Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
1 To whom correspondence should be addressed at Department of Biochemistry, K121, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Fax: (617) 638-5339. E-mail: wandeli{at}bu.edu.
Received March 25, 2005; accepted May 23, 2005
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ABSTRACT |
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Key Words: cigarette smoke condensate; lysyl oxidase relative transcriptional rate; lysyl oxidase mRNA stability; lysyl oxidase promoter activity; collagen type I; elastin.
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INTRODUCTION |
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Cigarette smoke (CS) contains more than 4,000 different constituents, which could act individually or collectively as pathogenic agents for pulmonary diseases (Hoffmann and Wynder, 1999). The particulate phase or cigarette smoke condensate (CSC) is composed of major toxicants such as nicotine, phenol, anthracyclic hydrocarbons, nitrosamines, heavy metals, and chemical carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CS accounted for 81.8% of deaths resulting from chronic obstructive pulmonary disease, mainly manifest as emphysema (American Lung Association of Washington, 2004
; Hoffmann et al., 2001
). Although clinical correlation studies have established a causal relationship between smoking and emphysema (Kimbel, 1985
), the precise mechanisms and cellular events leading to the abnormal enlargement of the airspace in the lung by CS remain unclear (D'Armiento et al., 1990; Wright and Churg, 1995
).
Our studies have shown that exposure of rat fetal lung fibroblasts (RFL6) to CSC induced downregulation of LO at protein and catalytic levels (Chen et al., 2005). LO deficiency is intrinsically relevant to the pathogenesis of emphysema, as evidenced by reduced levels of LO activities in rats, hamsters, or chicks fed with food deficient in copper, a cofactor of LO, associated with the reduction of elastin deposition and evolution of emphysematous lesions in the lung (Harris, 1986
; O'Dell et al., 1978
; Soskel et al., 1984
).
To further define cellular and molecular events in downregulation of LO by CSC in an effort to understand mechanisms for CS-induced emphysema, we have examined modulation of the mRNA expression of LO as well as its substrates, i.e., collagen and elastin by CSC in cultured rat fetal lung fibroblasts. In this paper, we report that CSC strongly inhibited mRNA expression of LO and its substrates in exposed cells and that CSC-induced reduction of LO mRNA stability combined with suppression of LO transcriptional initiation, collectively leading to downregulation of LO at the mRNA level.
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MATERIALS AND METHODS |
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Cell culture.
Rat fetal lung fibroblasts (RFL6) obtained from ATCC were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) as previously described (Chen et al., 2005). Stock cultures were derived from the frozen cell line and passaged every 4 days. A total of 6 passages were used for experiments. To obtain quiescent cultures, cells were plated at 34 x 103 cells/cm2 in 10% FBS/DMEM, incubated for 24 h and then growth-arrested by incubation of cells in DMEM containing 0.3% FBS for 3 days (Li et al., 1995
). Cells were then changed to fresh 0.3% FBS/DMEM before experimental use.
The cell viability assay.
Growth-arrested cells were exposed to CSC for 24 h at indicated doses. Control cells were treated only with vehicle at a final concentration of 0.003 (v/v), and the same control was included in all assays in this study. Cell viability was determined by the trypan blue exclusion assay. Briefly, control and treated cells were detached from dishes by trypsinization and resuspended in PBS at the concentration of 106 cells/ml. Aliquots of cell suspension mixed with the same volume of 0.4% trypan blue/PBS (Sigma, St. Louis, MO) were transferred into the counting chamber of the hemocytometer. The number of membrane-injured cells stained by trypan blue and the total number of cells were counted under the microscope as described (Elia et al., 1993).
Northern blot analysis.
Total RNA was isolated from control and CSC-treated cells by TRIzol reagent (Invitrogen, Carlsbad, CA). The steady-state concentrations of mRNA for LO, collagen type I, tropoelastin, ß-actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Northern blot, using the corresponding 32P-labeled cDNA probe as described (Li et al., 1995). Briefly, equal amounts of RNA (15 µg), quantified by spectrophotometry at 260 nm, were size-fractionated by electrophoresis on a 1% (wt/vol) agarose gel. RNA was transferred to a GeneScreen nylon membrane (NEN, Boston, MA) and immobilized by ultraviolet crosslinking. Approximately 25 ng of the cDNA probe for LO, collagen type I, tropoelastin, ß-actin, or GAPDH were labeled with 32P using Ready-to-Go DNA labeling beads (dCTP) from Amersham Biosciences (Piscataway, NJ). The RNA blots on the membrane were hybridized with 32P-labeled cDNA at 68°C in an ExpressHyb hybridization buffer (Clontech, Palo Alto, CA). The membrane was washed with 2x SSC at room temperature, then with 0.2x SSC at 50°C and exposed to Kodak XAR-5 film at 80°C with an intensifying screen. Autoradiograms were analyzed with the 1D Scan EX software for densities of mRNA bands (Scananalytics, Fairfax, VA) (Chen et al., 2005
).
The nuclear run-on assay.
The nuclear run-on assay was employed to determine relative rates of LO transcription in control and CSC-treated cells as described (Schubeler and Bode, 2005). For isolation of nuclei, cell pellets were gently resuspended in a lysis buffer (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM TrisHCl, pH 8.0, 1 mM DTT, 0.5 mM PMSF, 0.5% [v/v] NP-40) and incubated on ice with intermittent microscopic examination for nuclear integrity. The nuclei were centrifuged at 500 x g and resuspended in a nuclear freezing buffer (50 mM TrisHCl, pH 8.0, 40% [v/v] glycerol, 5 mM MgCl2, 0.1mM EDTA, 1 mM DTT, 0.5 mM PMSF) either for direct use or for storage in liquid nitrogen (Dyer and Herzog, 1995
). For the nuclear run-on reaction, 100 µl of thawed nuclei were mixed with 30 µl of a 5x run-on buffer (25 mM TrisHCl, pH 8.0, 12.5 mM MgCl2, 0.75 M KCl, 1.25 mM each of ATP, GTP, and CTP) containing 100 µCi
-32P-UTP and 5 µl of the Sarkosyl stock to give a final concentration of 0.06%. The mixture was incubated for 30 min at 30°C; then 15 µl of DNase I (1U/µl) were added, and the incubation continued for another 15 min. RNA was isolated by a single-step TRIzol extraction, and the incorporation of 32P determined by
counting. Plasmids containing LO cDNA, GAPDH cDNA, or histone 3B cDNA, or without insert, were slot-blotted onto the nitrocellulose membrane using a BioRad BioDot SF apparatus. The blots were prehybridized in 1% SDS/10% dextran sulfate, 1.4 M NaCl and 325 µg/ml each of herring sperm DNA and yeast tRNA for 2 h at 60°C, followed by treatment with RNasin plus DTT. Radiolabeled RNAs were hybridized onto filters for 2 days. The filters were then washed, dried, and autoradiographed on preflashed film.
The assay for LO mRNA stability.
The half-life of LO mRNAs was assessed by an inhibitor-chase assay (Dani et al., 1984). Cells were incubated with 5 µg/ml of actinomycin D (Sigma, Saint Louis, MO) in the absence or presence of CSC at indicated doses. Cultures were harvested at various times during the actinomycin D-chase. Total RNA was isolated and analyzed by Northern blots using the 32P-labeled rat LO cDNA probe. After hybridization and autoradiography, band densities were measured by analysis with the 1D Scan EX software. For time-course studies, using mRNA levels at time zero of actinomycin D treatment as 100%, the t1/2 values for mRNA decay were determined from semilog plots of integrated densities versus time. For dose-response assays, using mRNA levels after a 24-h actinomycin D-chase in cells incubated in the absence of CSC as a control, mRNA levels in cells treated with various doses of CSC were measured and expressed as percent of the control.
LO promoter-luciferase construct.
To make LO promoter-reporter gene construct, using the rat genomic DNA extracted from RFL6 cells as a template, PCR amplifications of the rat LO promoter were carried out with following primers: (sense, 5'-GACTGAGCTCGAATGCACTAGGAAAGTCTGGAGGA-3') and (antisense, 5'-GTCACCCGGGATGATGCTCCCGGCTCGTCCCTTCT-3') in the presence of high-fidelity Taq DNA polymerase (Invitrogen, Carlsbad, CA), yielding an approximately 1.9 kb fragment of the LO promoter. The LO-specific sequences for the primer design as described here are based on the rattus norvegicus chromosome 18 WGS supercontig NW_047513 (Rat Genome Sequencing Consortium, 2003). The amplified LO promoter fragment was restricted with Sac I and Xma I and ligated into similarly restricted plasmid pGL3-basic (Promaga, Madison, WI) upstream of the luciferase gene. The predicted initiation site (CTT) for transcription was designated as +1, whereas the start codon (ATG) for translation was located at +287. This fragment of the LO promoter covers 1,579 bp before the predicted initiation site of transcription (Fig. 4A).
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Statistical analysis.
In cell viability and LO promoter activity assays, data are presented as mean ± standard deviation (SD) of three separate experiments, in which each control or variable was assessed in triplicate dishes. A one-way analysis of variance, followed by Dunnett test was performed. Differences were considered significant at p < 0.05.
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RESULTS |
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Steady-State mRNA Levels in RFL6 Cells Treated with CSC
To assess LO transcriptional expression in RFL6 cells exposed to CSC, steady-state mRNA levels of LO were determined by Northern blot. As shown (Fig. 1), consistent with prior observations, two LO transcripts were seen in the blot at approximately 5.8 and 4.5 kb, presumably due to alternative polyadenylation of the transcript (Li et al., 1995). Treatment of cells with CSC induced a dose-dependent inhibition of LO steady-state mRNAs. As revealed by density analysis, after 24 h incubation with CSC at 20, 40, 80, and 120 µg/ml, LO transcript levels normalized to internal GAPDH controls were reduced to 65.3, 37.4, 3.6, and 0.8% of the control, respectively, with IC50 = 30 µg/ml of CSC. Thus, CSC specifically inhibited LO expression at the transcriptional level.
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The Expression of Collagen Type I and Elastin, Substrates of LO, in RFL6 Cells Treated with CSC
Since collagen and elastin are substrates of LO (Kagan and Li, 2003), downregulation of LO mRNAs by CSC may be accompanied by altered metabolism of collagen and elastin substrates. To test this possibility, we examined the expression of collagen type I, selected as a representative of collagen protein family, and elastin at mRNA levels in control and CSC-treated cells. Northern blot analysis demonstrated that steady-state mRNA levels of collagen type I and tropoelastin were both decreased dramatically in CSC treated cells (Figs. 5A and 5B). The density analysis showed that CSC at 20, 40, 80, and 120 µg/ml reduced transcript levels of collagen type I to 90, 75, 50, and 10% of the control (Fig. 5A) and of tropoelastin to 90, 78, 61, and 33% of the control (Fig.5B), respectively. These results indicate that downregulation of LO by CSC was concomitant with inhibition of the expression of collagen and elastin substrates, major components of the lung ECM.
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DISCUSSION |
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Apparently, CSC-induced downregulation of LO mRNA as shown (Fig. 1) did not result from overt CSC toxicity, since doses of CSC used in this study ranging from 20 to 120 µg/ml did not induce significant damage to the cell membrane as revealed by trypan blue staining. Notably, is the maximum exposure dose of CSC relevant to the human exposure situation? Yes, according to the following calculation. There are 5 x 109 particles/ml with diameters of 0.11 µm in the mainstream CS aerosol. Thus, all of the particles in cigarette smoke are within the size range reaching the alveolar space, known to be 0.13 µm (Hoffmann and Wynder, 1999). Furthermore, each cigarette yields 26.1 mg CSC, of which at least 50% presumably deposit in the respiratory tract (IARC, 1985
). At a half pack to one pack per day (1020 cigarettes), the inhaled CSC amounts to 130.5261 mg. Since the wet weight of a human lung approximates 1200 g, upon homogenization, it could produce 1.2 L lung homogenate. Thus, the 24 h-adjusted lung burden of CSC of such a smoker is 130.5261 mg/1.2 L (= 108.8217.5 µg/ml) in comparison to the maximum dose of 120 µg/ml CSC exposure for 24 h to RFL6 cells. This is the rationale for using the range from 20 to 120 µg/ml of CSC in this cell culture study.
LO is a copper-dependent enzyme secreted by fibrogenic cells such as fibroblasts. This enzyme oxidizes peptidyl lysine residues to form peptidyl -aminoadipic-
-semialdehyde, resulting in spontaneous, covalent crosslinking of collagen and elastin in the ECM. Therefore, LO plays a critical role in ECM morphogenesis, stabilization, and repair (Kagan and Li, 2003
). LO is synthesized by fibroblasts as a 46 kD preproenzyme. Following signal peptide cleavage and N-glycosylation, the resulting 50 kD proenzyme is secreted and then proteolytically cleaved to the 32 kD functional species in the extracellular space (Li et al., 1995
; Trackman et al., 1992
). CS, a complex chemical mixture, contains more than 4,000 different compounds, including oxidants, heavy metals, and carcinogens that individually or collectively initiate or promote lung injuries. Our recent studies have indicated that LO is an important cellular target for CS insult. Exposure of rat fetal lung fibroblasts to CSC, the particulate phase of CS, induced decreased levels of all LO protein species, including the 46 kDa preproenzyme, the 50 kDa proenzyme, and the 32 kDa mature enzyme, as well as the catalytic activity, in a dose-dependent manner (Chen et al., 2005
). Consistent with these findings, the present data indicate that transcriptional and posttranscriptional regulation of LO are important targets for CSC.
The steady-state concentrations of mRNA as determined by Northern blot are dependent on both the transcriptional rate and the mRNA stability (Schubeler and Bode, 2005). The nuclear run-on assay (Fig. 2) showed that CSC treatment induced decreased transcriptional rates of LO such that the level of nascent LO transcripts was decreased to 27.7% of the control in cells exposed to 120 µg/ml CSC. Such inhibition of LO transcriptional initiation may result from perturbing by CSC the processes of transcription complex assembly and/or binding of RNA polymerase II as well as elongation of RNAs (Howe, 2002
; Parent et al., 2004
). Furthermore, the actinomycin D chase assay indicated that CSC also inhibited LO mRNA stability in a dose-dependent manner with an IC50 = 45 µg/ml CSC (Fig. 3C). The half-life of LO mRNA decay decreased from 24 h in the control to 4.5 h in cells incubated in the presence of 120 µg/ml of CSC (Figs. 3A and 3B). It is established that mammalian mRNAs contain stability elements at either termini including the 5'm7G cap and the poly (A) tail. Specific protein factors such as the cap binding proteins and the poly (A) binding proteins bind to these corresponding cis- elements, preventing an mRNA from exonucleolytic degradation (Guhaniyogi and Brewer, 2001
). CSC reduced LO mRNA stability as shown in this study possibly by blocking the binding and the interaction of these stability-related cis- elements with their protein factors. By further data analysis, we found that, under our cell culture conditions, for example, 40 µg/ml CSC decreased the LO mRNA stability to 57.5% of the control (Fig. 3C), while the same dose of CSC reduced the LO relative transcription rate only to 79.7% of the control (Fig. 2). Furthermore, 80 µg/ml CSC induced the mRNA decay to 26.3% of the control (Fig. 3C), whereas the same dose of CSC resulted in the transcription initiation only to 38.9% of the control (Fig. 2). Based on these parameters, the calculated overall decrements induced by 40 and 80 µg/ml CSC in LO mRNA levels reached 45.8% (79.7% x 57.7%) and 10.2% (38.9% x 26.3%) of the controls, respectively, that are close to the actual steady-state LO mRNA levels, i.e., 37.4% and 3.6% of the controls, respectively, measured by Northern blots under the same conditions (Fig. 1). These results suggest that lower doses of CSC were sufficient to perturb the LO mRNA stability, whereas higher doses of CSC were required to interfere with its transcriptional initiation. Thus, the decreased transcriptional rate and the enhanced mRNA instability both collectively led to decreased levels of steady-state concentrations of LO transcripts in cells exposed to CSC.
To further investigate CSC modulation of LO transcription, we cloned the rat LO promoter into the reporter gene construct, pGL3-basic (Promega), upstream of the luciferase gene. The predicted initiation site (CTT) for transcription (to be determined in future) was designated as +1, whereas the start codon (ATG) for translation was located at +287 (Fig. 4A). The sequence analysis for the approximately 1.9 kb LO promoter indicated that it contains 286 bp downstream of the predicted transcriptional initiation site, the 5'-untranslation region (5'UTR), with 1,579 bp upstream of the predicted transcription initiation site, the putative cis-element-containing region. In addition to TATA-like (TF II), GC (SP1), and GGG(A/C)GGGG (AP2) boxes, the cloned rat LO promoter also contains the metal response element (MRE, core sequence = TGCRCNC; where R = purine, N = any nucleotide) (Lichtlen et al., 2001; Stuart et al., 1984
), the hypoxia response element (HRE, core sequence = RCGTG) (Semenza, 2000
), and the antioxidant response element (ARE, core sequence =RTGACNNNGC) (Chen et al., 2003
; Hayes et al., 2000
). As shown here, CSC inhibited LO promoter-directed luciferase expression in treated cells such that 120 µg/ml of CSC reduced the reporter gene expression to 33% of the control (Fig. 4B). This could result from perturbation by CSC of the interaction of cis-elements with their corresponding transcription factors. Note that upregulation of metallothionein (MT) as a major phenotypic change was accompanied with downregulation of LO in CSC-treated cells (Chen et al., 2005
). Many MRE-related transcription factors would be recruited to bind to MREs in the MT promoters for gene activation, thus competing with their binding to the LO promoter. As reported, MT-I and MT-II genes contain six MREs in their promoter regions, respectively. In addition, inactivation of HRE-related transcription factors by oxidant-containing CSC may also inhibit LO promoter activity. These possibilities are under study. Results presented suggest that CSC inhibition of LO promoter activity may be a critical mechanism leading to decreased levels in LO relative transcriptional rates as shown by the nuclear run-on assay (Fig. 2) and in LO steady-state mRNAs revealed by Northern blot analysis (Fig. 1) in cells treated with CSC under the same conditions.
The lung ECM is a dynamic structure composed of a number of functionally diverse elements that were integrated mainly by interstitial cells, e.g., fibroblasts (Campa et al., 1993; Davidson, 1990
). The overall pattern of the lung ECM results from an intricate balance between the synthesis and degradation of its major structural components such as collagen and elastin. Emphysema is currently defined as "a condition of the lung characterized by abnormal enlargement of respiratory airspaces with destruction of alveolar walls and the loss of elasticity of the lung" (Snider et al., 1986
). The importance of LO in emphysema pathogenesis was demonstrated by the fact that chicks, rats, and hamsters fed with a copper-deficient diet developed lung lesions in these animals similar to panlobular emphysema in humans (Harris, 1986
; O'Dell et al., 1978
; Soskel et al., 1984
). ß-Aminoproprionitrile (BAPN), an irreversible inhibitor of LO, can limit the fibrogenic response in certain surgical procedures and decrease collagen/elastin deposition in models of lung fibrosis. Feeding of BAPN markedly enhanced elastase-induced emphysema in hamsters (Kuhn and Starcher, 1980
). Moreover, LO regulation of the substrate gene expression is further supported by the observation that treatment of rat neonatal aortic smooth muscle cells with BAPN induced downregulation of tropoelastin mRNAs (Jackson et al., 1991
), and that overexpression of LO in COS7 cells by gene transfection enhanced collagen type III promoter activity (Giampuzzi et al., 2000
). In this study, we indicated inhibition of LO mRNA expression by CSC associated with downregulation of collagen type I and tropoelastin transcripts, consistent with our previous findings that CSC suppressed production of collagen and elastin proteins (Chen et al., 2005
). In this case, the solubilized collagen and elastin as a result of LO deficiency may inhibit their own gene expression by activation of the feedback regulation mechanism (Ivarsson et al., 1993
; Jackson et al., 1991
; Riikonen et al., 1995
). Thus, LO may play a critical role in regulation of its substrate expression. Loss of ECM components, which may induce emphysema development, may result from inhibition of synthesis of ECM precursors important for the repair process. Briefly, our findings as shown in this study indicate perturbation of LO transcription and posttranscriptional modification by CSC coupled with downregulation of transcripts of collagen type I and tropoelastin, substrates of LO, enhancing our understanding of the molecular mechanism of CS damage to the lung ECM relevant to emphysema pathogenesis.
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ACKNOWLEDGMENTS |
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