* Institute of Biochemistry and
Departments of Applied Chemistry and
Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan
Received June 6, 2003; accepted August 11, 2003
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ABSTRACT |
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Key Words: gaseous nitrogen oxides; proliferation; cdk inhibitor (CKI); Rb phosphorylation; cell cycle progression.
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INTRODUCTION |
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Eukaryotic cells have developed precise and well-regulated mechanisms to control progression through the cell cycle (Pardee, 1989). Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of cyclin (regulatory) and cyclin-dependent kinase (cdk; catalytic) subunits. The associations of cyclin D1 and cdk4, cyclin E, and cdk2, and cyclin A and cdk2 have also been shown to phosphorylate rubidium (Rb) in the G0/G1 and the G1/S-phase transitions of the cell cycle (Weinberg, 1995
). Upon phosphorylation, pRb releases and activates a number of proteins such as the E2F family of transcription factors at the G1/S transition phase (Nevins et al., 1997
; Wang et al., 1994
), which in turn regulates the expression of several genes involved in DNA replication, such as dihydrofolate reductase, thymidine kinase, and DNA polymerase
(Izumi et al., 2000
). Regulation of G1 cyclin-cdk activity is also dependent on cdk inhibitory proteins (CKIs), which can bind and inactivate cyclin-cdk complexes (Hunter, 1993
; Hunter and Pines, 1994
; Peters and Herskowitz, 1994
). Several inhibitory proteins have been identified, including p27, p16, and p21, which have been reported to mediate G1 cell-cycle arrest (Hall et al., 1995
).
Our previous work on the action of NOx gas to induce human lung fibroblast cell MRC-5 proliferation demonstrated a relationship between iNOS expression and cell proliferation (Chou et al., 2002). In this current study, we clarify the effect of gaseous NOx on human lung fibroblast cell proliferation by cell cycle-regulatory proteins in order to explore the mechanism of gaseous NOx-mediated lung fibrosis.
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MATERIALS AND METHODS |
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Cell culture.
Human lung fibroblast cells (MRC-5) were maintained as monolayers in MEM supplemented with 10% heat-inactivated fetal-calf serum and PSN (100 units/ml penicillin and 10 µg/ml streptomycin) at 37°C in a humidified atmosphere of 95% air/5% CO2.
Treatment of gaseous NOx.
A gaseous NOx-saturated buffer was prepared by bubbling through phosphate-buffered saline (PBS) with N2 gas for 30 min to deoxygenate the solution, and then bubbling with authentic NO gas (10,000 ppm) for another 30 min. The above-surface space was briefly spurted with N2. This saturated solution had an NO concentration of approximately 1.25 mM, as measured by the ISO-NO analyzer, and was used for cell treatment. MRC-5 cells seeded on 60-mm plastic dishes (5 x 106 cells/dish) were treated with NOx gas-saturated PBS at various dilutions for the indicated time points (024 h) (as a gas-liquid interface culture system). After exposure, the cells were harvested, and cell extracts were prepared for immunoblot and immunoprecipitation analysis.
Preparation of cell extract and immunoblot analysis.
To prepare whole-cell extract, cells were washed with PBS containing zinc ion (1 mM), and then suspended in a lysis buffer (50 mM Tris, 5 mM EDTA, 150 µM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 µg/ml aprotinin, 170 µg/ml leupeptin, and 100 µg/ml phenylsulfonyl fluoride; pH7.5). After 30 min of mixing at 4°C, the mixture was centrifuged at 10,000 x g for 10 min, and the supernatant was collected as whole-cell extract. Protein content of the samples was determined with the Bio-Rad protein assay reagent using BSA as a standard. For Western-blotting analysis, whole-cell extracts (20 µg protein) from control and gaseous NOx-treated samples were resolved on 10% SDSPAGE gels along with pre-stained protein molecular weight standards (Bio-Rad). The separated proteins were then blotted onto NC membrane (0.45 µm, Bio-Rad) and reacted with primary antibodies (against cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, cdk2, cdk4, p27, p21, p16, Rb, phospho-Rb, E2F, and ß-actin as internal control). After washing, the blots were incubated with peroxidase-conjugated goat anti-mouse antibody. Immunodetection was carried out using the ECL Western-blotting detection kit (Amersham Corp., U.K.). Relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized with values of ß-actin.
Immunoprecipitation.
Cell lysates were prepared using lysis buffer: 50 mM Tris, 5 mM EDTA, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 µg/ml aprotinin, 170 µg/ml leupeptin, and 100 µg/ml phenylsulfonyl fluoride; pH7.5. 500 µg of protein from cell lysates was pre-cleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using monoclonal anti-cdk2, -cdk4, and -E2F (Santa Cruz Biotech) antibodies. Immune complexes were harvested with protein A, and immunoprecipitated proteins were analyzed by SDSPAGE, as above. Immunodetection was carried out using monoclonal anti-cdk2, -cdk4, -E2F, and -cyclin D1; polyclonal anti-cyclin A, -cyclin E, and -Rb antibodies.
Assessment of cell viability.
Cells were seeded at a density of 4 x 104 cells/well and exposed to NOx (1 µm) for various periods of time (0, 24, 48, and 72 h). Thereafter, the medium was removed and replaced with 3-(4, 5-dimethylthiazol-2-xl)-2,5-diphenyltetrazolium bromide [MTT, 0.1 mg/ml] for 4 h. The numbers of viable cells was directly proportional to the production of formazan, which was solubilized in isopropanol and measured spectrophotometrically at 563 nm (Mosmann, 1983).
Cell cycle analysis.
To analyze the cell cycle distribution, cells were treated with gaseous NOx-saturated buffer, and then trypsinized and resuspended in 70% ethanol. After incubation on ice for at least 1 h, the cells were resuspended in 1 ml of cell cycle assay buffer (0.38 mM sodium citrate, 0.5 mg/ml RNAse A, and 0.01 mg/ml propidium iodide) at a concentration of 5 x 105 cells/ml. Cell cycle analysis was carried out by use of a flow cytometer and ModFit LT 3.0 software (Verity Software, Topsham, ME).
Statistical analysis.
Results were reported as means ± SD, and statistical analysis was obtained using an unpaired t-test. A value of p < 0.05 was considered statistically significant.
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RESULTS |
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Effect of NOx Gas Enhances Cyclin/Cdk Association
Cell cycle transition from G1 to S requires the temporal activation of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 (Weinberg, 1995). To investigate how NOx gas induces cyclin (Fig. 2A
) and cdk (Fig. 2B
) activation and how it can promote cell-cycle progression, we used immunoprecipitation to ensure that cyclin-cdk complexes were activated to promote cell cycles. As shown in Figure 3
, cyclin D1-cdk4 and cyclin E-cdk2, which control the progression through the G1-phase, increased induction by 2.63- and 2.05-fold at 9 h. Cyclin A-cdk2 induced cell cycles from the S phase to initiation of DNA synthesis, and this was increased by 3.55-fold at 9 h. This result implies that the NOx gas activation of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 complexes did promote cell-cycle transition from G1 to the S phase.
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DISCUSSION |
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In addition, interaction between airway epithelial cells and mesenchymal fibroblast cells has been investigated in human and animal studies of lung development, injury, and repair. (Sanders, 1988; Young and Adamson, 1993
). After lung transplantation, iNOS immunoreactivity could be seen in damaged bronchiolar epithelium that initiated mesenchymal fibroblast cell proliferation (Mason et al., 1998
). It is possible that, after transplantation, the injured epithelial and the destroyed basement membrane could directly expose fibroblasts to the injured epithelial cells, and induce tissue repairing by activation of inflammatory cytokines and growth factors that can stimulate fibroblast proliferation (Nakamura et al., 1995
; Saleh et al., 1997
; Young and Adamson, 1993
). Therefore, NO derived from epithelial and inflammatory cells maybe a key mediator of fibroblast activation. This hypothesis is supported by the finding of this present study, using an in vitro treatment of gaseous NOx to induce the proliferation of human lung fibroblast cells.
Pulmonary fibrosis includes an inflammatory constituent that implicates iNOS expression in the fibrotic lung (MacNee and Rahman, 1995; Saleh et al., 1997
). Gaseous NOx-induced iNOS expression should occur in response to lung fibrosis (Paredi et al., 1999
). Thus, iNOS activation and NO release may be involved in the proliferative response of fibroblasts to NOx gas exposure. It has been proven that NO synthesis activity can regulate NF-
B (Marshall and Stamler, 1999
). NF-
B functions in controlling cell growth by regulation of cyclin D1 expression and G0/G1-to-S phase transition (Hinz et al., 1999
). Hence, our previous data demonstrating that gaseous NOx could activate NF-
B (Chou et al., 2002
) might also relate to the regulation of NOx gas-induced cell cycle progression by Rb phosphorylation via activation of cyclin/cdk complexes.
In conclusion, our previous results demonstrated that gaseous NOx stimulated proliferation via direct and indirect activation of MEKK1, JNK, and p38 signaling pathways (Chou et al., 2002). In this current study, NOx-saturated solution induced cell proliferation through inhibition of cdk inhibitory proteins p27 and p21, and then activated cyclin D1-cdk4 and cyclin E-cdk2 complexes to induce Rb phosphorylation and to promote cell-cycle transition from the G1 to the S phase. Therefore, the results of this study support the hypothesis that NOx and NO may act as profibrotic mediators, thus contributing to the pathogenesis of IPF.
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ACKNOWLEDGMENTS |
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NOTES |
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