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Nicotine-responsive genes in cultured embryonic mouse lung buds: interaction of nicotine and superoxide dismutase
Pharmacological Research 50 (2004) 341–350
Nicotine-responsive genes in cultured embryonic mouse lung buds: interaction of nicotine and superoxide dismutase Carol Wuenschell a,∗ , Masao Kunimi a , Carmenza Castillo a , Paul Marjoram b a
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, CA 90033-9062, USA b Division of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9062, USA Accepted 5 February 2004
Abstract Nicotine exposure during prenatal development may be a cause of the abnormal lung function seen in infants born to smoking women. Previously we used an organ culture system to demonstrate that nicotine directly affects branching morphogenesis and gene expression in embryonic mouse lung buds. Here we attempt to identify genes potentially involved in the nicotine response and explore the relationship between gene expression changes and stimulation of branching. DNA microarray technology, analyzed by DChip software, and semi-quantitative RT–PCR were applied to RNA samples from embryonic lung buds grown in presence or absence of nicotine. Four genes, BAX, calcyclin, osteopontin and Cu–Zn superoxide dismutase (SOD1), identified by the microarray as showing changes in mRNA level with nicotine treatment were investigated in detail. RT–PCR showed that nicotine exposure resulted in significant decreases in mRNA levels for BAX, calcyclin and osteopontin, but nicotine did not affect the mRNA level of SOD1. Nicotine-induced changes in BAX, calcyclin and osteopontin mRNAs showed a general correlation with stimulation of branching, implying a common mechanism for effects of nicotine on branching and on gene expression. BAX, calcyclin and osteopontin mRNA levels were found to be developmentally regulated, but only the effect of nicotine on BAX mRNA was parallel to the developmental change in vivo, suggesting that nicotine action cannot be explained simply as a stimulation of the embryonic lung’s developmental program. Addition of exogenous SOD to the culture medium resulted in increased branching similar to that caused by nicotine, but, unexpectedly, branching was not increased relative to control when nicotine and SOD were co-administered, suggesting interfering mechanisms of action of the two agents. Exogenous SOD was found to alter mRNA levels of BAX, calcyclin and osteopontin in a pattern that differed from that seen in response to nicotine. Gene expression changes seen with co-administration of nicotine and SOD yielded further evidence of interaction between these agents. In conclusion, three putative nicotine-responsive genes were identified whose expression was also influenced by developmental stage and by exogenously added SOD. A common mechanism likely underlies nicotine’s effects on both branching and gene expression in this system. Our evidence also suggests that nicotine and SOD stimulate branching by distinct but interacting mechanisms. © 2004 Elsevier Ltd. All rights reserved. Keywords: Branching morphogenesis; BAX; Calcylcin; Osteopontin
1. Introduction Although acetylcholine is known primarily as a neurotransmitter, it is present at many sites outside the nervous system. It appears to be a very old signalling molecule in evolutionary terms, although its function and mode of action in extra-neuronal contexts are generally not clear [1]. In recent years there have been a number of reports of expression Abbreviations: BAX, Bcl2-associated protein X; EST, expressed sequence tag; nAChR, nicotinic acetylcholine receptor; Nic, nicotine; ROS, reactive oxygen species; SOD, superoxide dismutase ∗ Corresponding author. Tel.: +1-323-442-3421; fax: +1-323-442-2981. E-mail address: [email protected] (C. Wuenschell). 1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.02.001
of so-called “neuronal” nicotinic acetylcholine receptors (nAChRs) in various types of non-neuronal cells including airway epithelial cells in the lung ([2,3], for review see [4]). These reports suggest that extra-neuronal nicotinic receptorbased signalling systems could be involved in autocrine or paracrine signalling in various tissues, including lung. Infants born to women who smoked during pregnancy have abnormal lung function that persists during the first years of life [5,6]. The diminished lung capacity and decreased expiratory flow rates seen in these children suggest that exposure to maternal smoking during the period of lung development in utero may have altered the structure or physiology of the airways. Nicotine crosses the placenta [7] and animal studies have shown that maternally
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administered nicotine can cause developmental changes in the lung in the absence of other constituents of tobacco smoke [8–11]. While maternally-administered nicotine could act on the fetus indirectly through maternally-derived signalling molecules or general fetal hypoxia, we have used an organ culture model system to demonstrate that nicotine exposure directly affects branching morphogenesis and gene expression in isolated embryonic mouse lung buds through an nAChR-mediated mechanism [12]. While the stimulation of branching and surfactant gene expression we have seen in the lung bud culture model appear paradoxical in light of the generally negative effects of maternal smoking on infant health and survival, these observations may be related to the equally paradoxical observation that infants born to smoking mothers have apparently more mature lungs as evidenced by a lower incidence of respiratory distress syndrome than unexposed infants of the same gestational age [13]. Based on the above considerations, it seems plausible that extra-neuronal nAChR-based signalling systems could come into play during development of the lung and that these systems could be involved in mediating the pulmonary effects of fetal exposure to maternal smoking. We have hypothesized that signalling through nAChRs during nicotine exposure of cultured embryonic lung buds leads to specific changes in gene expression that could be used to further study the nicotine response. As a part of our effort to study the extra-neuronal nAChR systems in the developing mouse lung, we have therefore used DNA microarray technology to explore the relationship of gene expression changes to the observed effect on branching morphogenesis, as well as to identify genes that are potentially involved in the response to nicotine in cultured embryonic mouse lung buds. In the course of these investigations we serendipitously uncovered effects of superoxide dismutase (SOD) on branching and gene expression in the organ culture system and found evidence of interactions between these effects and the effects of nicotine.
2. Materials and methods 2.1. Animals and organ culture Timed pregnant female Swiss–Webster or C3H/HeN mice (Simonsen), were sacrificed on the 11th day of gestation (E11) (day of plug is E0). Lung buds were dissected and staged based on the number of airway side-branches in the left lung. Organ cultures were set up as previously described [12]. Briefly, lung buds were cultured for 4 days on Millapore filters at the air/medium interface in chemically defined BGJb medium (Gibco) under an atmosphere of 5% CO2 at 37 ◦ C. Lung buds subjected to nicotine treatment were stage-matched to control lung buds grown in the absence of nicotine. For treatments, 1 uM [−]nicotine hydrogen tartrate and/or 150 U ml−1 SOD (Sigma) were added to the culture medium.
2.2. Quantification of branching morphogenesis Lung buds were removed from the filters, and terminal branches counted for all lobes of each lung. Data are presented as percent of the average control value for the same experiment. Lung buds having at least a 25% increase in branches relative to the matched control were considered to show stimulation of branching. 2.3. DNA microarray gene expression analysis Lung buds used for the microarray analysis were selected from six independent organ culture experiments and pooled to form three samples, each containing a total of 11– 15 lungs. Two of the samples were composed of nicotinetreated lungs while the third contained matched controls. The two nicotine-treated samples were chosen to represent low response and high response to nicotine, respectively. Nicotine-treated lungs were assigned to these samples based on branch counts, compared to stage-matched control lungs from the same experiment, to control for differences arising from variation in the initial stage of development. Total RNA extracted from each sample was submitted to the Childrens Hospital of Los Angeles Microarray Core Facility for analysis using Affymetrix MG-U74A microarrays (Santa Clara, CA). RNA sample preparation and hybridization were performed according to procedures provided by Affymetrix. Computer analysis of the resulting data was performed using the DChip software package [14]. Samples were run on triplicate microarrays and the resulting data combined into subsets and compared using DChip. Defective probes sets on the MG-U74A microarray were identified and eliminated as directed by Affymetrix. A total of 89 genes were identified as showing changes in gene expression using a value of 1.25 for the lower bound of the 95% confidence interval for the fold change as a cutoff. Of the 89 genes, 82 were scored present on all microarrays, and the remainder were scored absent on no more than one of the microarrays. For the purposes of reporting, approximate fold changes in expression level were calculated from the averages of the signals obtained from the arrays in each subset. Increases are reported as the direct ratio, decreases as the negative reciprocal. The lower bound of the 95% confidence interval reflects the confidence in the fold change estimates. The DChip statistical analysis yields a 97.5% probability that the actual fold change is at least as great as the value of the lower bound. 2.4. Semi-quantitative RT–PCR Relative message levels of selected genes were measured by RT–PCR employing 18S rDNA as an internal standard (QuantumRNA 18S Internal Standards, Ambion). Total RNA was extracted from cultured lung buds (RNAwiz Total RNA Isolation Kit, Ambion). RNA was treated with DNase I (Ambion) to eliminate genomic contamination.
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2.5. Statistical analysis Unless otherwise indicated, data presented are means ± S.D. of values from three or more replicate samples for each condition. Significance of differences between means was evaluated by t-test for pairs of samples (criterion: P < 0.05) or by analysis of variance (ANOVA) for multiple sample comparison.
3. Results 3.1. Microarray samples and analysis Stimulation of branching morphogenesis in response to nicotine is a variably expressed trait in outbred Swiss–Webster mice. While the full reasons for this are not known, a genetic contribution is likely since lung buds from the inbred strain C3H exhibit much less variation and no stimulation of branching in response to nicotine (Fig. 1). Swiss–Webster mouse embryos were chosen for the present study because the greatest magnitude of nicotine branching response was observed in lung buds from these animals, suggesting they might also show the largest changes in gene expression. To investigate the relationship between stimulation of branching morphogenesis and changes in gene expression, lung buds were collected after 4 days in culture in
Individual branch counts
(% of avg. control for each exp.)
250
(a)
200 150 100 50 0
Individual branch counts
250
(b)
(% of avg. control for each exp.)
Reverse transcription was performed using MLV reverse transcriptase and random decamer primers (Ambion). The cDNA was subjected to PCR using specific primer pairs for each gene of interest based on GenBank sequence data. Identities of amplified genes were confirmed by sequencing. Primer sequences were as follows: BAX (accession no. L22472): 5 -CAGGGTTTCATCCAGGATCGAG-3 , 5 -TGTCCAGCCCATGATGGTTCTGA-3 ; Calcyclin (accession no. M37761): 5 -CTCGTGGCCATCTTCCACAAGTA-3 , 5 -CATTGTAGATCAAAGCCAAGGCC-3 ; Osteopontin (accession no. X13986): 5 -ACCATGAGATTGGCAGTGATTTGC-3 , 5 -ATCAGACTCATCCGAATGGTGAGA-3 ; SOD1 (accession no. M35725): 5 -TGAAAGCGGTGTGCGTGCTGAAG-3 , 5 -GGAATGCTCTCCTGAGAGTGAGA-3 . PCR reactions lacking RT product were run to control for reagent contamination. Amplification was done in a GeneAmp PCR System 9700 (Applied Biosystems). Optimal cycle number, denaturation, annealing, and extension temperatures were determined empirically to achieve amplification within the linear range. Amplification of 18S cDNA was adjusted by the ratio of 18S primer to competitor template to achieve amplification in a range similar to that of each specific cDNA. Reaction products were analyzed by agarose gel electrophoresis and densitometric analysis performed on electronically captured digital images of stained gels. Results are presented as a ratio of the specific cDNA (target)/adjusted 18S band intensities, or as percent of control.
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200 150 100 50 0
Fig. 1. Distribution of numbers of terminal branches from individual nicotine-exposed embryonic mouse lung buds of outbred Swiss–Webster vs. inbred C3H mice. Swiss–Webster (a) data combined from seven experiments (n = 3–5). C3H (b) data combined from five experiments (n = 3–6). Each bar represents the number of terminal branches for a single lung bud grown in culture for 4 days in the presence of 1 uM nicotine, expressed as a percent of the average control from the same experiment. Values are ranked from least to greatest to facilitate comparisons.
the presence or absence of nicotine, and the nicotine-treated lungs divided into samples showing a low branching response or a high branching response. Microarray analysis was performed as described in the methods section. Since nicotine exposure might have effects on developing lung buds unrelated to branching morphogenesis, we attempted to identify genes that might be involved in a general nicotine response as well as genes whose expression changes might be related to branching. To find putative general nicotine response genes, we selected genes whose expression appeared to be altered in the nicotine-treated lung samples relative to the control by means of three analyses: Control arrays were compared to either the high response (Analysis I), the low response (Analysis II), or to both high and low response nicotine arrays combined (Analysis III). The 27 genes identified by all three analyses were considered the most likely candidates for general nicotine response genes and are listed in Table 1. To identify genes with expression changes possibly related to the nicotine-induced stimulation of branching, we selected genes whose expression was differentially altered in the high response nicotine sample relative to the low response and control samples using three analyses: The high response nicotine arrays were compared to the control (Analysis I, same as above), the low
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Table 1 Genes possibly associated with nicotine exposure independent of branching GenBank accession no.
Gene name and description
Expression fold change
Low bound confidence interval
L22472 M83749 AF038939 AW124902 M35725 AI851740 M12481 X12973 M15501 U34259 AW227650 AI844532 L04961 AI846739 U05809 X05546 Y12713 AA815831 AI848479 AI854006 AV363696 AI853444 AI848522 AW124028 AW125079 AW123408 AI505453
BAX, Bcl2-associated X protein Cyclin D2 Paternally expressed gene 3, Peg3 EST similar to calcium binding protein P22 Cu–Zn superoxide dismutase 1, SOD1 EST similar to actin related protein 2/3 complex, subunit 3 Actin, beta, cytoplasmic Myosin alkali light chain (fast skeletal muscle isoform) Actin, alpha, cardiac Lysosomal-associated protein transmembrane 4 alpha TRAP gamma (translocon-associated protein gamma subunit) EST similar to murine sf3b1 pre-mRNA splicing factor SF3b Inactive X specific transcript EST similar to rat glycogen phosphorylase brain isozyme Transketolase (pentose phosphate pathway) mRNA fragment for gag related peptide Mu ERV-L gag, pol, and dUTPase genes (retroviral) EST similar to multiple genes including intracisternal A particle EST similar to multiple genes including D-like retrovirus D1 EST similar to human SET translocon (myeloid leukemia) EST similar to FGF receptor 2, 3 untranslated Unknown EST Unknown EST Unknown EST Unknown EST Unknown EST Unknown EST
−1.59 2.11 1.50 −1.59 2.12 −1.50 2.19 −2.05 −1.67 −1.48 −2.04 1.56 2.24 −1.86 −1.76 −1.87 −2.00 −2.00 −1.81 1.60 1.90 −1.86 2.05 1.69 −1.66 1.70 1.65
−1.47 1.67 1.37 −1.35 1.85 −1.38 1.63 −1.85 −1.45 −1.35 −1.82 1.42 1.92 −1.55 −1.47 −1.69 −1.75 −1.68 −1.57 1.33 1.69 −1.51 −1.78 1.42 −1.46 1.40 1.49
response nicotine arrays (Analysis IV), or to control and low response arrays combined (Analysis V). The 10 genes identified by all three of these analyses were considered the best candidates for genes whose expression changes were related to stimulation of branching and these genes are listed in Table 2. 3.2. RT–PCR analysis of selected putative nicotine-responsive genes: relationship of gene expression to stimulation of branching Two genes from the putative general response category, encoding Bcl2-associated protein X (BAX) and Cu–Zn
superoxide dismutase (SOD1), were selected for quantitative RT–PCR analysis. BAX is a pro-apoptotic mediator (for review see [15]) chosen for further study because the down-regulation of its message by nicotine, shown in the microarray data, appeared to be consistent with the observation that nicotine is anti-apoptotic in small cell lung carcinoma lines [16,17]. SOD1 is an antioxidant enzyme that dismutes superoxide anion, O2 − , to form hydrogen peroxide, H2 O2 , which is then destroyed by catalase [18]. Since the culture system is hyperoxic relative to normal fetal tissue, we hypothesized that up-regulation of SOD1 in response to nicotine might cause increased branching in the culture system as a result of moderation of oxidative stress.
Table 2 Genes possibly associated with nicotine-induced stimulation of branching GenBank accession no.
Gene name and description
Expression fold change
Low bound confidence interval
X66449 X13986 U79550 Z12171 M28845 X13297 M77497 AA921489 AF038995 AW123810
Calcium binding protein A6 (S100A6 = calcyclin) Osteopontin (minopontin, secreted phosphoprotein 1) Slugh (slug, chicken homolog) Dlk 1 (delta-like homolog, Drosophila) Early growth response 1 (Egr1, NGF I-A, Krox 24) Actin, alpha, vascular smooth muscle Cytochrome P450, 2f2 (naphthalene hydrogenase) EST similar to surfactant associated protein C Putative RNA helicase RCK (DEAD box helicase) Unknown EST
1.58 −1.84 −1.57 −2.05 −1.46 1.40 1.49 −7.20 1.92 1.71
1.45 −1.60 −1.33 −1.96 −1.26 1.26 1.28 −6.09 1.70 1.40
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Two additional genes, those encoding calcyclin and osteopontin, were chosen from among the putative branchingrelated genes. Calcyclin (S100A6) is a calcium binding protein whose function is not clearly defined. Calcyclin expression is induced as part of the serum mediated growth response, suggesting the possibility of responsiveness to growth factors [19]. Roles have also been suggested in the response of lung fibroblasts to mechanical strain, and in mucus secretion [20,21]. Calcyclin was also chosen in part because changes in intracellular calcium may be involved in signalling via nAChRs which are ligand gated cation channels. Osteopontin is a secreted, phosphorylated glycoprotein implicated in diverse processes. It is expressed in bronchial epithelium and smooth muscle cells and its proposed functions include involvement in bone development and resorption, inflammation, calcium regulation, cell adhesion, and signalling through binding to ␣v3 integrin (for review see [22,23]). The role of osteopontin in lung development is not clear. New lung culture experiments were performed to test whether the DChip analyses had identified general nicotine response genes or genes whose expression correlated with stimulation of branching by nicotine. Lung buds to be treated with nicotine were matched with lung buds of the same stage of development that were grown in control medium. At the end of each experiment, branches were counted and the lung buds were pooled into samples and separated into two groups based on whether or not the nicotine-treated lungs showed a stimulation of branching. RT–PCR analysis was performed for all four genes and results from multiple experiments were normalized by expressing the values as a percent of the average control for each experiment. For each of the four genes, the results were then combined and averaged for the control and nicotine-treated samples in each of the two groups (branching stimulated by nicotine and branching not stimulated by nicotine). The results, shown in Fig. 2, were compared with the microarray results and the relationship between stimulation of branching and changes in gene expression was assessed. Fig. 2a represents data from paired control and nicotine-treated samples showing a stimulation of branching, while Fig. 2b represents data from samples showing no such stimulation. The RT–PCR results in Fig. 2a show a significant decrease with nicotine treatment in the levels of mRNAs encoding BAX, calcyclin and osteopontin, but no change in SOD1 mRNA. Fig. 2b shows the same trends although the changes in mRNA levels are less marked and there is a loss of statistical significance. Although we designed the DChip analyses to differentiate between genes whose expression changes were associated with the nicotine stimulation of branching and genes that might respond to nicotine in a more general way, our findings do not suggest that such a dichotomy exists. The DChip analyses of the microarray data had suggested that the changes in expression of calcyclin and osteopontin messages were correlated with stimulation of branching, whereas the changes in the
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Fig. 2. RT–PCR analysis of selected individual genes: effect of nicotine treatment and relation to stimulation of branching. Lung buds were grown for 4 days in the presence or absence of nicotine and pooled into samples (3–6 lungs per sample) based on whether branching was increased (a) or not increased (b) in the nicotine-treated sample relative to control. Message levels for BAX, calcyclin, osteopontin, and SOD1 were assessed by semi-quantitative RT–PCR normalized to adjusted 18S rRNA for each sample. Shown are means ± S.D. for each gene in five to seven samples combined from several experiments by expressing the data as a percent of the average control for each experiment. (*) Nicotine-treated samples in (a) differ significantly from control for BAX (P = 0.01), calcyclin (P = 0.01), and osteopontin (P = 0.009).
levels of BAX and SOD1 mRNAs might be independent of branching. SOD1 mRNA did not change in the RT–PCR analysis, of course, and in that sense is not correlated with branching, but not in the way we had anticipated. The extent of change in BAX mRNA does not appear to be any more similar between Fig. 2a and b than is the case for calcyclin and osteopontin. The most likely interpretation now appears to be that a common mechanism is driving both the stimulation of branching and the changes in gene expression, with those nicotine-treated lungs that are the most strongly affected with respect to gene expression also being the most likely to be identifiable as showing branching stimulation. The lack of complete correspondence between the microarray and RT–PCR results was of concern. The two sets of data are in agreement for BAX and osteopontin, but SOD1 did not change in the RT–PCR analysis and calcyclin mRNA actually changed in opposite directions in the two data sets. Occasional false positives or anomalous results can be expected in any microarray study because of the large number of genes involved and the inherent complexity of biological
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systems. The opposite RT–PCR result observed for calcyclin was so unexpected, however, that it led us to consider whether there could be factors other than nicotine that could have affected expression of this gene. We considered possible confounding effects from the lung buds’ developmental program, and a hypothetical elevation of SOD1 as detailed in the following sections. 3.3. Relationship of nicotine response to developmental changes in gene expression Changes in gene expression in the high response sample might reflect a more advanced stage of development since many of these lung buds were larger and more branched at the end of the culture period than those in the other two samples. Differential gene expression in this sample might, therefore, be only indirectly related to the mechanism of nicotine action. We therefore examined the developmental patterns of expression of BAX, calcyclin and osteopontin mRNAs in lungs developing in vivo by RT–PCR. The results are presented in Fig. 3. Message levels for each gene were assessed in lungs from E12, E14, and E18 embryos/fetuses and from postnatal day 2 (P2) mouse pups. All three of these genes appeared to be developmentally regulated, but the patterns of regulation differed substantially. BAX mRNA level was down-regulated between E12 and E18 with E14 apparently representing a transitional stage. Calcyclin mRNA increased dramatically with development, with the greatest change between E14 and P2. Osteopontin mRNA increased modestly from E12 to a plateau at E14. Developmental changes and nicotine-induced changes were thus parallel only for BAX mRNA, being opposite for calcyclin and osteopontin. Taken together with the findings
Fig. 3. Developmental patterns of expression of BAX, calcyclin and osteopontin during lung development in vivo. Lungs were harvested on days 12, 14 and 18 of gestation (E12, E14, E18) and on postnatal day 2 (P2). Samples from three individuals were analyzed for each stage, except E12 for which three pooled samples of three lungs per pool were analyzed. Message levels for BAX, calcyclin, and osteopontin were assessed by semi-quantitative RT–PCR, normalized to adjusted 18S rRNA, for each sample. All calcyclin values were divided by 3 to facilitate use of a common scale. Shown are means ± S.D. Significant differences between stages were observed for all three genes (determined by ANOVA).
presented in Fig. 2, these results suggest that nicotine can perturb the developmental program of individual genes, specifically calcyclin and osteopontin. The results also suggest that developmental stage could be a confounding factor in studies of the effect of nicotine on expression of these genes. In our microarray experiment, however, the increase in calcyclin mRNA level cannot be explained in terms of the developmental program overriding the nicotine effect. If this had occurred, both calcyclin and osteopontin should have been affected, but osteopontin mRNA was not increased in the microarray data. 3.4. Effect of exogenously added SOD and interaction with nicotine While the identification of SOD1 by the microarray analysis could represent a technical false positive of unknown origin on the microarray, the fact that our results were obtained from triplicate arrays makes it relatively more likely that there was a real difference in SOD1 mRNA levels between the samples applied to the arrays. We therefore considered the possibility that SOD1 mRNA could have been elevated in the nicotine-treated samples for reasons unrelated to nicotine exposure. SOD1 activity has the potential to affect three molecules that are implicated in signalling in biological systems, superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), and nitric oxide (NO) (for review see [18]). We therefore speculated that a change in SOD1 expression could potentially have affected expression of other genes or development of the lung buds through one or more of these signalling pathways. To assess whether an elevation of SOD1 in the microarray samples could account for the increase in calcyclin mRNA in those same samples, we examined the effect of exogenously added SOD on calcyclin gene expression in the culture system. Fig. 4 shows the combined results from several such experiments. In all of the experiments,
Fig. 4. Effect of exogenous SOD on calcyclin mRNA levels in cultured lung buds. Lung buds were grown for 4 days in control medium or medium containing 150 U ml−1 SOD. Lung buds (4–5 per condition) were pooled for each condition and processed for determination of mRNA levels by semi-quantitative RT–PCR, normalized to adjusted 18S rRNA, for each sample. Shown are means ± S.D. for five samples combined from several experiments by expressing the data as a percent of the average control for each experiment.
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calcyclin mRNA was increased in the SOD-treated samples relative to control, although statistical significance was not achieved owing to the wide range in magnitude of the effect. These results are broadly consistent with the hypothesis that the elevated calcyclin mRNA seen in the nicotine-treated microarray samples could be related to the increased SOD1 mRNA that was seen in these same samples. To further explore the effects of SOD, in the context of the relationship of gene expression to branching, several experiments were performed in which exogenous SOD was added alone or co-administered with nicotine in the culture system, and the effects on gene expression were examined. The resultant effects on branching morphogenesis are shown in Fig. 5a. Both nicotine alone and SOD alone caused a significant stimulation of branching. Paradoxically, no stimu-
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lation of branching was seen when nicotine and SOD were co-administered. This suggests that nicotine and SOD act on branching by different mechanisms that interfere when activated simultaneously. The effects of these treatments on gene expression are presented in Fig. 5b–f. The results for osteopontin (Fig. 5b) mirror the effects on branching. In three experiments, osteopontin mRNA was significantly decreased following exposure to either nicotine or SOD alone. When nicotine and SOD were co-administered, however, the resulting level of osteopontin mRNA was similar to control in two of the three experiments and similar to the level obtained with SOD alone in the third. The combined results from all three experiments are shown graphed together. The first two experiments are consistent with an interference between the
Fig. 5. Effect of exogenous SOD on gene expression in cultured lung buds: interaction with nicotine. Lung buds were grown for 4 days in control medium or medium with 1 uM nicotine alone, 150 U ml−1 SOD alone, or nicotine plus SOD. Terminal branches were counted and presented as mean ± S.D. in (a) (n = 4–5). (*) Nicotine- and SOD-treated lungs had significantly more branches than control or lungs treated with nicotine plus SOD (P = 0.045 and 0.001, respectively, relative to control and 0.05 and 0.008, respectively, relative to nic + SOD). For gene expression, lung buds were pooled (3–5 per sample) and mRNA levels determined by semi-quantitative RT–PCR for osteopontin (b), calcyclin (c and e), and BAX (d and f). Values are expressed as percent of control from the same experiment. Mean ± S.D. is shown in (b) (n = 3). (*) Values for nicotine and SOD differ significantly from control (P = 0.003 and 0.01, respectively). Means with error bars representing high and low values from two experiments are shown in (c) and (f). Bar heights in (d) and (e) represent values from single experiments.
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actions of nicotine and SOD as was observed for branching (Fig. 5a), while the third suggests a lack of effect of nicotine on the lungs in the co-administration group. The results were more complex for calcyclin and BAX. For both of these genes, the effect of SOD alone was opposite to that previously observed for nicotine. In some experiments, two in the case of calcyclin (Fig. 5c) and one in the case of BAX (Fig. 5d), co-administration changed the message level seen with SOD alone back towards the control level. This pattern is again suggestive of interference between nicotine and SOD. In other cases, however, the opposite appeared to occur and there was a synergistic increase in the message level with co-administration of the two agents. Fig. 5e shows this result for calcyclin in a single experiment. For BAX, only one experiment showed a clear synergistic increase with co-administration, while in the third experiment the message levels were similar for SOD alone and SOD plus nicotine. The results of these last two experiments are graphed together (Fig. 5f). The effect on BAX expression of SOD alone was quite variable and this may have affected the interaction with nicotine with respect to this gene. We do not have an explanation of the causes of the different patterns of gene expression. We can speculate that there may be an interplay of opposing factors (see Section 4). Alternatively, there could be genetic variants within the outbred Swiss–Webster mouse population. The results for all three genes taken together are generally consistent with the interpretation that there is interaction between the effects of nicotine and SOD in this system, which can manifest as interference. The observation that both branching and osteopontin mRNA expression are affected similarly by nicotine and SOD alone, but show a partial or complete reversal of these effects upon co-administration, suggests that, at least for these two parameters, the effects of nicotine and SOD alone are probably produced through different mechanisms.
4. Discussion Our RT–PCR analysis successfully identified, from among the microarray results, three genes, BAX, calcyclin and osteopontin, whose mRNA levels appear to be influenced by nicotine exposure in embryonic lung buds developing in culture. The data further suggest that a common mechanism underlies the effect of nicotine on branching morphogenesis and on changes in expression of these genes. Further study of these genes may increase our understanding of the ways in which prenatal nicotine exposure can affect the developing lung. We believe the discrepancy between the RT–PCR and microarray data with respect to expression of calcyclin and SOD1 may reflect an unusual event that may have increased expression of SOD1 in the nicotine-treated samples applied to the microarrays. We speculate that factors affecting the expression of SOD1 might include oxidative stress and cellular
immune response or inflammation. Alternatively, there could be a genetic variant in the outbred Swiss–Webster mouse population with higher SOD1 expression. Our suggestion that elevated SOD1 could be responsible for the increase in calcyclin mRNA seen on the microarray is supported by our observation that addition of exogenous SOD tended to increase calcyclin mRNA levels. It is also supported by the observation of Hoyaux et al., who found that calcyclin was the only one of a dozen calcium binding proteins whose expression was elevated in a mouse model for familial amyotrophic lateral sclerosis bearing a gain of function mutation in SOD1 [24]. There is thus reason to believe that expression of calcyclin might be linked to SOD1 activity. We cannot, of course, rule out the possibility that whatever affected SOD or calcyclin expression in the microarray data could have also caused changes in some of the other genes in Tables 1 and 2 that we have not yet examined. Before each of these genes can be stated to be nicotine-responsive, its expression change with nicotine exposure must be independently verified. Our study of the developmental expression of the identified genes clearly shows that the effect of nicotine on gene expression in this system cannot be explained by a global stimulation of the developmental program, as the stimulation of branching might suggest. Our data indicate that nicotine can, in fact, override the developmental program in the cases of calcyclin and osteopontin gene expression. The effect of nicotine on BAX mRNA level could, however, be linked to development through an effect on developmentally programmed cell death. Heusch and Maneckjee showed that the anti-apoptotic effect of nicotine in a small cell lung carcinoma line involved an increase in expression of Bcl-2 [17]. BAX and Bcl-2 are related proteins that are thought to titrate one another with the balance of their relative expression levels determining the balance between pro- and anti-apoptotic influences in the cell (for review see [15]). This does not conflict with our results since a decrease in apoptotic potential could result from either a decrease in BAX expression or an increase in Bcl-2 expression, or from both changes occurring simultaneously. We did not independently investigate Bcl-2 in our system, and Bcl-2 is not represented on the MG-U74A microarray. The finding of an apparent interaction between nicotine and SOD is a potentially interesting observation. Although the underlying mechanism is not apparent from our studies, work by other investigators leads us to suggest a hypothetical mechanism for the effect of nicotine and SOD on branching morphogenesis involving the level of intracellular H2 O2 . Mayhan and Sharpe observed that exogenously added SOD antagonized the nicotine blockade of NO-mediated cholinergic vasodilation in the hamster cheek pouch [25], and suggested that this action of SOD was due to removal of reactive oxygen species (ROS) produced by nicotine. Recent unpublished data from the laboratory of Philip Tsao at Stanford University have indicated that nicotine can cause increased production of ROS by stimulation of the plasma membrane
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NAD(P)H oxidase complex in cultured endothelial cells. This NAD(P)H oxidase has also been demonstrated in lung fibroblasts and airway smooth muscle cells [26,27]. The O2 − produced by NAD(P)H oxidase is dismuted to H2 O2 by cellular SODs. Since such production of H2 O2 is required for growth factor signalling [18], the finding that nicotine stimulates NAD(P)H oxidase may provide a possible explanation for nicotine’s stimulation of branching and also for the interaction of nicotine and SOD. We suggest that nicotine may increase branching by potentiating growth factor signalling through increased production of intracellular H2 O2 . There is an effective concentration range of H2 O2 needed for growth factor signalling, however, with too little being ineffective and too much being toxic to the cell. This limited effective range could explain the variation that we have seen in the effect of nicotine on branching in the culture system. We know that the culture system is hyperoxic relative to fetal tissues and the positive effect of SOD on branching could therefore be due to moderation of oxidative damage through removal of ROS. The effectiveness of SOD as an antioxidant, however, depends upon the activity of catalase, since the action of SOD is to convert one ROS (O2 − ) to another (H2 O2 ), with the remainder of the antioxidant pathway being carried out by catalase [18]. An excess of H2 O2 could possibly occur if the combined effects of exogenous SOD and activation of NAD(P)H oxidase by nicotine were sufficient to overwhelm the system’s catalase activity since H2 O2 produced in the culture medium by added SOD could presumably diffuse into the tissue. The toxic effects of excess H2 O2 could explain the apparent interfering action of nicotine and SOD with respect to branching. There is a substantial literature on the involvement of ROS in apoptosis (for review see [18,28]). Since BAX is a proapoptotic mediator, it is possible that elevations of BAX mRNA in our system could reflect activation of apoptosis. We observed increases in BAX mRNA sometimes when SOD was added alone to the cultures and sometimes when nicotine and SOD were added together. Pervaiz and Clement have proposed that apoptosis can be triggered when there is a shift in the balance of ROS in the cell from O2 − to H2 O2 . Since added SOD could be expected to have this effect on the system, it is easy to imagine that adding SOD could trigger apoptosis, depending upon the extent of catalase action. Adding nicotine, which may generate O2 − , could make the situation worse in the presence of excess SOD activity. The interplay of the various effects of nicotine and SOD on growth and cell death could be quite complex and variation in the observed responses would not be surprising. In conclusion, three putative nicotine-responsive genes, BAX, calcyclin and osteopontin, were identified by microarray and RT–PCR analysis in embryonic mouse lung buds developing in culture. It appears that a common mechanism may underlie the nicotine effects on both gene expression and branching morphogenesis in this system. Expression of
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all three genes was developmentally regulated and also found to be affected by exogenously administered SOD, with patterns that differed from that of the nicotine response. Finally, our evidence suggests that the mechanisms underlying the effects of nicotine and SOD can interfere with one another when activated simultaneously. More studies are planned to better define the biology of the nicotine responses, including further examination of inbred strains of mice and use of an in utero nicotine exposure model.
Acknowledgements This work was supported by the California TobaccoRelated Disease Research Program of the University of California, grant nos. 4KT-0339 and 9RT-0238, and by NHLBI grant nos. HL54960 and K02-HL03786. We thank Dr. David Warburton for the generous gift of microarrays. We acknowledge Betty Schaub and the Childrens Hospital, Los Angeles Microarray Core facility for preparation and hybridization of the microarray samples, and Dr. Hosun Cheong for many helpful discussions.
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[12] Wuenschell CW, Zhao J, Tefft JD, Warburton D. Nicotine stimulates branching and expression of SP-A and SP-C mRNAs in embryonic mouse lung culture. Am J Physiol 1998;274:L165–70. [13] White E, Shy KK, Daling JR, Guthrie RD. Maternal smoking and respiratory distress syndrome. Obstet Gynecol 1986;67:365–70. [14] Li C, Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 2001;98:31–6. [15] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322–6. [16] Maneckjee R, Minna JD. Opioids induce while nicotine suppresses apoptosis in human lung cancer cells. Cell Growth Diff 1994;5:1033– 40. [17] Heusch WL, Maneckjee R. Signalling pathways involved in nicotine regulation of apoptosis of human lung cancer cells. Carcinogenesis 1998;19:551–6. [18] Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47–95. [19] Gou X, Chambers AF, Parfett CLJ, Waterhouse P, Murphy LC, Reid RE, et al. Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin. Tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells. Cell Growth Diff 1990;1:333–8. [20] Breen EC, Fu Z, Normand H. Calcyclin gene expression is increased by mechanical strain in fibroblasts and lung. Am J Respir Cell Mol Biol 1999;21:746–52.
[21] Timmons PM, Chan C-TJ, Rigby PWJ, Poirier F. The gene encoding the calcium binding protein calcyclin is expressed at sites of exocytosis in the mouse. J Cell Sci 1993;104:187–96. [22] Denhardt DT, Guo X. Osteopontin: a protein with diverse functions. FASEB J 1993;7:1475–82. [23] O’Regan A, Berman JS. Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation. Int J Exp Pathol 2000;81:373– 90. [24] Hoyaux D, Alao J, Fuchs J, Kiss R, Keller B, Heizmann CW, et al. S100A6, a calcium- and zinc-binding protein, is overexpressed in SOD1 mutant mice, a model for amyotrophic lateral sclerosis. Biochim Biophys Acta 2000;1498:264–72. [25] Mayhan WG, Sharpe GM. Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase. J Appl Physiol 1999;86:1126–34. [26] Thannickal VJ, Fanburg BL. Activation of an H2 O2 -generating NADH oxidase in human lung fibroblasts by transforming growth factor 1. J Biol Chem 1995;270:30334–8. [27] Brar SS, Kennedy TP, Sturrock AB, Huecksteadt TP, Quinn MT, Murphy TM, et al. NADPH oxidase promotes NF-B activation and proliferation in human airway smooth muscle. Am J Pysiol 2002;282:L782–95. [28] Pervaiz S, Clement M-V. A permissive apoptotic environment: function of a decrease in intracellular superoxide anion and cytosolic acidification. Biochem Biophys Res Commun 2002;290:1145– 50.
Nicotine-responsive genes in cultured embryonic mouse lung buds: interaction of nicotine and superoxide dismutase Carol Wuenschell a,∗ , Masao Kunimi a , Carmenza Castillo a , Paul Marjoram b a
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, CA 90033-9062, USA b Division of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9062, USA Accepted 5 February 2004
Abstract Nicotine exposure during prenatal development may be a cause of the abnormal lung function seen in infants born to smoking women. Previously we used an organ culture system to demonstrate that nicotine directly affects branching morphogenesis and gene expression in embryonic mouse lung buds. Here we attempt to identify genes potentially involved in the nicotine response and explore the relationship between gene expression changes and stimulation of branching. DNA microarray technology, analyzed by DChip software, and semi-quantitative RT–PCR were applied to RNA samples from embryonic lung buds grown in presence or absence of nicotine. Four genes, BAX, calcyclin, osteopontin and Cu–Zn superoxide dismutase (SOD1), identified by the microarray as showing changes in mRNA level with nicotine treatment were investigated in detail. RT–PCR showed that nicotine exposure resulted in significant decreases in mRNA levels for BAX, calcyclin and osteopontin, but nicotine did not affect the mRNA level of SOD1. Nicotine-induced changes in BAX, calcyclin and osteopontin mRNAs showed a general correlation with stimulation of branching, implying a common mechanism for effects of nicotine on branching and on gene expression. BAX, calcyclin and osteopontin mRNA levels were found to be developmentally regulated, but only the effect of nicotine on BAX mRNA was parallel to the developmental change in vivo, suggesting that nicotine action cannot be explained simply as a stimulation of the embryonic lung’s developmental program. Addition of exogenous SOD to the culture medium resulted in increased branching similar to that caused by nicotine, but, unexpectedly, branching was not increased relative to control when nicotine and SOD were co-administered, suggesting interfering mechanisms of action of the two agents. Exogenous SOD was found to alter mRNA levels of BAX, calcyclin and osteopontin in a pattern that differed from that seen in response to nicotine. Gene expression changes seen with co-administration of nicotine and SOD yielded further evidence of interaction between these agents. In conclusion, three putative nicotine-responsive genes were identified whose expression was also influenced by developmental stage and by exogenously added SOD. A common mechanism likely underlies nicotine’s effects on both branching and gene expression in this system. Our evidence also suggests that nicotine and SOD stimulate branching by distinct but interacting mechanisms. © 2004 Elsevier Ltd. All rights reserved. Keywords: Branching morphogenesis; BAX; Calcylcin; Osteopontin
1. Introduction Although acetylcholine is known primarily as a neurotransmitter, it is present at many sites outside the nervous system. It appears to be a very old signalling molecule in evolutionary terms, although its function and mode of action in extra-neuronal contexts are generally not clear [1]. In recent years there have been a number of reports of expression Abbreviations: BAX, Bcl2-associated protein X; EST, expressed sequence tag; nAChR, nicotinic acetylcholine receptor; Nic, nicotine; ROS, reactive oxygen species; SOD, superoxide dismutase ∗ Corresponding author. Tel.: +1-323-442-3421; fax: +1-323-442-2981. E-mail address: [email protected] (C. Wuenschell). 1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.02.001
of so-called “neuronal” nicotinic acetylcholine receptors (nAChRs) in various types of non-neuronal cells including airway epithelial cells in the lung ([2,3], for review see [4]). These reports suggest that extra-neuronal nicotinic receptorbased signalling systems could be involved in autocrine or paracrine signalling in various tissues, including lung. Infants born to women who smoked during pregnancy have abnormal lung function that persists during the first years of life [5,6]. The diminished lung capacity and decreased expiratory flow rates seen in these children suggest that exposure to maternal smoking during the period of lung development in utero may have altered the structure or physiology of the airways. Nicotine crosses the placenta [7] and animal studies have shown that maternally
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administered nicotine can cause developmental changes in the lung in the absence of other constituents of tobacco smoke [8–11]. While maternally-administered nicotine could act on the fetus indirectly through maternally-derived signalling molecules or general fetal hypoxia, we have used an organ culture model system to demonstrate that nicotine exposure directly affects branching morphogenesis and gene expression in isolated embryonic mouse lung buds through an nAChR-mediated mechanism [12]. While the stimulation of branching and surfactant gene expression we have seen in the lung bud culture model appear paradoxical in light of the generally negative effects of maternal smoking on infant health and survival, these observations may be related to the equally paradoxical observation that infants born to smoking mothers have apparently more mature lungs as evidenced by a lower incidence of respiratory distress syndrome than unexposed infants of the same gestational age [13]. Based on the above considerations, it seems plausible that extra-neuronal nAChR-based signalling systems could come into play during development of the lung and that these systems could be involved in mediating the pulmonary effects of fetal exposure to maternal smoking. We have hypothesized that signalling through nAChRs during nicotine exposure of cultured embryonic lung buds leads to specific changes in gene expression that could be used to further study the nicotine response. As a part of our effort to study the extra-neuronal nAChR systems in the developing mouse lung, we have therefore used DNA microarray technology to explore the relationship of gene expression changes to the observed effect on branching morphogenesis, as well as to identify genes that are potentially involved in the response to nicotine in cultured embryonic mouse lung buds. In the course of these investigations we serendipitously uncovered effects of superoxide dismutase (SOD) on branching and gene expression in the organ culture system and found evidence of interactions between these effects and the effects of nicotine.
2. Materials and methods 2.1. Animals and organ culture Timed pregnant female Swiss–Webster or C3H/HeN mice (Simonsen), were sacrificed on the 11th day of gestation (E11) (day of plug is E0). Lung buds were dissected and staged based on the number of airway side-branches in the left lung. Organ cultures were set up as previously described [12]. Briefly, lung buds were cultured for 4 days on Millapore filters at the air/medium interface in chemically defined BGJb medium (Gibco) under an atmosphere of 5% CO2 at 37 ◦ C. Lung buds subjected to nicotine treatment were stage-matched to control lung buds grown in the absence of nicotine. For treatments, 1 uM [−]nicotine hydrogen tartrate and/or 150 U ml−1 SOD (Sigma) were added to the culture medium.
2.2. Quantification of branching morphogenesis Lung buds were removed from the filters, and terminal branches counted for all lobes of each lung. Data are presented as percent of the average control value for the same experiment. Lung buds having at least a 25% increase in branches relative to the matched control were considered to show stimulation of branching. 2.3. DNA microarray gene expression analysis Lung buds used for the microarray analysis were selected from six independent organ culture experiments and pooled to form three samples, each containing a total of 11– 15 lungs. Two of the samples were composed of nicotinetreated lungs while the third contained matched controls. The two nicotine-treated samples were chosen to represent low response and high response to nicotine, respectively. Nicotine-treated lungs were assigned to these samples based on branch counts, compared to stage-matched control lungs from the same experiment, to control for differences arising from variation in the initial stage of development. Total RNA extracted from each sample was submitted to the Childrens Hospital of Los Angeles Microarray Core Facility for analysis using Affymetrix MG-U74A microarrays (Santa Clara, CA). RNA sample preparation and hybridization were performed according to procedures provided by Affymetrix. Computer analysis of the resulting data was performed using the DChip software package [14]. Samples were run on triplicate microarrays and the resulting data combined into subsets and compared using DChip. Defective probes sets on the MG-U74A microarray were identified and eliminated as directed by Affymetrix. A total of 89 genes were identified as showing changes in gene expression using a value of 1.25 for the lower bound of the 95% confidence interval for the fold change as a cutoff. Of the 89 genes, 82 were scored present on all microarrays, and the remainder were scored absent on no more than one of the microarrays. For the purposes of reporting, approximate fold changes in expression level were calculated from the averages of the signals obtained from the arrays in each subset. Increases are reported as the direct ratio, decreases as the negative reciprocal. The lower bound of the 95% confidence interval reflects the confidence in the fold change estimates. The DChip statistical analysis yields a 97.5% probability that the actual fold change is at least as great as the value of the lower bound. 2.4. Semi-quantitative RT–PCR Relative message levels of selected genes were measured by RT–PCR employing 18S rDNA as an internal standard (QuantumRNA 18S Internal Standards, Ambion). Total RNA was extracted from cultured lung buds (RNAwiz Total RNA Isolation Kit, Ambion). RNA was treated with DNase I (Ambion) to eliminate genomic contamination.
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2.5. Statistical analysis Unless otherwise indicated, data presented are means ± S.D. of values from three or more replicate samples for each condition. Significance of differences between means was evaluated by t-test for pairs of samples (criterion: P < 0.05) or by analysis of variance (ANOVA) for multiple sample comparison.
3. Results 3.1. Microarray samples and analysis Stimulation of branching morphogenesis in response to nicotine is a variably expressed trait in outbred Swiss–Webster mice. While the full reasons for this are not known, a genetic contribution is likely since lung buds from the inbred strain C3H exhibit much less variation and no stimulation of branching in response to nicotine (Fig. 1). Swiss–Webster mouse embryos were chosen for the present study because the greatest magnitude of nicotine branching response was observed in lung buds from these animals, suggesting they might also show the largest changes in gene expression. To investigate the relationship between stimulation of branching morphogenesis and changes in gene expression, lung buds were collected after 4 days in culture in
Individual branch counts
(% of avg. control for each exp.)
250
(a)
200 150 100 50 0
Individual branch counts
250
(b)
(% of avg. control for each exp.)
Reverse transcription was performed using MLV reverse transcriptase and random decamer primers (Ambion). The cDNA was subjected to PCR using specific primer pairs for each gene of interest based on GenBank sequence data. Identities of amplified genes were confirmed by sequencing. Primer sequences were as follows: BAX (accession no. L22472): 5 -CAGGGTTTCATCCAGGATCGAG-3 , 5 -TGTCCAGCCCATGATGGTTCTGA-3 ; Calcyclin (accession no. M37761): 5 -CTCGTGGCCATCTTCCACAAGTA-3 , 5 -CATTGTAGATCAAAGCCAAGGCC-3 ; Osteopontin (accession no. X13986): 5 -ACCATGAGATTGGCAGTGATTTGC-3 , 5 -ATCAGACTCATCCGAATGGTGAGA-3 ; SOD1 (accession no. M35725): 5 -TGAAAGCGGTGTGCGTGCTGAAG-3 , 5 -GGAATGCTCTCCTGAGAGTGAGA-3 . PCR reactions lacking RT product were run to control for reagent contamination. Amplification was done in a GeneAmp PCR System 9700 (Applied Biosystems). Optimal cycle number, denaturation, annealing, and extension temperatures were determined empirically to achieve amplification within the linear range. Amplification of 18S cDNA was adjusted by the ratio of 18S primer to competitor template to achieve amplification in a range similar to that of each specific cDNA. Reaction products were analyzed by agarose gel electrophoresis and densitometric analysis performed on electronically captured digital images of stained gels. Results are presented as a ratio of the specific cDNA (target)/adjusted 18S band intensities, or as percent of control.
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200 150 100 50 0
Fig. 1. Distribution of numbers of terminal branches from individual nicotine-exposed embryonic mouse lung buds of outbred Swiss–Webster vs. inbred C3H mice. Swiss–Webster (a) data combined from seven experiments (n = 3–5). C3H (b) data combined from five experiments (n = 3–6). Each bar represents the number of terminal branches for a single lung bud grown in culture for 4 days in the presence of 1 uM nicotine, expressed as a percent of the average control from the same experiment. Values are ranked from least to greatest to facilitate comparisons.
the presence or absence of nicotine, and the nicotine-treated lungs divided into samples showing a low branching response or a high branching response. Microarray analysis was performed as described in the methods section. Since nicotine exposure might have effects on developing lung buds unrelated to branching morphogenesis, we attempted to identify genes that might be involved in a general nicotine response as well as genes whose expression changes might be related to branching. To find putative general nicotine response genes, we selected genes whose expression appeared to be altered in the nicotine-treated lung samples relative to the control by means of three analyses: Control arrays were compared to either the high response (Analysis I), the low response (Analysis II), or to both high and low response nicotine arrays combined (Analysis III). The 27 genes identified by all three analyses were considered the most likely candidates for general nicotine response genes and are listed in Table 1. To identify genes with expression changes possibly related to the nicotine-induced stimulation of branching, we selected genes whose expression was differentially altered in the high response nicotine sample relative to the low response and control samples using three analyses: The high response nicotine arrays were compared to the control (Analysis I, same as above), the low
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Table 1 Genes possibly associated with nicotine exposure independent of branching GenBank accession no.
Gene name and description
Expression fold change
Low bound confidence interval
L22472 M83749 AF038939 AW124902 M35725 AI851740 M12481 X12973 M15501 U34259 AW227650 AI844532 L04961 AI846739 U05809 X05546 Y12713 AA815831 AI848479 AI854006 AV363696 AI853444 AI848522 AW124028 AW125079 AW123408 AI505453
BAX, Bcl2-associated X protein Cyclin D2 Paternally expressed gene 3, Peg3 EST similar to calcium binding protein P22 Cu–Zn superoxide dismutase 1, SOD1 EST similar to actin related protein 2/3 complex, subunit 3 Actin, beta, cytoplasmic Myosin alkali light chain (fast skeletal muscle isoform) Actin, alpha, cardiac Lysosomal-associated protein transmembrane 4 alpha TRAP gamma (translocon-associated protein gamma subunit) EST similar to murine sf3b1 pre-mRNA splicing factor SF3b Inactive X specific transcript EST similar to rat glycogen phosphorylase brain isozyme Transketolase (pentose phosphate pathway) mRNA fragment for gag related peptide Mu ERV-L gag, pol, and dUTPase genes (retroviral) EST similar to multiple genes including intracisternal A particle EST similar to multiple genes including D-like retrovirus D1 EST similar to human SET translocon (myeloid leukemia) EST similar to FGF receptor 2, 3 untranslated Unknown EST Unknown EST Unknown EST Unknown EST Unknown EST Unknown EST
−1.59 2.11 1.50 −1.59 2.12 −1.50 2.19 −2.05 −1.67 −1.48 −2.04 1.56 2.24 −1.86 −1.76 −1.87 −2.00 −2.00 −1.81 1.60 1.90 −1.86 2.05 1.69 −1.66 1.70 1.65
−1.47 1.67 1.37 −1.35 1.85 −1.38 1.63 −1.85 −1.45 −1.35 −1.82 1.42 1.92 −1.55 −1.47 −1.69 −1.75 −1.68 −1.57 1.33 1.69 −1.51 −1.78 1.42 −1.46 1.40 1.49
response nicotine arrays (Analysis IV), or to control and low response arrays combined (Analysis V). The 10 genes identified by all three of these analyses were considered the best candidates for genes whose expression changes were related to stimulation of branching and these genes are listed in Table 2. 3.2. RT–PCR analysis of selected putative nicotine-responsive genes: relationship of gene expression to stimulation of branching Two genes from the putative general response category, encoding Bcl2-associated protein X (BAX) and Cu–Zn
superoxide dismutase (SOD1), were selected for quantitative RT–PCR analysis. BAX is a pro-apoptotic mediator (for review see [15]) chosen for further study because the down-regulation of its message by nicotine, shown in the microarray data, appeared to be consistent with the observation that nicotine is anti-apoptotic in small cell lung carcinoma lines [16,17]. SOD1 is an antioxidant enzyme that dismutes superoxide anion, O2 − , to form hydrogen peroxide, H2 O2 , which is then destroyed by catalase [18]. Since the culture system is hyperoxic relative to normal fetal tissue, we hypothesized that up-regulation of SOD1 in response to nicotine might cause increased branching in the culture system as a result of moderation of oxidative stress.
Table 2 Genes possibly associated with nicotine-induced stimulation of branching GenBank accession no.
Gene name and description
Expression fold change
Low bound confidence interval
X66449 X13986 U79550 Z12171 M28845 X13297 M77497 AA921489 AF038995 AW123810
Calcium binding protein A6 (S100A6 = calcyclin) Osteopontin (minopontin, secreted phosphoprotein 1) Slugh (slug, chicken homolog) Dlk 1 (delta-like homolog, Drosophila) Early growth response 1 (Egr1, NGF I-A, Krox 24) Actin, alpha, vascular smooth muscle Cytochrome P450, 2f2 (naphthalene hydrogenase) EST similar to surfactant associated protein C Putative RNA helicase RCK (DEAD box helicase) Unknown EST
1.58 −1.84 −1.57 −2.05 −1.46 1.40 1.49 −7.20 1.92 1.71
1.45 −1.60 −1.33 −1.96 −1.26 1.26 1.28 −6.09 1.70 1.40
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Two additional genes, those encoding calcyclin and osteopontin, were chosen from among the putative branchingrelated genes. Calcyclin (S100A6) is a calcium binding protein whose function is not clearly defined. Calcyclin expression is induced as part of the serum mediated growth response, suggesting the possibility of responsiveness to growth factors [19]. Roles have also been suggested in the response of lung fibroblasts to mechanical strain, and in mucus secretion [20,21]. Calcyclin was also chosen in part because changes in intracellular calcium may be involved in signalling via nAChRs which are ligand gated cation channels. Osteopontin is a secreted, phosphorylated glycoprotein implicated in diverse processes. It is expressed in bronchial epithelium and smooth muscle cells and its proposed functions include involvement in bone development and resorption, inflammation, calcium regulation, cell adhesion, and signalling through binding to ␣v3 integrin (for review see [22,23]). The role of osteopontin in lung development is not clear. New lung culture experiments were performed to test whether the DChip analyses had identified general nicotine response genes or genes whose expression correlated with stimulation of branching by nicotine. Lung buds to be treated with nicotine were matched with lung buds of the same stage of development that were grown in control medium. At the end of each experiment, branches were counted and the lung buds were pooled into samples and separated into two groups based on whether or not the nicotine-treated lungs showed a stimulation of branching. RT–PCR analysis was performed for all four genes and results from multiple experiments were normalized by expressing the values as a percent of the average control for each experiment. For each of the four genes, the results were then combined and averaged for the control and nicotine-treated samples in each of the two groups (branching stimulated by nicotine and branching not stimulated by nicotine). The results, shown in Fig. 2, were compared with the microarray results and the relationship between stimulation of branching and changes in gene expression was assessed. Fig. 2a represents data from paired control and nicotine-treated samples showing a stimulation of branching, while Fig. 2b represents data from samples showing no such stimulation. The RT–PCR results in Fig. 2a show a significant decrease with nicotine treatment in the levels of mRNAs encoding BAX, calcyclin and osteopontin, but no change in SOD1 mRNA. Fig. 2b shows the same trends although the changes in mRNA levels are less marked and there is a loss of statistical significance. Although we designed the DChip analyses to differentiate between genes whose expression changes were associated with the nicotine stimulation of branching and genes that might respond to nicotine in a more general way, our findings do not suggest that such a dichotomy exists. The DChip analyses of the microarray data had suggested that the changes in expression of calcyclin and osteopontin messages were correlated with stimulation of branching, whereas the changes in the
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Fig. 2. RT–PCR analysis of selected individual genes: effect of nicotine treatment and relation to stimulation of branching. Lung buds were grown for 4 days in the presence or absence of nicotine and pooled into samples (3–6 lungs per sample) based on whether branching was increased (a) or not increased (b) in the nicotine-treated sample relative to control. Message levels for BAX, calcyclin, osteopontin, and SOD1 were assessed by semi-quantitative RT–PCR normalized to adjusted 18S rRNA for each sample. Shown are means ± S.D. for each gene in five to seven samples combined from several experiments by expressing the data as a percent of the average control for each experiment. (*) Nicotine-treated samples in (a) differ significantly from control for BAX (P = 0.01), calcyclin (P = 0.01), and osteopontin (P = 0.009).
levels of BAX and SOD1 mRNAs might be independent of branching. SOD1 mRNA did not change in the RT–PCR analysis, of course, and in that sense is not correlated with branching, but not in the way we had anticipated. The extent of change in BAX mRNA does not appear to be any more similar between Fig. 2a and b than is the case for calcyclin and osteopontin. The most likely interpretation now appears to be that a common mechanism is driving both the stimulation of branching and the changes in gene expression, with those nicotine-treated lungs that are the most strongly affected with respect to gene expression also being the most likely to be identifiable as showing branching stimulation. The lack of complete correspondence between the microarray and RT–PCR results was of concern. The two sets of data are in agreement for BAX and osteopontin, but SOD1 did not change in the RT–PCR analysis and calcyclin mRNA actually changed in opposite directions in the two data sets. Occasional false positives or anomalous results can be expected in any microarray study because of the large number of genes involved and the inherent complexity of biological
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systems. The opposite RT–PCR result observed for calcyclin was so unexpected, however, that it led us to consider whether there could be factors other than nicotine that could have affected expression of this gene. We considered possible confounding effects from the lung buds’ developmental program, and a hypothetical elevation of SOD1 as detailed in the following sections. 3.3. Relationship of nicotine response to developmental changes in gene expression Changes in gene expression in the high response sample might reflect a more advanced stage of development since many of these lung buds were larger and more branched at the end of the culture period than those in the other two samples. Differential gene expression in this sample might, therefore, be only indirectly related to the mechanism of nicotine action. We therefore examined the developmental patterns of expression of BAX, calcyclin and osteopontin mRNAs in lungs developing in vivo by RT–PCR. The results are presented in Fig. 3. Message levels for each gene were assessed in lungs from E12, E14, and E18 embryos/fetuses and from postnatal day 2 (P2) mouse pups. All three of these genes appeared to be developmentally regulated, but the patterns of regulation differed substantially. BAX mRNA level was down-regulated between E12 and E18 with E14 apparently representing a transitional stage. Calcyclin mRNA increased dramatically with development, with the greatest change between E14 and P2. Osteopontin mRNA increased modestly from E12 to a plateau at E14. Developmental changes and nicotine-induced changes were thus parallel only for BAX mRNA, being opposite for calcyclin and osteopontin. Taken together with the findings
Fig. 3. Developmental patterns of expression of BAX, calcyclin and osteopontin during lung development in vivo. Lungs were harvested on days 12, 14 and 18 of gestation (E12, E14, E18) and on postnatal day 2 (P2). Samples from three individuals were analyzed for each stage, except E12 for which three pooled samples of three lungs per pool were analyzed. Message levels for BAX, calcyclin, and osteopontin were assessed by semi-quantitative RT–PCR, normalized to adjusted 18S rRNA, for each sample. All calcyclin values were divided by 3 to facilitate use of a common scale. Shown are means ± S.D. Significant differences between stages were observed for all three genes (determined by ANOVA).
presented in Fig. 2, these results suggest that nicotine can perturb the developmental program of individual genes, specifically calcyclin and osteopontin. The results also suggest that developmental stage could be a confounding factor in studies of the effect of nicotine on expression of these genes. In our microarray experiment, however, the increase in calcyclin mRNA level cannot be explained in terms of the developmental program overriding the nicotine effect. If this had occurred, both calcyclin and osteopontin should have been affected, but osteopontin mRNA was not increased in the microarray data. 3.4. Effect of exogenously added SOD and interaction with nicotine While the identification of SOD1 by the microarray analysis could represent a technical false positive of unknown origin on the microarray, the fact that our results were obtained from triplicate arrays makes it relatively more likely that there was a real difference in SOD1 mRNA levels between the samples applied to the arrays. We therefore considered the possibility that SOD1 mRNA could have been elevated in the nicotine-treated samples for reasons unrelated to nicotine exposure. SOD1 activity has the potential to affect three molecules that are implicated in signalling in biological systems, superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), and nitric oxide (NO) (for review see [18]). We therefore speculated that a change in SOD1 expression could potentially have affected expression of other genes or development of the lung buds through one or more of these signalling pathways. To assess whether an elevation of SOD1 in the microarray samples could account for the increase in calcyclin mRNA in those same samples, we examined the effect of exogenously added SOD on calcyclin gene expression in the culture system. Fig. 4 shows the combined results from several such experiments. In all of the experiments,
Fig. 4. Effect of exogenous SOD on calcyclin mRNA levels in cultured lung buds. Lung buds were grown for 4 days in control medium or medium containing 150 U ml−1 SOD. Lung buds (4–5 per condition) were pooled for each condition and processed for determination of mRNA levels by semi-quantitative RT–PCR, normalized to adjusted 18S rRNA, for each sample. Shown are means ± S.D. for five samples combined from several experiments by expressing the data as a percent of the average control for each experiment.
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calcyclin mRNA was increased in the SOD-treated samples relative to control, although statistical significance was not achieved owing to the wide range in magnitude of the effect. These results are broadly consistent with the hypothesis that the elevated calcyclin mRNA seen in the nicotine-treated microarray samples could be related to the increased SOD1 mRNA that was seen in these same samples. To further explore the effects of SOD, in the context of the relationship of gene expression to branching, several experiments were performed in which exogenous SOD was added alone or co-administered with nicotine in the culture system, and the effects on gene expression were examined. The resultant effects on branching morphogenesis are shown in Fig. 5a. Both nicotine alone and SOD alone caused a significant stimulation of branching. Paradoxically, no stimu-
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lation of branching was seen when nicotine and SOD were co-administered. This suggests that nicotine and SOD act on branching by different mechanisms that interfere when activated simultaneously. The effects of these treatments on gene expression are presented in Fig. 5b–f. The results for osteopontin (Fig. 5b) mirror the effects on branching. In three experiments, osteopontin mRNA was significantly decreased following exposure to either nicotine or SOD alone. When nicotine and SOD were co-administered, however, the resulting level of osteopontin mRNA was similar to control in two of the three experiments and similar to the level obtained with SOD alone in the third. The combined results from all three experiments are shown graphed together. The first two experiments are consistent with an interference between the
Fig. 5. Effect of exogenous SOD on gene expression in cultured lung buds: interaction with nicotine. Lung buds were grown for 4 days in control medium or medium with 1 uM nicotine alone, 150 U ml−1 SOD alone, or nicotine plus SOD. Terminal branches were counted and presented as mean ± S.D. in (a) (n = 4–5). (*) Nicotine- and SOD-treated lungs had significantly more branches than control or lungs treated with nicotine plus SOD (P = 0.045 and 0.001, respectively, relative to control and 0.05 and 0.008, respectively, relative to nic + SOD). For gene expression, lung buds were pooled (3–5 per sample) and mRNA levels determined by semi-quantitative RT–PCR for osteopontin (b), calcyclin (c and e), and BAX (d and f). Values are expressed as percent of control from the same experiment. Mean ± S.D. is shown in (b) (n = 3). (*) Values for nicotine and SOD differ significantly from control (P = 0.003 and 0.01, respectively). Means with error bars representing high and low values from two experiments are shown in (c) and (f). Bar heights in (d) and (e) represent values from single experiments.
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actions of nicotine and SOD as was observed for branching (Fig. 5a), while the third suggests a lack of effect of nicotine on the lungs in the co-administration group. The results were more complex for calcyclin and BAX. For both of these genes, the effect of SOD alone was opposite to that previously observed for nicotine. In some experiments, two in the case of calcyclin (Fig. 5c) and one in the case of BAX (Fig. 5d), co-administration changed the message level seen with SOD alone back towards the control level. This pattern is again suggestive of interference between nicotine and SOD. In other cases, however, the opposite appeared to occur and there was a synergistic increase in the message level with co-administration of the two agents. Fig. 5e shows this result for calcyclin in a single experiment. For BAX, only one experiment showed a clear synergistic increase with co-administration, while in the third experiment the message levels were similar for SOD alone and SOD plus nicotine. The results of these last two experiments are graphed together (Fig. 5f). The effect on BAX expression of SOD alone was quite variable and this may have affected the interaction with nicotine with respect to this gene. We do not have an explanation of the causes of the different patterns of gene expression. We can speculate that there may be an interplay of opposing factors (see Section 4). Alternatively, there could be genetic variants within the outbred Swiss–Webster mouse population. The results for all three genes taken together are generally consistent with the interpretation that there is interaction between the effects of nicotine and SOD in this system, which can manifest as interference. The observation that both branching and osteopontin mRNA expression are affected similarly by nicotine and SOD alone, but show a partial or complete reversal of these effects upon co-administration, suggests that, at least for these two parameters, the effects of nicotine and SOD alone are probably produced through different mechanisms.
4. Discussion Our RT–PCR analysis successfully identified, from among the microarray results, three genes, BAX, calcyclin and osteopontin, whose mRNA levels appear to be influenced by nicotine exposure in embryonic lung buds developing in culture. The data further suggest that a common mechanism underlies the effect of nicotine on branching morphogenesis and on changes in expression of these genes. Further study of these genes may increase our understanding of the ways in which prenatal nicotine exposure can affect the developing lung. We believe the discrepancy between the RT–PCR and microarray data with respect to expression of calcyclin and SOD1 may reflect an unusual event that may have increased expression of SOD1 in the nicotine-treated samples applied to the microarrays. We speculate that factors affecting the expression of SOD1 might include oxidative stress and cellular
immune response or inflammation. Alternatively, there could be a genetic variant in the outbred Swiss–Webster mouse population with higher SOD1 expression. Our suggestion that elevated SOD1 could be responsible for the increase in calcyclin mRNA seen on the microarray is supported by our observation that addition of exogenous SOD tended to increase calcyclin mRNA levels. It is also supported by the observation of Hoyaux et al., who found that calcyclin was the only one of a dozen calcium binding proteins whose expression was elevated in a mouse model for familial amyotrophic lateral sclerosis bearing a gain of function mutation in SOD1 [24]. There is thus reason to believe that expression of calcyclin might be linked to SOD1 activity. We cannot, of course, rule out the possibility that whatever affected SOD or calcyclin expression in the microarray data could have also caused changes in some of the other genes in Tables 1 and 2 that we have not yet examined. Before each of these genes can be stated to be nicotine-responsive, its expression change with nicotine exposure must be independently verified. Our study of the developmental expression of the identified genes clearly shows that the effect of nicotine on gene expression in this system cannot be explained by a global stimulation of the developmental program, as the stimulation of branching might suggest. Our data indicate that nicotine can, in fact, override the developmental program in the cases of calcyclin and osteopontin gene expression. The effect of nicotine on BAX mRNA level could, however, be linked to development through an effect on developmentally programmed cell death. Heusch and Maneckjee showed that the anti-apoptotic effect of nicotine in a small cell lung carcinoma line involved an increase in expression of Bcl-2 [17]. BAX and Bcl-2 are related proteins that are thought to titrate one another with the balance of their relative expression levels determining the balance between pro- and anti-apoptotic influences in the cell (for review see [15]). This does not conflict with our results since a decrease in apoptotic potential could result from either a decrease in BAX expression or an increase in Bcl-2 expression, or from both changes occurring simultaneously. We did not independently investigate Bcl-2 in our system, and Bcl-2 is not represented on the MG-U74A microarray. The finding of an apparent interaction between nicotine and SOD is a potentially interesting observation. Although the underlying mechanism is not apparent from our studies, work by other investigators leads us to suggest a hypothetical mechanism for the effect of nicotine and SOD on branching morphogenesis involving the level of intracellular H2 O2 . Mayhan and Sharpe observed that exogenously added SOD antagonized the nicotine blockade of NO-mediated cholinergic vasodilation in the hamster cheek pouch [25], and suggested that this action of SOD was due to removal of reactive oxygen species (ROS) produced by nicotine. Recent unpublished data from the laboratory of Philip Tsao at Stanford University have indicated that nicotine can cause increased production of ROS by stimulation of the plasma membrane
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NAD(P)H oxidase complex in cultured endothelial cells. This NAD(P)H oxidase has also been demonstrated in lung fibroblasts and airway smooth muscle cells [26,27]. The O2 − produced by NAD(P)H oxidase is dismuted to H2 O2 by cellular SODs. Since such production of H2 O2 is required for growth factor signalling [18], the finding that nicotine stimulates NAD(P)H oxidase may provide a possible explanation for nicotine’s stimulation of branching and also for the interaction of nicotine and SOD. We suggest that nicotine may increase branching by potentiating growth factor signalling through increased production of intracellular H2 O2 . There is an effective concentration range of H2 O2 needed for growth factor signalling, however, with too little being ineffective and too much being toxic to the cell. This limited effective range could explain the variation that we have seen in the effect of nicotine on branching in the culture system. We know that the culture system is hyperoxic relative to fetal tissues and the positive effect of SOD on branching could therefore be due to moderation of oxidative damage through removal of ROS. The effectiveness of SOD as an antioxidant, however, depends upon the activity of catalase, since the action of SOD is to convert one ROS (O2 − ) to another (H2 O2 ), with the remainder of the antioxidant pathway being carried out by catalase [18]. An excess of H2 O2 could possibly occur if the combined effects of exogenous SOD and activation of NAD(P)H oxidase by nicotine were sufficient to overwhelm the system’s catalase activity since H2 O2 produced in the culture medium by added SOD could presumably diffuse into the tissue. The toxic effects of excess H2 O2 could explain the apparent interfering action of nicotine and SOD with respect to branching. There is a substantial literature on the involvement of ROS in apoptosis (for review see [18,28]). Since BAX is a proapoptotic mediator, it is possible that elevations of BAX mRNA in our system could reflect activation of apoptosis. We observed increases in BAX mRNA sometimes when SOD was added alone to the cultures and sometimes when nicotine and SOD were added together. Pervaiz and Clement have proposed that apoptosis can be triggered when there is a shift in the balance of ROS in the cell from O2 − to H2 O2 . Since added SOD could be expected to have this effect on the system, it is easy to imagine that adding SOD could trigger apoptosis, depending upon the extent of catalase action. Adding nicotine, which may generate O2 − , could make the situation worse in the presence of excess SOD activity. The interplay of the various effects of nicotine and SOD on growth and cell death could be quite complex and variation in the observed responses would not be surprising. In conclusion, three putative nicotine-responsive genes, BAX, calcyclin and osteopontin, were identified by microarray and RT–PCR analysis in embryonic mouse lung buds developing in culture. It appears that a common mechanism may underlie the nicotine effects on both gene expression and branching morphogenesis in this system. Expression of
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all three genes was developmentally regulated and also found to be affected by exogenously administered SOD, with patterns that differed from that of the nicotine response. Finally, our evidence suggests that the mechanisms underlying the effects of nicotine and SOD can interfere with one another when activated simultaneously. More studies are planned to better define the biology of the nicotine responses, including further examination of inbred strains of mice and use of an in utero nicotine exposure model.
Acknowledgements This work was supported by the California TobaccoRelated Disease Research Program of the University of California, grant nos. 4KT-0339 and 9RT-0238, and by NHLBI grant nos. HL54960 and K02-HL03786. We thank Dr. David Warburton for the generous gift of microarrays. We acknowledge Betty Schaub and the Childrens Hospital, Los Angeles Microarray Core facility for preparation and hybridization of the microarray samples, and Dr. Hosun Cheong for many helpful discussions.
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