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Published ahead of print on June 21, 2007, doi:10.1165/rcmb.2006-0402OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 438-446, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0402OC

Neurotropin Demonstrates Cytoprotective Effects in Lung Cells through the Induction of Thioredoxin-1

Yuma Hoshino1, Takayuki Nakamura2,{dagger}, Atsuyasu Sato3, Michiaki Mishima3, Junji Yodoi4 and Hajime Nakamura1

1 Thioredoxin Project, Department of Experimental Therapeutics, Translational Research Center; 2 Department of Organ Preservation Technology, Graduate School of Medicine, Kyoto University Hospital; 3 Department of Respiratory Medicine, Graduate School of Medicine; and 4 Laboratory of Infection and Prevention, Department of Biological Response, Institute for Virus Research, Kyoto University, Kyoto, Japan

Correspondence and requests for reprints should be addressed to Yuma Hoshino, M.D., Ph.D., 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 6068507 Japan. E-mail: yuma{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Neurotropin, a nonprotein extract from inflamed rabbit skin inoculated with vaccinia virus, is well known as an analgesic drug, but its cytoprotective effects have not been explored. Because infection by viruses, such as human T-cell leukemia virus type I and Epstein-Barr virus, induces expression of the redox-regulating molecule, thioredoxin (TRX), we hypothesized that neurotropin would also be capable of regulating the redox balance and could be applied for the therapeutics of lung diseases caused by oxidative stress, such as chronic obstructive pulmonary disease. Neurotropin enhanced mRNA expression of the redox-regulating molecules, glutathione peroxidase and catalase and, particularly, TRX, in human lung adenocarcinoma A549 cells. Neurotropin also increased the cellular TRX content and regulated TRX release from cells. The cytoprotective effects of neurotropin against hydrogen peroxide and cigarette smoke extracts was demonstrated by an attenuation of lactate dehydrogenase release from oxidant-exposed A549 cells and the inhibition of apoptosis. This cytoprotection was linked with reduced activity of intracellular oxidants. Furthermore, neurotropin enhanced TRX expression in mouse lungs and ameliorated cigarette smoke–induced lung injury in mice, suggesting that its cytoprotective effects in lung epithelial cells are mediated through the induction of redox-regulating molecules that reduce intracellular oxidative activity.

Key Words: antioxidants • chronic obstructive pulmonary disease • cytoprotection • therapeutics



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The authors demonstrate antioxidative and cytoprotective properties of an analgesic drug, neurotropin, and associated them with thioredoxin induction. Neurotropin could provide another option for chronic obstructive pulmonary disease therapeutics.

 
Chronic obstructive pulmonary disease (COPD) is a slowly progressive chronic lung disease mainly caused by cigarette smoking. It is the fourth leading cause of death in the United States and its incidence is expected to rise over the course of the next few decades, reflecting the recent increase in cigarette smokers (1). The established therapeutic intervention is bronchodilator administration of beta-adrenergic receptor agonists, anticholinergics, and theophyllines. Glucocorticoid therapy and long-term home oxygen therapy are limited for patients with severe COPD. These interventions temporarily improve the disease status, but do not substantially slow disease progression.

Although smoking cessation dramatically improves the clinical course in most cases, a large proportion of smokers fail to stop smoking. Moreover, it is also reported that lung inflammation persists in patients with COPD even if they quit smoking (2). A "pathogenesis-based" strategy is therefore of interest to slow disease progression. It is widely recognized that inflammation, oxidative stress, and apoptosis, followed by matrix breakdown through enhanced proteolytic activity, are the major pathogenetic processes of COPD (3). Several interventions of these processes have been explored with limited success: the potent antiinflammatory agent, corticosteroid, failed to arrest the rate of decline in COPD lung function, although it decreased the frequency of exacerbation (4). Specific antiinflammatory agents, such as phosphodiesterase-4 inhibitors, are now under development (5–7). In addition, the clinical effectiveness of antioxidants, such as N-acetylcysteine, is also being investigated, but has not yet been established (8).

Thioredoxin (TRX)-1 is a ubiquitous, redox-acting small protein of 105 amino acids with a conserved -CXXC- construct in its active site that exchanges dithiol to disulfide to maintain the redox status of other molecules. TRX is inducible under various stress conditions (9, 10) and, when present in the serum/plasma, is a useful biomarker for inflammatory diseases, such as rheumatoid arthritis, acute lung injury, and asthmatic attack (11–13). Cigarette smoking is also associated with elevated serum TRX (14).

Administration or overexpression of recombinant TRX is effective in a wide variety of in vivo disease models, such as viral pneumonia, acute lung injury, myocarditis, and pancreatitis (15–18). Indeed, we recently found that TRX administration ameliorated cigarette smoke–induced lung inflammation and injury in mice (unpublished data). Although it has the potential for COPD therapeutics, prolonged TRX administration to humans would be impractical. A TRX inducing agent is therefore a more suitable alternative, and several have been reported (19–21), but their level of TRX induction in the lung is either minimal or unknown.

Neurotropin is a nonprotein extract from inflamed rabbit skin inoculated with vaccinia virus. It has widely been used in clinical settings as an analgesic drug against lumbago, shoulder, arm, and neck syndrome, sequelae of subacute myelo-opticoneuropathy, and postherpetic neuralgia. As an analgesic, it inhibits the kallikrein–kinin system (22), but its cytoprotective effects have not been explored. As infection by viruses, such as human T-cell leukemia virus type 1 and Epstein-Barr virus, induces TRX expression (23), we hypothesized that neurotropin could be used as a novel TRX inducer in a therapeutic strategy against lung diseases caused by oxidative stress, such as COPD. We therefore explored the cytoprotective effects and antioxidant-inducing capacity of neurotropin in lung cells both in vitro and in vivo. We observed cytoprotective effects, mainly through induction of TRX, against a wide variety of oxidative injuries.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
In Vitro Cytotoxicity
A549 lung epithelial cells were cultured until confluent and then cultured in a serum-free, thiol-depleted Dulbecco's modified Eagle's medium (GIBCO/Invitrogen, Gaithersburg, MD). Neurotropin was added to the medium at specified doses and cells were incubated for 16 hours. Cells were exposed to 0.3 mM hydrogen peroxide for 6 hours, after which lactate dehydrogenase (LDH) release to the culture medium was assessed using the Cytotoxicity Detection kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Absorbance of the final solution was detected using a microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA). LDH release was expressed as a ratio of the total cellular LDH content.

Apoptosis Assay by Flow Cytometry
Apoptotic cell death of A549 cells was assessed using annexin V–FITC Apoptosis Detection kit I (BD Biosciences, San Jose, CA) according to manufacturer's instruction. After 6-hour exposure to hydrogen peroxide, cells were trypsinized and 105 cells were incubated with annexin V–FITC and propidium iodide (PI) for 15 minutes. The cell suspension was applied to a flow cytometer (FACSCalibur; BD, Franklin Lakes, NJ).

Morphological Assessment of Cell Death
A549 cells were cultured on chamber slides (LabTek ChamberSlide System; Nalge Nunc International, Tokyo, Japan). Cells were treated with 0.3 mM hydrogen peroxide in the presence or absence of neurotropin for 24 hours, then stained with 1 µg/ml PI and Hoechst 33,342 (Sigma-Aldrich, St. Louis, MO) and observed under a fluorescent microscope (BZ-8000; Keyence, Osaka, Japan).

Conditioning of Cigarette Smoke Extract
Cigarette smoke extract was prepared using a smoke generator (SG-200; Shibata Scientific, Tokyo, Japan). Ten research-standard cigarettes (2R4F; Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY) were lit and their smoke was bubbled through 10 ml HEPES-buffered Dulbecco's modified Eagle's medium (GIBCO/Invitrogen). The extract was filtered through a 22-µm filter (Millipore, Billerica, MA).

Intracellular Oxidative Activity
Intracellular oxidative activity was assessed as described previously (24). Briefly, A549 cells were pretreated with neurotropin for 16 hours and then exposed to 0.3 mM hydrogen peroxide for 3 hours. Cells were trypsinized and the cell suspension was conditioned with 106 cells. Cells were then incubated with 1 µM CM-H2DCFDA, an oxidant-reactive fluorogenic dye (Molecular Probes/Invitrogen, Carlsbad, CA), for 20 minutes at 37°C. Cells were washed with PBS and green fluorescence was analyzed using a FACSCalibur flow cytometer as an index of oxidative stress.

Real-Time PCR of Antioxidative Molecules
mRNA expression levels of catalase (CAT), glutathione peroxidase (GPX), glutamate–cysteine ligase catalytic subunit (GCLC), glutamate–cysteine ligase modifier subunit (GCLM), and TRX was assessed using real-time PCR. Total RNA was isolated from cultured A549 cells using TRIzol (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) and cDNA was amplified with specific primers and fluorogenic probes in a Taqman gene expression assay (Applied Biosystems, Foster City, CA) using an ABI7300 Real Type PCR system (Applied Biosystems). The probes used in this study for CAT, GPX, GCLC, GCLM, TRX, and 18S were Hs00156308_m1, Hs00829989_gH, Hs00155249_m1, Hs00157694_m1, Hs00828652_m1, and Hs99999901_s1, respectively. The expression of each antioxidative molecule was given as the ratio to the level of 18S expression.

Western Blot of Human TRX
TRX protein levels in A549 cells were evaluated by Western blot. Cell lysates were run on SDS-PAGE followed by Western blotting with a mouse monoclonal antibody to human TRX (ADF-11; Redox Bioscience, Kyoto, Japan) as described previously (25).

Glutathione Recycling Assay
Cellular glutathione content was evaluated using BIOXYTECH GSH/GSSG-412 (OxisResearch, Portland, OR) according to manufacturer's instruction. After trypsinization, cells were sonicated in 5% metaphosphatic acid. The supernatant was incubated with chromogen (5,5'-dithiobis-2-nitrobenzoic acid) and glutathione reductase for 5 minutes. Immediately after an addition of nicotinamide adenine dinucleotide phosphate reduced, changes of absorbance at 412 nm were recorded using a microplate reader (VERSAmax; Molecular Devices). The concentration of glutathione was expressed per milligram protein.

Animal Experiments
The 6- to 8-wk-old male C57BL6/J mice (Japan SLC, Shizuoka, Japan) were injected intraperitoneally with neurotropin (gift from Nippon Zoki Pharmaceutical, Osaka, Japan). After 24 hours, mice were anesthetized and killed by exsanguination. Lungs were extracted, inflated, and fixed with formalin using tracheal cannulation.

To assess the protective effects of neurotropin in vivo, mice were exposed to 3% diluted main-stream cigarette smoke in a plexiglass box for 60 minutes daily for 3 days. Mice were intraperitoneally injected with 1 NU/kg neurotropin or saline on Days 0–3. The smoking apparatus and cigarettes used were the same as in the in vitro study. To assess lung inflammation, bronchoalveolar lavage (BAL) was performed with 1 ml cold saline five times. Lungs were then extracted as before for histologic examination.

The collected BAL fluid was used for the preparation of cytospin using a Shandon Cytospin 4 Cytocentrifuge (Thermo Scientific, Waltham, MA). The cytospin slides were stained with DiffQuik (Sysmex International Reagents, Kobe, Japan). At least 500 inflammatory cells were counted under a light microscope for each slide.

All procedures involving animals were performed in accordance with protocols approved by the Institute for Virus Research at Kyoto University, Kyoto, Japan.

Immunohistochemistry
Immunohistochemistry for mouse TRX was performed using a modification of the previously published method (25). Paraffin-embedded lung sections were prepared for histologic analysis, including TRX and ssDNA expression. Rabbit antisera to mouse TRX (1:500 dilution; Redox Bioscience) was used as a primary antibody and immunostaining was performed using the VECTASTAIN ABC kit (AK-5001; Vector Laboratories, Burlingame, CA) followed by development with the Alkaliphosphatase Substrate kit I (SK-5100; Vector Lab). TRX immunoreactivity was quantified with a modified method of De and colleagues (26). Briefly, a full-color microscopic image (Figure 7E, left panel) was split into RGB and green channel was subtracted from red channel using ImageJ software (http://rsb.info.nih.gov/ij/). The mean gray value of the resulting gray-scale image (Figure 7E, right panel) was calculated as an index of TRX immunoreactivity.


Figure 1
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  		Figure 1. Neurotropin treatment induced antioxidative molecules in A549 cells. (A) A549 cells were treated with different concentrations of neurotropin (white bars, 0 NO/ml; light gray bars, 0.001 NO/ml; dark gray bars, 0.010 NO/ml; black bars, 0.100 NO/ml) for 9 hours. mRNA expression levels of thioredoxin (TRX), catalase (CAT), and glutathione peroxidase (GPX) were significantly enhanced, as quantified by real-time PCR. *P < 0.05 compared with medium-only conditions. (B) TRX induction was time-dependent with a peak at 9 hours of incubation. {dagger}P < 0.05 compared with 0-hour incubation. GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modifier subunit.

 

Figure 2
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  		Figure 2. Neurotropin enhanced TRX protein production in A549 cells. (A) A549 cells were treated with indicated concentrations of neurotropin for 24 hours. Concentration-dependent TRX induction was observed. *P < 0.05 compared with medium-only conditions. Representative immunoblots are shown below each graph. (B) A549 cells were treated with 0.01 NU/ml neurotropin for the indicated time. TRX induction in the cell lysate was assessed by Western blotting, peaked after 9-hour incubation, and then gradually decreased. {dagger}P < 0.05 compared with 0-hour incubation. (C) A549 cells were treated with indicated concentrations of neurotropin for 24 hours. The neurotropin treatment did not affect cellular glutathione content.

 

Figure 3
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  		Figure 3. Neurotropin regulated TRX release from A549 cells. A549 cells were incubated with neurotropin (white bars, 0 NO/ml; light gray bars, 0.001 NO/ml; dark gray bars, 0.010 NO/ml; black bars, 0.100 NO/ml) in the presence or absence of hydrogen peroxide for 6 hours. TRX release into the culture supernatant was evaluated using ELISA, and was unaffected by hydrogen peroxide. Neurotropin treatment suppressed TRX release from the cells.

 

Figure 4
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  		Figure 4. Neurotropin (white bars, 0 NO/ml; light gray bars, 0.001 NO/ml; dark gray bars, 0.010 NO/ml; black bars, 0.100 NO/ml) attenuated cytotoxicity induced by various oxidative stresses. (A) A549 cells were cultured in thiol- and serum-depleted conditions (left), with hydrogen peroxide (right), or with cigarette smoke extract (CSE) (B) in the presence of neurotropin. After 6-hour treatment, lactate dehydrogenase release into the media was assessed as an index of cytotoxicity. *P < 0.05 compared with neurotropin-free conditions; {dagger}P < 0.05 compared with hydrogen peroxide or CSE treatments.

 

Figure 5
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  		Figure 5. Neurotropin attenuated apoptosis induced by oxidative stimuli. A549 cells were cultured with serum (A), in serum- and thiol-depleted conditions (B and D), or with hydrogen peroxide (C and E) for 24 hours with (D and E) or without (A–C) 0.01 NU/ml neurotropin. Cells were stained with propidium iodide (PI) and Hoechst 33,342 and observed under a fluoroscent microscope. Both oxidative stresses induced apoptosis, as shown by condensed, intensely fluorescent nuclei. Neurotropin treatment reduced the number of apoptotic cells. A549 cells were cultured with serum (F) or in serum- and thiol-depleted conditions with (I and J) or without (G and H) 0.01 NU/ml neurotropin. The cells were then exposed to hydrogen peroxide for 6 hours (H and J). Cells were stained with annexin V–FITC and PI, and apoptosis was assessed by flow cytometry. Neurotropin treatment reduced the number of apoptotic cells.

 

Figure 6
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  		Figure 6. Neurotropin attenuated intracellular oxidative activity. (A) A549 cells were cultured either under serum- and thiol-depleted conditions or with 0.3 mM hydrogen peroxide (gray area, control [58%]; white area with black outline, neurotropin [51%]) (B) with or without 0.01 NU/ml neurotropin (gray area, control [69%]; white area with black outline, neurotropin [57%]). Cells were trypsinized and treated with CM-H2DCFDA for 20 minutes, and cellular green fluorescence was measured as an index of oxidative stress using a flow cytometer. Percentages of positive cells for CM-H2DCFDA are indicated in parenthesis.

 

Figure 7
Figure 7
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  		Figure 7. Neurotropin induced TRX and cytoprotection in mice in vivo. Neurotropin was injected intraperitoneally into C57BL/6 mice (A–D: 0, 0.1, 1.0, and 10.0 NU/kg, respectively); 1 NU/kg neurotropin enhanced TRX expression in the lungs, particularly in the epithelial cells of terminal bronchioles and alveoli (C). The immunoreactivity was quantified as described in MATERIALS AND METHODS (E and F). *P < 0.05 compared with saline-injected mice. Mice were exposed to cigarette smoke for 3 days after intraperitoneal injection of neurotropin. At 24 hours after the last exposure, ssDNA immunostaining (G–I) and cell differential in bronchoalveolar lavage fluid (J) was assessed. Neurotropin treatment attenuated cigarette smoke–induced apoptosis and neutrophil inflammation. *P < 0.05 compared with saline-injected mice.

 
Immunostaining for ssDNA was performed using an anti-ssDNA antibody (1:100 dilution; DAKO, Glostrup, Denmark) followed by development with the EmVision+ system (DAKO). The number of positively stained cells was counted and expressed per millimeter alveolar wall.

Statistical Analysis
Data were expressed as mean values (± SD). Statistical analysis was performed using StatView software (SAS Institute, Cary, NC). An analysis of variance was performed followed by Fisher's post hoc test. A P value less than 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
In Vitro Neurotropin Induction of Antioxidative Molecules
We examined the antioxidant-inducing capacity of neurotropin in A549 cells and found that it significantly enhanced the transcription of TRX, CAT, and GPX (Figure 1A), all of which are capable of scavenging hydrogen peroxide. The induction was concentration- and time-dependent (Figures 1A and 1B), and was most prominent on TRX expression. Western blot analysis revealed that TRX induction also occurred at the protein level (Figure 2), with maximum induction occurring after 9 hours of treatment (Figure 2A) and at 0.01 NU/ml neurotropin (Figure 2B). As shown in Figure 3, neurotropin also enhanced the cellular retention of TRX in a concentration-dependent manner. The amount of TRX released is approximately equal with or without exposure to hydrogen peroxide. In contrast to TRX induction, neurotropin did not affect GSH synthesis. Neurotropin did not enhance mRNA expression of glutamate–cysteine ligase (GCLC and GCLM), a rate-limiting enzyme of glutathione synthesis (Figure 1A), or cellular glutathione content (Figure 2C).

In Vitro Cytoprotection by Neurotropin
In thiol-depleted conditions, neurotropin suppressed LDH release from A549 cells induced by exposure to hydrogen peroxide (Figure 4A). The effect was dose-dependent and significant at concentrations above 0.001 NU/ml. A similar effect was observed in cells exposed to cigarette smoke extract (CSE), where 10% CSE induced the same level of cytotoxicity as 0.3 mM hydrogen peroxide; 0.01 NU/ml neurotropin significantly attenuated CSE-induced cytotoxicity (Figure 4B).

As shown in Figures 5F–5J, the exposure to hydrogen peroxide enhanced both apoptotic and necrotic cell death. Treatment with neurotropin suppressed early apoptosis (lower-right quadrant, from 5.21 to 2.34%), but was less effective for suppression of late apoptosis and necrosis (propidium iodide [PI]-positive, from 13.0 to 11.99%). When exposed to the same degree of oxidative stress for a longer period (24 hours), most A549 cells underwent apoptosis, as observed by fluorescent microscopy (Figure 5). However, neurotropin inhibited the apoptosis caused by oxidative conditions of 0.3 mM hydrogen peroxide.

We next examined whether neurotropin attenuated intracellular reactive oxygen species (ROS). Depletion of serum and thiol in A549 media or treatment with 0.3 mM hydrogen peroxide increased cellular oxidative stress, as shown by fluorescence levels of the oxidant-reactive fluorogenic dye, CM-H2DCFDA (Figure 6). Neurotropin treatment decreased the percentage of fluorescence-positive cells in both oxidative conditions, from 58 to 51% and from 69 to 57%, respectively.

In Vivo Cytoprotective Effect and TRX Induction by Neurotropin
TRX is constitutively but weakly expressed in mouse lungs and is localized to airway and alveolar epithelial cells and alveolar macrophages (Figure 7A). To examine the TRX induction by neurotropin in vivo, we injected various concentrations into mice and observed that TRX expression was enhanced, especially in the epithelial cells of terminal bronchioles (Figures 7B–7D). The enhancement was maximal at the dosage of 1 NU/kg up to 5.2-fold (Figures 7E–7F).

Mice were treated with neurotropin or saline and exposed to cigarette smoke to determine the cytoprotective effects of neurotropin. The ssDNA immunostaining revealed that apoptosis was markedly suppressed by neurotropin treatment, but was diffusely exhibited in bronchial epithelia, vascular endothelial cells, and alveolar pneumocytes of saline-treated mice (Figures 7G–7I). Moreover, BAL fluid neutrophilia induced by the same 3-day smoke exposure was also significantly attenuated by neurotropin treatment (Figure 7J).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we demonstrated novel properties of the analgesic, neurotropin. In addition to its analgesic and antiallergic effects previously reported, this drug offered cytoprotective effects against a wide variety of oxidative stresses placed on lung cells. The effects were mediated by the induction of antioxidative molecules, mainly TRX, which attenuated intracellular ROS production.

Neurotropin is an analgesic drug currently used in Japan and China for the treatment of chronic pain refractory to a standard nonsteroidal antiinflammatory drug therapy. Its clinical value has become recognized in western countries, and a clinical trial of neurotropin against chronic pain with complex regional pain syndromes I and II or reflex sympathetic dystrophy and causalgia is now underway in the United States (protocol no. 00-NR-0200; http://clinicalstudies.info.nih.gov/detail/A_2000-NR-0200.html).

The mechanism of neurotropin pain relief is believed to be an inhibition of the kallikrein–kinin system, but without affecting prostaglandins (22, 27). Neurotropin also has antiallergic effects through its suppression of eosinophilic infiltration and the regulation of cholinergic receptors on the nasal mucosa (28, 29). For this reason, it is clinically applied to several allergic diseases, such as allergic rhinitis and pruritus in patients on hemodialysis. Because neurotropin is a mixture of nonprotein molecules that originated from inflamed tissue, it is likely to contain many antioxidative and antiinflammatory molecules involved in acute-phase defense responses. To our knowledge, this study is the first to investigate the antioxidative and cytoprotective properties of neurotropin.

Neurotropin treatment decreased intracellular ROS and inhibited apoptotic cell death, which is not secondary to necrosis, because a subset of cells exhibited early apoptosis on flow cytometry (Figure 5H). The induction of the antioxidative molecules, catalase, superoxide dismutase, GPX, and TRX, after neurotropin treatment likely contributed to apoptosis inhibition. Among these, TRX induction was found to be most prominent. Because superoxide dismutase does not scavenge hydrogen peroxide, we did not examine its induction further. The protective effects of TRX against oxidative lung injury have already been shown in several lung injury models using either TRX-transgenic mice or injection of recombinant protein (16, 30). Taken together with our observation that neurotropin did not alter GSH synthesis, it is likely that the TRX induction accounts for a large part of cytoprotection by neurotropin.

The induction of TRX peaked at 0.01 NU/ml neurotropin in vitro, whereas the cytoprotective effects remained at higher doses of neurotropin. Under the intense oxidative stress of hydrogen peroxide treatment, the degree of TRX induction might differ from that in less oxidative conditions. Moreover, 0.1 NU/ml neurotropin exceeds the physiologic dosage, and we believe that TRX would be a major component of the cytoprotective effects provided at physiologic dosages of neurotropin. Indeed, TRX was reported to be exclusively important for cytoprotection of bronchial epithelial cells against oxidative injury (31). The inhibition of apoptosis signal–regulating kinase-1 (ASK-1) by TRX is a well-known mechanism of its antiapoptotic effect (32). We can therefore assume that antiapoptosis achieved by neurotropin would also be associated with the regulation of the TRX–ASK-1 interaction.

The cytoprotective and antioxidative effects of neurotropin suggest that it might have therapeutic applications for lung diseases in which oxidative stress is involved, such as COPD, bronchial asthma, cystic fibrosis, and interstitial lung diseases (33). As the drug can be administered orally or intravenously, it is applicable for the treatment of both chronic and acute lung diseases. Moreover, it has already proved safe for long-term use, whereas TRX has not yet been administered to humans. Small molecules, such as TRX inducers, are also usually less expensive than larger protein compounds.

TRX is a multifunctional protein with antiinflammatory, antiapoptotic, and antioxidative effects. Interestingly, recent studies suggest that its antiinflammatory effects are not necessarily dependent on its reducing function (34). As shown in Figure 7, neurotropin has antichemotactic effects in smoke-exposed mice, which we observed previously using TRX overexpression or TRX injection (unpublished data). We can therefore speculate that, aside from its antioxidative and antiapoptotic effects, neurotropin could also be used in an antiinflammatory strategy in the therapeutics of pulmonary disorders.

Recently, several studies have used alternative TRX-inducing agents, such as geranylgeranylacetone, temocapril, and sulforaphane, for therapeutic purposes (19–21). Geranylgeranylacetone, an antiulcer drug, suppresses ethanol-induced cytotoxicity in hepatocytes through induction of TRX (19). Sulforaphane, a naturally occurring isothiocyanate that is highly concentrated in broccoli sprouts, induces TRX through the antioxidant-responsive element and attenuates retinal light damage in mice (21). Temocapril, an angiotensin-converting enzyme inhibitor, also has the potential to induce TRX and ameliorates autoimmune myocarditis in mice (20). Although statins are not TRX inducers, they enhance TRX activity by S-nitrosylation of cysteine 69 of the TRX protein (35); however, this has not been clearly shown in the lungs. Lung TRX expression is enhanced in pathologic conditions, such as interstitial lung diseases or lung cancer, reflecting the stress response and accompanying structural damage of lung tissue (36, 37). Our in vivo experiments demonstrated that TRX was constitutively but weakly expressed by bronchial epithelial cells, alveolar pneumocytes, and alveolar macrophages, consistent with previous reports and typical of a first-line defense against airborne particles. TRX expression was augmented by neurotropin treatment in the same cell types, but was not inducible in other cell types. It is of note that the TRX protein induced by neurotropin was distributed in the cytoplasm and not the nuclei, and that the cell shape remained intact. To our knowledge, this is the first report of TRX induction in the lungs in the absence of pathologic conditions.

As shown in Figure 7, TRX attenuated apoptosis of airway epithelial cells and endothelial cells exposed to cigarette smoke. Because lung cell apoptosis is a crucial step in the development of emphysema (38, 39), our observations strongly suggest a potential for neurotropin in COPD therapeutics. It is likely that TRX-associated antiapoptotic mechanisms, such as ASK-1/TRX interactions, were involved in TRX induction in airway epithelia, although this is not the case for endothelial cells in which TRX was not inducible. In these cells, two mechanisms are possible: first, TRX secreted from macrophages or epithelial cells might block apoptotic signals directing endothelial cells. Second, antioxidative molecules other than TRX could be induced by endothelial cells for cytoprotection. In our experiment, neurotropin treatment attenuated neutrophil inflammation to the lungs, which could also mediate lung cell death. This might explain the first mechanism. Further investigation is required to clarify the precise mechanisms of antiapoptosis.

In the present study, TRX induction peaked at 0.01 NU/ml neurotropin in vitro and at 1 NU/kg in vivo. The concentrations in the two settings are comparable given that the extracellular fluid volume is 20% of body weight and that injected neurotropin is completely absorbed and equally distributed. In a rat model of chronic pain, an analgesic effect of neurotropin was achieved at dosages ranging from 10 to 100 NU/kg (40). By contrast, in clinical settings for pain management, 0.1 NU/kg neurotropin was injected daily, suggesting that a lower dosage is sufficient for humans but not for animal models. It is therefore assumed that the concentrations of neurotropin required for TRX induction in the present study are attainable in clinical settings.

As shown in both in vitro and in vivo studies, TRX induction efficacy fell at higher doses of neurotropin. This could be because neurotropin is a mixture of nonprotein molecules containing both TRX-inducible and -inhibitory molecules. At higher doses, the inhibitors might override the effect of the inducers, so further work is required to determine which fractions of this nonprotein mixture offer maximum induction efficacy.

The thiol-depleted conditions used for in vitro experiments in the present study aimed to mimic conditions of chronic oxidative stress, such as chronic smoking (41–43). However, even under such conditions, we observed enhanced TRX production after neurotropin treatment. The reason for this is unclear, but it is unlikely to be enabled by the cysteine contained by neurotropin, because TRX induction was observed at a transcriptional level.

TRX induction is regulated by transcription factor binding to specific protein (SP)-1 sites, antioxidant responsive elements or cyclic AMP responsive elements in its 5' flanking sequence (44–46). To identify the binding site involved, we performed a luciferase promoter assay, but were unable to identify the promoters involved. It is conceivable that transcriptional regulation of TRX by neurotropin is more complex than regulation of a single transcription factor. Alternatively, neurotropin treatment might increase the mRNA stability of TRX. However, our time course experiments demonstrated that 9 hours was the optimal time for TRX induction, suggesting that de novo protein synthesis is required. Further examination is required to determine the precise mechanism.

Finally, neurotropin appears to enhance the cellular retention of TRX through mechanisms that are not based solely on cytoprotection, because TRX release was not significantly enhanced by hydrogen peroxide treatment. It is arguable that only a short-term induction of TRX mRNA is sufficient for the cytoprotection by neurotropin. It is possible that TRX induction lasts longer than observed in the time-course study because the serum- and thiol-free condition itself decreased basal TRX expression. Alternatively, these unknown mechanisms (enhanced TRX retention) might also facilitate cellular antioxidative defenses. Interestingly, it was recently reported that extracellular TRX enhances the retention of intracellular TRX under oxidative conditions (47).

In conclusion, we suggest that neurotropin has the potential for COPD therapeutics by means of its cytoprotective effects against oxidative lung injury, mainly through TRX induction.


   Acknowledgments
 
The authors thank A. Yamada, M. Takenaka, S. Furukawa, and A. Teratani for excellent technical assistance. This study is dedicated to the memory of Dr. Takayuki Nakamura, who passed away during manuscript review.


   Footnotes
 
{dagger} Deceased. Back

This work was supported by funds from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Redox Bioscience, Inc. (Kyoto, Japan).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0402OC on June 21, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 26, 2006

Accepted in final form March 2, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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