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Effect of Thiol-Containing Molecule Cysteamine on the Induction of Inducible Nitric Oxide Synthase in Hepatocytes

Takashi Ozaki, MD, PhD^*, Masaki Kaibori, MD, PhD^*, Kosuke Matsui, MD, PhD^*, Katsuji Tokuhara, MD^*, Hironori Tanaka, MD^*, Yasuo Kamiyama, MD, PhD^*, Mikio Nishizawa, MD, PhD, Seiji Ito, MD, PhD and Tadayoshi Okumura, PhD

From the ^* Department of Surgery and the Department of Medical Chemistry, Kansai Medical University, Moriguchi, Osaka, Japan

Correspondence: Tadayoshi Okumura, PhD, Department of Medical Chemistry, Kansai Medical University, 10–15 Fumizonocho, Moriguchi, Osaka 570-8506, Japan. Electronic mail may be sent to okumura{at}takii.kmu.ac.jp.

Background: Cysteamine, which is a known antioxidant and anti-inflammatory agent, is believed to be a key regulator of essential metabolic pathways in organisms. Cysteamine has beneficial effects in liver damaged by a variety of insults. During liver injury, inducible nitric oxide synthase (iNOS) is induced by lipopolysaccharide or proinflammatory cytokines, leading to excessive nitric oxide (NO) production. Accumulated evidence indicates that NO is an important factor associated with hepatic dysfunction. We examined whether cysteamine influences the induction of iNOS in hepatocytes. Methods: Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence and absence of cysteamine. NO production, iNOS induction, and iNOS signal were analyzed. Results: IL-1β stimulated the inhibitory protein B (IB)/nuclear factor B (NFB) pathway, resulting in the activation of NFB (nuclear translocation and DNA binding), which was followed by the induction of iNOS and NO production. The addition of IL-1β and cysteamine (1–4 mmol/L) markedly inhibited NO production, with a maximal effect at 4 mmol/L (80%–90% inhibition). Cysteamine also decreased the levels of iNOS protein and mRNA. Transfection experiments revealed that cysteamine decreased the transactivation activity of the iNOS promoter. An electrophoretic mobility shift assay demonstrated that cysteamine inhibited the activation of NFB. Furthermore, cysteamine decreased the mRNA levels of the NFB subunit p65 but increased those of the inhibitory protein IB. Conclusions: These findings suggest that cysteamine inhibits iNOS induction at the step of NFB activation. Further study is necessary to define the molecular basis of this effect of cysteamine on the regulation of NFB and its pharmacologic implications.

Cysteamine, which is a known antioxidant and anti-inflammatory agent, is broadly distributed in organisms and is believed to be a key regulator of essential metabolic pathways. It acts through sulfhydryl-disulfide exchange reactions in vivo. Cysteamine is used to prevent damage caused by the buildup of cystine crystals in various organs, as in the therapy for nephropathic cystinosis. Cysteamine also has beneficial effects in liver damaged by a variety of insults, including ischemia-reperfusion, endotoxin, and hemorrhagic shock.^1–3

Recent accumulated evidence indicates that nitric oxide (NO) is an important factor associated with hepatic dysfunction. The level of inducible nitric oxide synthase (iNOS) in the liver is negligible under physiologic conditions. During liver inflammation, lipopolysaccharide (LPS) or proinflammatory cytokines, such as tumor necrosis factor (TNF)- and interleukin-1β (IL-1β), stimulate the induction of iNOS gene expression, which is followed by the production of excessive levels of NO. The promoters of the mouse, rat, and human genes encoding iNOS contain a consensus sequence for the binding of a transcription factor, nuclear factor B (NFB),^4–7 which is necessary for the induction of iNOS by proinflammatory cytokines including IL-1β, TNF-, and interferon-.⁸ NFB typically exists in the form of a p50/p65 heterodimer attached to its inhibitory protein (inhibitory protein B, IB) in the cytoplasm of cells.⁹ Activation of NFB involves (i) proteolytic degradation of IB in the proteasome after phosphorylation by IB kinase,^10,11 (ii) translocation of NFB to the nucleus, and (iii) its binding to the B site in the promoter of the target gene.

In this study, we investigated whether cysteamine influences the induction of iNOS in primary cultures of rat hepatocytes and, if so, what its mechanism of action is.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Materials
Recombinant human IL-1β (2 x 10⁷ units/mg protein) was provided by Otsuka Pharmaceutical Co (Tokushima, Japan). Cysteamine was purchased from Sigma (St. Louis, MO). All other chemicals were of reagent grade. Animal experiments were approved by the Animal Care Committee of Kansai Medical University.

Cultures
Hepatocytes were harvested from male Wistar rats (200–250 g) by collagenase (Wako Pure Chemicals, Osaka, Japan) perfusion, as described previously.¹² The isolated hepatocytes were suspended in culture medium at 6 x 10⁵/mL, seeded into plastic dishes (2 mL/35 mm; Falcon Plastic, Oxnard, CA), and then cultured at 37°C in a CO₂ incubator under a humidified atmosphere of 5% CO₂ in air. The culture medium was Williams' medium E supplemented with 10% newborn calf serum, Hepes (5 mmol/L), penicillin (100 units/mL), streptomycin (0.1 mg/mL), dexamethasone (10 nmol/L), and insulin (10 nmol/L). After 7 hours, the medium was replaced with fresh serum- and hormone-free medium; cells were cultured overnight and then used in experiments. The purity of isolated hepatocytes was >99% by microscopic observation.¹³ The number of cells attached to the dishes was calculated by counting the nuclei¹⁴ and using a ratio of 1.37 ± 0.04 nuclei per cell (mean ± SE; n = 7).

Treatment of Cells With Cysteamine and Measurement of NO Production
On day 1, cultured hepatocytes were washed with fresh serum- and hormone-free medium and then treated with IL-1β in the same medium in the presence or absence of cysteamine. Cells were placed on ice after the indicated times, and the accumulation of nitrite (NO ^–₂), a stable metabolite of NO, in the medium was determined by the method of Green et al.¹⁵

Western Blot Analysis
Cells (35-mm dish) were lysed in 100–200 µL of solubilizing buffer (10 mmol/L Tris-HCl, pH 7.4, containing 1% Triton X-100, 0.5% Nonidet P-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonylfluoride (PMSF), and 100 U/mL Trasylol (Bayer, Leverkusen, Germany), passed through a 26-gauge needle, and incubated on ice for 30 minutes, followed by centrifugation (16,000 x g for 15 minutes). The supernatant (total cell lysate) was mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, subjected to SDS-PAGE, and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Immunostaining was performed using rabbit polyclonal antibodies directed against mouse iNOS (Affinity BioReagents, Golden, CO), human IB, and human IBβ (Santa Cruz Biotech, Santa Cruz, CA) as the primary antibodies and an ECL blotting detection agent (Amersham Co, Amersham, Bucks, United Kingdom).

Northern Blot Analysis
Total RNA was extracted from cultured hepatocytes using the acid guanidinium–phenol–chloroform method.¹⁶ Total RNA (10 µg) was fractionated by 1% agarose-formaldehyde gel electrophoresis, transferred to nylon membranes (Nytran; Schleicher & Schuell, Dassel, Germany), and immobilized by baking at 80°C for 1 hour before hybridization with DNA probes. A cDNA for rat iNOS (830 bp) was provided.¹⁷ A cDNA encoding mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)¹⁸ was prepared by PCR.¹⁹ These cDNAs were radiolabeled with [-³²P]dCTP by the random priming method.

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared at 4°C according to the methods of Schreiber et al,²⁰ with minor modifications,²¹ unless otherwise stated. Briefly, approximately 2 x 10⁶ cells (two 35-mm dishes) were placed on ice, washed with Tris-buffered saline, harvested in the same buffer using a rubber policeman, and centrifuged (1840 x g for 1 minute). The cell pellet was resuspended in 400 µL of lysis buffer (10 mmol/L Hepes, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 500 U/mL aprotinin, 0.5 mmol/L PMSF, and 1 mmol/L dithiothreitol), and cells were allowed to swell on ice for 15 minutes. Twenty-five microliters of 10% Nonident P-40 was added to the lysis buffer, and the tube was vigorously vortexed for 1 minute at room temperature and then centrifuged (15,000 x g for 1 minute). After removal of the supernatant, the nuclear pellet was resuspended in 75 µL of nuclear extraction buffer (20 mmol/L Hepes, pH 7.9, 0.4 M NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 500 U/mL aprotinin, 1 mmol/L PMSF, and 1 mmol/L dithiothreitol). The tube was incubated on ice for 20 minutes with continuous mixing and centrifuged (15,000 x g for 5 minutes). Aliquots of the supernatant were frozen with liquid nitrogen and stored at –80°C until use.

Binding reactions (15 µL total) were performed by incubating an amount of nuclear extract containing 4 µg of protein with reaction buffer (20 mmol/L Hepes, pH 7.9, 1 mmol/L EDTA, 60 mmol/L KCl, 10% glycerol, 1 mg of poly (dI-dC)) in the absence or presence of anti-p50 and anti-p65 antibodies (against NFBp50 [NLS] and NFBp65 [H286]; Santa Cruz Biotech) or cold probe as a competitor (250-fold excess), for 30 minutes, followed by a 20-minute incubation at room temperature with cDNA probes (approximately 40,000 cpm). Products were electrophoresed at 100 V on a 4.8% polyacrylamide gel in high-ionic-strength buffer (50 mmol/L Tris-HCl, 380 mmol/L glycine, 2 mmol/L EDTA, pH 8.5), and dried gels were analyzed by autoradiography. An NFB consensus oligonucleotide (5'-AGTTGAGGGGA-CTTTCCCAGGC) from mouse immunoglobulin light chain was purchased (Promega, Madison, WI) and labeled with [-³²P]ATP using T4 polynucleotide kinase. The protein concentration was measured by the method of Bradford²² with a dye-binding assay kit (Bio-Rad) using bovine serum albumin as a standard.

Transfection and Luciferase Assay
Transfection of cultured hepatocytes was performed according to published methods.^23,24 Briefly, hepatocytes were cultured at 4 x 10⁵ cells per dish (35 x 10 mm) in Williams' medium E supplemented with serum, dexamethasone, and insulin for 7 hours. Then, cells were subjected to magnet-assisted transfection (MATra). The 1.2-kb 5'-flanking region of the rat iNOS gene, including a TATA box, was inserted into pGL3-Basic vector (Promega) to create pRiNOS-Luc.²¹ Reporter plasmid pRiNOS-Luc (1 µg) and CMV promoter-driven β-galactosidase plasmid pCMV-LacZ (1 ng) as an internal control were mixed with MATra-A reagent (1 µL; IBA GmbH, Göttingen, Germany). After a 15-minute incubation on a magnetic plate at room temperature, the medium was replaced by fresh medium without serum. Then, cells were cultured overnight, treated with or without IL-1β, and the luciferase and β-galactosidase activities of cell extracts were measured using PicaGene (Wako Pure Chemicals) and β-Glo kits (Promega), respectively.

Statistical Analysis
Results in the figures are representative of 3–4 independent experiments yielding similar findings. Differences were analyzed by the Bonferroni/Dunn test, and p < .05 was considered to indicate statistical significance.

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Effect of Cysteamine on the Induction of NO Production and iNOS in Hepatocytes
The proinflammatory cytokine IL-1β stimulated the production of NO time-dependently in cultured hepatocytes, as reported previously.^25,26 Simultaneous addition of cysteamine with IL-1β markedly inhibited the production of NO (Figure 1A). The inhibition of NO production with cysteamine was concentration dependent, showing the maximal effect at 4 mmol/L (Figure 1B). Cysteamine alone had little effect on the production of NO. Cysteamine (1–4 mmol/L) was not toxic to cells within the incubation periods, irrespective of the presence of IL-1β, as tested by trypan blue exclusion and the release of lactate dehydrogenase (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Effect of cysteamine on the production of nitric oxide stimulated by interleukin-1β in hepatocytes. A, Time course: cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (CysA, 4 mmol/L) for the indicated times. B, Dose dependence: cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (0.5–4 mmol/L) for 8 hours. NO production was measured as nitrite in the culture medium. Control (without IL-1β and cysteamine, x), Cysteamine (), IL-1β (), IL-1β plus cysteamine (•), mean ± SD (n = 3 dishes), *p < .01 vs IL-1β alone.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Effect of cysteamine on the induction of iNOS protein and mRNA in hepatocytes. Cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (1–4 mmol/L) for the indicated times. A, Western blotting. Cell lysates (50 µg of protein) were subjected to SDS–PAGE through 7.5% gels, followed by immunoblotting with an anti-iNOS antibody; time course (upper panel) and dose dependency (lower panel). Molecular mass markers are shown in kDa on the left. B, Northern blotting. Total RNA (10 µg) was analyzed as described in Materials and Methods. Filters were hybridized with radiolabeled iNOS and glyceraldehyde-3-phosphate dehydrogenase cDNAs. Representative results of 4 independent experiments are shown.

Effects of Cysteamine on the Activation of NFB and the iNOS Promoter in Hepatocytes

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Effect of cysteamine on the activation of NFB in hepatocytes. Cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (4 mmol/L) for the indicated times. Nuclear extracts (4 µg of protein) were analyzed by an electrophoretic mobility shift assay (EMSA). A representative result of 3 independent experiments is shown.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Effect of cysteamine on the transactivation of the iNOS promoter in hepatocytes. The 1.4-kb iNOS promoter construct (pRNOS-Luc-N) was introduced into hepatocytes. Transfected cells were treated with or without IL-1β (1 nmol/L) in the presence or absence of cysteamine (2.5 mmol/L) for 8 hours. Assays of luciferase activity were performed and normalized to β-galactosidase activity. Fold activation is calculated by dividing the normalized luciferase activity with cysteamine and IL-1β by that with reporter alone. Data represent the mean ± SD (n = 3 independent experiments). *p < .01 vs without cysteamine.

Effects of Cysteamine on the Induction of IB (Inhibitory Subunit of NFB) and p65 (Subunit of NFB) in Hepatocytes
IL-1β stimulated the degradation of IB and IBβ (inhibitory proteins of NFB), which was followed by the recovery of each protein to basal levels at 2 hours and 4 hours, respectively. Cysteamine had no effect on the degradation and recovery of IB and IBβ proteins (Figure 5). After stimulation with IL-1β, the mRNA levels of both IB and p65 increased with time, coincident with the activation of NFB. Cysteamine further enhanced the levels of IB mRNA, but, by contrast, it inhibited the increases in the level of p65 mRNA (Figure 6), consistent with the inhibitory effect of cysteamine on NFB activation (Figure 3).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Effects of cysteamine on the degradation of IB protein in hepatocytes. Cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (CysA, 4 mmol/L) for the indicated times. Cell lysates (50 µg of protein) were subjected to SDS-PAGE through 12.5% gels and followed by immunoblotting with anti-IB (top) and IBβ (bottom) antibodies. Representative results of 3 independent experiments are shown.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Effect of cysteamine on the induction of IB and p65 mRNA in hepatocytes. Cells were treated with IL-1β (1 nmol/L) in the presence or absence of cysteamine (CysA, 4 mmol/L) for the indicated times. Total RNA (10 µg) was analyzed by Northern blotting. Filters were hybridized with radiolabeled IB, p65, and glyceraldehyde-3-phosphate dehydrogenase cDNAs. Representative results of 3 independent experiments are shown.

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
In the present study, we found that cysteamine inhibited the induction of iNOS stimulated by the proinflammatory cytokine IL-1β in primary cultured rat hepatocytes. Cysteamine inhibited the activation of NFB signal (Figure 3), which is essential for the transcriptional activation of iNOS, as mentioned before. Transfection experiments with an iNOS promoter construct also demonstrated that cysteamine decreased the transactivation activity of the iNOS promoter (Figure 4). It has been reported that the mRNA levels for NFB inhibitory proteins (IB; IB and IBβ) and NFB subunits (p50 and p65) are immediately increased after stimulation with IL-1β and that the levels of the "inactive complex" of IB/NFB recover to basal levels in the cytoplasm. In the presence of cysteamine, levels of IB mRNA are further increased; by contrast, levels of p65 mRNA decreased (Figure 6), suggesting that cysteamine may accelerate the reconstitution of the inactive complex of IB/NFB and block the translocation of NFB from the cytoplasm to the nucleus.

Western blot analysis showed that there was no significant change in the levels of IB and IBβ proteins (Figure 5), the reason for which remains unclear at present. However, the possibility exists that cysteamine has no effect on the translocation of NFB to the nucleus, but inhibits its binding to B sites in the iNOS promoter, as we have found in our previous reports.^27,28 Pirfenidone, an antifibrotic agent, could not prevent the degradation of IB and the nuclear translocation of NFB (p50/p65 subunits) but inhibited the induction of iNOS stimulated by IL-1β at the step of NFB DNA binding.²⁸ Further, cysteamine probably contributes to the cellular redox state of hepatocytes, as cysteamine acts through sulfhydryl-disulfide exchange reactions in cells. We also reported that a vicinal dithiol-binding regent, phenylarsine oxide, inhibits iNOS gene expression at the step of NFB DNA binding in hepatocytes.²¹ This suggests that the sulfhydryl groups of NFB play a crucial role in the activation of NFB. It is possible that cysteamine regulates the activation of NFB via its thiol group, either directly or indirectly.

Accordingly, cysteamine can prevent the transcriptional activation of the iNOS promoter through the inhibition of NFB activation; this in turn inhibits the induction of iNOS mRNA and protein, resulting in a reduction in NO production. Further investigation is needed to define the mechanisms involved in this effect of cysteamine on iNOS expression in the liver. Animal models of liver injuries are also needed to clarify the effects of cysteamine on the iNOS induction and hepatic protection. The fact that iNOS blockade is not beneficial in critical care patients should be considered. Such studies may provide the foundation for novel pharmacologic approaches to prevent hepatic injury.

The study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan and by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.

Received for publication December 12, 2006. Accepted for publication February 15, 2007.

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Discussant

1. Why did you specifically choose to investigate cysteamine, and how did you administer it to the hepatocytes (eg, chemical/biochemical properties, solvent, solubility, practicalities)?
   
2. You describe cysteamine as a "key regulator of essential metabolic pathways." Please elaborate on these metabolic pathways and how you think cysteamine acts as a key regulator.
   
3. You suggest that cysteamine may regulate activation of NFB via its thiol group. Which additional experiments would you suggest to throw more light onto this proposed mechanism?
   
4. Your paper describes several elegant experimental procedures that are also described in the companion paper by Kosuke Matsui and 11 other authors, including yourself. Which of these methods did you personally validate and what was your (%) contribution to the research work carried out in the cysteamine study?
   

Author's Response

1. Sulfhydryl (HS)-group–containing molecules such as cysteamine, D-cysteine and N-acetyl-D-cysteine have been reported to have protective effects against liver damage. Their antioxidant property is believed to be involved in the protective effects.
   We make 10 mmol/L of cysteamine as a stock solution (Figure A) as follows: cysteamine powder (Sigma) is dissolved in culture medium (Williams' medium E; 0.7715 mg/mL), adjusted to pH 7.4 with 1-N HCl, and followed by a filtration with a 0.45-µm filter (bacteria elimination). Cysteamine (10 mmol/L) is stable for at least 1 month at 4°C. Cysteamine (10 mmol/L) is diluted to various concentrations (0.5–4 mmol/L) with culture medium just before use.
   
2. As shown in Figure B, cysteamine is a key regulator of essential metabolic pathways. Cysteamine is a component of coenzyme A, which plays a crucial role for various metabolic intermediates, including lipid metabolism. Cysteamine is involved in sulfhydryl-disulfide exchange such as glutathione and its disulfide. Cysteamine has been reported to be an antioxidant and O ^–₂ scavenger during inflammation. It is also known that taurine, which is an oxidized product of cysteamine and is metabolized to bile acids, improves liver function.
   
3. We are going to investigate whether cysteamine regulates NFB activation via its thiol group. We will have a plan for additional EMSA experiments with nuclear extracts as follows: we prepare nuclear extracts from IL-1β-treated cells and incubate the extracts in the presence and absence of cysteamine in vitro. Then we examine whether cysteamine directly influences the NFB activation. Other SH-group containing agents such as D-cysteine and N-acetyl-D-cysteine can be compared.
   
4. Most methods involved in iNOS induction have been developed in our laboratory in the last 1–5 years by coworkers, as mentioned in this paper. My coworkers and I developed several methods, including transfection experiments with iNOS promoter constructs. Most of the work in cysteamine study has been done by me.
   

View larger version (23K): [in this window] [in a new window]   FIGURE A.

View larger version (20K): [in this window] [in a new window]   FIGURE B.

Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 5, 366-372 (2007)
DOI: 10.1177/0148607107031005366


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