RAPID COMMUNICATION
Nicotine stimulates branching and expression of SP-A and SP-C mRNAs in embryonic mouse lung culture

Carol W. Wuenschell1, Jingsong Zhao1, J. Denise Tefft1, and David Warburton1,2

1 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles 90033; and 2 Developmental Biology Program, Department of Surgery, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Although the effects of maternal smoking on fetal growth and viability are overwhelmingly negative, there is a paradoxical enhancement of lung maturation as evidenced, in part, by a lower incidence of respiratory distress syndrome in infants of smoking mothers. Other epidemiologic and experimental evidence further support the view that a tobacco smoke constituent, possibly nicotine, affects the development of the lung in utero. We are studying the direct effects of nicotine on murine lung development using a serumless organ culture system. We have found that embryonic lungs explanted at 11 days gestation showed a 32% increase in branching after 4 days in culture in the presence of 1 µM nicotine and 7- to 15-fold increases in mRNAs encoding surfactant proteins A and C after 11 days. The effect of nicotine exposure on surfactant gene expression is apparently mediated by nicotinic acetylcholine receptors because it was blocked by D-tubocurarine. The nicotine-induced stimulation of surfactant gene expression could, in part, account for the effect of maternal smoking on the incidence of respiratory distress syndrome.

surfactant protein A; surfactant protein C; messenger ribonucleic acid; respiratory distress syndrome; nicotinic acetylcholine receptors; branching morphogenesis; lung development

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE NEGATIVE EFFECTS of maternal smoking on pregnancy outcome are well known. Maternal smoking during pregnancy is associated with a higher incidence of spontaneous abortion, low birth weight, and neonatal mortality (for reviews, see Refs. 1, 26). Immediate and long-term deleterious effects on postnatal lung development have also been associated with prenatal exposure to maternal smoking (8, 12). This latter observation indicates that maternal smoking affects the developmental program of the lung in utero. Nicotine is a possible causative agent for these effects because it is a major pharmacological constituent of tobacco smoke that crosses the placenta and is concentrated in the fetal compartment (14). Animal studies show that both nicotine and tobacco smoke have similar adverse effects on fetal lung growth (7, 16). In addition, nicotine exposure in rabbits leads to hyperplasia of pulmonary neuroendocrine cells (4), and a similar result has been observed in the lungs of human fetuses exposed to maternal smoking during pregnancy (5).

Although the overall effects of tobacco smoke exposure on fetal growth and viability are negative, evidence suggests that prenatal exposure to maternal smoking paradoxically enhances lung maturation. It is well known that infants born to smoking mothers have a lower incidence of respiratory distress syndrome (RDS) (9, 28), a condition resulting from lung immaturity primarily due to inadequate production of pulmonary surfactant (23). In addition, smoking-exposed fetuses have been found to have saturated phosphatidylcholine levels and lecithin-to-sphingomyelin ratios consistent with an increase in functional lung maturity of ~1.5 wk relative to unexposed fetuses (13).

We are examining the hypothesis that nicotine from maternal smoking might perturb the developmental program of the lung, initially accelerating growth but eventually causing premature differentiational changes that could result in impaired growth and function after birth. In these studies, the use of embryonic mouse lung buds grown in serumless culture allows us to isolate direct, local effects of nicotine from effects due to systemic mechanisms. Here we report that nicotine exposure stimulates lung branching morphogenesis and increases expression of mRNAs encoding the surfactant-associated protein (SP) A and SP-C.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Organ culture. The serumless organ culture system has been previously described (22). Briefly, timed pregnant Swiss-Webster mice were obtained from Simonsen (Gilroy, CA). Embryos were dissected on embryonic day 11 or 12. Lung buds were removed from the embryos in sterile Hanks' balanced salt solution, placed on Millipore filters resting on stainless steel wire mesh, and cultured at the air-medium interface in chemically defined medium (Fitton-Jackson modified BGJb, GIBCO, Grand Island, NY) containing 0.2 mg/ml of ascorbic acid. The cultures were incubated under 5% CO2 at 37°C for up to 11 days, with the medium changed every second or third day. Drugs [(-)-nicotine di-tartrate and/or D-tubocurarine chloride (D-TC); Sigma Chemical, St. Louis, MO] at the indicated concentrations were added to the medium at the time the cultures were established and at each subsequent medium change.

Quantification of branching morphogenesis. Quantification was done on lungs by counting the number of terminal branches present in each explant after 4 days in culture. The terminal branches were visualized in whole mounts, and the number of terminal branches for all the lobes of each lung pair was counted with transillumination on a dissecting microscope. Three to five lungs were counted for each data point. The means ± SD were calculated, and the significance of differences between means was evaluated by t-test (criterion for significance, P < 0.05). Changes in the complexity of branching in response to nicotine are expressed as a percent difference in the number of terminal branches in treated versus control lungs.

Competitive reverse transcription-polymerase chain reaction. Total RNA was extracted from cultured lungs in guanidinium thiocyanate with the total RNA isolation kit from 5 Prime-3 Prime (West Chester, PA) and was reverse transcribed with Moloney leukemia virus reverse transcriptase (RT) and oligo deoxythymidine primers. Primers for SP-A, SP-C, Clara cell 10-kDa protein (CC10), and beta -actin were synthesized at the University of Southern California Health Science Campus Microchemical Core Facility on the basis of published mouse sequences (GenBank accession nos. are S48768, M38314, S56696, and X03672, respectively). Amplification was done in a Robocycler (Stratagene, La Jolla, CA) for 35 cycles at appropriate denaturation, annealing, and extension temperatures. Reaction products were analyzed by electrophoresis on 3% agarose gels, stained with ethidium bromide, and photographed. Polymerase chain reactions (PCRs) with RNA in place of the RT product were run to control for DNA contamination. Validity of the PCR products was checked by sequence determination. Quantitation by competitive RT-PCR was performed as previously described (30). Briefly, the method involves comparison of the intensity of the band amplified from the target cDNA with the intensity of a band amplified from a known mass of competitor template DNA added to the same tube and amplified by the same primers. A competitor template with appropriate specific primer sequences flanking a stretch of heterologous DNA sequence was constructed for each target mRNA. The competitor template was designed in each case to be slightly longer than the intended target sequence to allow resolution by gel electrophoresis of the PCR products generated from the competitor template and from the target sequence. Yields of RNA obtained from each lung were determined by ultraviolet spectrophotometry. Equal masses of RNA were reverse transcribed for each sample to be compared, and equal volumes of the RT reaction product were loaded into each PCR tube together with a known amount of competitor template DNA. After PCR amplification and gel electrophoresis, quantification was accomplished by densitometric scanning of the bands on photographs of the electrophoretic gels. A standard curve was generated for each sequence under study by PCR amplification of serial dilutions of a known concentration of the specific cDNA sequence together with a constant amount of the appropriate competitor template DNA. A straight-line standard curve for each sequence was generated by plotting log (densitometric value of the target band divided by the densitometric value of competitor band) versus the amount of specific cDNA for each tube. From the log of the ratio of the two bands for each sample lane, the amount of target cDNA in each sample was obtained by using the computer-generated linear regression equation. This approach controls for differences in priming efficiency in the PCRs because the amount of target PCR product is always determined relative to the amount of product generated from a known amount of a competitor template in the same tube with the same pair of primers. To control for possible inaccuracies in spectrophotometric RNA determinations as well as for differences in the efficiencies of the RT reactions, values for each sample studied were normalized to values for beta -actin obtained from matched samples by the same competitive PCR technique. Graphed results are presented as means ± SD of values from three replicate lungs for each condition graphed as a percentage of the control value. Significance of differences between means was evaluated by t-test, with the criterion for significance being P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of nicotine on branching morphogenesis. We initially observed that lung buds from 11-day embryos cultured for 4 days in the presence of 1 µM nicotine appeared to be more highly branched than lung buds grown in control medium (Fig. 1, A and B). At the time of explant, lung buds were at an early stage of branching, exhibiting primary bronchi and, in some cases, the beginnings of the next generation of branches. Lower concentrations of nicotine yielded inconsistent results (100 nM nicotine: stimulation in one experiment, no change in two experiments) or had no effect on branching (10 nM nicotine in three experiments). Nicotine concentrations > 1 µM were not tested out of concern that this would clearly exceed the physiologically relevant level of fetal exposure from maternal smoking, although direct comparison of nicotine exposure in vivo with exposure in the culture system is not possible. Luck et al. (14) measured levels of nicotine up to 23 ng/ml in amniotic fluid at 12-16 wk gestation and up to 25 ng/ml in umbilical vein serum at delivery (slightly in excess of 0.1 µM). The level of exposure of a fetus in vivo would be expected to vary depending on how recently the mother had smoked because the rate of transfer to the fetal compartment is rapid relative to the half-life of nicotine in the body (14). The concentration of nicotine in our cultures also probably varied over time but in a different way because nicotine was introduced only at 2-day intervals when the medium was changed and was presumably lost at an unknown rate during the intervening periods due to such processes as chemical breakdown, volatilization, and metabolism in the lung tissue. Future studies will be needed to address the stability of nicotine in the culture system.


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Fig. 1.   Effect of nicotine exposure on branching morphogenesis. Lung buds were grown for 4 days in presence or absence of 1 µM nicotine. A: representative control lung. B: nicotine-treated lung. Bars, 250 µm. C: quantitation of terminal branches. Values are means ± SD; n = 4 lungs. Nicotine exposure significantly stimulates branching, P < 0.05.

The stimulation of branching by 1 µM nicotine was quantified by counting terminal branches and found to be significant in three separate experiments. The mean increase in the number of terminal branches ranged from 20 to 53% and averaged 32% over all three experiments. Results from one experiment are shown in Fig. 1C.

Effects of nicotine on specific gene expression. To assess the effect of nicotine exposure on differentiation of the lung epithelium, we decided to examine the expression of genes, the products of which are characteristic of specific differentiated epithelial cell types. To date, we have looked at CC10, a specific marker for Clara cells of the bronchiolar epithelium (21), and at two of the four known surfactant-associated proteins, SP-A and SP-C. Increasing expression of the SP genes is known to correlate with increasing functional maturity of the fetal lung (for reviews, see Refs. 3, 20, 27). SP-C was chosen because it is the only SP that is a specific marker for type II pneumocytes, the surfactant-secreting cells of the alveolar epithelium. SP-A is expressed by both Clara and type II cells (see references in Ref. 27) and was chosen because in humans it increases later in gestation than SP-B or SP-C and might yield more information about changes in the timing of differentiation.

We have used a sensitive competitive RT-PCR assay to measure the relative levels of SP-A, SP-C, and CC10 mRNAs in control and nicotine-treated lung buds dissected from day 11-12 mouse embryos and maintained for 11 days in culture. Previously, Wuenschell et al. (29) and Slavkin et al. (22) showed that differentiation of various epithelial cell types begins within this culture period. We found that exposure to 1 µM nicotine significantly increased the levels of mRNA encoding the two SPs but had no effect on the level of CC10 mRNA (Fig. 2). A preliminary quantification of SP-B mRNA using recently designed primers and competitor template showed no change in the level of this mRNA in nicotine-treated versus control lungs in one experiment (data not shown). More work will be required to confirm this result. Interestingly, we noted that 10-fold less cDNA was required for optimal quantitation of SP-B mRNA than for SP-C mRNA and 25-fold less than for SP-A mRNA. Although some of this difference could be due to differences in priming efficiency of the different primer pairs, this observation strongly suggests that the control lungs contain substantially more SP-B than SP-C or SP-A mRNA.


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Fig. 2.   Effect of nicotine (Nic) exposure on expression of surfactant protein (SP) A, SP-C, and Clara cell 10-kDa protein (CC10) mRNAs. RNA was extracted from lung buds grown for 11 days in presence or absence [control (Ctrl)] of 1 µM Nic and subjected to competitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitation. A: examples of competitive RT-PCR gels showing lanes corresponding to 1 control and 1 nicotine-treated lung assayed for each gene of interest and for beta -actin. An increase in intensity of bottom band relative to top band indicates an increase in specific mRNA in the sample. B: quantitation of competitive RT-PCR data similar to those shown in A normalized to beta -actin. Values are means ± SD; n = 3 lungs. Nicotine exposure significantly increases expression of SP-A and SP-C mRNAs (P < 0.05) but not of CC10 mRNA.

Because an increase in the levels of SP-A and SP-C mRNAs could reflect either an increase in the amount of message per cell or an increase in the fraction of cells expressing these genes, we used immunocytochemical staining with a polyclonal antibody directed against SP-C precursor protein (25) to obtain a preliminary assessment of the relative numbers of SP-C-positive cells. We did not observe any apparent difference in the number of stained cells in control versus nicotine-treated lungs in a small number of lungs taken from two separate experiments (data not shown). Although more extensive morphometric analysis will be required to determine whether there might be a small change in the number of type II cell precursors, we conclude that the majority of the effect of nicotine on SP-A and SP-C mRNA levels probably represents a change in gene expression (via either transcription or message stability) rather than an effect on type II cell differentiation.

To determine whether the effect of nicotine on expression of SP-A and SP-C mRNA was mediated by a member of the family of nicotinic acetylcholine receptors (nAChRs), we attempted to block the effect by coadministration of an equimolar concentration of the nAChR antagonist D-TC. As shown in Fig. 3, D-TC significantly blocked the nicotine-induced increase in SP-A and SP-C mRNA levels, demonstrating the involvement of nAChRs.


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Fig. 3.   Blocking of nicotine stimulation of SP-A and SP-C mRNA expression by nicotinic acetylcholine receptor antagonist D-tubocurarine (D-TC). Lung buds were grown for 11 days in control medium (no added drug), medium + 1 µM Nic alone, medium + 1 µM Nic and 1 µM D-TC, or medium + 1 µM D-TC alone. A: typical examples of competitive RT-PCR gels showing lanes corresponding to 1 sample lung from each of the 4 experimental conditions assayed for SP-A, SP-C, and beta -actin mRNAs. Interpretation as in Fig. 2. +, Presence; -, absence. B: quantitation of competitive RT-PCR data similar to those shown in A (also including data for CC10). Values are means ± SD; n = 3 lungs. Stimulation of SP-A and SP-C mRNA expression by nicotine is significant relative to control, Nic + D-TC, or D-TC alone, P < 0.05. Differences among control, Nic + D-TC, and D-TC alone are not significant for any of these mRNAs.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The finding that nicotine exposure stimulates branching morphogenesis is consistent with the idea that nicotine may accelerate the developmental program of the lung. In contrast, the fact that expression of SP-A and SP-C mRNAs is stimulated by nicotine, whereas the expression of CC10 mRNA is unaffected, indicates that the effect on the SP-A and SP-C genes is not the result of a global stimulation of differentiation-associated genes or of differentiation in general. Thus, although we have demonstrated a direct stimulatory effect of nicotine on some indexes of lung development, it appears that the overall effect of nicotine exposure is more complicated than we had originally hypothesized.

We would like to know the identity of the cells bearing the nAChRs that we have detected. We do not know whether the precursors of parasympathetic ganglionic neurons are present in the embryonic lungs at the time of explant. Abundant evidence suggests that mature pulmonary neuroendocrine cells possess nAChRs (2, 18, 19). However, an earlier study by Wuenschell et al. (29) showed that these cells do not become overtly differentiated until 11 days in culture, whereas we saw an effect of nicotine on branching after just 4 days. The possibility that there could be nAChRs on undifferentiated epithelial cells is of great interest because a number of lung epithelial tumor lines have been shown to express nAChR subunits (6, 24) and because nicotine is known to be mitogenic for some of these cell lines (15).

The negative effects of smoking during pregnancy on fetal growth and viability are thought to be primarily due to fetal hypoxia (1). The mechanisms underlying the positive effect of maternal smoking on fetal lung maturation are less well understood. It has been proposed that chronic fetal stress, from a variety of causes, can lead to accelerated maturation of the lung and other organ systems as some sort of compensatory response (11). This has been the prevailing hypothesis to explain enhanced lung maturation in the smoking-exposed fetus (9, 28), although the exact mechanism is not clear (13). It is possible that the effect is mediated, at least in part, by glucocorticoids because these hormones are a major factor in normal fetal lung maturation (reviewed in Ref. 3) and because cortisol levels are elevated in the amniotic fluid of smokers (10). Increased glucocorticoid secretion due to stress-related release of adrenocorticotropic hormone could be a general mechanism for the stimulation of lung maturation in chronically stressed fetuses, and nicotine could play a direct role in the specific case of tobacco smoke exposure because it has been shown that nicotine acts centrally to increase secretion of adrenocorticotropic hormone (17).

Our new findings suggest that, in addition to the probable hormonal mechanisms acting to enhance lung maturation, there may be a direct, local effect of nicotine on the developing lungs of tobacco smoke-exposed fetuses. We hypothesize that the effect of nicotine on SP-A and SP-C mRNA expression could at least partially explain the effect of maternal smoking on the incidence of RDS even in the absence of a corresponding stimulation of SP-B mRNA production. SP-A, SP-B, and SP-C all contribute to the functional properties of pulmonary surfactant (for reviews, see Refs. 3, 20, 27), and identification of mutations in the human SP-B gene has shown that normal SP-B gene function is essential for viability (25). It has also been shown, however, that normal fetuses at 24 wk gestation already have SP-B mRNA levels that are 50% of the adult level, whereas SP-A mRNA is still barely detectable at this stage (3). Because both SP-A and SP-B are thought to be necessary for the formation of tubular myelin, the presumed structural intermediate between the lamellar bodies and the functional phospholipid monolayer (20, 27), it appears at least possible that a premature infant could develop RDS specifically due to a lack of SP-A and the consequent inability to form tubular myelin. Such an infant might be capable of producing enough SP-B to avoid developing RDS if SP-A expression were stimulated in some way.

Additional studies are required to further explore the effect of nicotine on lung maturation as well as to determine the nature and location of the nAChRs in the developing lung.

    ACKNOWLEDGEMENTS

This work was supported in part by funds from the Cigarette and Tobacco Surtax Fund of the State of California; by Grant 4KT-0339 from the Tobacco-Related Disease Research Program of the University of California (to C. W. Wuenschell); and by National Heart, Lung, and Blood Institute Grants HL-44977 and HL-44060 (to D. Warburton).

    FOOTNOTES

Address for reprint requests: C. W. Wuenschell, Center for Craniofacial Molecular Biology, Univ. of Southern California, 2250 Alcazar St., CSA 1st Floor, Los Angeles, CA 90033.

Received 25 April 1997; accepted in final form 26 September 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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