MyD88-dependent signaling pathways by CDr10b, NF-κB activation was induced by
the overexpression of MyD88, IKKβ, or p65 in 293T cells. CDr10b suppressed the agonist-independent activation of NF-κB induced by MyD88 (Fig. 5A), IKKβ (Fig. 5B) or p65 (Fig. 5C), demonstrating that CDr10b suppresses MyD88-dependent signaling pathways.
4. Discussion Toll-like receptor 4 (TLR4) is the first TLR identified that recognizes LPS, which is a cell wall component of gram-negative bacteria (Miyake, 2004). The LPS-induced TLR4 activation leads to both early and late NF-κB activation through myeloid differential factor 88 (MyD88)- or Toll/iInterleukin-1 receptor (TIR) domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways, respectively (Takeda and Akira, 2005). MyD88 is the immediate adapter molecule which is common to all TLRs, with the exception of TLR3. The MyD88 recruits the IL-1 receptor-associated kinase (IRAK), tumor necrosis factor (TNF), and
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receptor-associated factor 6 (TRAF6), leading to the activation of the canonical IKK complex. In the un-stimulated cells, NF-κB is sequestered in the cytoplasm as an inactive form by interaction with an inhibitor IκBα proteins, preventing its nuclear accumulation and transcriptional activation of the target genes. The IKK complex phosphorylates IκBα which is degraded by the 26S proteasome, allowing NF-κB to translocate into the nucleus. The translocated NF-κB binds to DNA and up-regulates the expressions of numerous pro-inflammatory gene products including COX-2, iNOS, and a wide variety of proinflammatory cytokines and chemokines (Surh et al., 2001). NF-κB, an important component of inflammatory responses, is a transcription factor that regulates over 150 genes, many of which are associated with inflammation and cancer (Baldwin, 1996). The LPS-induced activation of TRIF/TICAM-1, an adapter molecule which functions independently of MyD88, also leads to the activation of the transcriptional regulator, IRF3, and the expression of IFNβ and IFN-inducible genes (Fitzgerald et al., 2003; Toshchakov et al., 2002). TRIF recruits TRAF family member-associated NF-κB activator-binding kinase 1 (TBK1) and inhibitor-κB kinase-ε (IKKε), leading to IRF3 activation (Fitzgerald et al., 2003). The activated IRF3 translocates into the nucleus and binds to its target DNA sequences of IFN-stimulated response elements that are found in the promoter regions of the genes, such as those encoding IFNβ and interferon inducible protein-10 (IP-10), and are regulated by the activation of normal T-cells expressed and secreted (Bjorkbacka et al., 2004; Gao et al., 1998; Kawai et al., 2001). The TRIF also induces the activation of receptor interacting protein-1, leading to a delayed NF-κB activation (Meylan et al., 2004). In this study, we investigated whether CDr10b could regulate the expression levels of COX-2 and iNOS, which are two of the most important enzymes for certain inflammatory diseases. In murine cells, COX-2 and iNOS expressions are regulated at the transcriptional level by NF-κB, with the promoter of both genes containing an NF-κB binding sequence. All of the results support the suggestion that CDr10b suppresses LPS-induced NF-κB activation, resulting in the induction of the expression of target genes such as COX-2 and iNOS. These results suggest that CDr10b can regulate TLR4 signaling pathways. The recent studies suggest that TLRs and their signaling components could become excellent therapeutic targets for chronic inflammatory diseases (Kawai and Akira, 2010). Therefore, our results provide new insights into the understanding of the mode of the anti-inflammatory action of CDr10b. Acknowledgments This study was supported by Soonchunhyang University research fund and by Basic Science Research Program, through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and, Technology (2010-0023637). References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511.
Alvarez, Y., Briones, A.M., Balfagon, G., Alonso, M.J., Salaices, M., 2005. Hypertension increases the participation of vasoconstrictor prostanoids from cyclooxygenase-2 in phenylephrine responses. J. Hypertens. 23, 767–777. Antman, E.M., DeMets, D., Loscalzo, J., 2005. Cyclooxygenase inhibition and cardiovascular risk. Circulation 112, 759–770. Baldwin Jr., A.S., 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–683. Bjorkbacka, H., Fitzgerald, K.A., Huet, F., Li, X., Gregory, J.A., Lee, M.A., Ordija, C.M., Dowley, N.E., Golenbock, D.T., Freeman, M.W., 2004. The induction of macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades. Physiol. Genomics 19, 319–330. Fitzgerald, K.A., McWhirter, S.M., Faia, K.L., Rowe, D.C., Latz, E., Golenbock, D.T., Coyle, A.J., Liao, S.M., Maniatis, T., 2003. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496. Gao, J.J., Filla, M.B., Fultz, M.J., Vogel, S.N., Russell, S.W., Murphy, W.J., 1998. Autocrine/paracrine IFN-alphabeta mediates the lipopolysaccharide-induced activation of transcription factor Stat1alpha in mouse macrophages: pivotal role of Stat1alpha in induction of the inducible nitric oxide synthase gene. J. Immunol. 161, 4803–4810. Hacker, H., Karin, M., 2006. Regulation and function of IKK and IKK-related kinases. Sci. STKE 2006, re13. Kawai, T., Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384. Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, P.F., Sato, S., Hoshino, K., Akira, S., 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167, 5887–5894. Leong, C., Lee, S.C., Ock, J., Li, X., See, P., Park, S.J., Ginhoux, F., Yun, S.W., Chang, Y.T., 2014. Microglia specific fluorescent probes for live cell imaging. Chem. Commun. (Camb.) 50, 1089–1091. Medzhitov, R., Preston-Hurlburt, P., Janeway Jr., C.A., 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397. Meylan, E., Burns, K., Hofmann, K., Blancheteau, V., Martinon, F., Kelliher, M., Tschopp, J., 2004. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation. Nat. Immunol. 5, 503–507. Miyake, K., 2004. Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2. Trends Microbiol. 12, 186–192. Moncada, S., 1999. Nitric oxide: discovery and impact on clinical medicine. J. R. Soc. Med. 92, 164–169. Murakami, A., Ohigashi, H., 2007. Targeting NOX, INOS and COX-2 in inflammatory cells: chemoprevention using food phytochemicals. Int. J. Cancer 121, 2357–2363. Rouzer, C.A., Marnett, L.J., 2009. Cyclooxygenases: structural and functional insights. J. Lipid Res. 50 (Suppl), S29-S34. Surh, Y.J., Chun, K.S., Cha, H.H., Han, S.S., Keum, Y.S., Park, K.K., Lee, S.S., 2001. Molecular mechanisms underlying chemopreventive activities of antiinflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat. Res. 480–481, 243–268. Takeda, K., Akira, S., 2005. Toll-like receptors in innate immunity. Int. Immunol. 17, 1–14. Toshchakov, V., Jones, B.W., Perera, P.Y., Thomas, K., Cody, M.J., Zhang, S., Williams, B.R., Major, J., Hamilton, T.A., Fenton, M.J., Vogel, S.N., 2002. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat. Immunol. 3, 392–398. Turini, M.E., DuBois, R.N., 2002. Cyclooxygenase-2: a therapeutic target. Annu. Rev. Med. 53, 35–57. Vallance, P., 2003. Nitric oxide: therapeutic opportunities. Fundam. Clin. Pharmacol. 17, 1–10. Youn, H.S., Lee, J.Y., Fitzgerald, K.A., Young, H.A., Akira, S., Hwang, D.H., 2005. Specific inhibition of MyD88-independent signaling pathways of TLR3 and TLR4 by resveratrol: molecular targets are TBK1 and RIP1 in TRIF complex. J. Immunol. 175, 3339–3346. Youn, H.S., Lee, J.Y., Saitoh, S.I., Miyake, K., Hwang, D.H., 2006a. Auranofin, as an anti-rheumatic gold compound, suppresses LPS-induced homodimerization of TLR4. Biochem. Biophys. Res. Commun. 350, 866–871. Youn, H.S., Lee, J.Y., Saitoh, S.I., Miyake, K., Kang, K.W., Choi, Y.J., Hwang, D.H., 2006b. Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea. Biochem. Pharmacol. 72, 850–859. Youn, H.S., Saitoh, S.I., Miyake, K., Hwang, D.H., 2006c. Inhibition of homodimerization of Toll-like receptor 4 by curcumin. Biochem. Pharmacol. 72, 62–69.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
CDr10b inhibits the expression of cyclooxygenase-2 and inducible nitric oxide synthase induced by lipopolysaccharide Gyo-Jeong Gu a,1, Se-Jin Lim b,1, Sang-il Ahn a, Sung-Chan Lee c, Young-Tae Chang d,e, Tae Hyun Choi f, Byoung Soo Kim f, Yong-Bin Eom b, Na Kyung Lee b, Hyung-Sun Youn a,b,n a
Departments of Medical Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea c Aptabio Therapeutics Inc., Kyunggi-do, Yong-in city 446-908, Republic of Korea d Department of Chemistry and MedChem Program, Life Sciences Institute, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore e Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium (SBIC), 11 Biopolis Way, #02-02 Helios, Agency for Science, Technology and Research (AnSTAR), Biopolis, Singapore 138667, Singapore f Department of Imaging, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2014 Received in revised form 19 August 2014 Accepted 24 August 2014 Available online 6 September 2014
The pathophysiological processes of inflammation can lead to a host of diseases, such as periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer. The dysregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activation play important roles in the development of certain inflammatory diseases. Here, we investigated the effects of CDr10b which is originally developed for a microglia staining probe on inflammation, by modulating NF-κB activation and iNOS and COX-2 expression induced by lipopolysaccharide (LPS) in murine macrophages. The CDr10b suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. All the results suggest that CDr10b is a promising novel agent for the treatment of inflammatory diseases. & 2014 Elsevier B.V. All rights reserved.
Keywords: Inflammation Nuclear factor-κB Cyclooxygenase-2 Inducible nitric oxide synthase CDr10b
1. Introduction Toll-like receptors (TLRs) play an important role in recognition of conserved pathogen-associated molecular patterns (PAMPs) derived from various microbial pathogens including viruses, bacteria, protozoa and fungi, and in the subsequent initiation of innate immune responses (Akira and Takeda, 2004; Medzhitov et al., 1997). The activation of TLRs by agonists can induce inflammatory responses that are key etiological conditions for the development of many chronic inflammatory diseases. Inflammation, which is mediated by multiple molecular mechanisms, refers to the pathological and physiological processes known to be involved in numerous diseases. Two of the most important enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2), have important roles in the development of certain inflammatory diseases (Moncada, 1999; Turini and DuBois, 2002).
n Corresponding author at : Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University, Chungnam, Asan-Si 336-745, Republic of Korea. Tel.: þ 82 41 530 3086; fax: þ 82 41 530 3085. E-mail address: [email protected] (H.-S. Youn). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2014.08.036 0014-2999/& 2014 Elsevier B.V. All rights reserved.
NOS, which catalyzes the conversion of L-arginine to nitric oxide, is classified into several isoforms, based on the cell type or the location and manner of expression, such as endothelial NOS (eNOS), neuronal NOS (nNOS), and a macrophage or inducible NOS (iNOS) (Murakami and Ohigashi, 2007). The eNOS and nNOS are constitutively active enzymes in vascular endothelial cells and neuronal tissues. On the other hand, iNOS enzymes are induced under both normal and pathological conditions in a variety of cell types, including macrophages, keratinocytes, hepatocytes, astrocytes, and microglial cells. Since dysregulated iNOS expression might be intimately involved in the development of certain inflammatory diseases, iNOS is a very important therapeutic target in the development of anti-inflammatory drugs (Vallance, 2003). Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids, is the molecular target for antiinflammatory remedies (Rouzer and Marnett, 2009). The COX consists of at least two isoforms: COX-1 and COX-2. The COX-1 enzyme is constitutively expressed in many normal tissues. In contrast, although COX-2 is undetectable in most of the normal mammalian tissues, it is an inducible enzyme expressed in them in response to pathophysiological stimuli including ultraviolet light, dioxin, and lipopolysaccharide (LPS) (Murakami and Ohigashi, 2007). It is reported that the expression of COX-2 is regulated at
G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
43
Fig. 1. (A) The structure of CDr10b. (B) Excitation/emission spectra of CDr10b.
transcriptional and post-transcriptional levels by pro-inflammatory agents, cytokines, growth factors, and tumor-promoters (Alvarez et al., 2005; Antman et al., 2005). Previously, we reported the development of a Compound of Designation red 10 binding (CDr10b) (Fig. 1) for the isolation and imaging of live microglia, when investigating their roles in neuroinflammatory diseases (Leong et al., 2014). Herein, we report immunomodulatory activity of CDr10b in the context of NF-κB activation induced by LPS (TLR4 agonist) in murine macrophages. The CDr10b suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. These results suggest that CDr10b can be developed as a potent anti-inflammatory drug.
2. Materials and methods
2.4. Plasmids The NF-κB (2x)-luciferase reporter construct was provided by Frank Mercurio (Signal Pharmaceuticals, San Diego, CA, USA). The heat shock protein 70 (HSP70)-β-galactosidase reporter plasmid was purchased from Robert Modlin (University of California, Los Angeles, CA, USA). The COX-2 and iNOS luciferase reporter constructs were obtained from Dr. Daniel Hwang (University of California, Davis, CA, USA). A wild type of MyD88 was provided by J. Tschopp (University of Lausanne, Lausanne, Switzerland). The wild-type IKKβ was obtained from M. Karin (University of California, San Diego, CA). The wild type of p65 was obtained from J. Ye (Pennington Biomedical Research Center, Baton Rouge, LA). All DNA constructs were prepared in large scale for transfection using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA, USA).
2.1. Synthetic procedure of CDr10b CDr10b was synthesized by following the established procedure (Leong et al., 2014). The 8-(4-Aminophenyl)-4,4-difluoro-1, 3-dimethyl-4-bora-3a,4a-diaza-s-indacene (aminophenyl bodipy) (100 mg, 320 mmole) and 3-ethoxy-4-methoxybenzaldehyde (86 mg, 480 mmole) were dissolved with acetonitrile (50 ml). Then, 3 eq. of pyrrolidine and acetic acid was added to the reaction mixture. The reaction mixture was heated to 60 1C for 3 h. After completion of the reaction, NaHCO3 saturated solution (500 ml) and chloroacetyl chloride 200 ml were added to the reaction mixture and stirred vigorously for 5 min. The crude compound was purified using column chromatography with MeOH/DCM eluent system (0–5% of MeOH), and 148 mg of product was obtained. The spectroscopic properties and analytical data were identical as the reference. 2.2. Reagents LPS was obtained from List Biological Laboratories (San Jose, CA, USA), and it was dissolved in endotoxin-free water. All other reagents were purchased from Sigma-Aldrich, unless otherwise indicated. 2.3. Cell culture RAW 264.7 cells (a murine monocytic cell line; ATCC TIB71) and 293T human embryonic kidney cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 units/ml Penicillin (Invitrogen, Carlsbad, CA, USA), and 100 μg/ml Streptomycin (Invitrogen). Cells were maintained at 37 1C in a 5% CO2/95% air environment.
2.5. Transfection and reporter gene luciferase assay The assays were performed as previously described (Youn et al., 2005, 2006b). RAW264.7 cells were co-transfected with a luciferase plasmid and a plasmid containing heat shock protein (HSP)70-βgalactosidase as an internal control, using SuperFect transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions and then pretreated with 20 or 50 μM CDr10b for 1 h and then treated with LPS (10 ng/ml) for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined using the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The luciferase activity was normalized by β-galactosidase activity.
2.6. Immunoblotting Immunoblotting was performed as previously described (Youn et al., 2006a, 2006c). Equal amounts of cell extracts were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked to prevent nonspecific binding of antibodies in phosphate-buffered saline containing 0.1% Tween-20 and 3% nonfat dry milk. The immunoblotting was performed with the indicated antibodies, and the secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences, Arlington Heights, IL, USA). The reactive bands were visualized with the enhanced chemiluminescence Western blot detection reagents (Intron, Seongnam, Gyeonggi-do, South Korea).
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2.7. Cell viability test
2.8. Data analysis
The cell viability was assessed using MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] based colorimetric assay. RAW264.7 cells were treated with CDr10b (20, 50, 100 μM) for 4 h. Twenty microliters of the CellTiter 96 AQueous One Solution Reagent was added directly to culture wells. The plate was incubated at 37 1C for 4 h in a humidified, 5% CO2 atmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader.
The data were obtained from triplicate experiments. The values are expressed as the mean 7standard error of the mean (S.E.M.).
3. Results 3.1. CDr10b inhibits LPS-induced NF-κB activation To evaluate the cytotoxic nature of CDr10b in RAW 264.7 cells, the MTS viability assay was used. It was apparent that CDr10b was not cytotoxic at the tested concentrations of 20–100 μM. Therefore, the cellular toxicity could be excluded from the possible reason for any observed changes in the subsequent study. Next, we determined the effect of CDr10b on LPS-induced NF-κB activation, by using the NF-κB luciferase reporter assay. The CDr10b inhibited NF-κB activation which was induced by LPS (Fig. 2). The NF-κB is activated in response to stimuli of various pathogens (Hacker and Karin, 2006). Therefore, the suppression of NF-κB activation is considered as an important strategy for antiinflammation and anti-cancer therapies. 3.2. CDr10b suppresses LPS-induced iNOS expression
Fig. 2. CDr10b inhibits LPS-induced NF-κB activation. NF-κB luciferase assay in RAW264.7 cells. Values are expressed as mean7 S.E.M. (n¼ 3). nn denotes significantly different from LPS alone, Po 0.01. Veh, vehicle.
The next experiment assessed the capability of CDr10b to regulate iNOS expression, which is one of the target genes regulated through NF-κB activation in macrophages. The CDr10b suppressed the iNOS expression that was induced by LPS in RAW264.7 cells, as determined by iNOS-luciferase reporter assay (Fig. 3A). The CDr10b also suppressed the iNOS protein that was induced by LPS, as determined by iNOS immunoblotting assay
Fig. 3. CDr10b inhibits LPS-induced iNOS expression. (A) iNOS luciferase assay in RAW264.7 cells. Values represent the mean 7S.E.M. (n¼3). nn Significantly different from LPS alone, P o 0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates were analyzed for iNOS and β-actin protein by immunoblots. Veh, vehicle.
Fig. 4. CDr10b inhibits LPS-induced COX-2 expression. (A) COX-2 luciferase assay in RAW264.7 cells. Values represent the mean7 S.E.M. (n ¼3). nn Significantly different from LPS alone, Po 0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates were analyzed for COX-2 and β-actin protein by immunoblots. Veh, vehicle.
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Fig. 5. CDr10b suppresses MyD88-dependent signaling pathway of TLR4. (A–C) 293T cells were transfected with NF-κB luciferase reporter plasmid and the expression plasmid of MyD88 (A), IKKβ (B) or p65 (C). Cells were further treated with 20 or 50 μM CDr10b for 18 h. Relative luciferase activity was normalized with β-gal activity. Values represent mean7 S.E.M. (n¼ 3). þdenotes a result that is significantly different from MyD88 alone, Po 0.01 ( þ þ) (A). # denotes a result that is significantly different from IKKβ alone, P o 0.01 (##) (B). n denotes a result that is significantly different from p65 alone, Po 0.01 (nn) (C). Veh, vehicle.
(Fig. 3B). All of the results suggested that CDr10b inhibited iNOS expression induced by LPS. 3.3. CDr10b suppresses LPS-induced COX-2 expression Next, we determined whether CDr10b could regulate COX-2 expression, which is another target gene regulated through NF-κB activation. The CDr10b suppressed the COX-2 expression that was induced by LPS in RAW264.7 cells, as determined by COX-2luciferase reporter assay (Fig. 4A). The CDr10b also suppressed the COX-2 protein that was induced by LPS, as determined by COX-2 immunoblotting assay (Fig. 4B). 3.4. CDr10b suppresses MyD88-dependent signaling pathway of TLRs To further investigate the regulation of MyD88-dependent signaling pathways by CDr10b, NF-κB activation was induced by
the overexpression of MyD88, IKKβ, or p65 in 293T cells. CDr10b suppressed the agonist-independent activation of NF-κB induced by MyD88 (Fig. 5A), IKKβ (Fig. 5B) or p65 (Fig. 5C), demonstrating that CDr10b suppresses MyD88-dependent signaling pathways.
4. Discussion Toll-like receptor 4 (TLR4) is the first TLR identified that recognizes LPS, which is a cell wall component of gram-negative bacteria (Miyake, 2004). The LPS-induced TLR4 activation leads to both early and late NF-κB activation through myeloid differential factor 88 (MyD88)- or Toll/iInterleukin-1 receptor (TIR) domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways, respectively (Takeda and Akira, 2005). MyD88 is the immediate adapter molecule which is common to all TLRs, with the exception of TLR3. The MyD88 recruits the IL-1 receptor-associated kinase (IRAK), tumor necrosis factor (TNF), and
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receptor-associated factor 6 (TRAF6), leading to the activation of the canonical IKK complex. In the un-stimulated cells, NF-κB is sequestered in the cytoplasm as an inactive form by interaction with an inhibitor IκBα proteins, preventing its nuclear accumulation and transcriptional activation of the target genes. The IKK complex phosphorylates IκBα which is degraded by the 26S proteasome, allowing NF-κB to translocate into the nucleus. The translocated NF-κB binds to DNA and up-regulates the expressions of numerous pro-inflammatory gene products including COX-2, iNOS, and a wide variety of proinflammatory cytokines and chemokines (Surh et al., 2001). NF-κB, an important component of inflammatory responses, is a transcription factor that regulates over 150 genes, many of which are associated with inflammation and cancer (Baldwin, 1996). The LPS-induced activation of TRIF/TICAM-1, an adapter molecule which functions independently of MyD88, also leads to the activation of the transcriptional regulator, IRF3, and the expression of IFNβ and IFN-inducible genes (Fitzgerald et al., 2003; Toshchakov et al., 2002). TRIF recruits TRAF family member-associated NF-κB activator-binding kinase 1 (TBK1) and inhibitor-κB kinase-ε (IKKε), leading to IRF3 activation (Fitzgerald et al., 2003). The activated IRF3 translocates into the nucleus and binds to its target DNA sequences of IFN-stimulated response elements that are found in the promoter regions of the genes, such as those encoding IFNβ and interferon inducible protein-10 (IP-10), and are regulated by the activation of normal T-cells expressed and secreted (Bjorkbacka et al., 2004; Gao et al., 1998; Kawai et al., 2001). The TRIF also induces the activation of receptor interacting protein-1, leading to a delayed NF-κB activation (Meylan et al., 2004). In this study, we investigated whether CDr10b could regulate the expression levels of COX-2 and iNOS, which are two of the most important enzymes for certain inflammatory diseases. In murine cells, COX-2 and iNOS expressions are regulated at the transcriptional level by NF-κB, with the promoter of both genes containing an NF-κB binding sequence. All of the results support the suggestion that CDr10b suppresses LPS-induced NF-κB activation, resulting in the induction of the expression of target genes such as COX-2 and iNOS. These results suggest that CDr10b can regulate TLR4 signaling pathways. The recent studies suggest that TLRs and their signaling components could become excellent therapeutic targets for chronic inflammatory diseases (Kawai and Akira, 2010). Therefore, our results provide new insights into the understanding of the mode of the anti-inflammatory action of CDr10b. Acknowledgments This study was supported by Soonchunhyang University research fund and by Basic Science Research Program, through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and, Technology (2010-0023637). References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511.
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