Nicotine exposure during differentiation causes inhibition of N-myc expression
© Ben-Yehudah et al.; licensee BioMed Central Ltd. 2013
Received: 25 January 2013
Accepted: 11 October 2013
Published: 5 November 2013
The ability of chemicals to disrupt neonatal development can be studied using embryonic stem cells (ESC). One such chemical is nicotine. Prenatal nicotine exposure is known to affect postnatal lung function, although the mechanisms by which it has this effect are not clear. Since fibroblasts are a critical component of the developing lung, providing structure and secreting paracrine factors that are essential to epithelialization, this study focuses on the differentiation of ESC into fibroblasts using a directed differentiation protocol.
Fibroblasts obtained from non-human primate ESC (nhpESC) differentiation were analyzed by immunohistochemistry, immunostaining, Affymetrix gene expression array, qPCR, and immunoblotting.
Results of these analyses demonstrated that although nhpESCs differentiate into fibroblasts in the presence of nicotine and appear normal by some measures, including H&E and SMA staining, they have an altered gene expression profile. Network analysis of expression changes demonstrated an over-representation of cell-cycle related genes with downregulation of N-myc as a central regulator in the pathway. Further investigation demonstrated that cells differentiated in the presence of nicotine had decreased N-myc mRNA and protein expression and longer doubling times, a biological effect consistent with downregulation of N-myc.
This study is the first to use primate ESC to demonstrate that nicotine can affect cellular differentiation from pluripotency into fibroblasts, and in particular, mediate N-myc expression in differentiating ESCs. Given the crucial role of fibroblasts throughout the body, this has important implications for the effect of cigarette smoke exposure on human development not only in the lung, but in organogenesis in general.
KeywordsEmbryonic stem cells Directed differentiation Lung development Nicotine Cell cycle
Maternal exposure to nicotine is known to lead to alterations in postnatal lung function. Nicotine freely passes through the placenta and can accumulate in amniotic fluid [1, 2]. Studies using non-human primates have shown that nicotine exposure during development leads to the overexpression of some nicotinic acetylcholine receptors (nAChR) as well as some specific genes and proteins such as collagens in the developed lung [3–5]. In addition, pulmonary function assessments of non-human primates exposed to prenatal nicotine demonstrated decreased forced expiratory flows that mimic the decreased forced expiratory flows measured in human infants who were exposed to maternal cigarette smoke , implying that among the many chemical present in cigarette smoke with the potential to affect development, nicotine may have a significant role in the decreased pulmonary function in infants exposed to maternal smoking. However, the mechanisms by which nicotine affects lung development are not well understood.
The ability to use embryonic stem cells (ESCs) as tools for the study of developmental biology and, in particular, as sensors to determine the adverse effects of chemical exposures during development was recognized even before primate embryonic stem cells were first derived . ESCs are now in use for the study of developmental cardiotoxicity and neurotoxicity [7–10], since studies of the earliest stages of embryonic development and the effect of environmental exposures are difficult in animals and impossible in humans. Directed-differentiation protocols shed insight into these early stages by allowing investigation of differentiation of ESCs as they move towards more mature cell types ( for review). As protocols for differentiating ESCs become more established, we can move our focus to using cells in these models to examine the effects of chemical exposures during the differentiation process. In this study, we focus on the differentiation of ESC into fibroblasts. Fibroblasts are a critical component of the lung and direct epithelialization of the lung through paracrine and other methods, and prenatal nicotine exposure has been demonstrated to increase collagen and airway wall thickness, affecting airway resistance . Thus, changes in fibroblast phenotype during development may affect lung function after birth.
Non-human primate embryonic stem cells (nhpESC) are available [12–21] and provide an opportunity to correlate in vivo physiologic data from previous studies with analysis of intracellular signaling. We have published a series of related nhpESC lines derived from Rhesus macaca[12, 22]. These lines are more than 95% identical in their gene expression patterns, making them ideal for array analysis of gene expression [22, 23]. In this study we use these lines as sensors to assess alterations in differentiation that result from nicotine exposure.
NAChR have been shown to be expressed on many cell types, and the endogenous ligand, acetylcholine, is expressed ubiquitously (reviews by [24–30]). Nicotine exposure in vitro leads to alterations in the differentiation of keratinocytes [31, 32] and causes release of chromogranin A from pheochromocytoma cells . Thus, nicotine has been previously demonstrated to affect cellular differentiation processes in some somatic tissues, although neither nhpESC nor hESC have been examined for the expression of nAChR or for the effect of nicotine on the differentiation process. In this study, the expression of nAChR was confirmed in nhpESC, and directed differentiation of nhpESC into fibroblasts was carried out in the presence or absence of nicotine. Microarray analysis of the resulting differentiated cells includes a number of previously unreported targets of nicotine regulation. These changes are specific to the differentiation process, and alterations of these genes by nicotine during development could contribute to the decreased lung function seen in infants exposed to maternal nicotine.
All experiments involving animals were approved by the Institutional Animal Care and Use Committees (IACUCs) from the Magee-Womens Research Institute and the University of Pittsburgh and experiments involving embryonic stem cells were approved by the University of Pittsburgh Human Stem Cell Research Oversight (hSCRO) committee.
NhpESC cell lines were a generous gift from Gerald Schatten (U. Pittsburgh). NhpESC lines were previously described (, personal communication). Primary adult non-human primate lung fibroblasts were obtained during necropsy by taking tissue biopsies. Tissues were minced and grown in DMEM with 15% serum, 1% penicillin/streptomycin, and 2 mM L-glutamine.
NhpESCs were cultured in 80% KO-DMEM (Invitrogen, Carlsbad, CA), 20% Knockout Serum Replacement (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), and 4 ng/ml basic human recombinant FGF (Invitrogen). NhpESCs were grown on inactivated mouse embryonic feeder cells (MEF’s) and manually passaged weekly. Media was replaced every 24 hours .
Pluripotency marker expression
NhpESCs were fully characterized to demonstrate pluripotency using a teratoma assay . To ensure nhpESCs maintained a pluripotent phenotype throughout the experiments, cells were stained for the positive pluripotency markers: Oct-4, SSEA-4, Tra 1–80 and Tra 1–61, as well as the negative nhpESC marker SSEA-1. Immunocytochemistry was performed as follows: Culture dishes containing undifferentiated colonies were fixed by addition of either 100% methanol at -20°C for 15 min, or 2% paraformaldehyde in PBS for 40 minutes followed by a 15 min wash in PBS + 1% Triton X-100 (PBS-Tx, Sigma, St. Louis MO). After fixation, non-specific binding of the primary and secondary antibodies was blocked by 30 minutes incubation in PBS with 0.5% goat serum. Primary antibodies were diluted in PBS-Tx and incubated on the coverslips for 40 min at 37°C in a humidified chamber, with the exception of Oct-4 which was incubated overnight at 4°C. Primary antibodies were detected with fluorescently labeled appropriate secondary antibodies. DNA was detected with 5 μM TOTO-3 (Molecular Probes, Eugene OR). Coverslips were inverted onto slides and mounted in Vectashield anti-fade medium (Vector Labs, Burlingame, NH) to prevent photobleaching .
In Vitro differentiation
Colonies were manually passaged and dissociated into small clusters. They were cultured in non-adherent dishes to form embryoid bodies in differentiation medium (80% KO-DMEM, 1 mM L-glutamine, 20% FBS, 1% non-essential amino acids). After 4 days in suspension, aggregates were transferred onto gelatin coated culture dishes and cultured for an additional 9 days in differentiation medium. Outgrowth cultures were manually passaged by scraping cells with a differentiated phenotype from the periphery of the colonies, and these differentiated cells were placed in a new culture plate in differentiation medium. After the first passage, cells were grown in standard fibroblast media (90% KO-DMEM, 10% FBS, 1% non-essential amino acids, 2 mM L-glutamine) and passaged at 80% confluency using trypsin. Cells were banked at each passage and were cultured until they stopped growing or through passage 10. Experimental groups were treated with 100 nM nicotine starting on day 1 of the differentiation protocol.
PCR for nAChR
RT-PCR for nAChR was done using primer sequences that have been previously published . All primers span introns and do not amplify DNA. GAPDH or actin was always used as a positive control for RNA integrity. Oligo dT12-18 (Invitrogen) was annealed to 1 μg total RNA and reverse transcribed with Superscript II (Invitrogen). The reaction contained 1 μg RNA, 500 ng Oligo dT12-18, 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each dNTP, 200 U superscript. Briefly, total RNA was incubated with oligo dT12-18 at 70.0°C for 10 min. The cDNA produced was then used as a template for PCR using specific primers. PCR amplification was performed in a 20 μl reaction containing 2 μl of the RT reaction, Taq DNA polymerase (Perkin Elmer), 1X PCR buffer, 1.5 mM MgCl2, 1 mM each dNTP and 1 μM primer. PCR was carried out in a Perkin-Elmer 9700 Thermocycler with 2 min, 95.0°C denaturation, followed by 30 cycles of 94.0°C for 30 s, 55.0-62.0°C (see ) for 30 s and 72.0°C for 30 s. Final extension was at 72.0°C for 5 min. 10 μl of each reaction was run on a 1% TBE gel for analysis. β2 and δ subunits were detected using nested PCR. Primary PCR reactions were carried out as described above. 2 μl of the primary reaction was used as the template for the secondary PCR reaction/second round PCR. Thirty rounds of PCR were carried out.
Differentiated fibroblasts were tested for their ability to form teratomas in the testes of mice. The analysis was done at the MWRIF Transgenic Core Facility according to their standard methods [12, 22, 35–38]. Pathology on the testes was performed by the MWRIF Histology Research Core Facility.
Gene expression analysis
Gene expression analysis was performed by the DNA Analysis group at the University of Pittsburgh Genomics and Proteomic Core Laboratory using cell pellets provided by D. Carlisle’s laboratory as described herein. RNA was isolated from in vitro differentiated fibroblasts from three different nhpESC lines, each differentiated in the presence and absence of nicotine: nhpESC2706, nhpESC3106 and nhpESC4706. Three technical replicates were done with each cell line and condition, and cultures with and without nicotine were matched for passage number after differentiation. RNA Isolation: RNA was purified using a modified Trizol extraction method (Invitrogen, Carlsbad, CA). Briefly, suspended cells were extracted in 1 mL of Trizol with the addition of 200 μg of GlycoBlue (Ambion Inc, Austin, TX) added to each sample as a nucleotide carrier. After aqueous phase separation, the samples were incubated overnight at 20°C in 500 μl of isopropanol (100%) to precipitate the RNA. The RNA was then pelleted by centrifugation (14,000 g × 15 min), washed in 1 mL of 75% ethanol, and resuspended in 20 μl nuclease-free water at 45°C for 5 minutes. The RNA concentration and quality was evaluated with criteria for inclusion in subsequent in vitro transcription (IVT) assays comprising spectrophotometric absorption ratio of 260/280 >1.8 (NanoDrop, Wilmington, DE) and a RIN value of >8.0 via electrophoretic analysis (Agilent Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA). Affymetrix Eukaryotic Target Preparation and Hybridization: In vitro transcription was performed using the Ambion Message Amp II-Biotin Enhanced Assay protocol starting with 100 ng of purified total RNA. Confirmation of cRNA diversity was obtained using the Bioanalyzer 2100 to generate an electrophoretogram for each IVT reaction regarding sample yield, integrity, and size diversity against a Universal Human Reference RNA (Stratagene, La Jolla, CA). Fifteen micrograms of purified, biotin labeled cRNA was fragmented and hybridized on Rhesus Macaque Whole Genome Arrays (Affymetrix Corp., Santa Clara, CA) for 18 hours. Washing, staining and scanning of arrays were performed on the Fluidics Station 450 and Scanner 3000 immediately after completion of hybridization. Microarray data was processed using GeneChip Operating Software (GCOS, Affymetrix) with signal intensity calculated by Microarray Suite version 5.0 (MAS 5.0).
Differential gene expression analysis was performed in consultation with the University of Pittsburgh Genomics Analysis Services using BRB Array Tools from NCI (http://linus.nci.nih.gov/BRB-ArrayTools.html), and genes were selected at p < 0.001 (Additional file 1: Table S1). Statistical analysis of qPCR and immunoblotting was done by t-test, and analysis of slope for doubling time was done using a two-factor ANOVA for interaction on log-transformed cell counts.
Cells were pelleted by centrifugation at 200 × g for 5 min. RNA was isolated using Trizol (Invitrogen). Briefly, 1 ml of Trizol was added per cell culture dish and vortexed to lyse cells to homogeneity. 200 μl of chloroform (Sigma) was added followed by mixing and centrifugation for five min at 25,000 × g. The RNA containing supernatant was removed and the RNA pelleted using 600 μl of 100% isopropanol added to the supernatant and incubated at -20°C for at least 4 hrs. The sample was centrifuged twice at 13000 × g for 30 min at 4°C, with an ethanol wash in between, followed by air-drying. The RNA pellet was reconstituted in nuclease free water and treated with 1 μl DNase I for 30 min at 37°C. cDNA was prepared using the Improm-II Reverse Transcription System (Promega, Madison WI), according to manufacturer’s directions. Validated primer sets were purchased from Applied Biosystems and were used on an ABI 7900HT real-time PCR machine. Real-time data was analyzed and fold change was calculated using the comparative CT method with either beta-actin or GADPH as the internal control .
Protein isolation and immunoblotting
Proliferating cells at passage 8, 9, or 13 were scraped from flasks in ice cold PBS/Phosphatase Inhibitors. Cell pellets were lysed in RIPA Buffer supplemented with a Protease Inhibitor Cocktail (Roche). The lysate was centrifuged for 20 minutes at 14,000 × g and 4°C in a precooled microcentrifuge. Protein concentrations of the clarified lysates were quantitated using the BCA Assay Kit (Thermo Scientific) and a BSA standard curve following the manufacturer’s instructions. Protein samples were stored at -80°C. Samples for Western blot analysis were diluted in 4 × Laemmli buffer (BioRad) supplemented with 10% β-mercaptoethanol to a concentration of 10 μg of protein in 20 μL. Samples were heated at 55°C for 5 minutes and were separated on 10% polyacrylamide gels by SDS-PAGE and transferred to Immobilon-FL PVDF membranes (Millipore) by BioRad wet transfer. The membranes were blocked with LiCor Odyssey blocking buffer for 1 hour at room temperature and were then probed with a primary N-myc antibody (NCM II 100, Millipore) diluted 1:100 in LiCor Odyssey blocking buffer with 0.05% Tween. Membranes were washed three times for 15 minutes each in PBS supplemented with 0.05% Tween (TBST) and were subsequently probed with goat anti-mouse IR-Dye 800cw labeled secondary antibody diluted 1:30000 in LiCor Odyssey blocking buffer. Washes were repeated after labeling with secondary antibody three times in PBST and then once in PBS. Membranes were imaged using LiCor scanner. Membranes were stripped using LiCor NewBlot stripping buffer for 15 minutes at room temperature. Membranes were reprobed with mouse anti-β-actin diluted 1:10000 in LiCor Odyssey blocking buffer with 0.05% Tween followed by washes and probing with the LiCor secondary antibody, then washed and imaged as described above.
Analysis of culture doubling time
For growth analysis, cells were plated in triplicate in 6-well plates (Corning Life Sciences, Corning, NY) at a concentration of 2 × 105 cells/well. After 2, 3, 4, 5, 6, and 7 days, cells were trypsinized and counted using the Countess Automated Cell Counter (Invitrogen). Cell counts were analyzed by logistic regression and doubling times were calculated using http://www.doubling-time.com/compute.php.
Nicotine receptors are expressed on stem cells before and after differentiation
Expression of stem cell markers after in vitro differentiation
Differentiated fibroblasts do not form teratomas
Although the parental undifferentiated nhpESC were able to form teratomas (; personal communication), the hallmark of pluripotent cells, we confirmed that in vitro differentiated fibroblasts cannot form teratomas, using the same Core facility and methods. Cells were injected into SCID mice in 6 separate injections for a 0% success rate, comparing with an historical success rate of 92% for teratoma formation after injection of pluripotent stem cells in the Core facility . Histological analysis of the injected testes four or more months after injection showed an apparent injection site with some inflammation, but no evidence of tumor formation (Additional file 1: Figure S1). Therefore, we conclude that these cells are differentiated and do not retain the ability to form teratomas.
Nicotine increases doubling time of differentiated nhpESC
The acetylcholine signaling pathway has been established as functional in mouse ESC (mESC), and publications have demonstrated that mESC express choline acetyltransferase and secrete acetylcholine into the culture medium [44–46]. There are currently a few studies that examine the effect of nicotine on ESC culture and differentiation; however, they have conflicting results. Nicotine increases the expression of OCT4 and Rex1 in undifferentiated mESC . However, in a separate study, nicotine inhibited attachment of undifferentiated hESC to matrigel and led to a corresponding decrease in OCT4 staining, although the cells remained pluripotent . Thus, the effect of nicotine on the expression of pluripotency markers in undifferentiated cells is unclear. Related studies have examined induced pluripotent stem cells (iPSC) from mice, and these studies found that nicotine increased undifferentiated stem cell proliferation [49, 50].
In this study, we document for the first time the presence of nAChR on primate pluripotent stem cells. Since ESC differentiation can serve as a model for development, these results are a critical discovery, because they imply that with the presence of these receptors it is possible for maternal nicotine to affect the earliest stages of embryonic development, including the ability to differentiate into all the cell types in the body.
During active smoking, smokers have been shown to obtain a nicotine concentration of 100 nM in the serum . In addition, smokers have a minimum serum-nicotine concentration that they maintain throughout the day, which has been shown to average 100 nM but ranges between 10 nM and 200 nM for light and heavy smokers, respectively . Furthermore, since nicotine is known to cross the placenta, this dose may also be relevant to fetal exposure . Our data show that nAChR are expressed on nhpESC, therefore, we tested the ability of a physiologically-relevent dose of nicotine, 100 nM, to affect differentiation. Using an in vitro differentiation method, we found that nhpESCs differentiated into fibroblasts in the presence of nicotine do not have any obvious differences in cell appearance (Figures 3 and 4); however, they show significant differences in gene expression patterns especially with respect to cell cycle-related genes, most notably N-myc. N-myc is decreased in expression in the differentiated fibroblasts. This effect is most dramatic in the early passages after differentiation (passages 1–4); and in some experiments remained significantly decreased through the final passage examined, passage 10. The decreased expression of N-myc is not mimicked by long-term exposure of adult lung fibroblasts to nicotine (Figure 7). These expression differences are unique to nhpESCs differentiated in the presence of nicotine, and are not changed in adult NHP fibroblasts passaged in nicotine for an equivalent period of time. This implies that they are disregulated during differentiation, and this disregulation is maintained multiple passages after differentiation.
The effects of nicotine exposure on adult fibroblasts has been studied previously [34, 53] and many others have examined the effect of nicotine on bronchial epithelial cells and lung cancer cells [54–62]. In the lung, normal fibroblasts and epithelium express functional nAChR and these receptors are overexpressed in lung cancer [34, 54]. Signaling through these receptors in the lung leads to activation of signaling pathways consistent with lung cancer, including the MAPKs and AKT [34, 63]. In addition, short term experiments, including those up to 48 hours, indicated that bronchial epithelial cells increased expression of nAChR and have increased nicotinic signaling after exposure  and fibroblasts increase fibronectin and nAChR expression . Thus, both in vivo and in vitro studies show that nAChR have an endogenous function in the lung, and that exposure to nicotine alters characteristics of the lung epithelium and the supporting fibroblasts. A murine lung explant model has also been used to examine nicotine toxicity, whereby embryonic lungs were isolated from normal mice halfway through gestation and then exposed to nicotine in culture . However, the published data, whereby lung explants, which are composed of already differentiated (although immature) cells, are exposed to nicotine after they are placed in culture, are not likely to reflect the effects of nicotine on the differentiation process. Thus, none of these in vivo or in vitro studies can model the effect of nicotine on the differentiation process itself, since these cells are already differentiated at the time that they are exposed.
Several cell cycle genes were significantly different between nicotine exposed and unexposed groups in this study. The determination that N-myc was decreased in expression was particularly interesting due to its role in lung organogenesis. N-myc has been established as an essential regulator of lung organogenesis using murine developmental models [64–68]. Our data suggest that nicotine downregulates N-myc through intracellular signaling initiated by the binding of nicotine to the muscle-type and/or α7 neuronal nAchR. Our data also show that nhpESC cultures differentiated into fibroblasts in the presence of nicotine have a slower doubling time, indicative of either a slower proliferation rate or an increased cell death rate in the culture. This data, together with publications that have shown that N-myc is a mediator of both cell proliferation and cell death, suggest that as a result of nicotine inhibition of N-myc expression, fibroblasts proceed through the cell cycle at a significantly slower pace than do fibroblasts that are not exposed to nicotine during differentiation.
In the lung, prenatal cigarette smoke exposure causes decreased lung function at birth and into childhood [69–74] as well as increased incidence of asthma and allergies . The mechanisms by which cigarette smoke has these effects are not known; however, it is modeled in non-human primates exposed during pregnancy to nicotine. Studies find that although there are thousands of chemicals in cigarette smoke, NHP infants subjected to nicotine alone via a maternal exposure during development have increased thickness of the inner airway wall and altered complexity of branching, decreased respiratory volumes, and altered response to airway challenges [4, 5, 40, 75]. The ability of nicotine to alter N-myc signaling in nhpESC may be one mechanism by which maternal cigarette smoking adversely affects embryonic lung development, leading to disease later in life.
Fetal bovine serum
Fibroblast growth factor
Human embryonic stem cell
Knockout dulbeccos modified eagle’s medium
Mouse embryonic fibroblast
Nicotinic acetylcholine receptor
nOn-human primate embryonic stem cell
Phosphate buffered saline
Quantitative real-time polymerase chain reaction
Reverse transcription polymerase chain reaction
We would like to thank Carrie Redinger, Jocelyn Mich-Basso, Christopher Navara, and Gerald Schatten for generously providing nhpESC lines, technical expertise, helpful discussions, and general support for the project. We thank William Framboise and Christin Sciulli of the Genomics Core Faciltiy of U. Pittsburgh, Uma Chandran of the Genomics Analysis Services at U. Pittsburgh and Kyle Orwig of the MWRIF Transgenic Core Facility for their assistance.
Grant Support: This work was supported by the Flight Attendants’ Medical Research Institute (FAMRI) #42232 YCSA Award, NIH R01HL10277, and PA Department of Health CURE grant to D.Carlisle; and NIH P01 HD047675 to G. Schatten.
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