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Clonidine Enhances Type-2 Cationic Amino Acid Transporter Transcription in Endotoxin-activated Murine Macrophages
Acta Anaesthesiol Taiwan 2008;46(3):118−123
O R IGINAL ART I CL E
Clonidine Enhances Type-2 Cationic Amino Acid Transporter Transcription in Endotoxin-activated Murine Macrophages Yen-Chun Lai1, Pei-Shan Tsai2, Chun-Jen Huang1,3,4* 1
Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan, R.O.C. College of Nursing, Taipei Medical University, Taipei, Taiwan, R.O.C. 3 College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C. 4 Mackay Medicine, Nursing and Management College, Taipei, Taiwan, R.O.C. 2
Received: Apr 10, 2008 Revised: Jun 12, 2008 Accepted: Jun 17, 2008 KEY WORDS: cationic amino acid transporter 2; clonidine; lipopolysaccharides; macrophages
Background: We sought to evaluate the effects of clonidine on type-2 cationic amino acid transporter (CAT-2) transcription in endotoxin-activated murine macrophages. Methods: To determine the effects of clonidine on CAT-2 transcription, confluent murine macrophages (RAW264.7 cells) were treated with 1× phosphate buffered saline, clonidine (1000 μM), lipopolysaccharide (LPS, 100 ng/mL), or LPS plus clonidine (10, 100, or 1000 μM). After reacting with LPS for 18 hours or a comparable duration in groups without LPS, cell cultures were harvested and the CAT-2 mRNA concentration was assayed. To determine the stability of CAT-2 mRNA, confluent macrophages were treated with LPS or LPS plus clonidine (100 μM). After reacting with LPS for 6 hours, CAT-2 transcription was terminated and the stability of CAT-2 mRNA was determined. Results: The CAT-2 mRNA concentration of cell cultures receiving LPS plus clonidine (100 μM) or LPS plus clonidine (1000 μM) were significantly higher than that of the cell cultures receiving LPS alone, whereas the CAT-2 mRNA concentrations of cell cultures receiving LPS plus clonidine (10 μM) was comparable to that of cell cultures receiving LPS alone. The data indicated that clonidine significantly enhanced LPS-induced CAT-2 transcription. The estimated half-life of CAT-2 mRNA of cell cultures receiving LPS was similar to that of cell cultures receiving LPS plus clonidine. These results indicated that clonidine did not affect CAT-2 mRNA stability. Conclusion: Clonidine enhances CAT-2 transcription in endotoxin-activated murine macrophages.
1. Introduction Induction of inducible nitric oxide (iNOS) and resultant overproduction of nitric oxide (NO) have been shown to play an important role in mediating the systemic inflammatory responses during sepsis.1,2
Type-2 cationic amino acid transporter (CAT-2) is the main enzyme that mediates cellular uptake of circulating L-arginine, i.e., the sole substrate for iNOS.3,4 Kakuda et al reported that CAT-2 mediates L-arginine transport and NO biosynthesis in activated murine macrophages.5 Using macrophages from
*Corresponding author. Department of Anesthesiology, Mackay Memorial Hospital, 92, Section 2, Chung Shan North Road, Taipei 104, Taiwan, R.O.C. E-mail: [email protected] ©2008 Taiwan Society of Anesthesiologists
Clonidine enhances CAT-2 transcription CAT-2 knockout mice, Nicholson et al also reported that sustained NO production in macrophages requires CAT-2.4 These results clearly indicate that CAT-2 plays a crucial role in regulating iNOS activity. Clonidine, an α2-adrenergic receptor agonist has been shown to have a potent antihypertensive effect.6 Previous data indicated that NO may play a role in mediating the effects of clonidine.7 Moreover, clonidine has been shown to significantly enhance the expression of iNOS in endotoxinstimulated C6 glioma cells.8 Nevertheless, the effects of clonidine on regulating the expression of CAT-2 remain undetermined. We thus conducted this cellular study based on the hypothesis that clonidine could significantly enhance CAT-2 transcription in endotoxin-activated murine macrophages. Moreover, the effects of clonidine on CAT-2 mRNA stability were also investigated in this study.
2. Methods 2.1. Cell culture Live murine macrophages (RAW264.7 cells) were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Life Technologies), as we previously reported.9 RAW264.7 cells were incubated in a humidified chamber at 37ºC in a mixture of 95% air and 5% CO2.
2.2. Experimental protocols RAW264.7 cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL; E. coli serotype 0127:B8; Sigma-Aldrich, St Louis, MO, USA) to induce CAT-2 transcription, as we previously reported.9 To determine whether the effects of clonidine on CAT-2 transcription were dose-dependent, six groups of confluent cells (n = 6 in each group) were included, of which two groups of cells whose culture media received 1× phosphate buffered saline (Life Technologies; denoted as the PBS group) or LPS (the LPS group) served as the negative or the positive control, respectively. Another three groups of cell cultures were treated with one of the three different doses of clonidine (10, 100, or 1000 μM) immediately after LPS administration—the LPS + clonidine (10), LPS + clonidine (100), and LPS + clonidine (1000) groups, respectively. Finally, the remaining group of cells whose cultures received 1000 μM of clonidine only served as the control of clonidine (the clonidine group). After exposure to LPS for 18 hours or a comparable duration in groups without LPS, cell cultures were harvested.
119 According to the preliminary results of the abovementioned experiment, we chose to employ the dose of 100 μM clonidine for the following experiments. Confluent cells were randomized to one of the four groups (n = 15): the PBS, clonidine, LPS, and LPS + clonidine groups. To elucidate the effects of clonidine on the time course of CAT-2 transcription induction, three culture dishes from each group were harvested after being exposed to LPS for 2, 4, 6, 12 and 18 hours or after similar durations in groups without LPS.
2.3. Reverse transcription and polymerase chain reaction (RT-PCR) Three independent RT-PCR assays were performed to determine the CAT-2 mRNA concentrations of each sample, as we previously reported.9 In brief, total RNA was isolated from cell cultures with TRIzol Reagent (Life Technologies). RNA samples were then extracted by a phenol-chloroform technique. The RNA concentrations were quantified by measuring ultraviolet light absorbance at a wavelength of 260 nm. Maloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready-To-Go RT-PCR Beads; Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) were used to reverse transcribe all messenger RNA species to complementary DNA (cDNA). The reaction was incubated at 42ºC for 30 minutes in a thermocycler. cDNA samples were then incubated at 95ºC for 5 minutes to inactivate the reverse transcriptase enzyme. Each sample was carried through the PCR procedure separately without adding reverse transcriptase to prevent genomic DNA contamination. The cDNA encoding CAT-2 and β-actin (as an internal standard) were then amplified using PCR. The primer sequences for each of the enzymes were designed in accordance with published rat DNA sequences and obtained from our previous reports.9 Amplification of CAT-2/β-actin was performed in 35 cycles at 94ºC for 1 minute, 60ºC for 1 minute, 72ºC for 2 minutes, and a final extension of the products at 72ºC for 7 minutes. PCR-amplified samples were electrophoretically separated on 1% ethidium bromide-stained agarose gels. The Gel Documentation System (Gel Doc 2000; Bio-Rad Laboratories, Hercules, CA, USA) was used to assay the PCR products. The cDNA band densities were quantified by using densitometric techniques with Scion Image for Windows (Scion Corp., Frederic, MD, USA).
2.4. CAT-2 mRNA stability assay and real-time PCR assay The protocols for the CAT-2 mRNA stability assay were developed according to a previous report.10
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In brief, confluent cells were randomized to the LPS or the LPS + clonidine group (n = 18). After reacting with LPS for 6 hours, actinomycin D (10 μg/mL, Sigma-Aldrich) was added to the cell cultures to terminate transcription. Three culture dishes from each group were harvested after being exposed to actinomycin D for 0, 2, 4, 6, 8 or 10 hours. The concentrations of CAT-2 mRNA were then assayed by quantitative real-time PCR assay according to our previous report.11 In brief, quantitative realtime PCR was performed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA), SYBR green core reagent, and Ampli-Taq gold polymerase. PCR was performed twice for each primer set using 25 μL reactions, and complementary DNA loading was normalized to rat 18s rRNA. The amount of gene transcript was then measured using the comparative method (2-⌬⌬CT) described by Applied Biosystems. The stability of CAT-2 mRNA (as evaluated by the percentage remaining) was then calculated by comparing the CAT-2 mRNA concentrations in macrophages harvested at different time points after actinomycin D to those in macrophages harvested at 0 hours after actinomycin D.
2.5. Statistical analysis One-way analysis of variance was used to determine the between-group differences. The Tukey test was used for multiple comparisons. Data were presented as mean ± standard deviation. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Inc., Chicago, IL, USA) was used for data analysis.
3. Results 3.1. Effects of various dosages of clonidine on LPS-induced CAT-2 transcription The concentrations of CAT-2 mRNA of the PBS and clonidine groups were almost undetectable (Figure 1). In contrast, the CAT-2 mRNA concentration of the LPS, LPS + clonidine (10), LPS + clonidine (100), and LPS + clonidine (1000) groups were significantly higher than that of the PBS group (all p < 0.001; Figure 1). The CAT-2 mRNA concentration of the LPS + clonidine (100) and LPS + clonidine (1000) groups were significantly higher than that of the LPS
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Figure 1 Effects of clonidine (10, 100, and 1000 μM) on type-2 cationic amino acid transporter (CAT-2) mRNA expression in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Representative gel photography illustrates the products of RT-PCR. PCR products were analyzed using the densitometric technique. Data were obtained from three independent analyses of freshly harvested cell samples and the CAT-2 mRNA concentrations were normalized by β-actin and expressed as mean ± standard deviation. Cell cultures were harvested after exposing to LPS for 18 hours or comparable duration in groups without LPS. PBS = phosphate buffered saline. *p < 0.05 vs. the LPS group; †p < 0.05 vs. the PBS group; ‡p < 0.05 vs. the LPS + clonidine (10) group; §p < 0.05 vs. the LPS + clonidine (100) group.
Clonidine enhances CAT-2 transcription
121 LPS + clonidine group harvested at 6 and 18 hours after LPS were significantly higher than those of the LPS group (p = 0.029 and 0.016, respectively; Figure 2), whereas the CAT-2 mRNA concentrations of the LPS + clonidine group harvested at 12 hours after LPS were comparable to that of the LPS group (Figure 2).
group (p = 0.025 and 0.001, respectively; Figure 1), whereas the CAT-2 mRNA concentrations of the LPS + clonidine (10) group was comparable to that of the LPS group (Figure 1). The CAT-2 mRNA concentrations of the LPS + clonidine (1000) and LPS + clonidine (100) groups were also significantly higher than that of the LPS + clonidine (10) group (p = 0.038 and 0.014, respectively; Figure 1). Moreover, the CAT-2 mRNA concentration of the LPS + clonidine (1000) group was higher than that of the LPS + clonidine (100) group (p = 0.033; Figure 1).
3.3. Effects of clonidine on the stability of CAT-2 mRNA Figure 3 illustrates the stability of CAT-2 mRNA as evaluated by the percentage remaining. The estimated half-life of the CAT-2 mRNA of the LPS group was approximately 8 hours (Figure 3). Similarly, the estimated half-life of the CAT-2 mRNA of the LPS + clonidine group was approximately 8 hours (Figure 3).
3.2. Effects of clonidine on the time course of CAT-2 transcription induction The CAT-2 mRNA concentrations of the PBS and clonidine groups harvested at 2, 4, 6, 12 and 18 hours were low (data not shown). The CAT-2 mRNA concentrations of the LPS and LPS + clonidine groups harvested at 2 and 4 hours after LPS were comparable to those of the PBS group. Nevertheless, the CAT-2 mRNA concentrations of the LPS and LPS + clonidine groups harvested at 6, 12 and 18 hours after LPS were significantly higher than those of the PBS group (all p < 0.001; Figure 2). Moreover, the CAT-2 mRNA concentrations of the
4. Discussion Data from this study confirmed our hypothesis and provided the first evidence to demonstrate that clonidine-induced enhancement of CAT-2 transcription involves increases in enzyme transcription
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Figure 2 Effects of lipopolysaccharide (LPS) and LPS + clonidine (100 μM) on type-2 cationic amino acid transporter (CAT-2) mRNA expression in stimulated murine macrophages (RAW264.7 cells) after exposure to LPS for 2, 4, 6, 12 and 18 hours. Representative gel photography illustrates the products of RT-PCR. PCR products were analyzed using the densitometric technique. Data were obtained from three independent analyses of the freshly harvested cell samples and the CAT-2 mRNA concentrations were normalized by β-actin and expressed as mean ± standard deviation. *p < 0.05 vs. the LPS group.
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Figure 3 The stability of type-2 cationic amino acid transporter (CAT-2) mRNA in murine macrophages (RAW264.7 cells) treated with lipopolysaccharide (LPS) or LPS + clonidine (100 μM). Actinomycin D was added to macrophages at 6 hours after LPS administration to terminate transcription. Three culture dishes from each group were then harvested after exposing them to actinomycin D for 0, 2, 4, 6, 8 and 10 hours, respectively. Total RNA was isolated and the CAT-2 mRNA concentrations were assayed by quantitative real-time PCR assay. The CAT-2 mRNA concentrations were then normalized by rat 18s rRNA. The stability of CAT-2 mRNA, as evaluated by the percentage remaining, was then calculated by comparing the CAT-2 mRNA concentrations in macrophages harvested at different time points after actinomycin D to those in macrophages harvested at 0 hours after actinomycin D, respectively.
induction in endotoxin-activated macrophages. It is well-established that nuclear factor-κB (NF-κB) is a crucial factor for maximal transcription of a wide array of inflammatory molecules, including intercellular adhesion molecular-1, iNOS, cycloxygenase-2, interleukin (IL)-1β, IL-6, and tumor necrosis factorα.12 In a recent report, we demonstrated that NF-κB is involved in clonidine-induced enhancement of iNOS expression.13 As we have previously shown that transcription induction of CAT-2 is mainly regulated by NF-κB,14,15 these results seem to suggest that NF-κB may very likely participate in this clonidine-induced enhancement of CAT-2 transcription induction in activated murine macrophages. Transcription of iNOS induced by endotoxins has also been shown to be regulated by the toll-like receptor (TLR) family, especially TLR-4.16,17 Furthermore, activation of TLR induces the recruitment of myeloid differential factor 88 (MyD88), an intracellular adaptor protein that links to the activation of NF-κB.18 Endotoxin-induced TLR-4 activation also requires the presence of cluster of differention-14 (CD14), a membrane-anchor protein that mediates the recognition of LPS, and myeloid differential-2
(MD-2), an extracellular protein that is associated with the extracellular domain of TLR-4.19−21 Although direct evidence to illustrate the involvement of the CD14/MD-2/TLR-4/MyD88 pathway in regulating the endotoxin-induced CAT-2 transcription is still lacking, existing data seems to support the concept that the CD14/MD-2/TLR-4/MyD88 pathway may also be involved in this clonidine-induced enhancement of CAT-2 transcription induction in activated macrophages. A series of studies are currently being performed in our laboratory to elucidate this further. Previous data indicated that the stability of shortlived mRNAs, including iNOS and cytokines, is regulated by the interaction between RNA-binding factors and the adenosine/uridine-rich (AU-rich) elements (AREs) within three untranslated regions of these short-lived mRNAs that often contain several copies of the motif AUUUA.22 Among the identified RNA-binding factors, the RNA stabilizing factor HuR23 and the RNA destabilizing factors, including ARE/ poly(U) binding factor-1 (AUF-1)24 and tristetraprolin (TTP),25 have been shown to be associated with the stability of iNOS mRNA. To the best of our knowledge, no data are available regarding the pathways that regulate the CAT-2 mRNA stability so far. Judging from the fact that CAT-2 mRNA is a shortlived mRNA, we thus speculate that CAT-2 mRNA stability may be regulated by HuR, AUF-1, and TTP. We have shown that clonidine had significant effects on enhancing the expression of AUF-1 and TTP but not HuR in activated murine macrophages, whereas clonidine did not have significant effects on iNOS mRNA stability.13 One possible explanation is that the enhancement of AUF-1 and TTP is secondary to the increases in iNOS mRNA induced by clonidine. Similar to previous studies,13 the data from this study also revealed that clonidine did not have significant effects on CAT-2 mRNA stability. If the stability of CAT-2 mRNA is subject to the regulation of the aforementioned RNA-binding factors, then it is possible that the enhancement in CAT-2 mRNA induced by clonidine may also have a role in clonidineinduced enhancement of AUF-1 and TTP expression. More studies are needed before further conclusions can be drawn. In summary, clonidine-induced enhancement of CAT-2 transcription in endotoxin-activated murine macrophages involves increases in transcription induction but not mRNA stability.
Acknowledgments This work was supported by a grant from the National Science Council, Taiwan (NSC 93-2314-B195-022) and a grant from Mackay Memorial Hospital (MMH 9415) awarded to Dr Chun-Jen Huang.
Clonidine enhances CAT-2 transcription
References 1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051−64. Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking J, Nathan CF, Peitzman AB, et al. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 1998;187:917−28. Bogle RG, Baydoun AR, Pearson JD, Moncada S, Mann GE. L-arginine transport is increased in macrophages generating nitric oxide. Biochem J 1992;284:15−8. Nicholson B, Manner CK, Kleeman J, MacLeod CL. Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J Biol Chem 2001;276:15881−5. Kakuda DK, Sweet MJ, MacLeod CL, Hume DA, Markovich D. CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J 1999;340: 549−53. Carabine UA, Wright PM, Moore J. Preanaesthetic medication with clonidine: a dose-response study. Br J Anaesth 1991;67:79−83. Soares DM, Rios AA, de Oliveira LF, Resende AC, de Lemos NM, Santos EJ, Correia ML, et al. The effects of nitric oxide synthase inhibitors on the sedative effect of clonidine. Anesth Analg 2001;93:1217−21. Venturini G, Colasanti M, Persichini T, Fioravanti E, Federico R, Ascenzi P. Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug. Biochem Pharmacol 2000;60:539−44. Lin WC, Tsai PS, Huang CJ. Catecholamines’ enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A. Anesth Analg 2005; 101:226−32. Lahti A, Jalonen U, Kankaanranta H, Moilanen E. c-Jun NH2-terminal kinase inhibitor anthra(1,9-cd)pyrazol-6(2H)one reduces inducible nitric-oxide synthase expression by destabilizing mRNA in activated macrophages. Mol Pharmacol 2003;64:308−15. Huang CJ, Tsai PS, Pan WH, Skimming JW. Microdialysis for measurement of hepatic and systemic nitric oxide biosynthesis in septic rats. Acta Anaesthesiol Scand 2005;49: 28−34. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3−9. Su NY, Tsai PS, Huang CJ. Clonidine-induced enhancement of iNOS expression involves NF-kappaB. J Surg Res 2008;149: 131−7.
123 14. Yang CH, Tsai PS, Lee JJ, Huang CH, Huang CJ. NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver. Acta Anaesthesiol Scand 2005;49:468−76. 15. Chu LC, Tsai PS, Lee JJ, Yen CH, Huang CJ. NF-kappaB inhibitors significantly attenuate the transcription of high affinity type-2 cationic amino acid transporter in LPSstimulated rat kidney. Acta Anaesthesiol Taiwan 2005;43: 23−32. 16. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274:10689−92. 17. Matsumura T, Ito A, Takii T, Hayashi H, Onozaki K. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J Interferon Cytokine Res 2000;20:915−21. 18. Janssens S, Beyaert R. A universal role for MyD88 in TLR/ IL-1R-mediated signaling. Trends Biochem Sci 2002;27: 474−82. 19. Shuto T, Kato K, Mori Y, Viriyakosol S, Oba M, Furuta T, Okiyoneda T, et al. Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells. Biochem Biophys Res Commun 2005;338:1402−9. 20. Nagai Y, Akashi S, Nagafuku M, Ogata M, Iwakura Y, Akira S, Kitamura T, et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 2002;3:667−72. 21. da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. Transfer from CD14 to TLR4 and MD-2. J Biol Chem 2001;276:21129−35. 22. Kawahara K, Gotoh T, Oyadomari S, Kajizono M, Kuniyasu A, Ohsawa K, Imai Y, et al. Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells. Brain Res Mol Brain Res 2001;90:165−73. 23. Brennan CM, Steitz JA. HuR and mRNA stability. Cell Mol Life Sci 2001;58:266−77. 24. Blaxall BC, Pende A, Wu SC, Port JD. Correlation between intrinsic mRNA stability and the affinity of AUF1 (hnRNP D) and HuR for A + U-rich mRNAs. Mol Cell Biochem 2002;232: 1−11. 25. Lai WS, Carballo E, Thorn JM, Kennington EA, Blackshear PJ. Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J Biol Chem 2000;275:17827−37.
O R IGINAL ART I CL E
Clonidine Enhances Type-2 Cationic Amino Acid Transporter Transcription in Endotoxin-activated Murine Macrophages Yen-Chun Lai1, Pei-Shan Tsai2, Chun-Jen Huang1,3,4* 1
Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan, R.O.C. College of Nursing, Taipei Medical University, Taipei, Taiwan, R.O.C. 3 College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C. 4 Mackay Medicine, Nursing and Management College, Taipei, Taiwan, R.O.C. 2
Received: Apr 10, 2008 Revised: Jun 12, 2008 Accepted: Jun 17, 2008 KEY WORDS: cationic amino acid transporter 2; clonidine; lipopolysaccharides; macrophages
Background: We sought to evaluate the effects of clonidine on type-2 cationic amino acid transporter (CAT-2) transcription in endotoxin-activated murine macrophages. Methods: To determine the effects of clonidine on CAT-2 transcription, confluent murine macrophages (RAW264.7 cells) were treated with 1× phosphate buffered saline, clonidine (1000 μM), lipopolysaccharide (LPS, 100 ng/mL), or LPS plus clonidine (10, 100, or 1000 μM). After reacting with LPS for 18 hours or a comparable duration in groups without LPS, cell cultures were harvested and the CAT-2 mRNA concentration was assayed. To determine the stability of CAT-2 mRNA, confluent macrophages were treated with LPS or LPS plus clonidine (100 μM). After reacting with LPS for 6 hours, CAT-2 transcription was terminated and the stability of CAT-2 mRNA was determined. Results: The CAT-2 mRNA concentration of cell cultures receiving LPS plus clonidine (100 μM) or LPS plus clonidine (1000 μM) were significantly higher than that of the cell cultures receiving LPS alone, whereas the CAT-2 mRNA concentrations of cell cultures receiving LPS plus clonidine (10 μM) was comparable to that of cell cultures receiving LPS alone. The data indicated that clonidine significantly enhanced LPS-induced CAT-2 transcription. The estimated half-life of CAT-2 mRNA of cell cultures receiving LPS was similar to that of cell cultures receiving LPS plus clonidine. These results indicated that clonidine did not affect CAT-2 mRNA stability. Conclusion: Clonidine enhances CAT-2 transcription in endotoxin-activated murine macrophages.
1. Introduction Induction of inducible nitric oxide (iNOS) and resultant overproduction of nitric oxide (NO) have been shown to play an important role in mediating the systemic inflammatory responses during sepsis.1,2
Type-2 cationic amino acid transporter (CAT-2) is the main enzyme that mediates cellular uptake of circulating L-arginine, i.e., the sole substrate for iNOS.3,4 Kakuda et al reported that CAT-2 mediates L-arginine transport and NO biosynthesis in activated murine macrophages.5 Using macrophages from
*Corresponding author. Department of Anesthesiology, Mackay Memorial Hospital, 92, Section 2, Chung Shan North Road, Taipei 104, Taiwan, R.O.C. E-mail: [email protected] ©2008 Taiwan Society of Anesthesiologists
Clonidine enhances CAT-2 transcription CAT-2 knockout mice, Nicholson et al also reported that sustained NO production in macrophages requires CAT-2.4 These results clearly indicate that CAT-2 plays a crucial role in regulating iNOS activity. Clonidine, an α2-adrenergic receptor agonist has been shown to have a potent antihypertensive effect.6 Previous data indicated that NO may play a role in mediating the effects of clonidine.7 Moreover, clonidine has been shown to significantly enhance the expression of iNOS in endotoxinstimulated C6 glioma cells.8 Nevertheless, the effects of clonidine on regulating the expression of CAT-2 remain undetermined. We thus conducted this cellular study based on the hypothesis that clonidine could significantly enhance CAT-2 transcription in endotoxin-activated murine macrophages. Moreover, the effects of clonidine on CAT-2 mRNA stability were also investigated in this study.
2. Methods 2.1. Cell culture Live murine macrophages (RAW264.7 cells) were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Life Technologies), as we previously reported.9 RAW264.7 cells were incubated in a humidified chamber at 37ºC in a mixture of 95% air and 5% CO2.
2.2. Experimental protocols RAW264.7 cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL; E. coli serotype 0127:B8; Sigma-Aldrich, St Louis, MO, USA) to induce CAT-2 transcription, as we previously reported.9 To determine whether the effects of clonidine on CAT-2 transcription were dose-dependent, six groups of confluent cells (n = 6 in each group) were included, of which two groups of cells whose culture media received 1× phosphate buffered saline (Life Technologies; denoted as the PBS group) or LPS (the LPS group) served as the negative or the positive control, respectively. Another three groups of cell cultures were treated with one of the three different doses of clonidine (10, 100, or 1000 μM) immediately after LPS administration—the LPS + clonidine (10), LPS + clonidine (100), and LPS + clonidine (1000) groups, respectively. Finally, the remaining group of cells whose cultures received 1000 μM of clonidine only served as the control of clonidine (the clonidine group). After exposure to LPS for 18 hours or a comparable duration in groups without LPS, cell cultures were harvested.
119 According to the preliminary results of the abovementioned experiment, we chose to employ the dose of 100 μM clonidine for the following experiments. Confluent cells were randomized to one of the four groups (n = 15): the PBS, clonidine, LPS, and LPS + clonidine groups. To elucidate the effects of clonidine on the time course of CAT-2 transcription induction, three culture dishes from each group were harvested after being exposed to LPS for 2, 4, 6, 12 and 18 hours or after similar durations in groups without LPS.
2.3. Reverse transcription and polymerase chain reaction (RT-PCR) Three independent RT-PCR assays were performed to determine the CAT-2 mRNA concentrations of each sample, as we previously reported.9 In brief, total RNA was isolated from cell cultures with TRIzol Reagent (Life Technologies). RNA samples were then extracted by a phenol-chloroform technique. The RNA concentrations were quantified by measuring ultraviolet light absorbance at a wavelength of 260 nm. Maloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready-To-Go RT-PCR Beads; Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) were used to reverse transcribe all messenger RNA species to complementary DNA (cDNA). The reaction was incubated at 42ºC for 30 minutes in a thermocycler. cDNA samples were then incubated at 95ºC for 5 minutes to inactivate the reverse transcriptase enzyme. Each sample was carried through the PCR procedure separately without adding reverse transcriptase to prevent genomic DNA contamination. The cDNA encoding CAT-2 and β-actin (as an internal standard) were then amplified using PCR. The primer sequences for each of the enzymes were designed in accordance with published rat DNA sequences and obtained from our previous reports.9 Amplification of CAT-2/β-actin was performed in 35 cycles at 94ºC for 1 minute, 60ºC for 1 minute, 72ºC for 2 minutes, and a final extension of the products at 72ºC for 7 minutes. PCR-amplified samples were electrophoretically separated on 1% ethidium bromide-stained agarose gels. The Gel Documentation System (Gel Doc 2000; Bio-Rad Laboratories, Hercules, CA, USA) was used to assay the PCR products. The cDNA band densities were quantified by using densitometric techniques with Scion Image for Windows (Scion Corp., Frederic, MD, USA).
2.4. CAT-2 mRNA stability assay and real-time PCR assay The protocols for the CAT-2 mRNA stability assay were developed according to a previous report.10
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In brief, confluent cells were randomized to the LPS or the LPS + clonidine group (n = 18). After reacting with LPS for 6 hours, actinomycin D (10 μg/mL, Sigma-Aldrich) was added to the cell cultures to terminate transcription. Three culture dishes from each group were harvested after being exposed to actinomycin D for 0, 2, 4, 6, 8 or 10 hours. The concentrations of CAT-2 mRNA were then assayed by quantitative real-time PCR assay according to our previous report.11 In brief, quantitative realtime PCR was performed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA), SYBR green core reagent, and Ampli-Taq gold polymerase. PCR was performed twice for each primer set using 25 μL reactions, and complementary DNA loading was normalized to rat 18s rRNA. The amount of gene transcript was then measured using the comparative method (2-⌬⌬CT) described by Applied Biosystems. The stability of CAT-2 mRNA (as evaluated by the percentage remaining) was then calculated by comparing the CAT-2 mRNA concentrations in macrophages harvested at different time points after actinomycin D to those in macrophages harvested at 0 hours after actinomycin D.
2.5. Statistical analysis One-way analysis of variance was used to determine the between-group differences. The Tukey test was used for multiple comparisons. Data were presented as mean ± standard deviation. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Inc., Chicago, IL, USA) was used for data analysis.
3. Results 3.1. Effects of various dosages of clonidine on LPS-induced CAT-2 transcription The concentrations of CAT-2 mRNA of the PBS and clonidine groups were almost undetectable (Figure 1). In contrast, the CAT-2 mRNA concentration of the LPS, LPS + clonidine (10), LPS + clonidine (100), and LPS + clonidine (1000) groups were significantly higher than that of the PBS group (all p < 0.001; Figure 1). The CAT-2 mRNA concentration of the LPS + clonidine (100) and LPS + clonidine (1000) groups were significantly higher than that of the LPS
CAT-2
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Figure 1 Effects of clonidine (10, 100, and 1000 μM) on type-2 cationic amino acid transporter (CAT-2) mRNA expression in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Representative gel photography illustrates the products of RT-PCR. PCR products were analyzed using the densitometric technique. Data were obtained from three independent analyses of freshly harvested cell samples and the CAT-2 mRNA concentrations were normalized by β-actin and expressed as mean ± standard deviation. Cell cultures were harvested after exposing to LPS for 18 hours or comparable duration in groups without LPS. PBS = phosphate buffered saline. *p < 0.05 vs. the LPS group; †p < 0.05 vs. the PBS group; ‡p < 0.05 vs. the LPS + clonidine (10) group; §p < 0.05 vs. the LPS + clonidine (100) group.
Clonidine enhances CAT-2 transcription
121 LPS + clonidine group harvested at 6 and 18 hours after LPS were significantly higher than those of the LPS group (p = 0.029 and 0.016, respectively; Figure 2), whereas the CAT-2 mRNA concentrations of the LPS + clonidine group harvested at 12 hours after LPS were comparable to that of the LPS group (Figure 2).
group (p = 0.025 and 0.001, respectively; Figure 1), whereas the CAT-2 mRNA concentrations of the LPS + clonidine (10) group was comparable to that of the LPS group (Figure 1). The CAT-2 mRNA concentrations of the LPS + clonidine (1000) and LPS + clonidine (100) groups were also significantly higher than that of the LPS + clonidine (10) group (p = 0.038 and 0.014, respectively; Figure 1). Moreover, the CAT-2 mRNA concentration of the LPS + clonidine (1000) group was higher than that of the LPS + clonidine (100) group (p = 0.033; Figure 1).
3.3. Effects of clonidine on the stability of CAT-2 mRNA Figure 3 illustrates the stability of CAT-2 mRNA as evaluated by the percentage remaining. The estimated half-life of the CAT-2 mRNA of the LPS group was approximately 8 hours (Figure 3). Similarly, the estimated half-life of the CAT-2 mRNA of the LPS + clonidine group was approximately 8 hours (Figure 3).
3.2. Effects of clonidine on the time course of CAT-2 transcription induction The CAT-2 mRNA concentrations of the PBS and clonidine groups harvested at 2, 4, 6, 12 and 18 hours were low (data not shown). The CAT-2 mRNA concentrations of the LPS and LPS + clonidine groups harvested at 2 and 4 hours after LPS were comparable to those of the PBS group. Nevertheless, the CAT-2 mRNA concentrations of the LPS and LPS + clonidine groups harvested at 6, 12 and 18 hours after LPS were significantly higher than those of the PBS group (all p < 0.001; Figure 2). Moreover, the CAT-2 mRNA concentrations of the
4. Discussion Data from this study confirmed our hypothesis and provided the first evidence to demonstrate that clonidine-induced enhancement of CAT-2 transcription involves increases in enzyme transcription
CAT-2 (613 bp)
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Figure 2 Effects of lipopolysaccharide (LPS) and LPS + clonidine (100 μM) on type-2 cationic amino acid transporter (CAT-2) mRNA expression in stimulated murine macrophages (RAW264.7 cells) after exposure to LPS for 2, 4, 6, 12 and 18 hours. Representative gel photography illustrates the products of RT-PCR. PCR products were analyzed using the densitometric technique. Data were obtained from three independent analyses of the freshly harvested cell samples and the CAT-2 mRNA concentrations were normalized by β-actin and expressed as mean ± standard deviation. *p < 0.05 vs. the LPS group.
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Figure 3 The stability of type-2 cationic amino acid transporter (CAT-2) mRNA in murine macrophages (RAW264.7 cells) treated with lipopolysaccharide (LPS) or LPS + clonidine (100 μM). Actinomycin D was added to macrophages at 6 hours after LPS administration to terminate transcription. Three culture dishes from each group were then harvested after exposing them to actinomycin D for 0, 2, 4, 6, 8 and 10 hours, respectively. Total RNA was isolated and the CAT-2 mRNA concentrations were assayed by quantitative real-time PCR assay. The CAT-2 mRNA concentrations were then normalized by rat 18s rRNA. The stability of CAT-2 mRNA, as evaluated by the percentage remaining, was then calculated by comparing the CAT-2 mRNA concentrations in macrophages harvested at different time points after actinomycin D to those in macrophages harvested at 0 hours after actinomycin D, respectively.
induction in endotoxin-activated macrophages. It is well-established that nuclear factor-κB (NF-κB) is a crucial factor for maximal transcription of a wide array of inflammatory molecules, including intercellular adhesion molecular-1, iNOS, cycloxygenase-2, interleukin (IL)-1β, IL-6, and tumor necrosis factorα.12 In a recent report, we demonstrated that NF-κB is involved in clonidine-induced enhancement of iNOS expression.13 As we have previously shown that transcription induction of CAT-2 is mainly regulated by NF-κB,14,15 these results seem to suggest that NF-κB may very likely participate in this clonidine-induced enhancement of CAT-2 transcription induction in activated murine macrophages. Transcription of iNOS induced by endotoxins has also been shown to be regulated by the toll-like receptor (TLR) family, especially TLR-4.16,17 Furthermore, activation of TLR induces the recruitment of myeloid differential factor 88 (MyD88), an intracellular adaptor protein that links to the activation of NF-κB.18 Endotoxin-induced TLR-4 activation also requires the presence of cluster of differention-14 (CD14), a membrane-anchor protein that mediates the recognition of LPS, and myeloid differential-2
(MD-2), an extracellular protein that is associated with the extracellular domain of TLR-4.19−21 Although direct evidence to illustrate the involvement of the CD14/MD-2/TLR-4/MyD88 pathway in regulating the endotoxin-induced CAT-2 transcription is still lacking, existing data seems to support the concept that the CD14/MD-2/TLR-4/MyD88 pathway may also be involved in this clonidine-induced enhancement of CAT-2 transcription induction in activated macrophages. A series of studies are currently being performed in our laboratory to elucidate this further. Previous data indicated that the stability of shortlived mRNAs, including iNOS and cytokines, is regulated by the interaction between RNA-binding factors and the adenosine/uridine-rich (AU-rich) elements (AREs) within three untranslated regions of these short-lived mRNAs that often contain several copies of the motif AUUUA.22 Among the identified RNA-binding factors, the RNA stabilizing factor HuR23 and the RNA destabilizing factors, including ARE/ poly(U) binding factor-1 (AUF-1)24 and tristetraprolin (TTP),25 have been shown to be associated with the stability of iNOS mRNA. To the best of our knowledge, no data are available regarding the pathways that regulate the CAT-2 mRNA stability so far. Judging from the fact that CAT-2 mRNA is a shortlived mRNA, we thus speculate that CAT-2 mRNA stability may be regulated by HuR, AUF-1, and TTP. We have shown that clonidine had significant effects on enhancing the expression of AUF-1 and TTP but not HuR in activated murine macrophages, whereas clonidine did not have significant effects on iNOS mRNA stability.13 One possible explanation is that the enhancement of AUF-1 and TTP is secondary to the increases in iNOS mRNA induced by clonidine. Similar to previous studies,13 the data from this study also revealed that clonidine did not have significant effects on CAT-2 mRNA stability. If the stability of CAT-2 mRNA is subject to the regulation of the aforementioned RNA-binding factors, then it is possible that the enhancement in CAT-2 mRNA induced by clonidine may also have a role in clonidineinduced enhancement of AUF-1 and TTP expression. More studies are needed before further conclusions can be drawn. In summary, clonidine-induced enhancement of CAT-2 transcription in endotoxin-activated murine macrophages involves increases in transcription induction but not mRNA stability.
Acknowledgments This work was supported by a grant from the National Science Council, Taiwan (NSC 93-2314-B195-022) and a grant from Mackay Memorial Hospital (MMH 9415) awarded to Dr Chun-Jen Huang.
Clonidine enhances CAT-2 transcription
References 1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051−64. Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking J, Nathan CF, Peitzman AB, et al. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 1998;187:917−28. Bogle RG, Baydoun AR, Pearson JD, Moncada S, Mann GE. L-arginine transport is increased in macrophages generating nitric oxide. Biochem J 1992;284:15−8. Nicholson B, Manner CK, Kleeman J, MacLeod CL. Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J Biol Chem 2001;276:15881−5. Kakuda DK, Sweet MJ, MacLeod CL, Hume DA, Markovich D. CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J 1999;340: 549−53. Carabine UA, Wright PM, Moore J. Preanaesthetic medication with clonidine: a dose-response study. Br J Anaesth 1991;67:79−83. Soares DM, Rios AA, de Oliveira LF, Resende AC, de Lemos NM, Santos EJ, Correia ML, et al. The effects of nitric oxide synthase inhibitors on the sedative effect of clonidine. Anesth Analg 2001;93:1217−21. Venturini G, Colasanti M, Persichini T, Fioravanti E, Federico R, Ascenzi P. Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug. Biochem Pharmacol 2000;60:539−44. Lin WC, Tsai PS, Huang CJ. Catecholamines’ enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A. Anesth Analg 2005; 101:226−32. Lahti A, Jalonen U, Kankaanranta H, Moilanen E. c-Jun NH2-terminal kinase inhibitor anthra(1,9-cd)pyrazol-6(2H)one reduces inducible nitric-oxide synthase expression by destabilizing mRNA in activated macrophages. Mol Pharmacol 2003;64:308−15. Huang CJ, Tsai PS, Pan WH, Skimming JW. Microdialysis for measurement of hepatic and systemic nitric oxide biosynthesis in septic rats. Acta Anaesthesiol Scand 2005;49: 28−34. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3−9. Su NY, Tsai PS, Huang CJ. Clonidine-induced enhancement of iNOS expression involves NF-kappaB. J Surg Res 2008;149: 131−7.
123 14. Yang CH, Tsai PS, Lee JJ, Huang CH, Huang CJ. NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver. Acta Anaesthesiol Scand 2005;49:468−76. 15. Chu LC, Tsai PS, Lee JJ, Yen CH, Huang CJ. NF-kappaB inhibitors significantly attenuate the transcription of high affinity type-2 cationic amino acid transporter in LPSstimulated rat kidney. Acta Anaesthesiol Taiwan 2005;43: 23−32. 16. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274:10689−92. 17. Matsumura T, Ito A, Takii T, Hayashi H, Onozaki K. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J Interferon Cytokine Res 2000;20:915−21. 18. Janssens S, Beyaert R. A universal role for MyD88 in TLR/ IL-1R-mediated signaling. Trends Biochem Sci 2002;27: 474−82. 19. Shuto T, Kato K, Mori Y, Viriyakosol S, Oba M, Furuta T, Okiyoneda T, et al. Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells. Biochem Biophys Res Commun 2005;338:1402−9. 20. Nagai Y, Akashi S, Nagafuku M, Ogata M, Iwakura Y, Akira S, Kitamura T, et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 2002;3:667−72. 21. da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. Transfer from CD14 to TLR4 and MD-2. J Biol Chem 2001;276:21129−35. 22. Kawahara K, Gotoh T, Oyadomari S, Kajizono M, Kuniyasu A, Ohsawa K, Imai Y, et al. Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells. Brain Res Mol Brain Res 2001;90:165−73. 23. Brennan CM, Steitz JA. HuR and mRNA stability. Cell Mol Life Sci 2001;58:266−77. 24. Blaxall BC, Pende A, Wu SC, Port JD. Correlation between intrinsic mRNA stability and the affinity of AUF1 (hnRNP D) and HuR for A + U-rich mRNAs. Mol Cell Biochem 2002;232: 1−11. 25. Lai WS, Carballo E, Thorn JM, Kennington EA, Blackshear PJ. Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J Biol Chem 2000;275:17827−37.