Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation

Virender K. Rehan,1 Ying Wang,1 Sharon Sugano,1 Sonia Romero,1 Xiaoru Chen,1 Jamie Santos,1 Aarti Khazanchi,1 and John S. Torday1,2

Departments of 1Pediatrics and 2Obstetrics and Gynecology, Harbor-University of California Los Angeles Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, David Geffen School of Medicine at UCLA, Torrance, California

Submitted 21 September 2004 ; accepted in final form 30 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPAR{gamma} stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPAR{gamma} expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPAR{gamma}. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested.

chronic lung disease; lipofibroblast; myofibroblast; peroxisome proliferator-activated receptor {gamma}


MATERNAL SMOKING DURING PREGNANCY has diverse adverse effects on fetal outcome, including increased risk of spontaneous abortion, stillbirth, premature delivery, low birth weight, early neonatal mortality, sudden infant death syndrome, and poor pulmonary outcome (7, 10, 1214). Maternal smoking during pregnancy adversely affects fetal lung growth that may result in adverse long-term consequences such as increased occurrence of lower respiratory illnesses and altered pulmonary mechanics on pulmonary function testing (3, 6, 10, 14, 19, 27, 38). The mechanisms underlying the general effects of maternal smoking on fetal viability and growth are generally thought to be due to fetal hypoxia (5). However, the mechanisms underlying pulmonary outcomes appear to be more complex and are poorly understood (27). On the one hand, there is evidence of increased surfactant production at birth, possibly contributing to a decrease in the incidence of respiratory distress syndrome (8, 11, 18, 39). On the other hand, there is strong evidence for deleterious effects on pre- and postnatal lung growth and development following in utero exposure to maternal smoking (3, 57, 10, 1214, 19, 2729, 38). The mechanisms underlying these seemingly paradoxical effects remain largely unknown. This work elucidates the possible mechanism of altered prenatal and postnatal lung development and function and provides a molecular approach to its prevention.

Both normal lung development and injury/repair utilize common mesenchymal-epithelial signaling pathways to maintain homeostasis (9). Epithelially derived parathyroid hormone-related protein (PTHrP) induces the differentiation of mesodermal alveolar interstitial fibroblasts to lipid-containing interstitial lipofibroblasts (LIF) via a PTHrP receptor-mediated, cAMP-dependent PKA pathway (31–33). Other important key proteins in this pathway include peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and adipocyte differentiation-related protein (ADRP). The lipid-containing LIFs produce factors that induce the growth and differentiation of the adjoining type II cells, culminating in alveolar homeostasis (31). Factors that disrupt this cellular homeostatic mechanism by causing the transdifferentiation of LIFs to myofibroblasts (MYFs) lead to abnormal lung development and function (22, 24, 31). Using embryonic WI38 human lung fibroblasts as a model, we tested the hypothesis that in vitro nicotine exposure specifically disrupts PTHrP-mediated alveolar epithelial-mesenchymal paracrine signaling that results in alveolar LIF-to-MYF transdifferentiation, resulting in altered pulmonary growth and differentiation. Furthermore, we reasoned that by targeting the specific molecular elements that maintain the LIF phenotype, nicotine-induced LIF-to-MYF transdifferentiation could be prevented.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents

Nicotinic acetylcholine (nACh) receptor antagonists (D-tubocurarine, {alpha}-bungarotoxin, and mecamylamine) and dibutyryl cAMP (DBcAMP) were acquired from Sigma (St. Louis, MO). PTHrP-(1-34) was obtained from Bachem (Torrance, CA), and rosiglitazone maleate (RGZ) was from SmithKline Beecham Pharmaceuticals. nACh receptors {alpha}3 and {alpha}7 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The PPAR{gamma} expression vector (pCMX-PPAR{gamma}) was kindly provided by Dr. P. Tontonoz (Univ. of California Los Angeles).

Cell Culture

The human embryonic cell line WI38 was obtained from American Type Culture Collection (Rockville, MD). Cells were grown in MEM plus 10% FBS at 37°C in six-well plates, four-well slides, and 60- and 100-mm culture dishes as needed. At 70–80% confluence, the cells were treated with nicotine (1 x 10–9 or 1 x 10–6 M) with or without the specific agonists of the alveolar fibroblast lipogenic pathway: PTHrP (1 x 10–7 M or 5 x 10–7 M), DBcAMP (1 x 10–5 M or 1 x 10–4 M), or the potent PPAR{gamma} stimulant RGZ (1 x 10–6 M or 1 x 10–5 M). Medium containing fresh chemicals was added daily, and at the end of 7 days, the cells were processed as needed.

Triglyceride Uptake Assay

The method used to quantitate triglyceride uptake by fetal rat lung fibroblasts has been described previously (34). Briefly, culture medium was replaced with DMEM containing 20% adult rat serum mixed with [3H]triolein (5 µCi/ml). The cells were incubated at 37°C in 5% CO2 + balance air for 4 h. At the termination of the incubation, the medium was decanted, the cells were rinsed twice with 1 ml of ice-cold MEM, and the cells were removed from the culture plate after a 5- to 10-min incubation with 2 ml of a 0.05% trypsin solution. An aliquot of the cell suspension was taken for protein assay (2), and the remaining cell suspension was extracted for neutral lipid content.

PTHrP Receptor Binding Assay

The receptor binding assay was carried out as previously described (32, 33). The assay mixture, in a total volume of 0.1 ml, contained 50 mM Tris·HCl (pH 7.4), 2 mM dithiothreitol, 10 mM EDTA, 10 µg/ml each of protease inhibitors (leupeptin, pepstatin, antipain, and aprotinin), 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml BSA, 5 mM MgCl2, 10–500 pmol [125I]-Tyr34-PTHrP-(1-34) (specific activity 1,064 Ci/mmol), 10–12 µg membrane protein, and 1 x 10–10 to 1 x 10–6 M PTHrP-(1-34). Triplicate samples were incubated for 30 min at 30°C. Reactions were stopped by the addition of 0.1 ml of homogenization buffer containing 20 mg/ml of BSA and placed in ice-cold water for 30 min, followed by centrifugation at 15,000 g for 1 min. The supernatant was aspirated, and the pellet was counted for radioactive content with a gamma counter (model 1470; Wallace, Gaithersburg, MD). Nonspecific binding was determined in the presence of 1 µM nonradioactive PTHrP-(1-34). Specific binding of [125I]-Tyr34-PTHrP-(1-34) was calculated as total binding minus nonspecific binding and expressed as femtomoles per milligrams of protein. Specificity of binding was determined in the presence of 1 µM PTHrP-(7-34) amide, a selective PTHrP receptor antagonist, showing that it inhibited the binding of the radioactive ligand. In preliminary studies, the binding was found to be linear with time for up to 60 min of incubation. The effect of nicotine (1 x 10–6 M) on PTHrP receptor binding was examined without and with the nACh receptor antagonists D-tubocurarine, {alpha}-bungarotoxin, or mecamylamine (1 x 10–9 to 1 x 10–6 M).

Isolation of Total Cellular RNA

Total RNA was isolated by lysing the cells in 4 M guanidinium thiocyanate followed by extraction with 2 M sodium acetate (pH 4.0), phenol, and chloroform/isoamyl alcohol. RNA was precipitated with isopropanol, collected by centrifugation, vacuum dried, and then dissolved in diethylpyrocarbonate-treated water (4). Integrity of RNA was assessed from the visual appearance of the ethidium bromide-stained ribosomal RNA bands following fractionation on a 1.2% (wt/vol) agarose-formaldehyde gel and quantitated by absorbance at 260 nm.

Semiquantitative RT-PCR

RT-PCR probes used included PTHrP receptor: 5'-ATGTGGATGTAGTTGCGCGTGCAGT-3' and 3'-GGGAAGCCCAGGAAAGATAAGGCAT-5' (445 bp); PPAR{gamma}: 5'-CCCTCATGGCAATTGAATGTCGTG and 3'-TCGCAGGCTCTTTAGAAACTCCCT-5' (757 bp); ADRP: 5'-GTTGCAGTTGATCCACAACCG-3' and 3'-TGGTAGACAGGGATCCCAGTC-5' (666 bp); {alpha}-smooth muscle actin ({alpha}-SMA): 5'-CGCAAATATTCTGTCTGGATCG-3' and 3'-TCACAGTTGTGTGCTAGAGACA-5' (167 bp); nACh receptor {alpha}3: 5'-AGGCTACAAACACGACATCAAGTA-3' and 3'-TGGCTTCTTTGATTTCTGGTGACA-5' (694 bp); nACh receptor {alpha}7: 5'-GGCTTCCGCGGCCTGGACGGCGTGCACTGT-3' and 3'-GGCTTCCGCGGCCTGGACGGCGTGCACTGT-5' (596 bp); and 18s: 5'-TTAAGCCATGCATGTCTAAGTAC-3' and 3'-TGTTATTTTTCGTCACTACCTCC-5' (489 bp). cDNA was synthesized from 1 µg of total RNA by RT using 100 units of SuperScript reverse transcriptase II (Invitrogen, Carlsbad, CA) and random primers (Invitrogen) in a 20-µl reaction mixture containing 1 x SuperScript buffer (Invitrogen), 1 mM dNTP mix, 10 mM dithiothreitol, and 40 units of RNase inhibitor. Total RNA and random primers were incubated at 65°C for 5 min followed by 42°C for 50 min. A denaturing enzyme at 70°C for 15 min terminated the reaction. For PCR amplification, 1 µl of cDNA was added to 25 µl of a reaction mix containing 0.2 µM of each primer, 0.2 mM dNTP mix, 0.5 units of AccuPrime Taq DNA polymerase (Invitrogen), and 1 x reaction buffer. PCR was performed in a RoboCycler (Stratagene, La Jolla, CA). Initially, we obtained standard curves for the cycle number and the absorbance optical density for each of the markers examined by RT-PCR. The cycle number (30–38) for each PCR reaction was chosen so that the absorbance of the amplified product was in the linear range. The PCR products were visualized on 2% agarose gels by ethidium bromide staining, and gels were photographed under UV lights. Band densities were quantified using the Eagle Eye II System (Stratagene). The expression of different mRNAs was normalized to 18s mRNA levels.

Protein Determination and Western Blot Analysis

Protein determination was made using the Bradford dye-binding method (2). Western blotting was performed with modifications of methods described previously (1). Briefly, cells were lysed using an extraction buffer [10 mM Tris (hydroxymethyl) aminomethane (Tris, pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin A, aprotinin, and leupeptin] and centrifuged at 140 g for 10 min (4°C). Equal amounts of the protein (25 µg) from the supernatant were dissolved in electrophoresis sample buffer and were subjected to SDS-PAGE (4–12% gradient) followed by electrophoretic transfer to a nitrocellulose membrane. Nonspecific binding of antibody was blocked by washing with Tris-buffered saline (TBS) containing 5% milk for 1 h. The blot was then subjected to two brief washes with TBS plus 0.5% Tween 20, incubated in TBS plus 0.1% Tween 20 and the specific primary antibodies (PPAR{gamma} 1:2,000, Alexis Biochemicals, San Diego, CA; {alpha}-SMA 1:50,000, Sigma; ADRP 1:3,000, a kind gift from Dr. Constantine Londos, National Institute of Diabetes and Digestive and Kidney Diseases) overnight at 4°C. Blots were then washed in TBS plus 0.1% Tween 20 and then incubated for 1 h in secondary antibody, washed, and developed with a chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) following the manufacturer's protocol. The densities of the specific protein bands were quantified using a scanning densitometer (Eagle Eye II still video system, Stratagene). The blots were subsequently stripped and reprobed with anti-GAPDH (1:5,000; Chemicon, Temecula, CA) antibody to confirm equal loading of samples.

Transfection Protocol

For transient transfection, WI38 cells were transfected by using Lipofectamine Plus Reagent (Invitrogen). Cells were trypsinized 1 day before transfection and plated on 100-mm-diameter dishes so that they were 50–80% confluent on the day of transfection. Four or 8 µg of pCMX-PPAR{gamma} cDNA were diluted in 800 µl of serum-free medium, and 20 µl of Lipofectamine Plus Reagent were added to the diluted DNA. The DNA solution was incubated at room temperature for 15 min to precomplex DNA with Plus Reagent. Another 30 µl of Lipofectamine Reagent diluted to 800 µl in serum-free medium were combined with precomplexed DNA, and then the mixture incubated for 15 min at room temperature. Cells were washed with serum-free medium twice, and then 6.4 ml of serum-free medium were added to each dish, followed by the addition of DNA-Lipofectamine Plus Reagent complexes. The complexes were mixed into the medium gently and further incubated at 37°C at 5% CO2 for 3 h. After incubation, transfection medium was replaced by complete medium containing serum and antibiotics. After incubation overnight, DNA and protein were periodically extracted and analyzed for DNA fragment test and Western blot analysis. Once transfection was confirmed, the cells were treated with nicotine (1 x 10–9 M or 1 x 10–6 M) for 7 days, and the cell extracts were analyzed for PPAR{gamma} and {alpha}-SMA proteins by Western blot analysis.

Immunofluorescence Double Staining

Lipogenic and myogenic status of cultured WI38 cells was assessed by simultaneous staining for lipid droplets and {alpha}-SMA. Lipids were stained using oil red O staining, and {alpha}-SMA expression was assessed by using anti-{alpha}-SMA (cat. no. A2547, 1:1,000, mouse monoclonal IgG2, Sigma) primary antibody. In brief, cells were cultured on Lab-Tek four-chamber slides under control and experimental conditions (nicotine treatment, 1 x 10–9 M for 7 days). At the end of the experimental period, slides were fixed in freshly prepared 4% paraformaldehyde. Fixed slides were washed in PBS, blocked with 3% normal goat serum (Jackson Immunoresearch Lab) in PBS for 30 min at room temperature to block nonspecific binding, and then incubated in primary antibody overnight at 4°C. Secondary biotinylated anti-mouse IgG2 was used at 1:200 dilution for 30 min. The slides were then washed 3x with PBS and with double-distilled water 2x and were then incubated with oil red O (Sigma) for 15–30 min. Slides were rinsed 3x for 5 min and then mounted and coverslipped with Vestashield mounting medium with 4',6'-diamidino-2-phenylindole (Vector Laboratories) visualization under a fluorescence microscope.

Statistical Analysis

Analysis of variance for multiple comparisons with Newman-Keuls post hoc test and Student's t-test, as indicated, were used to analyze the experimental data. P < 0.05 was considered to indicate significant differences in the expression of lipogenic and myogenic markers among the control, nicotine, and nicotine plus treatment groups.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
nACh Receptor Expression by WI38 Cells

Initially, we examined the expression of nACh receptors {alpha}3 and {alpha}7 by WI38 cells. By RT-PCR (Fig. 1A) and Western blot analysis (Fig. 1B), we found that nACh receptors {alpha}3 and {alpha}7 were well expressed by WI38 cells. Upon nicotine stimulation (for 7 days), there were significant increases in the expressions of both nACh receptors {alpha}3 and {alpha}7 (P < 0.05 vs. control).



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Fig. 1. A and B: expression of nicotine acetylcholine (nACh) receptors {alpha}3 and {alpha}7 by control and nicotine-stimulated WI38 cells by RT-PCR and Western blot analysis, respectively. On nicotine (1 x 10–9 or 1 x 10–6 M) stimulation for 7 days, there were significant increases in the expressions of both nACh receptors {alpha}3 and {alpha}7 (*P < 0.05 vs. control by ANOVA, n = 3).

 
Nicotine-Induced LIF-to-MYF Transdifferentiation

Our previous studies have demonstrated that cultured developing pulmonary alveolar interstitial LIFs, exposed to stimuli that disrupt fetal lung development, e.g., hyperoxia or volutrauma, transdifferentiate to MYFs via downregulation of PTHrP-mediated cAMP-dependent PKA signaling (22, 33). In the present study, we examined whether cultured WI38 human embryonic lung fibroblasts exposed to nicotine demonstrate a similar effect. Furthermore, we determined whether stimulants of the PTHrP receptor-mediated cAMP-dependent PKA pathway would prevent nicotine-induced alveolar LIF-to-MYF transdifferentiation.

Effect of nicotine on mRNA expression of markers for the lung fibroblast phenotype (LIF vs. MYF). Exposure to nicotine (1 x 10–9 or 1 x 10–6 M) for 7 days resulted in dose-dependent decreases in PTHrP receptor (–33 ± 8% and –40 ± 3%, respectively; means ± SE), PPAR{gamma} (–26 ± 7% and –37 ± 3%), and ADRP (–41 ± 8% and –47 ± 4%) mRNA expression, as determined by RT-PCR (*P < 0.05 for all, nicotine vs. control; Fig. 2). This was accompanied by a concomitant dose-dependent increase in {alpha}-SMA (+38 ± 4% and +140 ± 5%) mRNA expression (*P < 0.05, nicotine vs. control).



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Fig. 2. Nicotine treatment of cultured WI38 cells for 7 days results in significant decreases in parathyroid hormone-related protein (PTHrP) receptor, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), and adipocyte differentiation-related protein (ADRP), and a significant increase in {alpha}-smooth muscle actin ({alpha}-SMA) mRNA expression (*P < 0.05 vs. control by ANOVA, n = 3). Representative RT-PCR blots (top) and densitometric histograms (bottom) for PTHrP receptor, PPAR{gamma}, ADRP, and {alpha}-SMA mRNA expression are shown.

 
Effect of nicotine on protein expression of markers for lung fibroblast phenotype (LIF vs. MYF). As assessed by Western blot analysis, the protein levels of PTHrP receptor (–31 ± 7%), PPAR{gamma} (–24 ± 2%), and ADRP (–20 ± 3%) decreased, and that of {alpha}-SMA (+100 ± 5%) increased significantly on exposure to nicotine (1 x 10–9) for 7 days (Fig. 3), indicating nicotine-induced LIF-to-MYF transdifferentiation. This was corroborated by double-immunofluorescence staining of WI38 cells for lipid droplets and {alpha}-SMA after 7 days of nicotine stimulation. Nicotine exposure clearly decreased lipid staining and increased {alpha}-SMA staining, the hallmarks of LIF-to-MYF transdifferentiation (Fig. 4).



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Fig. 3. Nicotine treatment of cultured WI38 cells for 7 days results in significant decreases in PTHrP receptor, PPAR{gamma}, and ADRP, and a significant increase in {alpha}-SMA protein expression (*P < 0.05 vs. control by ANOVA, n = 3). Representative Western blots (top) and densitometric histograms (bottom) for PTHrP receptor, PPAR{gamma}, ADRP, and {alpha}-SMA mRNA expression are shown.

 


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Fig. 4. Representative immunofluorescence staining for lipid droplets (red staining) and {alpha}-SMA (green staining) in cultured WI38 cells with and without nicotine (1 x 10–9 M) treatment for 7 days is shown. Cultured WI38 cells were stained for lipid droplets alone using oil red O, {alpha}-SMA using specific monoclonal antibody, and nuclei using 4',6'-diamidino-2-phenylindole (DAPI). Bottom: shows triple staining using oil red O, {alpha}-SMA-specific antibody, and DAPI. Nicotine treatment markedly reduced staining for lipid droplets and markedly increased {alpha}-SMA expression. This can be clearly seen in the bottom panel that shows cytoplasmic colocalization of staining for lipid droplets and {alpha}-SMA.

 
Effect of Nicotine on Triolein Uptake by WI38 Cells

To assess the effect of nicotine on LIF function, [3H]triolein uptake by cultured WI38 cells under control and experimental conditions was measured. Nicotine treatment (1 x 10–9 M for 7 days) caused an almost 50% decrease in phenotypic triglyceride uptake (Fig. 5), which was effectively prevented by concomitant treatment of WI38 cells with RGZ (1 x 10–5 M), PTHrP (5 x 10–7 M), or DBcAMP (1 x 10–4 M).



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Fig. 5. Nicotine treatment (1 x 10–9 M for 7 days) caused an almost 50% decrease in triglyceride uptake (*P < 0.05 vs. control by ANOVA, n = 6), which was completely prevented by concomitant treatment of WI38 cells with rosiglitazone (RGZ; 1 x 10–5 M), PTHrP (5 x 10–7 M), or dibutyryl cAMP (DBcAMP) (1 x 10–4 M).

 
Prevention of LIF-to-MYF Transdifferentiation by Stimulants of PTHrP-Mediated, cAMP-Dependent PKA Signaling Lipogenic Pathway

The effect of specific stimulants of PTHrP-mediated, cAMP-dependent PKA lipogenic pathway on nicotine-induced LIF-to-MYF transdifferentiation was assessed by pretreating WI38 cells with PTHrP (5 x 10–7 M), DBcAMP (1 x 10–4 M), or the potent PPAR{gamma} stimulant RGZ (1 x 10–5 M). Pretreatment with PTHrP, DBcAMP, or RGZ completely prevented the nicotine-induced decreases in PTHrP receptor (Fig. 6A) and PPAR{gamma} (Fig. 6B) and an increase in {alpha}-SMA (Fig. 6C) protein expression, indicating prevention of nicotine-induced LIF-to-MYF transdifferentiation. The prevention of nicotine-induced LIF-to-MYF transdifferentiation by stimulation of the PTHrP-driven lipogenic pathway is also supported by prevention of the nicotine-induced decrease in phenotypic triglyceride uptake by the stimulants of the PTHrP-driven PKA-mediated fibroblast lipogenic pathway (Fig. 5). As PPAR{gamma} expression is central to the maintenance of fibroblast lipogenic phenotype, we next examined the effect of transfection of WI38 cells with PPAR{gamma} expression vector on nicotine-induced LIF-to-MYF transdifferentiation (Fig. 7). Transfected cells were treated with nicotine (1 x 10–9 or 1 x 10–6 M) for 7 days, and the expressions of PPAR{gamma} and {alpha}-SMA were assessed by Western blot analysis. As shown in Fig. 7, under control conditions (without transfection), there was a significant decrease in PPAR{gamma} and a significant increase in {alpha}-SMA protein expression. However, with PPAR{gamma} transfection, these nicotine-induced changes were completely prevented.



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Fig. 6. Concomitant treatment with specific stimulants of PTHrP-mediated, cAMP-dependent PKA lipogenic pathway, i.e., PTHrP (5 x 10–7 M), DBcAMP (1 x 10–4 M), or RGZ (1 x 10–5 M), completely prevents the nicotine-induced decreases in PTHrP receptor (A) and PPAR{gamma} (B) protein, and increase in {alpha}-SMA (C) protein expressions (*P < 0.05 vs. control by ANOVA, n = 3), indicating prevention of nicotine-induced lipofibroblast-to-myofibroblast transdifferentiation by these agents.

 


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Fig. 7. Transfection of WI38 cells with PPAR{gamma} expression vector completely prevented the nicotine-induced lipofibroblast-to-myofibroblast transdifferentiation. WI38 cells were transfected cells with either 4 or 8 µg of pCMX-PPAR{gamma} cDNA. Then, the cells were treated with nicotine (1 x 10–9 or 1 x 10–6 M) for 7 days, following which the expressions of PPAR{gamma} and {alpha}-SMA were assessed by Western blot analyses. Under control conditions (without transfection), there was a significant decrease in PPAR{gamma} and a significant increase in {alpha}-SMA protein expression. However, after PPAR{gamma} transfection, these changes were completely prevented. Representative blots out of 2 independent experiments are shown.

 
Effect of Nicotine on PTHrP Binding to its Receptor

To elucidate the mechanism of nicotine-induced LIF-to-MYF transdifferentiation, the effect of nicotine on PTHrP binding to its receptor was examined. Nicotine (1 x 10–6 M) treatment caused a 30% decrease in PTHrP binding to its receptor (fmol·90 min–1·mg protein–1), and this effect was prevented by pretreatment with either D-tubocurarine (1 x 10–6 M), a nonspecific nicotine receptor antagonist, or {alpha}-bungarotoxin, a specific {alpha}7 nACh receptor antagonist (1 x 10–6 M), but not mecamylamine, an {alpha}3 nACh receptor antagonist (Fig. 8). To determine the functional significance of the differential effects of the {alpha}7 and {alpha}3 nACh receptor antagonists on PTHrP binding, the effect of nicotine (1 x 10–6 M) on triolein uptake by WI38 cells with and without D-tubocurarine (1 x 10–9 or 1 x 10–6 M), {alpha}-bungarotoxin (1 x 10–9 or 1 x 10–6 M), or mecamylamine (1 x 10–9 or 1 x 10–6 M) was examined. Similar to their differential effects on PTHrP receptor binding, the nicotine-induced decrease in triolein uptake was completely prevented by D-tubocurarine or {alpha}-bungarotoxin, but not by mecamylamine (Fig. 9).



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Fig. 8. Nicotine (1 x 10–6 M) treatment caused a 30% decrease in PTHrP binding to its receptor (fmol·90 min–1·mg protein–1; *P < 0.05 vs. control by ANOVA, n = 6), which was completely prevented by pretreatment with either D-tubocurarine (1 x 10–6 M), a nonspecific nACh receptor antagonist, or {alpha}-bungarotoxin (1 x 10–6 M), an {alpha}7 nACh receptor antagonist, but not by mecamylamine (1 x 10–9 or 1 x 10–6 M), an {alpha}3 nACh receptor antagonist.

 


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Fig. 9. Nicotine treatment (1 x 10–9 M for 24 h) caused an almost 30% decrease in triglyceride uptake (*P < 0.05 vs. control by ANOVA, n = 6), which was completely prevented by pretreatment with either D-tubocurarine (1 x 10–6 M), a nonspecific nACh receptor antagonist, or {alpha}-bungarotoxin (1 x 10–6 M), an {alpha}7 nACh receptor antagonist, but not by mecamylamine (1 x 10–9 or 1 x 10–6 M), an {alpha}3 nACh receptor antagonist.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using the WI38 human embryonic lung fibroblast cell line as a model, we tested the hypothesis that in vitro nicotine exposure disrupts the specific paracrine signaling pathway that results in pulmonary LIF-to-MYF transdifferentiation and through manipulation of specific molecular targets, this process could be prevented. First, we documented the expression of the specific nACh receptors {alpha}3 and {alpha}7 by WI38 cells, which, at least in part, have been implicated in mediating nicotine-induced effects on developing lung structure and function (28, 30). We, for the first time, demonstrate the presence of {alpha}3 and {alpha}7 nACh receptors in the cultured lung mesenchymal cells of human embryonic origin. On nicotine stimulation, the expressions of both nACh receptors {alpha}3 and {alpha}7 increased significantly. Furthermore, the exposure of cultured WI38 cells to nicotine for 7 days resulted in significant decreases in the expression of the key fibroblast lipogenic markers PTHrP receptor and PPAR{gamma}, along with their downstream targets ADRP and triglyceride uptake, and an increase in the expression of the key fibroblast myogenic marker, {alpha}-SMA. These molecular changes were accompanied by immunohistochemical changes that were also consistent with LIF-to-MYF transdifferentiation. More importantly, the nicotine-induced molecular and functional changes were completely prevented by concomitant treatment with PTHrP, cAMP, or a specific PPAR{gamma} agonist, RGZ, all of which are stimulants for the fibroblast lipogenic pathway. Moreover, the nicotine effect was completely blocked in WI38 cells transfected with PPAR{gamma}. Finally, the use of nonspecific (D-tubocurarine) and specific ({alpha}-bungarotoxin and mecamylamine) antagonists of nACh receptors suggested the specific involvement of the {alpha}7 nACh receptor in the nicotine-induced decrease in triglyceride lipid uptake by the lung fibroblasts. To our knowledge, these data provide the first evidence of nicotine-induced pulmonary alveolar LIF-to-MYF transdifferentiation and its complete prevention by concomitant treatment with stimulants of the fibroblast lipogenic pathway. These data provide a molecular pathway for the nicotine-induced disruption of lung development and offer an opportunity to test targeted molecular strategies to prevent it.

Until now, there has been no specific intervention to prevent nicotine-induced morbidity in the developing fetus. This is mainly because of failure to eliminate maternal smoking during pregnancy coupled with a lack of understanding of the molecular mechanisms involved in nicotine-induced morbidity (15, 21). We have recently proposed that specific disruption of pulmonary alveolar epithelial-mesenchymal interactions results in interstitial LIF-to-MYF transdifferentiation, which may be the final common pathway through which various noninflammatory and inflammatory triggers lead to chronic lung damage in the premature infant (31). Alveolar interstitial LIF-to-MYF transdifferentiation results in failed alveolarization in the developing lung, which leads to an arrest in pulmonary growth and development, the hallmarks of in utero nicotine-induced lung damage (6, 19, 24, 31). Our data suggest that the likely molecular mechanisms involved include decreased PTHrP binding to its receptor with resultant downregulation of the PTHrP-stimulated cAMP-dependent PKA pathway, which normally induces the LIF phenotype, characterized by expression of such lipogenic features as triglyceride accumulation and expression of PPAR{gamma} and ADRP. Our data indicating LIF-to-MYF transdifferentiation are also supported by previous observation of lower cellular lipid content in the lung tissue of both 8- and 21-day-old rat pups following in utero nicotine exposure (19).

PTHrP is a stretch-sensitive protein expressed by the developing lung epithelium and is upregulated during late fetal lung development (24, 25, 35). It signals to the neighboring alveolar mesenchymal cells through its seven-transmembrane-spanning G protein-dependent receptor, stimulating their lipogenic phenotype (26). The critical downstream target for PTHrP/PTHrP receptor signaling is PPAR{gamma}, which in turn controls other lipogenic regulatory genes such as ADRP and leptin (32, 33). Therefore, stimulation of PPAR{gamma} induces the lipogenic phenotype, which is necessary for maintaining alveolar homeostasis through its autocrine effect on interstitial fibroblasts and its paracrine effect on alveolar type II cells (32, 33). Specifically, the interstitial LIF phenotype is of functional importance as it provides cytoprotection against oxygen free radicals (36), traffics neutral lipid substrate to alveolar type II cells for surfactant phospholipid synthesis (34), and causes alveolar type II cell proliferation (31). Although MYFs also seem to be important for normal lung development, these cells are also the hallmark of chronic lung diseases in both the neonate and adult (17, 20, 37). In the developing lung, MYFs are fewer in number and are predominantly located at the periphery of the alveolar septa, where they very likely participate in the formation of new septa (17, 37). However, in chronic lung diseases, MYFs not only increase in number but also are located in the center of the alveolar septum in great abundance (37). In line with these observations, using both molecular and metabolic profiling, we have previously observed that upon hyperoxic exposure, fetal rat lung LIFs transdifferentiate to MYFs (16, 22). Our present data also imply LIF-to-MYF transdifferentiation as the potential underlying mechanism for the nicotine-induced lung damage in the developing fetus.

However, the effects of in utero nicotine exposure on the developing lung are extremely complex. On the one hand, there is evidence of enhanced functional pulmonary maturity at birth, possibly contributing to a decrease in the incidence of respiratory distress syndrome (8, 11, 18, 39). In contrast, clearly, reduction in both prenatal and postnatal lung growth occurs in children of women who smoke (3, 57, 10, 1214, 19, 2729, 38). Significant suppression of lung alveolarization, functional residual capacity, and tidal flow volumes has been demonstrated in the offspring of women exposed to smoke during pregnancy. So far, the molecular mechanisms underlying these paradoxical effects remain largely unknown. Although acceleration of the lung developmental program, including surfactant phospholipid synthesis and an increase in surfactant protein expression (8, 11, 18, 39), have been observed to explain enhanced functional pulmonary maturity at birth, the mechanisms underlying suppression of lung alveolarization and its functional consequences remain far less clear. Our data provide a plausible mechanism that explains not only failed alveolarization but also the functional pulmonary consequences following in utero nicotine exposure, including an increased predisposition to reactive airways disease. Our data complement and extend the extensive work done by Sekhon and colleagues (28, 29), who, using a rhesus monkey model, have reported that maternal nicotine exposure from day 26 to day 134 of gestation (term = 165 days) alters fetal lung development, resulting in smaller lung volume and decreased alveolar surface area with an accompanying increase in the size of gas exchanging units. More importantly, concomitant with these changes, they reported a significant upregulation of the lung {alpha}7 nACh receptor and collagen I and III expression. In association with these changes, they also observed alterations in pulmonary function as measured by increased pulmonary resistance and decreased expiratory flows (28, 29). These studies, for the first time, suggested that the observed alterations in lung mechanics in the infants of mothers who smoke during pregnancy could be linked to the passage of nicotine across the placenta, which causes increased collagen deposition and increased airway wall dimensions in the fetal lung. We speculate that a shift in lung mesenchyme phenotype from a lipogenic to a myogenic type, as predicted by our findings, not only explains the increased collagen expression but also provides a molecular mechanism for the altered postnatal pulmonary mechanics observed by Sekhon and colleagues (28, 29).

The complexity of the in utero effects of smoke exposure on the developing lung is further suggested by the fact that even though direct nicotine exposure might induce LIF-to-MYF transdifferentiation, in utero fetal smoke exposure is also accompanied by relative fetal hypoxia, which may prevent LIF-to-MYF transdifferentiation. As we have previously demonstrated that exposure to hyperoxia augments the spontaneously occurring pulmonary LIF-to-MYF transdifferentiation, it is tempting to speculate that the relative fetal hypoxia occurring with in utero smoke exposure may in fact have a protective effect on nicotine-induced LIF-to-MYF transdifferentiation. The exact mechanism(s) by which nicotine induces LIF-to-MYF transdifferentiation, in particular, the decrease in PTHrP receptor expression and PTHrP/PTHrP receptor binding, remains to be determined. The understanding of this mechanism is likely to be the key for designing specific preventive and therapeutic strategies. However, the decreases in both PTHrP/PTHrP receptor binding and its functional downstream effect, i.e., triolein uptake, were completely blocked by either tubocurarine or {alpha}-bungarotoxin, but not by mecamylamine, suggesting the specific involvement of the {alpha}7 nACh receptor subtype in this effect.

In summary, in addition to previously proposed mechanisms for in utero nicotine-induced lung effects, our data for the first time provide evidence for a mechanism for the direct effects of nicotine on the developing mesenchyme that could permanently alter the "developmental program" of the developing lung by disrupting critically important epithelial-mesenchymal interactions. More importantly, specific interventions that augment the pulmonary mesenchymal lipogenic pathway could at least partially ameliorate the very complex nicotine-induced in utero lung injury.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported by American Heart Association Grant 0265127Y, National Institutes of Health (NIH) Grant HL-075405, a Philip Morris USA grant (to V. K. Rehan), and NIH Grant HL-55268 (to J. S. Torday and V. K. Rehan). S. Sugano and S. Romero were supported by NIH Grant 1R25-GM-056902 (Cal State Dominiguez Hills, Torrance, CA).


    ACKNOWLEDGMENTS
 
The authors thank Dr. Srinivas Dhanakoti for technical help with some of the assays. This work was presented in part at the Pediatric Academic Societies Meeting, San Francisco, CA, May 2004.


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. K. Rehan, Dept. of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, David Geffen School of Medicine at UCLA, 1124 W. Carson St., Torrance, CA 90502 (e-mail: vrehan{at}labiomed.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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